WO2001051632A9 - Odorant receptor polypeptides and nucleic acids encoding same - Google Patents

Odorant receptor polypeptides and nucleic acids encoding same

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Publication number
WO2001051632A9
WO2001051632A9 PCT/US2001/001513 US0101513W WO0151632A9 WO 2001051632 A9 WO2001051632 A9 WO 2001051632A9 US 0101513 W US0101513 W US 0101513W WO 0151632 A9 WO0151632 A9 WO 0151632A9
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WIPO (PCT)
Prior art keywords
ofthe
polypeptide
nucleic acid
seq
novx
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PCT/US2001/001513
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French (fr)
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WO2001051632A3 (en
WO2001051632A2 (en
Inventor
Muralidhar Padigaru
Sudhirdas K Prayaga
Raymond J Taupier Jr
Vishnu Mishra
Velizar T Tchernev
Kimberley A Spytek
Li Li
Original Assignee
Curagen Corp
Muralidhar Padigaru
Sudhirdas K Prayaga
Raymond J Taupier Jr
Vishnu Mishra
Velizar T Tchernev
Kimberley A Spytek
Li Li
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Application filed by Curagen Corp, Muralidhar Padigaru, Sudhirdas K Prayaga, Raymond J Taupier Jr, Vishnu Mishra, Velizar T Tchernev, Kimberley A Spytek, Li Li filed Critical Curagen Corp
Priority to AU2001227925A priority Critical patent/AU2001227925A1/en
Publication of WO2001051632A2 publication Critical patent/WO2001051632A2/en
Publication of WO2001051632A3 publication Critical patent/WO2001051632A3/en
Publication of WO2001051632A9 publication Critical patent/WO2001051632A9/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention generally relates to nucleic acids and polypeptides encoded therefrom.
  • odor detection is a universal tool used for social interaction, predation, and reproduction.
  • Chemosensitivity in vertebrates is modulated by bipolar sensory neurons located in the olfactory epithelium, which extend a single, highly arborized dendrite into the mucosa while projecting axons to relay neurons within the olfactory bulb.
  • the many ciliae on the neurons bear odorant (or olfactory) receptors (ORs), which cause depolarization and formation of action potentials upon contact with specific odorants.
  • ORs may also function as axonal guidance molecules, a necessary function as the sensory neurons are normally renewed continuously through adulthood by underlying populations of basal cells.
  • Odorant receptors are believed to be encoded by an extremely large subfamily of G protein-coupled receptors. These receptors share a 7-transmembrane domain structure with many neurotransmitter and hormone receptors and are likely to underlie the recognition and G-protein- mediated transduction of odorant signals and possibly other chemosensing responses as well.
  • the genes encoding these receptors are devoid of introns within their coding regions.
  • Schurmans and co-workers cloned a member of this family of genes, OLFR1, from a genomic library by cross-hybridization with a gene fragment obtained by PCR. See Schurmans et al, Cyto genet. Cell Genet.. 1993, 63(3):200. By isotopic in situ hybridization, they mapped the gene to 17pl3- pl2 with a peak at band 17pl3. A minor peak was detected on chromosome 3, with a maximum in the region 3ql3-q21. After Mspl digestion, a restriction fragment length polymorphism (RFLP) was demonstrated.
  • RFLP restriction fragment length polymorphism
  • the OLFR genes in the cluster belong to 4 different gene subfamilies, displaying as much sequence variability as any randomly selected group of OLFRs. This suggested that the cluster may be one of several copies of an ancestral OLFR gene repertoire whose existence may have predated the divergence of mammals. Localization to 17pl3.3 was performed by fluorescence in situ hybridization as well as by somatic cell hybrid mapping.
  • OR genes cloned in different species were from disparate locations in the respective genomes.
  • the human OR genes on the other hand, lack introns and may be segregated into four different gene subfamilies, displaying great sequence variability. These genes are primarily expressed in olfactory epithelium, but may be found in other chemoresponsive cells and tissues as well.
  • OL2 polymerase chain reaction
  • PCR analysis reveals that the transcript is present mainly in the rat spleen and in a mouse insulin- secreting cell line (MIN6). No correlation was found between the tissue distribution of OL2 and that ofthe olfaction-related GTP-binding protein Golf alpha subunit.
  • Olfactory loss may be induced by trauma or by neoplastic growths in the olfactory neuroepithelium. There is currently no treatment available that effectively restores olfaction in the case of sensorineural olfactory losses. See Harrison's Principles of Internal Medicine, 14 th Ed., Fauci, AS et al. (eds.), McGraw-Hill, New York, 1998, 173. There thus remains a need for effective treatment to restore olfaction in pathologies related to neural olfactory loss.
  • the invention is based, in part, upon the discovery of novel polynucleotide sequences encoding novel polypeptides.
  • the invention provides an isolated nucleic acid molecule that includes the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can include, e.g. , a nucleic acid sequence encoding a polypeptide at least 85% identical to a polypeptide that includes the amino acid sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • the nucleic acid can be, e.g., a genomic DNA fragment, or a cDNA molecule.
  • Also included in the invention is a vector containing one or more ofthe nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein.
  • the invention is also directed to host cells transformed with a vector comprising any of the nucleic acid molecules described above.
  • the invention includes a pharmaceutical composition that includes a NOVX nucleic acid and a pharmaceutically acceptable carrier or diluent.
  • the invention includes a substantially purified NOVX polypeptide, e.g., any ofthe NOVX polypeptides encoded by an NONX nucleic acid, and fragments, homologs, analogs, and derivatives thereof.
  • the invention also includes a pharmaceutical composition that includes an ⁇ ONX polypeptide and a pharmaceutically acceptable carrier or diluent.
  • the invention provides an antibody that binds specifically to an
  • the antibody can be, e.g., a monoclonal or polyclonal antibody, and fragments, homologs, analogs, and derivatives thereof.
  • the invention also includes a pharmaceutical composition including ⁇ ONX antibody and a pharmaceutically acceptable carrier or diluent.
  • the invention is also directed to isolated antibodies that bind to an epitope on a polypeptide encoded by any ofthe nucleic acid molecules described above.
  • the invention also includes kits comprising any ofthe pharmaceutical compositions described above.
  • the invention further provides a method for producing an NONX polypeptide by providing a cell containing an ⁇ ONX nucleic acid, e.g., a vector that includes an ⁇ OVX nucleic acid, and culturing the cell under conditions sufficient to express the ⁇ ONX polypeptide encoded by the nucleic acid.
  • the expressed ⁇ ONX polypeptide is then recovered from the cell.
  • the cell produces little or no endogenous ⁇ ONX polypeptide.
  • the cell can be, e.g., a prokaryotic cell or eukaryotic cell.
  • the invention is also directed to methods of identifying an ⁇ ONX polypeptide or nucleic acid in a sample by contacting the sample with a compound that specifically binds to the polypeptide or nucleic acid, and detecting complex formation, if present.
  • the invention further provides methods of identifying a compound that modulates the activity of an ⁇ ONX polypeptide by contacting an ⁇ ONX polypeptide with a compound and determining whether the ⁇ ONX polypeptide activity is modified.
  • the invention is also directed to compounds that modulate ⁇ ONX polypeptide activity identified by contacting an ⁇ OVX polypeptide with the compound and determining whether the compound modifies activity ofthe ⁇ ONX polypeptide, binds to the ⁇ OVX polypeptide, or binds to a nucleic acid molecule encoding an ⁇ OVX polypeptide.
  • the invention provides a method of determining the presence of or predisposition of an ⁇ OVX-associated disorder in a subject.
  • the method includes providing a sample from the subject and measuring the amount of ⁇ OVX polypeptide in the subject sample.
  • the amount of ⁇ OVX polypeptide in the subject sample is then compared to the amount of ⁇ OVX polypeptide in a control sample.
  • An alteration in the amount of ⁇ OVX polypeptide in the subject protein sample relative to the amount of ⁇ OVX polypeptide in the control protein sample indicates the subject has a tissue proliferation-associated condition.
  • a control sample is preferably taken from a matched individual, i.e., an individual of similar age, sex, or other general condition but who is not suspected of having a tissue proliferation-associated condition.
  • the control sample may be taken from the subject at a time when the subject is not suspected of having a tissue proliferation-associated disorder.
  • the ⁇ OVX is detected using an ⁇ OVX antibody.
  • the invention provides a method of determining the presence of or predisposition of an ⁇ OVX-associated disorder in a subject.
  • the method includes providing a nucleic acid sample, e.g., R ⁇ A or D ⁇ A, or both, from the subject and measuring the amount of the ⁇ OVX nucleic acid in the subject nucleic acid sample.
  • the amount of ⁇ OVX nucleic acid sample in the subject nucleic acid is then compared to the amount of an NOVX nucleic acid in a control sample.
  • An alteration in the amount of NOVX nucleic acid in the sample relative to the amount of NOVX in the control sample indicates the subject has a NOVX-associated disorder.
  • the invention provides a method of treating or preventing or delaying an NOVX-associated disorder.
  • the method includes administering to a subject in which such treatment or prevention or delay is desired an NOVX nucleic acid, an NOVX polypeptide, or an NOVX antibody in an amount sufficient to treat, prevent, or delay a NOVX- associated disorder in the subject.
  • Olfactory receptors are the largest family of G-protein-coupled receptors (GPCRs) and belong to the first family (Class A) of GPCRs, along with catecholamine receptors and opsins.
  • GPCRs G-protein-coupled receptors
  • Class A the first family of GPCRs
  • the OR family contains over 1,000 members that traverse the phylogenetic spectrum from C. elegans to mammals. ORs most likely emerged from prototypic GPCRs several times independently, extending the structural diversity necessary both within and between species in order to differentiate the multitude of ligands. Individual olfactory sensory neurons are predicted to express a single, or at most a few, ORs.
  • ORs are believed to contain seven ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus.
  • the pocket of OR ligand binding is expected to be between the second and sixth transmembrane domains ofthe proteins.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%, and genes greater than 80% identical to one another at the amino acid level are considered to belong to the same subfamily.
  • ORs Since the first ORs were cloned in 1991, outstanding progress has been made into their mechanisms of action and potential dysregulation during disease and disorder. It is understood that some human diseases result from rare mutations within GPCRs. Drug discovery avenues could be used to produce highly specific compounds on the basis of minute structural differences of OR subtypes, which are now being appreciated with in vivo manipulation of OR levels in transgenic and knock-out animals. Furthermore, due to the intracellular homogeneity and ligand specificity of ORs, renewal of specific odorant-sensing neurons lost in disease or disorder is possible by the introduction of individual ORs into basal cells. Additionally, new therapeutic strategies may be elucidated by further study of so-called orphan receptors, whose ligand(s) remain to be discovered.
  • OR proteins bind odorant ligands and transmit a G-protein-mediated intracellular signal, resulting in generation of an action potential.
  • the accumulation of DNA sequences of hundreds of OR genes provides an opportunity to predict features related to their structure, function and evolutionary diversification. See Pilpel Y, etal., Essays Biochem 1998;33:93-104.
  • the OR repertoire has evolved a variable ligand-binding site that ascertains recognition of multiple odorants, coupled to constant regions that mediate the cAMP-mediated signal transduction.
  • the cellular second messenger underlies the responses to diverse odorants through the direct gating of olfactory-specific cation channels.
  • ORs odorant receptors
  • ORs are expressed by nasal olfactory sensory neurons, and each neuron expresses only 1 allele of a single OR gene.
  • different sets of ORs are expressed in distinct spatial zones. Neurons that express the same OR gene are located in the same zone; however, in that zone they are randomly interspersed with neurons expressing other ORs.
  • the cell chooses an OR gene for expression, it may be restricted to a specific zonal gene set, but it may select from that set by a stochastic mechanism.
  • Proposed models of OR gene choice fall into 2 classes: locus-dependent and locus-independent.
  • Locus-dependent models posit that OR genes are clustered in the genome, perhaps with members of different zonal gene sets clustered at distinct loci. In contrast, locus-independent models do not require that OR genes be clustered.
  • OR genes have been mapped to 11 different regions on 7 chromosomes. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Studies have shown that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones.
  • Issel-Tarver and Rine characterized 4 members ofthe canine olfactory receptor gene family.
  • the 4 subfamilies comprised genes expressed exclusively in olfactory epithelium.
  • Analysis of large DNA fragments using Southern blots of pulsed field gels indicated that subfamily members were clustered together, and that two ofthe subfamilies were closely linked in the dog genome.
  • Analysis ofthe four olfactory receptor gene subfamilies in 26 breeds of dog provided evidence that the number of genes per subfamily was stable in spite of differential selection on the basis of olfactory acuity in scent hounds, sight hounds, and toy breeds.
  • Issel-Tarver and Rine performed a comparative study of four subfamilies of olfactory receptor genes first identified in the dog to assess changes in the gene family during mammalian evolution, and to begin linking the dog genetic map to that of humans.
  • These four families were designated by them OLFl, OLF2, OLF3, and OLF4 in the canine genome.
  • the subfamilies represented by these four genes range in size from 2 to 20 genes. They are all expressed in canine olfactory epithelium but were not detectably expressed in canine lung, liver, ovary, spleen, testis, or tongue.
  • the OLFl and OLF2 subfamilies are tightly linked in the dog genome and also in the human genome.
  • the smallest family is represented by the canine OLFl gene.
  • dog gene probes individually to hybridize to Southern blots of genomic DNA from 24 somatic cell hybrid lines. They showed that the human homologous OLFl subfamily maps to human chromosome 11. The human gene with the strongest similarity to the canine OLF2 gene also mapped to chromosome 11. Both members ofthe human subfamily that hybridized to canine OLF3 were located on chromosome 7. It was difficult to determine to which chromosome or chromosomes the human genes that hybridized to the canine OLF4 probe mapped. This subfamily is large in mouse and hamster as well as human, so the rodent background largely obscured the human cross-hybridizing bands.
  • Issel-Tarver and Rine demonstrated that in the human OLFl and OLF2 homologs are likewise closely linked.
  • Issel-Tarver and Rine found that the human OLF3 homolog maps to 7q35.
  • a chromosome 19-specific cosmid library was screened by hybridization with the canine OLF4 gene probe, and clones that hybridized strongly to the probe even at high stringency were localized to 19p 13.1 and 19pl3.2. These clones accounted, however, for a small fraction ofthe homologous human bands.
  • Rouquier et al. demonstrated that members ofthe olfactory receptor gene family are distributed on all but a few human chromosomes. Through fluorescence in situ hybridization analysis, they showed that OR sequences reside at more than 25 locations in the human genome. Their distribution was biased for terminal bands of chromosome arms. Flow-sorted chromosomes were used to isolate 87 OR sequences derived from 16 chromosomes. Their sequence relationships indicated the inter- and intrachromosomal duplications responsible for OR family expansion. Rouquier et al. (1998) determined that the human genome has accumulated a striking number of dysfunctional copies: 72% of these sequences were found to be pseudogenes. ORF-containing sequences predominate on chromosomes 7, 16, and 17.
  • telomere a subtelomeric DNA duplication that provided insight into the variability, complexity, and evolutionary history of that unusual region ofthe human genome, the telomere.
  • ORs are likely G protein-coupled receptors, which characteristically are 7-transmembrane proteins.
  • ORs are likely members of a multigene family of considerable size, because an immense number of chemicals with vastly different structures can be detected and discriminated by the vertebrate olfactory system.
  • ORs are likely expressed selectively in olfactory sensory neurons. Ben-Arie et al. (1994) focused attention on a cluster of human OR genes on 17p, to which the first human OR gene, OR1D2, had been mapped by Schurmans et al. (1993). According to Mombaerts (1999), the sequences of more than 150 human OR clones had been reported.
  • the human OR genes differ markedly from their counterparts in other species by their high frequency of pseudogenes, except the testicular OR genes. Research showed that individual olfactory sensory neurons express a small subset ofthe OR repertoire. In rat and mouse, axons of neurons expressing the same OR converge onto defined glomeruli in the olfactory bulb.
  • the present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences and their polypeptides. The sequences are collectively referred to as “NOVX nucleic acids” or “NOVX polynucleotides” and the corresponding encoded polypeptides are referred to as “NOVX polypeptides” or “NOVX proteins.” Unless indicated otherwise, “NOVX” is meant to refer to any ofthe novel sequences disclosed herein. Table 1 provides a summary ofthe NOVX nucleic acids and their encoded polypeptides. Example 1 provides a description of how the novel nucleic acids were identified.
  • OR GPCR is an odorant receptor ofthe G-protein coupled-receptor family.
  • NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts.
  • the various NOVX nucleic acids and polypeptides according to the invention are useful as novel members ofthe protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members ofthe family to which the NOVX polypeptides belong.
  • NOVl-10 are homologous to members ofthe odorant receptor (OR) family ofthe human G-protein coupled receptor (GPCR) superfamily of proteins, as shown in Table 56.
  • OR odorant receptor
  • GPCR human G-protein coupled receptor
  • the NOVl-10 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in disorders of olfactory loss, e.g., trauma, HIV illness, neoplastic growth and neurological disorders e.g. Parkinson's disease and Alzheimer's disease.
  • the NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function.
  • nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., neurogenesis, cell differentiation, cell motility, cell proliferation and angiogenesis. Additional utilities for the NOVX nucleic acids and polypeptides according to the invention are disclosed herein. NOV1
  • a NO VI sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV1 nucleic acid and its encoded polypeptide includes the sequences shown in Table 2.
  • the disclosed nucleic acid (SEQ ID NO:l) is 1,071 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 42-44 and ends with a TAA stop codon at nucleotides 1,053-1,055.
  • the representative ORF encodes a 337 amino acid polypeptide (SEQ ID NO:2). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 1.
  • the NOVl nucleic acid sequence has homology with several fragments ofthe human olfactory receptor 17-93 (OLFR) (GenBank Accession No.: HSU76377), as shown in Table 3. Also, the NOVl polypeptide has homology (approximately 61% identity, 74% similarity) to human olfactory receptor, family 1, subfamily F, member 8 (OLFR) (GenBank Accession No.: XP007973), as is shown in Table 4. Furthermore, the NOVl polypeptide has homology (approximately 61% identity, 75% similarity) to a human olfactory protein (OLFR)(EMBL Accession No.: 043749), as is shown in Table 5.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy- terminus. Multiple sequence augment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOVl is predicted to have a seven transmembrane region and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288), as is shown in Table 6.
  • GPCR dopamine
  • NOVl 1034 GGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCAT 1093
  • OLFR 41260 GAGGTCAGTTGTTCGAGACCAACCTGGTCAAC 41291 (SEQ ID NO. 37)
  • NOVl 1032 CCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATC 1091
  • OLFR 1 CTGGGCTCGGTGGCTCACACGTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATC 60 NOVl: 1092 A--TGAGGTCAGGAGATCGAGACCATCCTGGCTAAC 1125 (SEQ ID NO. 41)
  • OLFR 61 ACATGAGGTCAGGAGTTCGAGACCAGCCTGGTCAAC 96 (SEQ ID NO. 47)
  • OLFR 4688 GTTAGCCAGGATGGTCTCAATCTCCTGACCTCGTGATCCGCCTGCCTTGGCCTCCCAAAG 4747 NOVl: 1065 TGCTGGGATTACAGGCGTGAGCCACCGCGCCCGG 1032 (SEQ ID NO. 48) OLFR: 4748 TGCTGGGATTACAGGCATGAGCCACTGCGCCCGG 4781 (SEQ ID NO. 52)
  • NOVl 1 MSGTNQSSVSEFLLLGLSRQPQQQHLLFVFFLSMYLATVLGNLLIILSVSIDSCLHTPMY 60 * * ** 4 .. .******* * *** ** _
  • OLFR 1 MEGKNQTNISEFLLLGFSS QQQQVLLFALFLCLYLTGLFGNLLILLAIGSDHCLHTPMY 60
  • OLFR 121 DRYVAICHPLHYSTIMALRLCASLVAAPWVIAILNPLLHTLi AHLHFCSDNVIHHFFCD 180
  • NOVl 181 VTPLLKLSCSDTHLNEVIILSEGALVMITPFLCILASYMHITCTVLKVPSTKGRWKAFST 240 . ** ****** **++ 4.*+ *4- 4. * 4.*** **4- * * ⁇ .**** +*+ *****
  • OLFR 181 INSLLPLSCSDTSLNQLSVLATVGLIFWPSVCILVSYILIVSAVMKVPSAQGKLKAFST 240 NOVl: 241 CGSHLAVVLLFYSTIIAVYFNPLSSHSAEKDT ATVLYTVVTP LNPFIYSLRNRYLKGA 300
  • NOVl 1 ⁇ GKNQTNISEFLLLGFSS QQQQVLLFALFLCLYLTGLFGNLLILLAIGSDHCLHTPMY 60 * * **++ 4 -******* * *** *** ** +** + *****+*-
  • OLFR 61 FFLSNLSFVDICFSFTTVPK LANHILETQTISFCGCLTQMYFVFMFVD DNFLLAVMAY 120 NOVl: 121 DRYVAICHPLHYSTIIVLALRLCASLVAAP VIAILNPLLHTLJMMAHLHFCSDNVIHHFFCD 180
  • OLFR 121 DHFVAVCHPLHYTAKMTHQLCALLVAGLWWANLNVLLHTLLMAPLSFCADNAITHFFCD 180
  • OLFR 181 VTPLLKLSCSDTHLNEVIILSEGALVMITPFLCILASYi HITCTVLKVPSTKGRWKAFST 240
  • NOVl 241 CGSHLALVILFYGAITGVYMSPLSNHSTEKDSAASVIFMVVAPVLNPFIYSLRNNELKG 299
  • NOVl 108 ANro FLLT MAYDRY/AICHPLHYS IM- LRLCASLVAAPWVIAILNP LH MAHL 166
  • NOVl can be used to detect nasal epithelial neuronal tissue.
  • NOVl Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing. NOV2
  • a NOV2 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV2 nucleic acid and its encoded polypeptide includes the sequences shown in Table 7.
  • the disclosed nucleic acid (SEQ ID NO:3) is 1,040 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 82-84 and ends with a TGA stop codon at nucleotides 1,012-1,014.
  • the representative ORF encodes a 310 amino acid polypeptide (SEQ ID NO:4). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 3.
  • NOV2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • a NOV2 nucleic acid was identified on human chromosome 6.
  • the NOV2 nucleic acid sequence has a high degree of homology (99% identity) with a human genomic clone corresponding to chromosome 6 (CHR6) (GenBank Accession No.: AL135904), as shown in Table 8. Additionally, the NOV2 polypeptide has a high degree of homology (approximately 95% identity) to a human olfactory receptor (OLFR) (GenBank Accession No.: AL135904), as shown in Table 9. Furthermore, the NOV2 polypeptide has a high degree of homology (approximately 91% identity) to a human olfactory protein (OLFR) (EMBL Accession No.: AC005587), as shown in Table 10.
  • OR proteins have seven transmembrane -helices separated by three extracellular and three cytoplasmic loops, along with an extracellular amino-terminus and a cytoplasmic carboxy- terminus. Multiple sequence augment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV2 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 11.
  • GPCR dopamine
  • N0V2 241 ctgcacgcccccatgtacttcttcctctcacacctggcggtcgtcgacatcgcctacgcc 300 CHR6 : 22339 ctgcacgcccccatgtacttcttcctctcacacctggcggtcgtcgacatcgcctacgcc 22280
  • N0V2 301 tgcaacacggtgccccggatgctggtgaacctcctgcatccagccaagcccatctccttt 360
  • N0V2 361 gcgggccgcatgatgcagacctttctgttttccacttttgctgtcacagaatgtctcctc 420
  • N0V2 421 ctggtggtgatgtcctatgatctgtacgtggccatctgccaccccctcgatatttggcc 480
  • N0V2 481 atcatgacctggagagtctgcatcaccctcgcggtgacttcctggaccactggagtcctt 540
  • CHR6 22099 atcatgacctggagagtctgcatcaccctcgcggtgacttcctggaccactggagtcctt 22040
  • N0V2 541 ttatccttgattcatcttgtgttacttctacctttacccttctgtaggccccagaaaatt 600
  • N0V2 601 tatcacniiiinnnngtgaaatcttggctgttctcaaacttgcctgtgcagatacccacatc 660
  • N0V2 661 aatgagaacatggtcttggccggagcaatttctgggctggtgggacccttgtccacaatt 720
  • N0V2 721 gtagtttcatatatgtgcatcctctgtgctatccttcagatccaatcaagggaagttcag 780
  • NOV2 901 ctgctgtttcacag ⁇ ctctttaatcccatgctcaatccccttatctgtagtcttaggaac 960 5 CHR6 : 21679 ctgctgtttcacagcctctttaatcccatgctcaatccccttatctgtagtcttaggaac 21620
  • N0V2 961 tcagaagtgaagaatactttgaagagagtgctgggagtagaaagggctttatgaaagga 1020 0 CHR6 : 21619 tcagaagtgaagaatactttgaagagagtgctgggagtagaaagggctttatgaaagga 21560 NOV2: 1021 ttatggcattgtgactgaca 1040 (SEQ ID NO. 3)
  • OLFR 73 CLLLWMSYDLYVAICHPLRYLAIMTWRVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRP 132 5 NOV2 : 171 QKIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSY CILCAILQIQSR 230
  • OLFR 133 QKIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSR 192 NOV2 : 231 EVQRKAFRTCFSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICS 290 (_) *** * * * ****** * **** **** *********** ** * * * ***** *
  • OLFR 193 EVQRKAFRTCFSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICS 252 NOV2: 291 LRNSEVKNTLKRVLGVERAL 310 (SEQ ID NO. 35)
  • NOV2 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFXXXXXXXXXXXXXXXXXXDSRLHAPMYF 60 0******************************************
  • OLFR 1 MGDNITSIR ⁇ FLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60 NOV2: 61 FLSHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGRiyiMQTFLFSTFAVTECLLLVVMSYD 120*******************************************
  • OLFR 121 LYVAICHPLRYLAI T RVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180
  • NOV2 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIVVSYMCILCAILQIQSREVQRKAFRTC 240************************************************
  • OLFR 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFRTC 240 NOV2: 241 FSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300
  • the OR family ofthe GPCR superfamily is involved in the initial steps ofthe olfactory signal transduction cascade. Therefore, the NOV2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV2 can be used to provide new diagnostic and/or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are also useful in the treatment of a variety of diseases and pathologies, including but not limited to, those involving neurogenesis, cancer, and wound healing.
  • NOV3 is also useful in the treatment of a variety of diseases and pathologies, including but not limited to, those involving neurogenesis, cancer, and wound healing.
  • a NOV3 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV3 nucleic acid and its encoded polypeptide includes the sequences shown in Table 12.
  • the disclosed nucleic acid (SEQ ID NO:5) is 1,090 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 15-17 and ends with a TAA stop codon at nucleotides 1,061-1,063.
  • the representative ORF encodes a 314 amino acid polypeptide (SEQ ID NO: 6). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 5.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • a NOV3 nucleic acid was identified on human chromosome 1.
  • the NOV3 nucleic acid sequence has a high degree of homology (99% identity) with a human genomic clone corresponding to chromosome 1 (CHR1) (GenBank Accession No.:AL121986), as is shown in Table 13.
  • NOV3 polypeptide has homology (approximately 50% identity, 70% similarity) to a human olfactory receptor (OLFR) (GenBank Accession No.: F20722), as is shown in Table 14.
  • OLFR human olfactory receptor
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy-terminus.
  • NOV3 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 15.
  • GPCR dopamine
  • NOV3 1 aagaagttcttcagatgcgaggtttcaacaaaccactgtggttacacagttcatcctgg 60
  • N0V3 121 acttgacaatcctggtggccaatgtgaccatcatggccgttattcgcttcagctggactc 180
  • NOV3 241 ttgtcatcatccctcagctgctggtccacctgctctcagacaccaagaccatctccctca 300 M l
  • NOV3 481 ttgctttggtggccaccaacctcatttgtgacatgcgtttttgtggccccaacagggtta 540
  • N0V3 601 aagagctggctttatttagcctcagcatcctggtaattatggtgccttttctgttaattc 660
  • NOV3 661 tcatatcctatggcttcatagtcaacaccatcctgaagatcccctcagctgagggcaaga 720 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
  • NOV3 781 ctatcatctatctgcggcccaagtccaagtctgcctcagacaaggatcagttggtggcag 840
  • N0V3 901 aggtaaaaactgcattgaaaagagttcttggaatgcctgtggcaaccaagatgagctaac 960
  • N0V3 961 aaaaaataataataaaattaactaggatagtcacagaagaaatcaaaggcataaaatttt 1020
  • NOV3 1021 ctgacctttaatgcatgtctcagacagtgtttccaaggattaagactactcttgcctttt 1080
  • N0V3 1081 tattttctcc 1090 (SEQ ID NO. 5) CHRl: 144815 tattttctcc 144806 (SEQ ID NO. 42)
  • NOV3 1 MRGFNKTTWTQFILVGFSSLGELQLLLFVIFLLLYLTILVANVTIMAVIRFS TLHTPM 59
  • OLFR 60 YLFLCVLSVSEILYTVAIIPRMLADLLSTQRSIAFLACASQMFFSFSFGFTHSFLLTVMG 119
  • OLFR 120 YDRYVAICHPLRYNVLMSPRGCACLVGCS AGGSVMGMWTSAIFQLTFCGSHEIQHFLC 179 NOV3: 180 DMAPVIKLAC-TDTHVKELALFSLSILVI VPFLLILISYGFIVNTILKIPSAEGK-KAF 239
  • OLFR 180 HVPPLLKLACGNNVPAVALGVGLVCIMALLGCFLLILLSYAFIVADILKIPSA ⁇ GRNKAF 239 NOV3: 240 VTCASHLTWFVHYGCASIIYLRPKSKSASDKDQLVAVTYTWTPLLNPLVYSLRNKEVK 299 ****** ** **** **_***.j_** . . * * ⁇ .* ** *-j-** *_j.* ⁇ .. ⁇ .****** ⁇ .*
  • OLFR 240 STCASHLIWIVHYGFASVIYLKPKGPHSQEGDTLMATTYAVLTPFLSPIIFSLRNKELK 299 NOV3: 300 TALKR 304 (SEQ ID NO. 43)
  • N0V3 222 IVNTILKI 229 (SEQ ID NO . 45 )
  • GPCR 162 IYIVLRRR 169 (SEQ ID NO . 46 )
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, in one embodiment, the NOV3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. ' Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV3 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV4 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • the NOV3 nucleic acid sequence (SEQ ID NO.: 5) was further analyzed by exon linking and the resulting sequence was identified as NOV4.
  • a NOV4 nucleic acid and its encoded polypeptide includes the sequences shown in Table 16.
  • the disclosed nucleic acid (SEQ ID NO:7) is 1,090 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 15-17 and ends with a TAA stop codon at nucleotides 1,061-1,063.
  • the representative ORF encodes a 314 amino acid polypeptide (SEQ ID NO: 8). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 7. TABLE 16.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV4 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • a NOV4 nucleic acid was identified on human chromosome 1.
  • the NOV4 nucleic acid sequence has a high degree of homology (99% identity) with a human genomic clone corresponding to chromosome 1 (CHRl) (GenBank Accession No.:AL121986), as is shown in Table 17.
  • the NOV4 nucleic acid sequence also has a high degree of homology with the NOV3 sequence (99% identity), as is shown in Table 18.
  • the NOV3 polypeptide has homology (approximately 53% identity, 71% similarity) to the human olfactory receptor 10J1 (OLFR) (GenBank Accession No.: P30954), as is shown in Table 19.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV4 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 20.
  • GPCR dopamine
  • NOV4 1 aagaagttcttcagatgcgaggtttcaacaaaccactgtggttacacagttcatcctgg 60
  • NOV4 61 tgggtttctccagcctgggggagct ⁇ cagctgctactttttgtcatctttcttctcctat 120
  • NOV4 121 acttgacaatcctggtggccaatgtgaccatcatggccgttattcgcttcagctggactc 180
  • NOV4 241 ttgtcatcatccctcagctgctggtccacctgctctcagacaccaagaccatctccctca 300
  • N0V4 541 accactatttctgtgacatggcacctgttatcaagttagcctgcactgacacccatgtga 600
  • N0V4 601 aagagctggctttatttagcctcagcatcctggtaattatggtgccttttctgttaattc 660
  • N0V4 661 tcatatcctatggcttcatagtcaacaccatcctgaagatcccctcagctgagggcaaga 720
  • N0V4 721 aggcctttgtcacctgtgcctcacatctcactgtggtctttgtccactatgactgtgcct 780 i I i 111111 M 11 ! 11 M 11 M ; ! I i ' 111 M 111111 i 111 i 11 ; i 11 IIIHI
  • N0V4 781 ctatcatctatctgcggcccaagtccaagtctgcctcagacaaggatcagttggtggcag 840
  • N0V4 841 tgacctacgcagtggttactcccttacttaatcctcttgtctacagtctgaggaacaag 900
  • N0V4 901 aggtaaaaactgcattgaaaagagttcttggaatgcctgtggcaaccaagatgagctaac 960
  • N0V4 961 aaaaaataataataaaattaactaggatagtcacagaagaaatcaaaggcataaaatttt 1020 llll II IIIMIII II II lllll III lllll lllll II I
  • N0V4 1021 ctgacctttaatgcatgtctcagacagtgtttccaaggattaagactactcttgcctttt 1080
  • N0V4 1081 tattttctcc 1090 (SEQ ID NO. 4)
  • NOV3 121 ACTTGACAATCCTGGTGGCCAATGTGACCATCATGGCCGTTATTCGCTTCAGCTGGACTC 180 NOV4: 181 TCCACACTCCCATGTATGGCTTTCTATTCATCCTTTCATTTTCTGAGTCCTGCTACACTT 240
  • N0V3 241 TTGTCATCATCCCTCAGCTGCTGGTCCACCTGCTCTCAGACACCAAGACCATCTCCTTCA 300
  • NOV4 301 TGGCCTGTGCCACCCAGCTGTTCTTTTTCCTTGGCTTTGCTTGCACCAACTGCCTCCTCA 360
  • N0V3 301 TGGCCTGTGCCACCCAGCTGTTCTTTTTCCTTGGCTTTGCTTGCACCAACTGCCTCCTCA 360
  • N0V4 361 TTGCTGTGATGGGATATGATCGCTATGTAGCAATTTGTCACCCTCTGAGGTACACACTCA 420
  • N0V3 361 TTGCTGTGATGGGATATGATCGCTATGTAGCAATTTGTCACCCTCTGAGGTACACACTCA 420
  • N0V4 421 TCATAAACAAAAGGCTGGGGTTGGAGTTGATTTCTCTCTCAGGGGCCACAGGTTTCTTTA 480 1 1 1 1 1 1 1 1 I I 1 1 1 1 1 1 1 1 1
  • N0V3 421 TCATAAACAAAAGGCTGGGGTTGGAGTTGATTTCTCTCTCAGGAGCCACAGGTTTCTTTA 480
  • N0V4 481 TTGCTTTGGTGGCCACCAACCTCATTTGTGACATGCGTTTTTGTGGCCCCAACAGGGTTA 540
  • N0V3 481 TTGCTTTGGTGGCCACCAACCTCATTTGTGACATGCGTTTTTGTGGCCCCAACAGGGTTA 540
  • N0V4 541 ACCACTATTTCTGTGACATGGCACCTGTTATCAAGTTAGCCTGCACTGACACCCATGTGA 600
  • N0V3 541 ACCACTATTTCTGTGACATGGCACCTGTTATCAAGTTAGCCTGCACTGACACCCATGTGA 600
  • N0V4 601 AAGAGCTGGCTTTATTTAGCCTCAGCATCCTGGTAATTATGGTGCCTTTTCTGTTAATTC 660
  • N0V3 601 AAGAGCTGGCTTTATTTAGCCTCAGCATCCTGGTAATTATGGTGCCTTTTCTGTTAATTC 660
  • N0V4 661 TCATATCCTATGGCTTCATAGTCAACACCATCCTGAAGATCCCCTCAGCTGAGGGCAAGA 720 11 II 111111111111 II 1111111111111111111
  • N0V3 661 TCATATCCTATGGCTTCATAGTTAACACCATCCTGAAGATCCCCTCAGCTGAGGGCAAGA 720 N0V4 : 721 AGGCCTTTGTCACCTGTGCCTCACATCTCACTGTGGTCTTTGTCCACTATGACTGTGCCT 780 M I N I M
  • N0V3 721 AGGCCTTTGTCACCTGTGCCTCACATCTCACTGTGGTCTTTGTCCACTATGGCTGTGCCT 780
  • N0V4 781 CTATCATCTATCTGCGGCCCAAGTCCAAGTCTGCCTCAGACAAGGATCAGTTGGTGGCAG 840
  • N0V3 781 CTATCATCTATCTGCGGCCCAAGTCCAAGTCTGCCTCAGACAAGGATCAGTTGGTGGCAG 840
  • N0V4 841 TGACCTACGCAGTGGTTACTCCCTTACTTAATCCTCTTGTCTACAGTCTGAGGAACAAAG 900
  • N0V3 841 TGACCTACACAGTGGTTACTCCCTTACTTAATCCTCTTGTCTACAGTCTGAGGAACAAAG 900
  • N0V4 901 AGGTAAAAACTGCATTGAAAAGAGTTCTTGGAATGCCTGTGGCAACCAAGATGAGCTAAC 960
  • N0V3 901 AGGTAAAAACTGCATTGAAAAGAGTTCTTGGAATGCCTGTGGCAACCAAGATGAGCTAAC 960 NOV4 : 961 AAAAAATAATAATAAAATTAACTAGGATAGTCACAGAAGAAATCAAAGGCATAAAATTTT 1020 lllll 11111111 II 111111111 M 111 ! I )! 1111111 N 1111111 lllll
  • NOV3 961 AAAAAATAATAATAAAATTAACTAGGATAGTCACAGAAGAAATCAAAGGCATAAAATTTT 1020
  • NOV4 1021 CTGACCTTTAATGCATGTCTCAGACAGTGTTTCCAAGGATTAAGACTACTCTTGCCTTTT 1080
  • NOV3 1021 CTGACCTTTAATGCATGTCTCAGACAGTGTTTCCAAGGATTAAGACTACTCTTGCCTTTTTT 1080
  • NOV4 1081 TATTTTCTCC 1090 (SEQ ID NO. 7)
  • NOV3 1081 TATTTTCTCC 1090 (SEQ ID NO. 5)
  • NOV4 18 TLITDFVFQGFSSFHEQQITLFGVFLALYILTLAGNIIIVTIIRIDLHLHTPMYFFLSML 77 *4-4.* * - **** * * * _ ** 4.** ** * * *4- * _ 4** ****** ** 4,* OLFR: 8 TWTQFILVGFSSLGELQLLLFVIFLLLYLTILVANVTI AVIRFSWTLHTPMYGFLFIL 67
  • NOV4 78 STSETVYTLVILPRMLSSLVGMSQP SLAGCATQMFFFVTFGITNCFLLTAMGYDRYVAI 137 * * *_ ** ** -*_ j ._ * *+ 4.4. 4.* * **** *** . * *** * -*******
  • OLFR 68 SFSESCYTFVIIPQLLVHLLSDTKTISLMACATQLFFFLGFACTNCLLIAVMGYDRYVAI 127
  • NOV4 138 CNPLRYMVIJNKRLRIQLVLGACSIGLIVAITQVTSVFRLPFCA-RKVPHFFCDIRPVMK 196 *4**** 4_* **** 44*4. 4 4 * 4*+ 4 4- ** 4-* *4-***4- **4*
  • OLFR 128 CHPLRYTLIINKRLGLELISLSGATGFFIALVATNLICDMRFCGPNRVNHYFCDMAPVIK 187 NOV4: 197 LSCIDTTVNEXXXXXXXXXXXPMGLVFISYVLIISTILKIASVEGRKKAFATCASHLT 256
  • OLFR 188 LACTDTHVKELALFSLSILVIMVPFLLILISYGFIVNTILKIPSAEG-KKAFVTCASHLT 246
  • N0V4 103 FFFLGFACTNCLLIAVMGYDRYVAICHPLRYTLIIN-KRLGLELISLSGATGFFIALVAT 161
  • NOV4 162 NLICDMRFCGPNRVNHYFCDMAPVIKLACTDTHVKELALFSLSILVIMVPFLLILISYGF 221 GPCR: 122 FGLNNTDQNEC IIANPAFWYSSIVSFYVPFIVTLLVYIK 161
  • NOV4 222 IVNTILKI 229 (SEQ ID NO. 51)
  • GPCR 162 IYIVLRRR 169 (SEQ ID NO. 46)
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV4 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV4 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV4 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in treating and/or diagnosing a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV5 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV5 nucleic acid and its encoded polypeptide includes the sequences shown in Table 21.
  • the disclosed nucleic acid (SEQ ID NO: 9) is 822 nucleotides in length and contains an open reading frame (ORF) that begins at nucleotide 6 and ends with a TGA stop codon at nucleotides 800-802.
  • ORF open reading frame
  • C indicates 'G' to 'C substitutions in the sequence to correct stop codons.
  • a representative ORF encodes a 265 amino acid polypeptide (SEQ ID NO: 10).
  • a putative untranslated region downstream ofthe coding sequence is underlined in SEQ ID NO: 9.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV5 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV5 nucleic acid sequence has a high degree of homology (94% identity) with a human genomic clone cotaining an OR pseudogene (OLFR) (GenBank Accession No.:AF065864), as is shown in Table 22.
  • the NOV5 polypeptide has homology (approximately 67% identity, 79% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No.:043789), as is shown in Table 23.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV5 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 24.
  • GPCR dopamine
  • OLFR 196 GCCACGGTTCCTAAGATGATTGTGGACATGCAGTCTCATACCAGAGTCATCTCTTATGAG 255
  • OLFR 256 GGCTGCCTGACACGGATATCTTTCTTGGTCCTTTTTGCATGTATAGAAGACATGCTCCTG 315
  • OLFR 316 ACTGTGATGGCCTATGACTGCTTTGTAGCCATCTGTCGCCCTCTGCACTACCCAGTCATC 375
  • NOV5 241 GTGAATCCTCACCTCTGTGTCTTCTTCGTCTTGGTGTCCTTTTTCCTTAGCCCGTTGGAT 300
  • OLFR 376 GTGAATCCTCACCTCTGTGTCTTCTTCCTTTTGGTATACTTTTTCCTTAGCTTGTTGGAT 435
  • NOV5 301 TCCCAGCTGCACAGTTGGATTGTGTTACTATTCACCATCATCAAGAATGTGGAAATCACT 360
  • OLFR 436 TCCCAGCTGCACAGTTGGATTGTGTTACAATTCACCATCATCAAGAATGTGGAAATCTCT 495
  • NOV5 421 AACATATTCATATATTTCGATAGTACTATGTTTGGTTTTCTTCCCATTTCAGGGATCCTT 480
  • NOV5 481 TTGTCTTACTATAAAATTGTCCCCTCCATTCTAAGGATGTCATCGTCAGATGGGAAGTAT 540
  • NOV5 721 GATATACAAAGTGTCCTGCGGAGGCTGTGCAGCAGAACAGTCGAATCTCATGATATGTTC 780
  • OLFR 916 CATCCTTT 923 (SEQ ID NO. 54)
  • OLFR 1 PiMYFFLSNLSLADIGFTSTTVPKMIVDMQTHSRVISYEGCLTQMSFFVLFACMDDMLLSV 60 NOV5: 187 MAYDCFVAICRPLHYPVIVNPHLCVFFVLVSFFLSPLDSQLHS IVLLFTIIKNVEITNF 366 **** ***** **** 4.* ⁇ .** ** * _ j _*4-***4-* ****** ⁇ _ *_ j _* * *4>*4.* .**
  • OLFR 61 MAYDRFVAICHPLHYRIIMNPRLCGFLILLSFFISLLDSQLHNLIMLQLTCFKDVDISNF 120
  • NOV5 367 VCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 546 *4-*****4-* ***4_ ** 4. *** 4.******* ****** *** - 4.******
  • OLFR 121 FCDPSQLLHLRCSDTFINEMVIYFMGAIFGCLPISGILFSYYKIVSPILRVPTSDGKYKA 180
  • NOV5 547 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNGWASV YAWTPMLNLFIYSLGKRDI 726 ******_ ⁇ .***** * ***4 > **_ ⁇ _*** * ** 4.*********** ***** 4.**
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade.
  • the NOV5 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV5 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV5 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV6 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV6 nucleic acid and its encoded polypeptide includes the sequences shown in Table 25.
  • the disclosed nucleic acid (SEQ ID NO:l 1) is 930 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 22-24 and ends with a TAA stop codon at nucleotides 907-909.
  • ORF open reading frame
  • C indicates 'G' to 'C substitutions in the sequence to correct stop codons.
  • the representative ORF encodes a 294 amino acid polypeptide (SEQ ID NO: 12). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 11.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade.
  • the NOV6 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV6 nucleic acid sequence has a high degree of homology (94% identity) with a human genomic clone cotaining an OR pseudogene (OLFR) (GenBank Accession No.:AF065864), as is shown in Table 26.
  • the NOV6 polypeptide has homology (approximately 67% identity, 79% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No.:043789), as is shown in Table 27.
  • NOV6 polypeptide also has a high degree of homology (99% identity) with the NOV5 polypeptide.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413.
  • NOV5 and NOV6 belong to the same OR subfamily.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy- terminus.
  • NOV6 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 29.
  • GPCR dopamine
  • NOV6 10 ctgtccctgtccatgtatatggtcacggtgctgaggaacctgctcagcatcctggctgtc 69
  • NOV6 70 agctctgactccccgctccacacccccatgtgcttcttcctctccaaactgtgctcagct 129 OLFR: 118 agctctgacccccacctccacacccccatgtgcttcttcctctccaacctgtgctgggct 177 NOV6 : 130 gacatcggtttcaccttggccatggttcccaagatgattgtgaacatgcagtcgcatagc 189
  • NOV6 190 agagtcatctcttatgagggctgcctgacacggatgtctttctttgtcctttttgcatgt 249 OLFR: 238 agagtcatctcttatgagggctgcctgacacggatatctttcttggtcctttttgcatgt 297
  • N0V6 250 atggaagacatgctcctgactgtgatggcctatgactgctttgtagccat ⁇ tgtcgccct 309
  • N0V6 310 ctgcactacccagtcatcgtgaatcctcacctctgtgtcttcttcgtcttggtgtccttt 369
  • NOV6 370 ttccttagcccgttggattcccagctgcacagttggattgtgttactattcaccatcatc 429 OLFR: 418 ttccttagcttgttggattcccagctgcacagttggattgtgttacaattcaccatcatc 477
  • NOV6 430 aagaatgtggaaatcactaattttgtctgtgaaccctctcaacttctcaaccttgcttgt 489
  • NOV6 550 cccatttcagggatccttttgtcttactataaaattgtccct ⁇ cattctaaggatgtca 609
  • NOV6 610 tcgtcagatgggaagtataaaggcttctcacctgtggct ⁇ ttacctggcagttgtttgc 669
  • NOV6 670 tcatttgatggaacaggcattggcatgtacctgacttcagctgtgtcaccaccccccagg 729 OLFR: 718 tgattttatggaacaggcattggcgtgtacctgacttcagctgtgtcaccaccccccagg 777
  • NOV6 7 P CFFLSKLCSADIGFTI-AMVPK IVNMQSHSRVISYEGCLTRMSFFVLFACMEDMLLTV 186 ** **** * ****** ******4** +********** +********4**** + *
  • NOV6 187 MAYDCFVAICRPLHYPVIWPHLCVFFVLVSFFLSPLDSQLHSWIVLLFTIIKJ EITNF 366 **** ***** **** 4*4** ** * 4*4***4* ****** + * * * * * + *4* + **
  • OLFR 61 MAYDRFVAICHPLHYRIIM PRLCGFLILLSFFISLLDSQLHNLIMLQLTCFKDVDISNF 120
  • NOV6 367 VCEPSQ LNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 546 *4*****4* *** + ** *** 4******* ****** ***4 + ******
  • OLFR 121 FCDPSQLLHLRCSDTFINEMVIYFMGAIFGCLPISGILFSYYKIVSPILRVPTSDGKYKA 180
  • NOV6 547 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNGWASVMYAWTPMLNLFIYSLGKRDI 726
  • NOV6 85 MAYDCFVAICRPLHYPVIVNPHLCXXXXXXXXXXXXXQLHS IVLLFTIIKNVEITNF 144*****************************************************
  • NOV5 61 MAYDCFVAICRPLHYPVIVNPHLCVFFVLVSFFLSPLDSQLHS IVLLFTIIKNVEITNF 120
  • NOV6 145 VCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 204
  • NOV5 121 VCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 180
  • NOV6 205 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNG-VASV YAWTPMLNLFILSLGKRDI 263
  • NOV5 181 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNGWASVMYAWTPMLNLFIYSLGKRDI 240
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV6 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV6 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV6 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV7 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV7 nucleic acid and its encoded polypeptide includes the sequences shown in Table 30.
  • the disclosed nucleic acid (SEQ ID NO: 13) is 930 nucleotides in length and contains an open reading frame (ORF) that begins with an ACG initiation codon at nucleotides 10-12 and ends with a TGA stop codon at nucleotides 882-884.
  • ORF open reading frame
  • C indicates 'G' to 'C substitutions in the sequence to correct stop codons.
  • the representative ORF encodes a 309 amino acid polypeptide (SEQ ID NO: 12). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 13
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade.
  • the NOV7 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV7 nucleic acid sequence has a high degree of homology (94% identity) with the human genomic clone pDJ392al7 from chromosome 11 (CHRl 1) (GenBank Accession No.:AC000385), as is shown in Table 31.
  • the NOV7 polypeptide has homology (approximately 68% identity, 78% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No..-043789), as is shown in Table 32.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV7 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 33.
  • GPCR dopamine
  • NOV7 1 cacagagccacggaatctcacaggtgtctcagaattcctcctgggactctcagagga 60
  • NOV7 241 tcccaagatgattgtggacatgcagtggtatagcagagtcatctctcatgcgggctgcct 300
  • N0V7 661 ctataaaattgtcccctccattctaaggatgtcatcgtcagatgggaagtataaaacttt 720
  • N0V7 781 gtacctggcttcagctatgtcaccaacccccaggaatggtgtggtggtgtcagtgatgta 840 MMMMIMMMMMM 111111 ; 1111
  • N0V7 841 agctgtggtcacccccatgctgaaccttttcatctacagcctgagaaacagggacataca 900
  • N0V7 179 PTYFFLSILCWADIGFTSATVPKMIVDMQWYSRVISHAGCLTQMSFLVLFACIEGMLLTV 358 * ***** ***********4*****4****** OLFR: 1 PMYFFLSNLSLADIGFTSTTVPKMIVDMQTHSRVISYEGCLTQMSFFVLFACMDDMLLSV 60 NOV7: 359 MAYDCFVGIYRPLHYPVIVNPHLCVFFVLVSFFLSLLDSQLHSWIVLQFTIIKNVEISNF 538 **** ** * * * * * ** _ j _* ** ** * 4*4***4********4.
  • the OR family ofthe GPCR superfamily is a group of related proteins that are specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV7 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV7 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV7 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV8 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV8 nucleic acid and its encoded polypeptide includes the sequences shown in Table 34.
  • the disclosed nucleic acid (SEQ ID NO: 15) is 994 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 27-29 and ends with a TGA stop codon at nucleotides 969-971.
  • the representative ORF encodes a 314 amino acid polypeptide (SEQ ID NO: 16). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 15.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade.
  • NOV8 nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV8 polypeptide has homology (approximately 44% identity, 65% similarity) to the human olfactory receptor family 2 subfamily F, member 1 (OLFR) (EMBL Accession No.:NP 036501), as is shown in Table 35.
  • the NOV8 polypeptide also has homology (44% identity, 65% similarity) to the rat olfactor receptor-like protein OLF3 (SwissProt Accession No.: Q13607), as is shown in Table 36.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV8 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 37.
  • GPCR dopamine
  • N0V8 1 MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY 60 ** **4- *4***4-** * 4.* ** * ** .*4- 4*** 4.4.4- ***4*********
  • OLFR 1 MGTDNQT VSEFILLGLSSDWDTRVSLFVLFLVMYWTVLGNCLIVLLIRLDSRLHTPMY 60
  • NOV8 61 FLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLMGVAEGVLLVLMSY 120 * * 4 - *** * 4 - 4 .*-_ 4 . ** * 4 . ***.** _ ⁇ .* * *** 4 -* 4 _*
  • OLFR 61 FFLTNLSLVDVSYATSWPQLLAHFLAEHKAIPFQSCAAQLFFSLALGGIEFVLLAVMAY 120
  • OLFR 121 DRYVAVCDALRYSAIMHGGLCARLAITS VSGFISSPVQTAITFQLPMCRNKFIDHISCE 180
  • OLFR 181 LLAWRLACVDTSSNEVTIMVSSIVLLMTPLCLVLLSYIQIISTILKIQSREGRKKAFHT 240 NOV8: 241 CSSHITVVGLFYGAAVFMYMVPCAYHSPQQDNVVSLFYSLVTPTLNPLIYSLRNPEV MA 300
  • NOV8 27 MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY 206 ** ** _ * _*** ⁇ .** * _* ** * **4-* - ⁇ .*** +4-4- ***4-********* OLFR: 1 MGTDNQT VS ⁇ FILLGLSSD DTRVSLFVLFLVMYWTVLGNCLIVLLIRLDSRLHTPMY 60
  • NOV8 207 FLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLMGVAEGVLLVLMSY 386 * *4_ *** *4. 4.*4.4- 4. ** * 4. ***4-** 4.* * *** -* -*
  • OLFR 61 FFLTNLSLVDVSYATSWPQLLAHFLAEHKAIPFQSCAAQLFFSLALGGI ⁇ FVLLAVMAY 120
  • NOV8 41 GNTVLLFLIRVDSRLHTPMYFLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQ 100 GPCR: 1 GNVLVCMAVSREKALQTTTIWLIVSIAVADLLVATLVMP VVYLEVVGE KFSRIHCDIF 60
  • the OR family ofthe GPCR superfamily is a group of related proteins located at the ciliated surface of olfactory sensory neurons in the nasal epithelium.
  • the OR family is involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV8 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV8 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV8 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV9 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV9 nucleic acid and its encoded polypeptide includes the sequences shown in Table 38.
  • the NOV8 nucleic acid sequence (SEQ ID NO.: 15) was further analyzed by exon linking, and the resulting sequence was identified as NOV9.
  • the disclosed nucleic acid (SEQ ID NO: 17) is 994 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 28-30 and ends with a TAG stop codon at nucleotides 979-981.
  • the representative ORF encodes a 317 amino acid polypeptide (SEQ ID NO: 18). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 17.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV9 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV9 polypeptide has homology (approximately 44% identity, 65% similarity) to the human olfactory receptor family 2 subfamily F, member 1 (OLFR) (EMBL Accession No.:NP 036501), as is shown in Table 39.
  • the NOV9 polypeptide also has a high degree of homology (99% identity) to the NOV8 polypeptide as shown in Table 40.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. Thus NOVS andNOV9 belong to the same subfamily of ORs.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy- terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV9 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 41.
  • GPCR dopamine
  • OLFR 1 MGTDNQTWVSEFILLGLSSD DTRVSLFVLFLVMYWTVLGNCLIVLLIRLDSRLHTPMY 60
  • OLFR 61 FFLTNLSLVDVSYATSWPQLLAHFLAEHKAIPFQSCAAQLFFSLALGGIEFVLLAVMAY 120
  • OLFR 121 DRYVAVCDALRYSAIMHGGLCARLAITS VSGFISSPVQTAITFQLPMCRNKFIDHISCE 180
  • OLFR 181 LLAWRLACVDTSSNEVTIMVSSIVLLMTPLCLVLLSYIQIISTILKIQSREGRKKAFHT 240
  • NOV8 1 MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY 60
  • N0V9 181 VPALLKLSCADTCAY ⁇ MALSTSGVLILMLPLSLIATSYGHVLQAVLSMRSE ⁇ ARHKAVTT 240*******************************************
  • NOV9 41 GNTVLLFLIRVDSRLHTPMYFLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQ 100 GPCR: 1 GNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMP WYL ⁇ WGE KFSRIHCDIF 60 NOV9: 101 IFFLTLMGVAEGVLLVLMSYDRYVAVCQPLQYPVLM-RRQVCLLMMGSSWWGVLNASIQ 159 GPCR: 61 VTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVWVLSFTISCPM 120
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV9 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV9 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV9 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing. NOV10
  • a NOV10 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV10 nucleic acid and its encoded polypeptide includes the sequences shown in Table 42.
  • the disclosed nucleic acid (SEQ ID NO: 19) is 1,077 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 31-33 and ends with a TAG stop codon at nucleotides 1,030- 1,032.
  • the representative ORF encodes a 318 amino acid polypeptide (SEQ ID NO:20). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 19. Exon linking was used to confirm the sequence.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV10 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV10 polypeptide has homology (approximately 55% identity, 72% similarity) to the olfactory receptor MOR83 (OLFR) (EMBL Accession No. :BAA86125), as is shown in Table 43.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus.
  • NOV10 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 44.
  • GPCR dopamine
  • NOVIO 79 MNPANHSQVAGFVLLGLSQV ELRFVFFTVFSAVYFMTWGNLLIWIVTSDPHLHTTMY 258
  • OLFR 1 MGALNQTRVTEFIFLGLTDNWVLEILFFVPFTVTYMLTLLGNFLIWTIVFTPRLHNPMY 60 NOVIO: 259 FLLGNLSFLDFCYSSITAPRMLVDLLSGNPTISFGGCLTQLFFFHFIGGIKIFLLTVMAY 438
  • OLFR 61 FFLSNLSFIDICHSSVTVPKMLEGLLLERKTISFDNCIAQLFFLHLFACSEIFLLTIMAY 120
  • OLFR 121 DRYVAICIPLHYSNVMNMKVCVQLVFALWLGGTIHSLVQTFLTIRLPYCGPNIIDSYFCD 180
  • GPCR 1 GNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWWYLEWGEWKFSRIHCDIF 60
  • NOVIO 101 LFFFHFIGGIKIFLLTVMAYDRYIAISQPLHYTLIMNQ-TVCALLMAAS VGGFIHSIVQ 159
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV10 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV10 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOVl 1 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • OR human odorant receptor
  • GPCR G-protein coupled receptor
  • a NOVl 1 nucleic acid was discovered by exon linking analysis of NOV2 (SEQ ID NO.: 3).
  • a NOVl 1 nucleic acid and its encoded polypeptide includes the sequences shown in Table 45.
  • the disclosed nucleic acid (SEQ ID NO:21) is 1,012 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 54-56 and ends with a TGA stop codon at nucleotides 984-986.
  • the representative ORF encodes a 310 amino acid polypeptide (SEQ ID NO:22). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 1 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOVl 1 polypeptide has a high degree of homology (approximately 99% identity) to a human olfactory receptor (OLFR) (EMBL Accession No.:095047), as is shown in Table 46.
  • the NOVl 1 polypeptide also has a high degree of homology (approximately 99% identity) to NOV2, as is shown in Table 47.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences. 1999, 20:413. Therefore, NOVl 1 and NOV2 are two members ofthe same OR subfamily.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus.
  • NOVl 1 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 48.
  • GPCR dopamine
  • OLFR 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60 NOVll: 61 FLSHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLVVMSYD 120*******************************************
  • OLFR 61 FLSHLAV ⁇ iAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLVVMSYD 120 NOVll: 121 LYVAICHPLRYLAIMT RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFC ⁇ I 180
  • OLFR 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFRTC 240 NOVll : 241 FSHLCVIGLFYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300
  • NOV2 1 MGDNITSIR ⁇ FLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60
  • NOV2 61 FLSHLAWDIAYACNTVPRMLVNLLHPAK ISFAGRMMQTFLFSTFAVTECLLLWMSYD 120 NOVll: 121 LYVAICHPLRYLAIMTWRVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180
  • NOVll 113 LLWMSYDLYVAICHPLRYLAIMT -RVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQ 171
  • GPCR 74 NLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVWVLSFTISCPMLFGLNNTDQNE- - 131
  • NOVll 172 KIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSRE 231
  • GPCR 132 - CIIANPAF WYSSIVSFYVPFIVTLLVYIKIYIVLRRKRV 173
  • the OR family of the GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 1 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOVl 1 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl 1 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOVl 2 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. .
  • a NOVl 2 nucleic acid was discovered by exon linking analysis of NOV2 (SEQ ID NO.: 3).
  • a NOV12 nucleic acid and its encoded polypeptide includes the sequences shown in Table 49.
  • the disclosed nucleic acid (SEQ ID NO:23) is 1,014 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 55-57 and ends with a TGA stop codon at nucleotides 985-987.
  • the representative ORF encodes a 310 amino acid polypeptide (SEQ ID NO:24). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 23.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV12 polypeptide has a high degree of homology (approximately 99% identity) to a human olfactory receptor (OLFR) (EMBL Accession No.:095047), as is shown in Table 50.
  • the NOVl 2 polypeptide also has a high degree of homology (approximately 99% identity) to NOV2, as is shown in Table 51.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. Therefore, NOVl 2 andNOV2 are two members ofthe same OR subfamily.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOVl 2 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 52.
  • GPCR dopamine
  • OLFR 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF
  • NOV12 61 FLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLWMSYD 120
  • OLFR 121 LYVAICHPLRYLAIMTWRVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180
  • OLFR 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFRTC 240
  • OLFR 241 FSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300
  • NOV12 113 LLWMSYDLYVAICHPLRYLAIMTW-RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQ 171
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOVl 2 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl 2 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOVl 3 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOVl 3 nucleic acid and its encoded polypeptide includes the sequences shown in Table 53.
  • the disclosed nucleic acid (SEQ ID NO:25) is 908 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 75-77 and ends with a TAA stop codon at nucleotides 901-903.
  • the representative ORF encodes a 270 amino acid polypeptide (SEQ ID NO:26). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 25.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV13 polypeptide has homology (approximately 73% identity, 83% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No.:Q9UPJl), as is shown in Table 54.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOVl 3 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 55.
  • GPCR dopamine
  • NOV12 1 MYFFLSNLC ADIGFTLATVPKMIVDMGSHSRVISYEGCLTQMSFFVLFACIEDMLLTVM 60****** ****************** 4.*****************44**** 4 **
  • NOV12 61 AYDQFVAICHPLHYPVIMNPHLCVFLVLVSFFLSLLDSQLHS IVLQFTFFKNVEISNFF 120 ***+********** 4 -**** ** ** 4 -* 4 _***+********+ *+** * **+*+*****
  • OLFR 61 AYDRFVAICHPLHYRIIMNPRLCGFLILLSFFISLLDSQLHNLIMLQLTCFKDVDISNFF 120 NOV12: 121 CDPSQLLNLACSDGIINSIFIYLDSILFSFLPISGILLSYYKIVPSILRISSSDGKYKAF 180
  • NOV13 1 MYFFLSNLC ADIGFTLATVPKMIVDMGSHSRVISY ⁇ GCLTQMSFFVLFACIEDMLLTVM 60 GPCR: 19 TNYLIVSLAVADLLVATLVMPWWYLEWGE KFSRIHCDIFVTLDVMMCTASILNLCAI 78
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOVl 3 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl 3 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • Table 56 shows a multiple sequence alignment of NOVl -13 polypeptides with the known human olfactory receptor 10J1 (GenBank Accession No.: P30954), indicating the homology between the present invention and known members of a protein family.
  • OR_10J1 is the known human olfactory receptor 10J1 (GenBank Accession No.: P30954).
  • nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in disorders ofthe neuro-olfactory system, such as those induced by trauma, surgery and/or neoplastic disorders.
  • a cDNA encoding the olfactory receptor protein may be useful in gene therapy for treating such disorders, and the olfactory receptor protein may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from disorders ofthe neuro-olfactory system.
  • novel nucleic acids encoding olfactory receptor protein, and the olfactory receptor protein ofthe invention, or fragments thereof, may further be useful in the treatment of adenocarcinoma; lymphoma; prostate cancer; uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, treatment of Albright hereditary ostoeodystrophy, development of powerful assay system for functional analysis of various human disorders which will help in understanding of pathology of the disease, and development of new drug targets for various disorders. They may also be used in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • nucleic acids ofthe invention include those that encode a NOVX polypeptide or protein.
  • polypeptide and protein are interchangeable.
  • a NOVX nucleic acid encodes a mature NOVX polypeptide.
  • a "mature" form of a polypeptide or protein described herein relates to the product of a naturally occurring polypeptide or precursor form or proprotein.
  • the naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame described herein.
  • the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the cell in which the gene product arises.
  • Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage ofthe N-terminal methionine residue encoded by the initiation codon of an open reading frame, or the proteolytic cleavage of a signal peptide or leader sequence.
  • a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine would have residues 2 through N remaining after removal ofthe N-terminal methionine.
  • a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining.
  • a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • NOVX nucleic acids is the nucleic acid whose sequence is provided in SEQ
  • the invention includes mutant or variant nucleic acids of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a fragment thereof, any of whose bases may be changed from the corresponding bases shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, while still encoding a protein that maintains at least one of its NOVX-like activities and physiological functions (i.e., modulating angiogenesis, neuronal development).
  • the invention further includes the complement ofthe nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, including fragments, derivatives, analogs and homologs thereof.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • nucleic acid molecules that encode NOVX proteins or biologically active portions thereof. Also included are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of NOVX nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs ofthe DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • Probes refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source ofthe nucleic acid.
  • isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived.
  • the isolated NOVX nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule ofthe present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a complement of any of this nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • NOVX nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et ⁇ l., eds., MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et ⁇ h, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid ofthe invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
  • an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at lease 6 contiguous nucleotides of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a complement thereof Oligonucleotides may be chemically synthesized and may be used as probes.
  • an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a portion of this nucleotide sequence.
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Von der Waals, hydrophobic interactions, etc.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • nucleic acid molecule ofthe invention can comprise only a portion ofthe nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, e.g., a fragment that can be used as a probe or primer, or a fragment encoding a biologically active portion of NOVX.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence.
  • Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.
  • Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution.
  • Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
  • Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • Derivatives or analogs ofthe nucleic acids or proteins ofthe invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions.
  • a “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above.
  • Homologous nucleotide sequences encode those sequences coding for isoforms of a NOVX polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous nucleotide sequences include nucleotide sequences encoding for a NOVX polypeptide of species other than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
  • homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations ofthe nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the nucleotide sequence encoding human NOVX protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, as well as a polypeptide having NOVX activity. Biological activities ofthe NOVX proteins are described below.
  • a homologous amino acid sequence does not encode the amino acid sequence of a human NOVX polypeptide.
  • the nucleotide sequence determined from the cloning ofthe human NOVX gene allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g., from other tissues, as well as NOVX homologues from other mammals.
  • the probe/primer typically comprises a substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 or more consecutive sense strand nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; or an anti-sense strand nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; or of a naturally occurring mutant of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.
  • Probes based on the human NOVX nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a NOVX protein, such as by measuring a level of a NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
  • polypeptide having a biologically active portion of NOVX refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide ofthe present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically active portion of NOVX” can be prepared by isolating a portion of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 that encodes a polypeptide having a NOVX biological activity (biological activities ofthe NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of NOVX.
  • a nucleic acid fragment encoding a biologically active portion of NOVX can optionally include an ATP-binding domain.
  • a nucleic acid fragment encoding a biologically active portion of NOVX includes one or more regions.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 due to the degeneracy ofthe genetic code.
  • These nucleic acids thus encode the same NOVX protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 e.g., the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of NOVX may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX gene may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a NOVX protein, preferably a mammalian NOVX protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe NOVX gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in NOVX that are the result of natural allelic variation and that do not alter the functional activity of NOVX are intended to be within the scope ofthe invention.
  • nucleic acid molecules encoding NOVX proteins from other species and thus that have a nucleotide sequence that differs from the human sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 are intended to be within the scope ofthe invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • a soluble human NOVX cDNA can be isolated based on its homology to human membrane-bound NOVX.
  • a membrane-bound human NOVX cDNA can be isolated based on its homology to soluble human NOVX.
  • an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.
  • the nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length.
  • an isolated nucleic acid molecule ofthe invention hybridizes to the coding region.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • Homologs /. e., nucleic acids encoding NOVX proteins derived from species other than human
  • other related sequences e.g., paralogs
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% ofthe probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • Stringent conditions are known to those skilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C. This hybridization is followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • An isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 corresponds to a naturally occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or fragments, analogs or derivatives thereof, under conditions of moderate stringency.
  • moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardf s solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • Other conditions of moderate stringency that may be used are well known in the art. See, e.g., Ausubel et al.
  • nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided.
  • low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
  • Other conditions of low stringency that may be used are well known in the art (e.g. , as employed for cross-species hybridizations).
  • allelic variants ofthe NOVX sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, thereby leading to changes in the amino acid sequence ofthe encoded NOVX protein, without altering the functional ability ofthe NOVX protein.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of NOVX without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the NONX proteins ofthe present invention are predicted to be particularly unamenable to alteration.
  • nucleic acid molecules encoding ⁇ OVX proteins that contain changes in amino acid residues that are not essential for activity. Such ⁇ OVX proteins differ in amino acid sequence from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, yet retain biological activity.
  • the isolated nucleic acid molecule comprises. a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8.
  • the protein encoded by the nucleic acid is at least about 80% homologous to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, more preferably at least about 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in NOVX is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined.
  • a mutant NOVX protein can be assayed for (1) the ability to form proteimprotein interactions with other NOVX proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant NOVX protein and a NOVX receptor; (3) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the ability to bind NOVX protein; or (5) the ability to specifically bind an anti-NOVX protein antibody.
  • Another aspect ofthe invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or fragments, analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • antisense nucleic acid molecules comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof.
  • Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a NOVX protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 are additionally provided.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding NOVX.
  • the term "coding region” refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of human NOVX corresponds to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding NOVX.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions). Given the coding strand sequences encoding NOVX disclosed herein (e.g., SEQ ID NO:
  • antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of NOVX mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid ofthe invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-me
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NOVX protein to thereby inhibit expression ofthe protein, e.g. , by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove ofthe double helix.
  • An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule ofthe invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) FEBS Lett 215: 327-330).
  • modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • NOVX Ribozymes and PNA moieties include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
  • an antisense nucleic acid ofthe invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.
  • ribozymes e. g. , hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)
  • a ribozyme having specificity for a NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of a NOVX DNA disclosed herein (i.e., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25).
  • a derivative of a Tetrahymena L- 19 IVS RNA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in a NOVX-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.
  • NOVX mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al, (1993) Science 261:1411-1418.
  • NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe NOVX (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe NOVX gene in target cells.
  • nucleotide sequences complementary to the regulatory region ofthe NOVX e.g., the NOVX promoter and/or enhancers
  • the NOVX promoter and/or enhancers e.g., the NOVX promoter and/or enhancers
  • the nucleic acids of ⁇ OVX can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23).
  • the terms "peptide nucleic acids" or "P ⁇ As” refer to nucleic acid mimics, e.g., D ⁇ A mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • PNAs of NOVX can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g. , inducing transcription or translation arrest or inhibiting replication.
  • PNAs of NOVX can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).
  • PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion ' would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl) amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al.
  • PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) above).
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre etal, 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre etal, 1987, Proc. Natl
  • oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al, 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
  • a NOVX polypeptide ofthe invention includes the NOVX-like protein whose sequence is provided in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 while still encoding a protein that maintains its NOVX-like activities and physiological functions, or a functional fragment thereof. In some embodiments, up to 20% or more ofthe residues may be so changed in the mutant or variant protein.
  • the NOVX polypeptide according to the invention is a mature polypeptide.
  • a NOVX -like variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues ofthe parent protein as well as the possibility of deleting one or more residues from the parent sequence.
  • Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
  • One aspect ofthe invention pertains to isolated NOVX proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies.
  • native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • NOVX proteins are produced by recombinant DNA techniques.
  • a NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of NOVX protein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of NOVX protein having less than about 30% (by dry weight) of non-NOVX protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX protein, still more preferably less than about 10% of non-NOVX protein, and most preferably less than about 5% non-NOVX protein.
  • non-NOVX protein also referred to herein as a "contaminating protein”
  • NOVX protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis ofthe protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX protein having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
  • Biologically active portions of a NOVX protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence ofthe NOVX protein, e.g., the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 that include fewer amino acids than the full length NOVX proteins, and exhibit at least one activity of a NOVX protein.
  • biologically active portions comprise a domain or motif with at least one activity ofthe NOVX protein.
  • a biologically active portion of a NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a biologically active portion of a NOVX protein ofthe present invention may contain at least one ofthe above-identified domains conserved between the NOVX proteins, e.g. TSR modules. Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native NOVX protein.
  • the NOVX protein has an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • the NOVX protein is substantially homologous to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 and retains the functional activity ofthe protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 and retains the functional activity ofthe NOVX proteins of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in either ofthe sequences being compared for optimal alignment between the sequences).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch 1970 J Mol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region ofthe analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part ofthe DNA sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • percentage of positive residues is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical and conservative amino acid substitutions, as defined above, occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of positive residues.
  • NOVX chimeric or fusion proteins As used herein, a NOVX "chimeric protein” or “fusion protein” comprises a NOVX polypeptide operatively linked to a non-NOVX polypeptide.
  • An "NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to NOVX
  • a non-NOVX polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism.
  • NOVX polypeptide can correspond to all or a portion of a NOVX protein.
  • a NOVX fusion protein comprises at least one biologically active portion of a NOVX protein.
  • a NOVX fusion protein comprises at least two biologically active portions of a NOVX protein.
  • the term "operatively linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame to each other.
  • the non-NOVX polypeptide can be fused to the N-terminus or C-terminus ofthe NOVX polypeptide.
  • a NOVX fusion protein comprises a NOVX polypeptide operably linked to the extracellular domain of a second protein.
  • fusion proteins can be further utilized in screening assays for compounds that modulate NOVX activity (such assays are described in detail below).
  • the fusion protein is a GST-NO VX fusion protein in which the NOVX sequences are fused to the C-terminus ofthe GST (i.e., glutathione S-transferase) sequences.
  • GST glutathione S-transferase
  • Such fusion proteins can facilitate the purification of recombinant NOVX.
  • the fusion protein is a NOVX-immunoglobulin fusion protein in which the NOVX sequences comprising one or more domains are fused to sequences derived from a member ofthe immunoglobulin protein family.
  • the NOVX-immunoglobulin fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a NOVX ligand and a NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo.
  • a contemplated NOVX ligand ofthe invention is the NOVX receptor.
  • the NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of a NOVX cognate ligand. Inhibition ofthe NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival, as well as acute and chronic inflammatory disorders and hyperplastic wound healing, e.g. hypertrophic scars and keloids.
  • NOVX-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with a NOVX ligand.
  • a NOVX chimeric or fusion protein ofthe invention can be produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
  • the present invention also pertains to variants ofthe NOVX proteins that function as either NOVX agonists (mimetics) or as NOVX antagonists.
  • Variants ofthe NOVX protein can be generated by mutagenesis, e.g., discrete point mutation or truncation ofthe NOVX protein.
  • An agonist ofthe NOVX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form ofthe NOVX protein.
  • An antagonist ofthe NOVX protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein.
  • treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe NOVX proteins.
  • Variants ofthe NOVX protein that function as either NOVX agonists (mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, ofthe NOVX protein for NOVX protein agonist or antagonist activity.
  • a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein.
  • a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein.
  • methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential NOVX sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.
  • libraries of fragments ofthe NOVX protein coding sequence can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of a NOVX protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal and internal fragments of various sizes ofthe NOVX protein.
  • Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of NOVX proteins.
  • the most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation ofthe vector encoding the gene whose product was detected.
  • Recrusive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
  • antibodies to NOVX proteins, or fragments of NOVX proteins.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin (Ig) molecules i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F ab , F ab , and F (ab . )2 fragments, and an F ab expression library.
  • an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature ofthe heavy chain present in the molecule.
  • Certain classes have subclasses as well, such as IgG l5 IgG 2 , and others.
  • the light chain may be a kappa chain or a lambda chain.
  • Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
  • An isolated NOVX-related protein ofthe invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments ofthe antigen for use as immunogens;
  • An antigenic peptide fragment comprises at least 6 amino acid residues ofthe amino acid sequence ofthe full length protein, such as an amino acid sequence shown in SEQ ID NO: 2, 4, 6 ,8 ,10, 12, 14, 16, 18, or 20, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.
  • the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions ofthe protein that are located on its surface; commonly these are hydrophilic regions.
  • At least one epitope encompassed by the antigenic peptide is a region of NOVX-related protein that is located on the surface ofthe protein, e.g., a hydrophilic region.
  • a hydrophobicity analysis ofthe human NOVX-related protein sequence will indicate which regions of a NOVX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.
  • a protein ofthe invention may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • an appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.
  • the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and
  • MPL-TDM adjuvant monophosphoryl Lipid A, synthetic trehalose dicorynomycolate.
  • the polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaff ⁇ nity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63). The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells can be grown iv vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies ofthe invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells ofthe invention serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody.
  • the antibodies directed against the protein antigens ofthe invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen- binding subsequences of antibodies) that are principally comprised ofthe sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice o the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol.. 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • the endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.
  • the human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement ofthe modifications.
  • nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
  • the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
  • 5,939,598 It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
  • a method for producing an antibody of interest such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein ofthe invention (see e.g., U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of F ab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
  • Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F (ab .
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one ofthe binding specificities is for an antigenic protein ofthe invention.
  • the second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one ofthe fusions.
  • CHI first heavy-chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part ofthe CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield ofthe heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One ofthe Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.
  • bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992).
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.
  • the "diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al, J. Immunol. 152:5368 (1994).
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
  • bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen ofthe invention.
  • an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc R), such as Fc RI (CD64), Fc RII (CD32) and Fc RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
  • Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.
  • antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
  • a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA.
  • Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
  • Heteroconjugate antibodies are also within the scope ofthe present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved intemalization capability and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992).
  • Homodimeric antibodies with enhanced anti- tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
  • PAP-S momordica charantia inhibitor
  • curcin crotin
  • sapaonaria officinalis inhibitor gelonin
  • mitogellin mitogellin
  • restrictocin phenomycin, enomycin, and the tricothecenes.
  • radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, ]31 I, ,31 In, 90 Y, and 186 Re.
  • Conjugates ofthe antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the antibody in another embodiment, can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
  • a "receptor” such streptavidin
  • a "ligand” e.g., avidin
  • vectors preferably expression vectors, containing a nucleic acid encoding a NOVX protein, or derivatives, fragments, analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors ofthe invention comprise a nucleic acid ofthe invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
  • the recombinant expression vectors ofthe invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells.
  • NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein.
  • Such fusion vectors typically serve three purposes: (/) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N. J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • E. coli expression vectors examples include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128.
  • Another strategy is to alter the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et ah, 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.
  • the NOVX expression vector is a yeast expression vector.
  • yeast Saccharomyces cerivisae examples include pYepSecl
  • NOVX can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • a nucleic acid ofthe invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBOJ. 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987. Genes Dev. 1 : 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
  • promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule ofthe invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription ofthe DNA molecule) of an RNA molecule that is antisense to NOVX mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression ofthe antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope ofthe term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • NOVX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as human, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

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Abstract

The present invention provides novel isolated NOVX polynucleotides and polypeptides encoded by the NOVX polynucleotides. Also provided are the antibodies that immunospecifically bind to an NOVX polypeptide or any derivative, variant, mutant or fragment of the NOVX polypeptide, polynucleotide or antibody. The invention additionally provides methods in which the NOVX polypeptide, polynucleotide and antibody are utilized in the detection and treatment of a broad range of pathological states, as well as to other uses.

Description

ODORANT RECEPTOR POLYPEPTIDES AND NUCLEIC ACIDS ENCODING SAME
RELATED APPLICATIONS
This application claims priority to USSN 60/177,839, filed January 25, 2000; USSN 60/176,134, filed January 14, 2000; USSN 60/175,989, filed January 13, 2000; USSN 60/218,324, filed July 14, 2000; USSN 60/220,253, filed July 24, 2000; USSN 60/178,191, filed January 26, 2000; USSN 60/178,227, filed January 26, 2000; and USSN 60/220,590, filed July 25, 2000, which are incorporated herein by reference in their entirety.
TECHNICAL FIELD OF THE INVENTION The invention generally relates to nucleic acids and polypeptides encoded therefrom.
BACKGROUND OF THE INVENTION
Within the animal kingdom, odor detection is a universal tool used for social interaction, predation, and reproduction. Chemosensitivity in vertebrates is modulated by bipolar sensory neurons located in the olfactory epithelium, which extend a single, highly arborized dendrite into the mucosa while projecting axons to relay neurons within the olfactory bulb. The many ciliae on the neurons bear odorant (or olfactory) receptors (ORs), which cause depolarization and formation of action potentials upon contact with specific odorants. ORs may also function as axonal guidance molecules, a necessary function as the sensory neurons are normally renewed continuously through adulthood by underlying populations of basal cells.
The mammalian olfactory system is able to distinguish several thousand odorant molecules. Odorant receptors are believed to be encoded by an extremely large subfamily of G protein-coupled receptors. These receptors share a 7-transmembrane domain structure with many neurotransmitter and hormone receptors and are likely to underlie the recognition and G-protein- mediated transduction of odorant signals and possibly other chemosensing responses as well.
The genes encoding these receptors are devoid of introns within their coding regions. Schurmans and co-workers cloned a member of this family of genes, OLFR1, from a genomic library by cross-hybridization with a gene fragment obtained by PCR. See Schurmans et al, Cyto genet. Cell Genet.. 1993, 63(3):200. By isotopic in situ hybridization, they mapped the gene to 17pl3- pl2 with a peak at band 17pl3. A minor peak was detected on chromosome 3, with a maximum in the region 3ql3-q21. After Mspl digestion, a restriction fragment length polymorphism (RFLP) was demonstrated. Using this in a study of 3 CEPH pedigrees, they demonstrated linkage with D17S126 at 17pter-pl2; maximum lod = 3.6 at theta = 0.0. Used as a probe on Southern blots under moderately stringent conditions, the cDNA hybridized to at least 3 closely related genes. Ben-Arie and colleagues cloned 16 human OLFR genes, all from 17pl3.3. See Ben-Arie et al, Hum. Mol. Genet., 1994, 3(2):229. The intronless coding regions are mapped to a 350-kb contiguous cluster, with an average intergenic separation of 15 kb. The OLFR genes in the cluster belong to 4 different gene subfamilies, displaying as much sequence variability as any randomly selected group of OLFRs. This suggested that the cluster may be one of several copies of an ancestral OLFR gene repertoire whose existence may have predated the divergence of mammals. Localization to 17pl3.3 was performed by fluorescence in situ hybridization as well as by somatic cell hybrid mapping. Previously, OR genes cloned in different species were from disparate locations in the respective genomes. The human OR genes, on the other hand, lack introns and may be segregated into four different gene subfamilies, displaying great sequence variability. These genes are primarily expressed in olfactory epithelium, but may be found in other chemoresponsive cells and tissues as well. Blache and co-workers used polymerase chain reaction (PCR) to clone an intronless cDNA encoding a new member (named OL2) ofthe G protein-coupled receptor superfamily.See Blache et al, Biochem. Biophvs. Res. Commun., 1998, 242(3):669. The coding region ofthe rat OL2 receptor gene predicts a seven transmembrane domain receptor of 315 amino acids. OL2 has 46.4 percent amino acid identity with OL1, an olfactory receptor expressed in the developing rat heart, and slightly lower percent identities with several other olfactory receptors. PCR analysis reveals that the transcript is present mainly in the rat spleen and in a mouse insulin- secreting cell line (MIN6). No correlation was found between the tissue distribution of OL2 and that ofthe olfaction-related GTP-binding protein Golf alpha subunit. These findings suggest a role for this new hypothetical G-protein coupled receptor and for its still unknown ligand in the spleen and in the insulin-secreting beta cells.
Olfactory loss may be induced by trauma or by neoplastic growths in the olfactory neuroepithelium. There is currently no treatment available that effectively restores olfaction in the case of sensorineural olfactory losses. See Harrison's Principles of Internal Medicine, 14th Ed., Fauci, AS et al. (eds.), McGraw-Hill, New York, 1998, 173. There thus remains a need for effective treatment to restore olfaction in pathologies related to neural olfactory loss.
SUMMARY OF THE INVENTION
The invention is based, in part, upon the discovery of novel polynucleotide sequences encoding novel polypeptides.
Accordingly, in one aspect, the invention provides an isolated nucleic acid molecule that includes the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 or a fragment, homolog, analog or derivative thereof. The nucleic acid can include, e.g. , a nucleic acid sequence encoding a polypeptide at least 85% identical to a polypeptide that includes the amino acid sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26. The nucleic acid can be, e.g., a genomic DNA fragment, or a cDNA molecule.
Also included in the invention is a vector containing one or more ofthe nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein.
The invention is also directed to host cells transformed with a vector comprising any of the nucleic acid molecules described above.
In another aspect, the invention includes a pharmaceutical composition that includes a NOVX nucleic acid and a pharmaceutically acceptable carrier or diluent. In a further aspect, the invention includes a substantially purified NOVX polypeptide, e.g., any ofthe NOVX polypeptides encoded by an NONX nucleic acid, and fragments, homologs, analogs, and derivatives thereof. The invention also includes a pharmaceutical composition that includes an ΝONX polypeptide and a pharmaceutically acceptable carrier or diluent. In still a further aspect, the invention provides an antibody that binds specifically to an
ΝONX polypeptide. The antibody can be, e.g., a monoclonal or polyclonal antibody, and fragments, homologs, analogs, and derivatives thereof. The invention also includes a pharmaceutical composition including ΝONX antibody and a pharmaceutically acceptable carrier or diluent. The invention is also directed to isolated antibodies that bind to an epitope on a polypeptide encoded by any ofthe nucleic acid molecules described above.
The invention also includes kits comprising any ofthe pharmaceutical compositions described above. The invention further provides a method for producing an NONX polypeptide by providing a cell containing an ΝONX nucleic acid, e.g., a vector that includes an ΝOVX nucleic acid, and culturing the cell under conditions sufficient to express the ΝONX polypeptide encoded by the nucleic acid. The expressed ΝONX polypeptide is then recovered from the cell. Preferably, the cell produces little or no endogenous ΝONX polypeptide. The cell can be, e.g., a prokaryotic cell or eukaryotic cell.
The invention is also directed to methods of identifying an ΝONX polypeptide or nucleic acid in a sample by contacting the sample with a compound that specifically binds to the polypeptide or nucleic acid, and detecting complex formation, if present. The invention further provides methods of identifying a compound that modulates the activity of an ΝONX polypeptide by contacting an ΝONX polypeptide with a compound and determining whether the ΝONX polypeptide activity is modified.
The invention is also directed to compounds that modulate ΝONX polypeptide activity identified by contacting an ΝOVX polypeptide with the compound and determining whether the compound modifies activity ofthe ΝONX polypeptide, binds to the ΝOVX polypeptide, or binds to a nucleic acid molecule encoding an ΝOVX polypeptide.
In another aspect, the invention provides a method of determining the presence of or predisposition of an ΝOVX-associated disorder in a subject. The method includes providing a sample from the subject and measuring the amount of ΝOVX polypeptide in the subject sample. The amount of ΝOVX polypeptide in the subject sample is then compared to the amount of ΝOVX polypeptide in a control sample. An alteration in the amount of ΝOVX polypeptide in the subject protein sample relative to the amount of ΝOVX polypeptide in the control protein sample indicates the subject has a tissue proliferation-associated condition. A control sample is preferably taken from a matched individual, i.e., an individual of similar age, sex, or other general condition but who is not suspected of having a tissue proliferation-associated condition. Alternatively, the control sample may be taken from the subject at a time when the subject is not suspected of having a tissue proliferation-associated disorder. In some embodiments, the ΝOVX is detected using an ΝOVX antibody.
In a further aspect, the invention provides a method of determining the presence of or predisposition of an ΝOVX-associated disorder in a subject. The method includes providing a nucleic acid sample, e.g., RΝA or DΝA, or both, from the subject and measuring the amount of the ΝOVX nucleic acid in the subject nucleic acid sample. The amount of ΝOVX nucleic acid sample in the subject nucleic acid is then compared to the amount of an NOVX nucleic acid in a control sample. An alteration in the amount of NOVX nucleic acid in the sample relative to the amount of NOVX in the control sample indicates the subject has a NOVX-associated disorder. In a still further aspect, the invention provides a method of treating or preventing or delaying an NOVX-associated disorder. The method includes administering to a subject in which such treatment or prevention or delay is desired an NOVX nucleic acid, an NOVX polypeptide, or an NOVX antibody in an amount sufficient to treat, prevent, or delay a NOVX- associated disorder in the subject.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing ofthe present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages ofthe invention will be apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
Olfactory receptors (ORs) are the largest family of G-protein-coupled receptors (GPCRs) and belong to the first family (Class A) of GPCRs, along with catecholamine receptors and opsins. The OR family contains over 1,000 members that traverse the phylogenetic spectrum from C. elegans to mammals. ORs most likely emerged from prototypic GPCRs several times independently, extending the structural diversity necessary both within and between species in order to differentiate the multitude of ligands. Individual olfactory sensory neurons are predicted to express a single, or at most a few, ORs. All ORs are believed to contain seven α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. The pocket of OR ligand binding is expected to be between the second and sixth transmembrane domains ofthe proteins. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%, and genes greater than 80% identical to one another at the amino acid level are considered to belong to the same subfamily.
Since the first ORs were cloned in 1991, outstanding progress has been made into their mechanisms of action and potential dysregulation during disease and disorder. It is understood that some human diseases result from rare mutations within GPCRs. Drug discovery avenues could be used to produce highly specific compounds on the basis of minute structural differences of OR subtypes, which are now being appreciated with in vivo manipulation of OR levels in transgenic and knock-out animals. Furthermore, due to the intracellular homogeneity and ligand specificity of ORs, renewal of specific odorant-sensing neurons lost in disease or disorder is possible by the introduction of individual ORs into basal cells. Additionally, new therapeutic strategies may be elucidated by further study of so-called orphan receptors, whose ligand(s) remain to be discovered.
OR proteins bind odorant ligands and transmit a G-protein-mediated intracellular signal, resulting in generation of an action potential. The accumulation of DNA sequences of hundreds of OR genes provides an opportunity to predict features related to their structure, function and evolutionary diversification. See Pilpel Y, etal., Essays Biochem 1998;33:93-104. The OR repertoire has evolved a variable ligand-binding site that ascertains recognition of multiple odorants, coupled to constant regions that mediate the cAMP-mediated signal transduction. The cellular second messenger underlies the responses to diverse odorants through the direct gating of olfactory-specific cation channels. This situation necessitates a mechanism of cellular exclusion, whereby each sensory neuron expresses only one receptor type, which in turn influences axonal projections. A 'synaptic image' ofthe OR repertoire thus encodes the detected odorant in the central nervous system.
The ability to distinguish different odors depends on a large number of different odorant receptors (ORs). ORs are expressed by nasal olfactory sensory neurons, and each neuron expresses only 1 allele of a single OR gene. In the nose, different sets of ORs are expressed in distinct spatial zones. Neurons that express the same OR gene are located in the same zone; however, in that zone they are randomly interspersed with neurons expressing other ORs. When the cell chooses an OR gene for expression, it may be restricted to a specific zonal gene set, but it may select from that set by a stochastic mechanism. Proposed models of OR gene choice fall into 2 classes: locus-dependent and locus-independent. Locus-dependent models posit that OR genes are clustered in the genome, perhaps with members of different zonal gene sets clustered at distinct loci. In contrast, locus-independent models do not require that OR genes be clustered. OR genes have been mapped to 11 different regions on 7 chromosomes. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Studies have shown that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones. These findings raised the possibility that OR gene choice is locus-independent or involved consecutive stochastic choices.
Issel-Tarver and Rine (1996) characterized 4 members ofthe canine olfactory receptor gene family. The 4 subfamilies comprised genes expressed exclusively in olfactory epithelium. Analysis of large DNA fragments using Southern blots of pulsed field gels indicated that subfamily members were clustered together, and that two ofthe subfamilies were closely linked in the dog genome. Analysis ofthe four olfactory receptor gene subfamilies in 26 breeds of dog provided evidence that the number of genes per subfamily was stable in spite of differential selection on the basis of olfactory acuity in scent hounds, sight hounds, and toy breeds.
Issel-Tarver and Rine (1997) performed a comparative study of four subfamilies of olfactory receptor genes first identified in the dog to assess changes in the gene family during mammalian evolution, and to begin linking the dog genetic map to that of humans. These four families were designated by them OLFl, OLF2, OLF3, and OLF4 in the canine genome. The subfamilies represented by these four genes range in size from 2 to 20 genes. They are all expressed in canine olfactory epithelium but were not detectably expressed in canine lung, liver, ovary, spleen, testis, or tongue. The OLFl and OLF2 subfamilies are tightly linked in the dog genome and also in the human genome. The smallest family is represented by the canine OLFl gene. Using dog gene probes individually to hybridize to Southern blots of genomic DNA from 24 somatic cell hybrid lines. They showed that the human homologous OLFl subfamily maps to human chromosome 11. The human gene with the strongest similarity to the canine OLF2 gene also mapped to chromosome 11. Both members ofthe human subfamily that hybridized to canine OLF3 were located on chromosome 7. It was difficult to determine to which chromosome or chromosomes the human genes that hybridized to the canine OLF4 probe mapped. This subfamily is large in mouse and hamster as well as human, so the rodent background largely obscured the human cross-hybridizing bands. It was possible, however, to discern some human- specific bands in blots corresponding to human chromosome 19. They refined the mapping ofthe human OLFl homolog by hybridization to YACs that map to 1 lql 1. In dogs, the OLFl and OLF2 subfamilies are within 45 kb of one another (Issel-Tarver and Rine (1996)).
Issel-Tarver and Rine (1997) demonstrated that in the human OLFl and OLF2 homologs are likewise closely linked. By studying YACs, Issel-Tarver and Rine (1997) found that the human OLF3 homolog maps to 7q35. A chromosome 19-specific cosmid library was screened by hybridization with the canine OLF4 gene probe, and clones that hybridized strongly to the probe even at high stringency were localized to 19p 13.1 and 19pl3.2. These clones accounted, however, for a small fraction ofthe homologous human bands.
Rouquier et al. (1998) demonstrated that members ofthe olfactory receptor gene family are distributed on all but a few human chromosomes. Through fluorescence in situ hybridization analysis, they showed that OR sequences reside at more than 25 locations in the human genome. Their distribution was biased for terminal bands of chromosome arms. Flow-sorted chromosomes were used to isolate 87 OR sequences derived from 16 chromosomes. Their sequence relationships indicated the inter- and intrachromosomal duplications responsible for OR family expansion. Rouquier et al. (1998) determined that the human genome has accumulated a striking number of dysfunctional copies: 72% of these sequences were found to be pseudogenes. ORF-containing sequences predominate on chromosomes 7, 16, and 17.
Trask et al. (1998) characterized a subtelomeric DNA duplication that provided insight into the variability, complexity, and evolutionary history of that unusual region ofthe human genome, the telomere. Using a DNA segment cloned from chromosome 19, they demonstrated that the blocks of DNA sequence shared by different chromosomes can be very large and highly similar. Three chromosomes appeared to have contained the sequence before humans migrated around the world. In contrast to its multicopy distribution in humans, this subtelomeric block maps predominantly to a single locus in chimpanzee and gorilla, that site being nonorthologous to any ofthe locations in the human genome. Three new members ofthe olfactory receptor (OR) gene family were found to be duplicated within this large segment of DNA, which was found to be present at 3q, 15q, and 19p in each of 45 unrelated humans sampled from various populations. From its sequence, one ofthe OR genes in this duplicated block appeared to be potentially functional. The findings raised the possibility that functional diversity in the OR family is generated in part through duplications and interchromosomal rearrangements ofthe DNA near human telomeres. Mombaerts (1999) reviewed the molecular biology ofthe odorant receptor (OR) genes in vertebrates. Buck and Axel (1991) discovered this large family of genes encoding putative odorant receptor genes. Zhao et al. (1998) provided functional proof that one OR gene encodes a receptor for odorants. The isolation of OR genes from the rat by Buck and Axel (1991) was based on three assumptions. First, ORs are likely G protein-coupled receptors, which characteristically are 7-transmembrane proteins. Second, ORs are likely members of a multigene family of considerable size, because an immense number of chemicals with vastly different structures can be detected and discriminated by the vertebrate olfactory system. Third, ORs are likely expressed selectively in olfactory sensory neurons. Ben-Arie et al. (1994) focused attention on a cluster of human OR genes on 17p, to which the first human OR gene, OR1D2, had been mapped by Schurmans et al. (1993). According to Mombaerts (1999), the sequences of more than 150 human OR clones had been reported.
The human OR genes differ markedly from their counterparts in other species by their high frequency of pseudogenes, except the testicular OR genes. Research showed that individual olfactory sensory neurons express a small subset ofthe OR repertoire. In rat and mouse, axons of neurons expressing the same OR converge onto defined glomeruli in the olfactory bulb.
The present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences and their polypeptides. The sequences are collectively referred to as "NOVX nucleic acids" or "NOVX polynucleotides" and the corresponding encoded polypeptides are referred to as "NOVX polypeptides" or "NOVX proteins." Unless indicated otherwise, "NOVX" is meant to refer to any ofthe novel sequences disclosed herein. Table 1 provides a summary ofthe NOVX nucleic acids and their encoded polypeptides. Example 1 provides a description of how the novel nucleic acids were identified.
TABLE 1. Sequences and Corresponding SEQ ID Numbers
SEQ ID
NOVX Internal NO SEQ ID NO
Assignment Identification (nucleic (polypeptide) Homology acid)
AL 121944 A 1 ORGPCR
AL135904 A ORGPCR
AL121986A ORGPCR
AL121986A1 ORGPCR
AC012661 A 10 ORGPCR
AGO 12661 B 11 12 ORGPCR
AF061779 A 13 14 ORGPCR
AC012616 A 15 16 ORGPCR
AC012616 A1 17 18 ORGPCR
10 AC019108 A 19 20 ORGPCR
11 AC012661 dal 21 22 ORGPCR
12 CG50381-01 23 24 ORGPCR
13 AC012661A_.0. 25 26 ORGPCR 46 EXT
Where OR GPCR is an odorant receptor ofthe G-protein coupled-receptor family.
NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members ofthe protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members ofthe family to which the NOVX polypeptides belong.
For example, NOVl-10 are homologous to members ofthe odorant receptor (OR) family ofthe human G-protein coupled receptor (GPCR) superfamily of proteins, as shown in Table 56. Thus, the NOVl-10 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in disorders of olfactory loss, e.g., trauma, HIV illness, neoplastic growth and neurological disorders e.g. Parkinson's disease and Alzheimer's disease. The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., neurogenesis, cell differentiation, cell motility, cell proliferation and angiogenesis. Additional utilities for the NOVX nucleic acids and polypeptides according to the invention are disclosed herein. NOV1
A NO VI sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. A NOV1 nucleic acid and its encoded polypeptide includes the sequences shown in Table 2. The disclosed nucleic acid (SEQ ID NO:l) is 1,071 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 42-44 and ends with a TAA stop codon at nucleotides 1,053-1,055. The representative ORF encodes a 337 amino acid polypeptide (SEQ ID NO:2). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 1.
TABLE 2.
ATATTTCATTCTCTGGGTCTTCATGCAGATATATTCAAGCAATGGAAGGGAAAAATC AAACCAATATCTCTGAATTTCTCCTCCTGGGCTTCTCAAGTTGGCAACAACAGCAGG TGCTACTCTTTGCACTTTTCCTGTGTCTCTATTTAACAGGGCTGTTTGGAAACTTACT CATCTTGCTGGCCATTGGCTCGGATCACTGCCTTCACACACCCATGTATTTCTTCCTT GCCAATCTGTCCTTGGTAGACCTCTGCCTTCCCTCAGCCACAGTCCCCAAGATGCTA CTGAACATCCAAACCCAAACCCAAACCATCTCCTATCCCGGCTGCCTGGCTCAGATG TATTTCTGTATGATGTTTGCCAATATGGACAATTTTCTTCTCACAGTGATGGCATATG ACCGTTACGTGGCCATCTGTCACCCTTTACATTACTCCACCATTATGGCCCTGCGCCT CTGTGCCTCTCTGGTAGCTGCACCTTGGGTCATTGCCATTTTGAACCCTCTCTTGCAC ACTCTTATGATGGCCCATCTGCACTTCTGCTCTGATAATGTTATCCACCATTTCTTCT GTGATATCAACTCTCTCCTCCCTCTGTCCTGTTCCGACACCAGTCTTAATCAGTTGAG TGTTCTGGCTACGGTGGGGCTGATCTTTGTGGTACCTTCAGTGTGTATCCTGGTATCC TATATCCTCATTGTTTCTGCTGTGATGAAAGTCCCTTCTGCCCAAGGAAAACTCAAG GCTTTCTCTACCTGTGGATCTCACCTTGCCTTGGTCATTCTTTTCTATGGAGCAATCA CAGGGGTCTATATGAGCCCCTTATCCAATCACTCTACTGAAAAAGACTCAGCCGCAT CAGTCATTTTTATGGTTGTAGCACCTGTGTTGAATCCATTCATTTACAGTTTAAGAAA CAATGAACTGAAGGGGACTTTAAAAAAGACCCTAAGCCGACCGGGCGCGGTGGCTC ACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCATGAGGTCAGGA GATCGAGACCATCCTGGCTAACAAGGTGAAACCCCGT (SEQ ID NO.: 1) MEGKNQTNISEFLLLGFSSWQQQQVLLFALFLCLYLTGLFGNLLILLAIGSDHCLHTPMY FFLANLSLVDLCLPSATVPKMLLNIQTQTQTISYPGCLAQMYFCMMFANMDNFLLTVM AYDRYVAICHPLHYSTIMALRLCASLVAAPWVIAILNPLLHTLMMAHLHFCSDNVIHHF FCDINSLLPLSCSDTSLNQLSVLATVGLIFVVPSVCILVSYILIVSAVMKVPSAQGKLKAFS TCGSHLALVILFYGAITGVYMSPLSNHSTEKDSAASVIFMWAPVLNPFIYSLRNNELKG TLKKTLSRPGAVAHACNPSTLGGRGGWIMRSGDRDHPG (SEQ ID NO.: 2)
The NOVl nucleic acid sequence has homology with several fragments ofthe human olfactory receptor 17-93 (OLFR) (GenBank Accession No.: HSU76377), as shown in Table 3. Also, the NOVl polypeptide has homology (approximately 61% identity, 74% similarity) to human olfactory receptor, family 1, subfamily F, member 8 (OLFR) (GenBank Accession No.: XP007973), as is shown in Table 4. Furthermore, the NOVl polypeptide has homology (approximately 61% identity, 75% similarity) to a human olfactory protein (OLFR)(EMBL Accession No.: 043749), as is shown in Table 5. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413.
OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy- terminus. Multiple sequence augment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
Thus, NOVl is predicted to have a seven transmembrane region and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288), as is shown in Table 6.
TABLE 3
NOVl: 1034 GGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCAT 1093
! ! I M I I I I M I M I I I I I I I i M l l l l l l i M M I I M I I I I M I I I I OLFR: 41200 GGATGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCGGATCAT 41259
NOVl: 1094 GAGGTCAGGAGATCGAGACCATCCTGGCTAAC 1125 (SEQ ID NO. 33)
OLFR: 41260 GAGGTCAGTTGTTCGAGACCAACCTGGTCAAC 41291 (SEQ ID NO. 37) NOVl: 1032 CCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATC 1091
OLFR: 1 CTGGGCTCGGTGGCTCACACGTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATC 60 NOVl: 1092 A--TGAGGTCAGGAGATCGAGACCATCCTGGCTAAC 1125 (SEQ ID NO. 41)
I MIIIMIIIM Mill III
OLFR: 61 ACATGAGGTCAGGAGTTCGAGACCAGCCTGGTCAAC 96 (SEQ ID NO. 47)
NOVl: 1125 GTTAGCCAGGATGGTCTCGATCTCCTGACCTCATGATCCACCCGCCTCGGCCTCCCAAAG 1066 II llll
OLFR: 4688 GTTAGCCAGGATGGTCTCAATCTCCTGACCTCGTGATCCGCCTGCCTTGGCCTCCCAAAG 4747 NOVl: 1065 TGCTGGGATTACAGGCGTGAGCCACCGCGCCCGG 1032 (SEQ ID NO. 48) OLFR: 4748 TGCTGGGATTACAGGCATGAGCCACTGCGCCCGG 4781 (SEQ ID NO. 52)
TABLE 4
NOVl: 1 MSGTNQSSVSEFLLLGLSRQPQQQHLLFVFFLSMYLATVLGNLLIILSVSIDSCLHTPMY 60 * * **4.. .******* * *** *** ** _|_** . *****.*,_|_ * *******
OLFR: 1 MEGKNQTNISEFLLLGFSS QQQQVLLFALFLCLYLTGLFGNLLILLAIGSDHCLHTPMY 60
NOVl: 61 FFLSNLSFVDICFSFTTVPKMLANHILETQTISFCGCLTQMYFVFMFVDMDNFLLAVMAY 120
** *.j.** * **-j-* -k-k-k-k-k-k - _j_**** *.j. *** **** * * _j_* * **** -k-k-k-k
OLFR: 61 FFLANLSLVDLCLPSATVPKMLLNIQTQTQTISYPGCLAQi^FCMMFANMDNFLLTVMAY 120
NOVl: 121 DHFVAVCHPLHYTAKMTHQLCALLVAGL VVANLNVLLHTLL APLSFCADNAITHFFCD 180
* ^- k^-k-k-k-k-k-k^. -k ic-k-k - k-k -k-k +- -k-k -k-k-k-k-k^-k-k * -k- + -k-k * -k-k-k-k-k
OLFR: 121 DRYVAICHPLHYSTIMALRLCASLVAAPWVIAILNPLLHTLi AHLHFCSDNVIHHFFCD 180
NOVl: 181 VTPLLKLSCSDTHLNEVIILSEGALVMITPFLCILASYMHITCTVLKVPSTKGRWKAFST 240 . ** ****** **++ 4.*+ *4- 4. * 4.*** **4- * *^.**** +*+ *****
OLFR: 181 INSLLPLSCSDTSLNQLSVLATVGLIFWPSVCILVSYILIVSAVMKVPSAQGKLKAFST 240 NOVl: 241 CGSHLAVVLLFYSTIIAVYFNPLSSHSAEKDT ATVLYTVVTP LNPFIYSLRNRYLKGA 300
******+*_|.*** * * * ψ***^.** ***_)_ *_j.*.ι.-+_ ** ■k^.kie-kk-k-k-k-kk'k -k-k-k
OLFR: 241 CGSHLALVILFYGAITGVYMSPLSNHSTEKDSAASVIFMWAPVLNPFIYSLRNNELKGT 300
NOVl: 301 LK WGR 307 (SEQ ID NO. 27) *** + *
OLFR: 301 LKKTLSR 307 (SEQ ID NO. 28)
Where * indicates identity and + indicates similarity.
TABLE 5
NOVl: 1 ΞGKNQTNISEFLLLGFSS QQQQVLLFALFLCLYLTGLFGNLLILLAIGSDHCLHTPMY 60 * * **++4-******* * *** *** ** +** + *****+*-|_.|. * *******
OLFR: 1 MSGTNQSSVSEFLLLGLSRQPQQQHLLFVFFLSMYLATVLGNLLIILSVSIDSCLHTPMY 60
NOVl: 61 FFLANLSLVDLCLPSATVPKMLLNIQTQTQTISYPGCLAQiiYFCMMFANMDNFLLTVMAY 120 ***+*** **+* ****** * 4*****4 *** **** ** +****** ****
OLFR: 61 FFLSNLSFVDICFSFTTVPK LANHILETQTISFCGCLTQMYFVFMFVD DNFLLAVMAY 120 NOVl: 121 DRYVAICHPLHYSTIIVLALRLCASLVAAP VIAILNPLLHTLJMMAHLHFCSDNVIHHFFCD 180
* _**4******4. * 4.*** *** **4_* ** *****4-** * **ψ** * *****
OLFR: 121 DHFVAVCHPLHYTAKMTHQLCALLVAGLWWANLNVLLHTLLMAPLSFCADNAITHFFCD 180
NOVl: 181 INSLLPLSCSDTSLNQLSVLATVGLIFWPSVCILVSYILIVSAV KVPSAQG LKAFST 240
4. ** ****** **4. _ 4-*4- *4 4- * 4.*** **4- * *4-**** 4-*4- *****
OLFR: 181 VTPLLKLSCSDTHLNEVIILSEGALVMITPFLCILASYi HITCTVLKVPSTKGRWKAFST 240
NOVl: 241 CGSHLALVILFYGAITGVYMSPLSNHSTEKDSAASVIFMVVAPVLNPFIYSLRNNELKG 299
(SEQ ID NO. 29)
•k -k -k -k -k -k - +-k -k -k -k k-k ^.-k-kk .$.** ***_)_ *^-*- ^- ** -k -k -k -k -k -k -k -k -k -k -k ***
OLFR: 241 CGSHLAWLLFYSTIIAVYFNPLSSHSAEKDTMATVLYTWTPMLNPFIYSLRNRYLKG 299
(SEQ ID NO. 30)
Where * indicates identity and + indicates similarity.
TABLE 6
NOVl: 48 AIGSDHCLHTPMYFFLANLSLVDLCLPSATVPKMLLNIQTQTQTISYPGCLAQMYFCMMF 107
GPCR: 8 AVSREKALQTTTNYLIVSLAVADLLVATLVMP VVYLEVVGEWKFSRIHCDIFVTLDVMM 67
NOVl: 108 ANro FLLT MAYDRY/AICHPLHYS IM- LRLCASLVAAPWVIAILNP LH MAHL 166
GPCR: 68 CTASILNLCAISIDRYTAVAMPMLYNTRYSS RRVTVMIAIVWVLSFTISCPMLFGLNNT 127
NOVl: 167 HFCSDNVIHHFFCDINSLLPLSCSDTSLNQLSVLATVGLIFWPSVCILVSYILIVSAVM 226
GPCR: 128 DQN ECIIANPAFWYSSIVS--FYVPFIVTLLVYIKIYIVLR 167
NOVl: 227 KVPSAQGKLK 236 (SEQ ID NO. 31) GPCR: 168 RRRKRVNTKR 177 (SEQ ID NO . 32)
Because the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade, NOVl can be used to detect nasal epithelial neuronal tissue.
Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing. NOV2
A NOV2 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. A NOV2 nucleic acid and its encoded polypeptide includes the sequences shown in Table 7. The disclosed nucleic acid (SEQ ID NO:3) is 1,040 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 82-84 and ends with a TGA stop codon at nucleotides 1,012-1,014. The representative ORF encodes a 310 amino acid polypeptide (SEQ ID NO:4). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 3.
TABLE 7.
CCGAACAAGTTAAAATGAATCTGTTTTTAAACACTTCTCCTAAACCATGAGCATTAA CTTGATTTCCTCTGTCATAGGGATATGGGAGACAATATAACATCCATCAGAGAGTTC CTCCTACTGGGATTTCCCGTTGGCCCAAGGATTCAGATGCTCCTCTTTGGGCTCTTCT CCCTGTTCTACGTCTTCACCCTGCTGGGGAACGGGACCATACTGGGGCTCATCTCAC TGGACTCCAGACTGCACGCCCCCATGTACTTCTTCCTCTCACACCTGGCGGTCGTCG ACATCGCCTACGCCTGCAACACGGTGCCCCGGATGCTGGTGAACCTCCTGCATCCAG CCAAGCCCATCTCCTTTGCGGGCCGCATGATGCAGACCTTTCTGTTTTCCACTTTTGC TGTCACAGAATGTCTCCTCCTGGTGGTGATGTCCTATGATCTGTACGTGGCCATCTGC CACCCCCTCCGATATTTGGCCATCATGACCTGGAGAGTCTGCATCACCCTCGCGGTG ACTTCCTGGACCACTGGAGTCCTTTTATCCTTGATTCATCTTGTGTTACTTCTACCTTT ACCCTTCTGTAGGCCCCAGAAAATTTATCACTTTTTTTGTGAAATCTTGGCTGTTCTC AAACTTGCCTGTGCAGATACCCACATCAATGAGAACATGGTCTTGGCCGGAGCAATT TCTGGGCTGGTGGGACCCTTGTCCACAATTGTAGTTTCATATATGTGCATCCTCTGTG CTATCCTTCAGATCCAATCAAGGGAAGTTCAGAGGAAAGCCTTCCGCACCTGCTTCT CCCACCTCTGTGTGATTGGACTCGTTTATGGCACAGCCATTATCATGTATGTTGGACC CAGATATGGGAACCCCAAGGAGCAGAAGAAATATCTCCTGCTGTTTCACAGCCTCTT TAATCCCATGCTCAATCCCCTTATCTGTAGTCTTAGGAACTCAGAAGTGAAGAATAC TTTGAAGAGAGTGCTGGGAGTAGAAAGGGCTTTATGAAAAGGATTATGGCATTGTG ACTGACA (SEQ ID NO.: 3) MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYFFL SHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLWMSYDL YVAICHPLRYLAIMTWRVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEILA VLKLACADTHINENMVLAGAISGLVGPLSTIVVSYMCILCAILQIQSREVQRKAFRTCFSH LCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTLKRV LGVERAL (SEQ ID NO.: 4)
The NOV2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. A NOV2 nucleic acid was identified on human chromosome 6.
The NOV2 nucleic acid sequence has a high degree of homology (99% identity) with a human genomic clone corresponding to chromosome 6 (CHR6) (GenBank Accession No.: AL135904), as shown in Table 8. Additionally, the NOV2 polypeptide has a high degree of homology (approximately 95% identity) to a human olfactory receptor (OLFR) (GenBank Accession No.: AL135904), as shown in Table 9. Furthermore, the NOV2 polypeptide has a high degree of homology (approximately 91% identity) to a human olfactory protein (OLFR) (EMBL Accession No.: AC005587), as shown in Table 10. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences. 1999, 20:413.
OR proteins have seven transmembrane -helices separated by three extracellular and three cytoplasmic loops, along with an extracellular amino-terminus and a cytoplasmic carboxy- terminus. Multiple sequence augment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains. Thus, NOV2 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 11.
TABLE 8
NOV2 : 1 ccgaacaagttaaaatgaatctgtttttaaacacttctcctaaaccatgagcattaactt 60 I I M I I I I I I I I I I I I I I I I I I I I I 1 1 I I I I I I I I I I I I I I I I I I I I I I 1 1 I I I 1 1 I I I I
CHR6 : 22579 ccgaacaagttaaaatgaatctgtttttaaacacttctcctaaaccatgagcattaactt 22520 NOV2 : 61 gatttcctctgtcatagggatatgggagacaatataacatccatcagagagttcctccta 120
CHR6 : 22519 gatttcctctgtcatagggatatgggagacaatataacatccatcagagagttcctccta 22460
NOV2 : 121 ctgggatttcccgttggcccaaggattcagatgctcctctttgggctcttctccctgttc 180
CHR6 : 22459 ctgggatttcccgttggcccaaggattcagatgctcctctttgggctcttctccctgttc 22400
NOV2 : 181 tacgtcttcaccctgctggggaacgggaccatactggggctcatctcactggactccaga 240
CHR6 : 22399 tacgtcttcaccctgctggggaacgggaccatactggggctcatctcactggactccaga 22340
N0V2 : 241 ctgcacgcccccatgtacttcttcctctcacacctggcggtcgtcgacatcgcctacgcc 300 CHR6 : 22339 ctgcacgcccccatgtacttcttcctctcacacctggcggtcgtcgacatcgcctacgcc 22280
N0V2 : 301 tgcaacacggtgccccggatgctggtgaacctcctgcatccagccaagcccatctccttt 360
111 II II 111 II 1111 II I II I II 11 II I II II III 11 II II II 1111 II 11 II 11 II 11
CHR6 : 22279 tgcaacacggtgccccggatgctggtgaacctcctgcatccagccaagcccatctccttt 22220
N0V2 : 361 gcgggccgcatgatgcagacctttctgttttccacttttgctgtcacagaatgtctcctc 420
CHR6 : 22219 gcgggccgcatgatgcagacctttctgttttccacttttgctgtcacagaatgtctcctc 22160
N0V2 : 421 ctggtggtgatgtcctatgatctgtacgtggccatctgccaccccctccgatatttggcc 480
CHR6 : 22159 ctggtggtgatgtcctatgatctgtacgtggccatctgccaccccctccgatatttggcc 22100
N0V2 : 481 atcatgacctggagagtctgcatcaccctcgcggtgacttcctggaccactggagtcctt 540 CHR6: 22099 atcatgacctggagagtctgcatcaccctcgcggtgacttcctggaccactggagtcctt 22040
N0V2 : 541 ttatccttgattcatcttgtgttacttctacctttacccttctgtaggccccagaaaatt 600
CHR6 : 22039 ttatccttgattcatcttgtgttacttctacctttacccttctgtaggccccagaaaatt 21980
N0V2 : 601 tatcacniiiinnnngtgaaatcttggctgttctcaaacttgcctgtgcagatacccacatc 660
M I N I
CHR6 : 21979 tatcactttttttgtgaaatcttggctgttctcaaacttgcctgtgcagatacccacatc 21920
N0V2 : 661 aatgagaacatggtcttggccggagcaatttctgggctggtgggacccttgtccacaatt 720
11 ) ] I i 111 ! I ) ! I ! ) i ] 11111111111 j I ] 1111 ! 111111 ] 11 ) 1111 i 1111111 )
CHR6 : 21919 aatgagaacatggtcttggccggagcaatttctgggctggtgggacccttgtccacaatt 21860
N0V2 : 721 gtagtttcatatatgtgcatcctctgtgctatccttcagatccaatcaagggaagttcag 780
CHR6 : 21859 gtagtttcatatatgtgcatcctctgtgctatccttcagatccaatcaagggaagttcag 21800 NOV2 : 781 aggaaagccttccgcacctgcttctcccacctctgtgtgattggactcgtttatggcaca 840
M N M i II i II 1111 II M 1111111! II 11 II 111 N I M 111 ! II 11 ! 1111 M 111
5 CHR6 : 21799 aggaaagccttccgcacctgcttctcccacctctgtgtgattggactcgtttatggcaca 21740
NOV2 : 841 gccattatcatgtatgttggacccagatatgggaaccccaaggagcagaagaaatatctc 900 0 CHR6 : 21739 gccattatcatgtatgttggacccagatatgggaaccccaaggagcagaagaaatatctc 21680
NOV2 : 901 ctgctgtttcacagσctctttaatcccatgctcaatccccttatctgtagtcttaggaac 960 5 CHR6 : 21679 ctgctgtttcacagcctctttaatcccatgctcaatccccttatctgtagtcttaggaac 21620
N0V2 : 961 tcagaagtgaagaatactttgaagagagtgctgggagtagaaagggctttatgaaaagga 1020 0 CHR6 : 21619 tcagaagtgaagaatactttgaagagagtgctgggagtagaaagggctttatgaaaagga 21560 NOV2: 1021 ttatggcattgtgactgaca 1040 (SEQ ID NO. 3)
CHR6: 21559 ttatggcattgtgactgaca 21540 (SEQ ID NO. 34) 5
TABLE 9
NOV2: 51 DSRLHAP YFFLSHLAVVDIAYACN VPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTE 110
******* ***************************
OLFR: 13 DSRLHAP YFFLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTE 72 0
NOV2 : 111 CLLLWMSYDLYVAICHPLRYLAI T RVCITLAVTSWTTGVXXXXXXXXXXXXXPFCRP 170
****************************************** *****
OLFR : 73 CLLLWMSYDLYVAICHPLRYLAIMTWRVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRP 132 5 NOV2 : 171 QKIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSY CILCAILQIQSR 230
** * ***** * * *** *** ***** * * ** * ** * * *** *** * ** ***** * * ** * * ******* * **
OLFR : 133 QKIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSR 192 NOV2 : 231 EVQRKAFRTCFSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICS 290 (_) ************************************************************
OLFR: 193 EVQRKAFRTCFSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICS 252 NOV2: 291 LRNSEVKNTLKRVLGVERAL 310 (SEQ ID NO. 35)
******************** 5 OLFR: 253 LRNSEVKNTLKRVLGVERAL 272 (SEQ ID NO . 36)
Where * indicates identity
TABLE 10
NOV2: 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFXXXXXXXXXXXXXXDSRLHAPMYF 60 0 ************************************ **********
OLFR: 1 MGDNITSIRΞFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60 NOV2: 61 FLSHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGRiyiMQTFLFSTFAVTECLLLVVMSYD 120 ************************************************************
OLFR: 61 FLSHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLVVMSYD 120 NOV2: 121 LYVAICHPLRYLAIMTWRVCITLAVTS TTGVXXXXXXXXXXXXXPFCRPQKIYHFFCEI 180 ******************************** ***************
OLFR: 121 LYVAICHPLRYLAI T RVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180
NOV2: 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIVVSYMCILCAILQIQSREVQRKAFRTC 240 ************************************************************
OLFR: 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFRTC 240 NOV2: 241 FSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300
************************************************************ OLFR: 241 FSHLCVIGLVYGTAIIMYVGPRYGNPKΞQKKYLLLFHSLFNP LNPLICSLRNSEVKNTL 300
NOV2: 301 KRVLGVΞRAL 310 (SEQ ID NO. 4)
**********
OLFR: 301 KRVLGVERAL 310 (SEQ ID NO. 38) Where * indicates identity TABLE 11
NOV2 : 53 RLHAP1 YFFLSHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECL 112 GPCR : 14 ALQTTTNYLIVSLAVADLLVATLVMPWVVYLEVVGEWKFSRIHCDIFVTLDVMMCTASIL 73 NOV2 : 113 LLWMSYDLYVAICHPLRYLAIMTW-RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQ 171
GPCR : 74 NLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIV VLSFTISCPMLFGLNMTDQNΞ- - 131
NOV2 : 172 KIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSRE 231 GPCR : 132 -CIIANPAF WYSSIVSFYVPFIVTLLVYIKIYIVLRRRRKRV 173
NOV2: 232 VQRK 235 (SEQ ID NO. 39) GPCR: 174 NTKR 177 (SEQ ID NO. 40)
The OR family ofthe GPCR superfamily is involved in the initial steps ofthe olfactory signal transduction cascade. Therefore, the NOV2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
Based on this relatedness to other known members ofthe OR family ofthe GPCR superfamily, NOV2 can be used to provide new diagnostic and/or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Moreover, nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are also useful in the treatment of a variety of diseases and pathologies, including but not limited to, those involving neurogenesis, cancer, and wound healing. NOV3
A NOV3 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. A NOV3 nucleic acid and its encoded polypeptide includes the sequences shown in Table 12. The disclosed nucleic acid (SEQ ID NO:5) is 1,090 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 15-17 and ends with a TAA stop codon at nucleotides 1,061-1,063. The representative ORF encodes a 314 amino acid polypeptide (SEQ ID NO: 6). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 5.
TABLE 12.
AAGAAGTTCTTCAGATGCGAGGTTTCAACAAAACCACTGTGGTTACACAGTTCATCC TGGTGGGTTTCTCCAGCCTGGGGGAGCTCCAGCTGCTGCTTTTTGTCATCTTTCTTCT CCTATACTTGACAATCCTGGTGGCCAATGTGACCATCATGGCCGTTATTCGCTTCAG CTGGACTCTCCACACTCCCATGTATGGCTTTCTATTCATCCTTTCATTTTCTGAGTCCT GCTACACTTTTGTCATCATCCCTCAGCTGCTGGTCCACCTGCTCTCAGACACCAAGA CCATCTCCTTCATGGCCTGTGCCACCCAGCTGTTCTTTTTCCTTGGCTTTGCTTGCACC AACTGCCTCCTCATTGCTGTGATGGGATATGATCGCTATGTAGCAATTTGTCACCCTC TGAGGTACACACTCATCATAAACAAAAGGCTGGGGTTGGAGTTGATTTCTCTCTCAG GAGCCACAGGTTTCTTTATTGCTTTGGTGGCCACCAACCTCATTTGTGACATGCGTTT TTGTGGCCCCAACAGGGTTAACCACTATTTCTGTGACATGGCACCTGTTATCAAGTT AGCCTGCACTGACACCCATGTGAAAGAGCTGGCTTTATTTAGCCTCAGCATCCTGGT AATTATGGTGCCTTTTCTGTTAATTCTCATATCCTATGGCTTCATAGTTAACACCATC CTGAAGATCCCCTCAGCTGAGGGCAAGAAGGCCTTTGTCACCTGTGCCTCACATCTC ACTGTGGTCTTTGTCCACTATGGCTGTGCCTCTATCATCTATCTGCGGCCCAAGTCCA AGTCTGCCTCAGACAAGGATCAGTTGGTGGCAGTGACCTACACAGTGGTTACTCCCT TACTTAATCCTCTTGTCTACAGTCTGAGGAACAAAGAGGTAAAAACTGCATTGAAAA GAGTTCTTGGAATGCCTGTGGCAACCAAGATGAGCTAACAAAAAATAATAATAAAA TTAACTAGGATAGTCACAGAAGAAATCAAAGGCATAAAATTTTCTGACCTTTAATGC ATGTCTCAGACAGTGTTTCCAAGGATTAAGACTACTCTTGCCTTTTTATTTTCTCC (SEQ ID NO.: 5) MRGFNKTTVVTQFILVGFSSLGELQLLLFVIFLLLYLTILVANVTIMAVIRFSWTLHTPMY GFLFILSFSESCYTFVIIPQLLVHLLSDTKTISFMACATQLFFFLGFACTNCLLIAVMGYDR YVAICHPLRYTLIiNKRLGLELISLSGATGFFIALVATNLICDMRFCGPNRVNHYFCDMAP VIKLACTDTHVKELALFSLSILVIMVPFLLILISYGFIVNTILKIPSAEGKKAFVTCASHLTV VFVHYGCASIIYLRPKSKSASDKDQLVAVTYTVVTPLLNPLVYSLRNKEVKTALKRVLG MPVATKMS (SEQ ID NO.: 6)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. A NOV3 nucleic acid was identified on human chromosome 1. The NOV3 nucleic acid sequence has a high degree of homology (99% identity) with a human genomic clone corresponding to chromosome 1 (CHR1) (GenBank Accession No.:AL121986), as is shown in Table 13. Also, the NOV3 polypeptide has homology (approximately 50% identity, 70% similarity) to a human olfactory receptor (OLFR) (GenBank Accession No.: F20722), as is shown in Table 14. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains. NOV3 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 15.
TABLE 13
NOV3 : 1 aagaagttcttcagatgcgaggtttcaacaaaaccactgtggttacacagttcatcctgg 60
I 1 1 1 1 1 ! : 1 1 1 1 1 1 M I '; 1 1 1 M : : M ' : I ' 1 1 1 1 M i 1 1 M M ' I
CHRl : 145895 aagaagttcttcagatgcgaggtttcaacaaaaccactgtggttacacagttcatcctgg 145836 N0V3 : 61 tgggtttctccagcctgggggagctccagctgctactttttgtcatctttcttctcctat 120 1 1 1 1 1 1 I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
CHRl: 145835 tgggtttctccagcctgggggagctccagctgctgctttttgtcatctttcttctcctat 145776
N0V3 : 121 acttgacaatcctggtggccaatgtgaccatcatggccgttattcgcttcagctggactc 180
CHRl: 145775 acttgacaatcctggtggccaatgtgaccatcatggccgttattcgcttcagctggactc 145716
N0V3 : 181 tccacactcccatgtatggctttctattcatcctttcattttctgagtcctgctacactt 240
CHRl: 145715 tccaoactcccatgtatggctttctattcatcctttcattttctgagtcctgctacactt 145656
NOV3 : 241 ttgtcatcatccctcagctgctggtccacctgctctcagacaccaagaccatctccctca 300 M l
CH l: 145655 ttgtcatcatccctcagctgctggtccacctgctctcagacaccaagaccatctccttca 145596
NOV3 : 301 tggcctgtgccacccagctgttctttttccttggctttgcttgcaccaactgcctcctca 360
11 i I ; 1111 i 111 i 11 i 111111 ϊ i 111111111111 : i i 1 : ; : I ; 11111 ; 111
CHRl: 145595 tggcctgtgccacccagctgttctttttccttggctttgcttgcaccaactgcctcctca 145536
NOV3 : 361 ttgctgtgatgggatatgatcgctatgtagcaatttgtcaccctctgaggtacacactca 420
CHRl: 145535 ttgctgtgatgggatatgatcgctatgtagcaatttgtcaccctctgaggtacacactca 145476
NOV3.- 421 tcataaacaaaaggctggggttggagttgatttctctctcaggggccacaggtttcttta 480
11 i 1111 ! 1111111 ! I i 1 : 1111 111111111 i ' 1111 ; i :.
CHRl: 145475 tcataaacaaaaggctggggttggagttgatttctctctcaggagccacaggtttcttta 145416
NOV3 : 481 ttgctttggtggccaccaacctcatttgtgacatgcgtttttgtggccccaacagggtta 540
CHRl: 145415 ttgctttggtggccaccaacctcatttgtgacatgcgtttttgtggocccaacagggtta 145356
N0V3 -. 541 accactatttctgtgacatggcacctgttatcaagttagcctgcactgacacccatgtga 600
CHRl: 145355 accactatttctgtgacatggcacctgttatcaagttagcctgcactgacacccatgtga 145296
N0V3 : 601 aagagctggctttatttagcctcagcatcctggtaattatggtgccttttctgttaattc 660
CHRl: 145295 aagagctggctttatttagcctcagcatcctggtaattatggtgccttttctgttaattc 145236
NOV3 : 661 tcatatcctatggcttcatagtcaacaccatcctgaagatcccctcagctgagggcaaga 720 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
CHRl: 145235 tcatatcctatggcttcatagttaacaccatcctgaagatcccctcagctgagggcaaga 145176
NOV3 : 721 aggcctttgtcacctgtgσctcacatctcactgtggtctttgtccactatgactgtgcct 780
CHRl: 145175 aggcctttgtcacctgtgcctcacatctcactgtggtctttgtccactatggctgtgcct 145116
NOV3 : 781 ctatcatctatctgcggcccaagtccaagtctgcctcagacaaggatcagttggtggcag 840
CHRl: 145115 ctatcatctatctgcggcccaagtccaagtctgcctcagacaaggatcagttggtggcag 145056 NOV3 : 841 tgacotacgcagtggttactcccttacttaatcctcttgtctacagtctgaggaacaaag 900
MINIM 111 Mil M 11 II 11 llli 111 Mil 11 II II 111 nm II 111111111 CHRl: 145055 tgacctacacagtggttactcccttacttaatcctcttgtctacagtctgaggaacaaag 144996
N0V3 : 901 aggtaaaaactgcattgaaaagagttcttggaatgcctgtggcaaccaagatgagctaac 960
MMMMMMIMMMIMMMMMIMMMMMIIMIMMMMIMIM CHRl: 144995 aggtaaaaactgcattgaaaagagttcttggaatgcctgtggcaaccaagatgagctaac 144936
N0V3 : 961 aaaaaataataataaaattaactaggatagtcacagaagaaatcaaaggcataaaatttt 1020
MM Mill I II Ml MM II I llll ill I III MINIMI CHRl: 144935 aaaaaataataataaaattaactaggatagtcacagaagaaatcaaaggcataaaatttt 144876
NOV3 : 1021 ctgacctttaatgcatgtctcagacagtgtttccaaggattaagactactcttgcctttt 1080
II II II II I II II II II II I II II II II I II I II II II 11 II II II II II II II 111 II I CHRl: 144875 ctgacctttaatgcatgtctcagacagtgtttccaaggattaagactactcttgcctttt 144816
N0V3 : 1081 tattttctcc 1090 (SEQ ID NO. 5) CHRl: 144815 tattttctcc 144806 (SEQ ID NO. 42)
TABLE 14
NOV3: 1 MRGFNKTTWTQFILVGFSSLGELQLLLFVIFLLLYLTILVANVTIMAVIRFS TLHTPM 59
* * * *++ -j--*******-- ***_j.**^.^.***^.** * _ * - *** .j. _***** OLFR: 1 MLGLNHTSM-SEFILVGFSAFPHLQLMLFLLFLLMYLFTLLGNLLIMATV SΞRSLHTPM 59
NOV3: 60 YGFLFILSFSESCYTFVIIPQLLVHLLSDTKTISFMACATQLFFFLGFACTNCLLIAV G 119
* ** 4.** ** ** *** _4.* *** 4.4.*4.* _***4.*4** * *4 * ***
OLFR: 60 YLFLCVLSVSEILYTVAIIPRMLADLLSTQRSIAFLACASQMFFSFSFGFTHSFLLTVMG 119
NOV3: 120 YDRYVAICHPLRYTLIINKRLGLELISLSGATGFFIALVATNLICDMRFCGPNRVNHYFC 179
************* _.+.}__£. * *_J. * * * .f. _|_* *^. * _J_ *** _J_ ^_ *^. *
OLFR: 120 YDRYVAICHPLRYNVLMSPRGCACLVGCS AGGSVMGMWTSAIFQLTFCGSHEIQHFLC 179 NOV3: 180 DMAPVIKLAC-TDTHVKELALFSLSILVI VPFLLILISYGFIVNTILKIPSAEGK-KAF 239
-J_ *_ ^.**** . * _J. *_|_ .(__. *****_r^.** *** *********+ ***
OLFR: 180 HVPPLLKLACGNNVPAVALGVGLVCIMALLGCFLLILLSYAFIVADILKIPSAΞGRNKAF 239 NOV3: 240 VTCASHLTWFVHYGCASIIYLRPKSKSASDKDQLVAVTYTWTPLLNPLVYSLRNKEVK 299 ****** ** **** **_***.j_** . . * *^.* ** *-j-** *_j.*^..^.******^.*
OLFR: 240 STCASHLIWIVHYGFASVIYLKPKGPHSQEGDTLMATTYAVLTPFLSPIIFSLRNKELK 299 NOV3: 300 TALKR 304 (SEQ ID NO. 43)
*_£.** OLFR: 300 VAMKR 304 (SEQ ID NO. 44)
Where * indicates identity and + indicates similarity
TABLE 15
NOV3 : 43 NVTIMAVIRFSWTLHTPMYGFLFILSFSESCYTFVIIPQLLVHLLSDTKTISFMACATQL 102 GPCR: 2 NVLVC AVSREKALQTTTNYLIVSLAVADLLVATLV P VVYLEVVGE KFSRIHCDIFV 61 NOV3 : 103 FFFLGFACTNCLLIAVMGYDRYVAICHPLRYTLIIN-KRLGLELISLSGATGFFIALVAT 161
GPCR : 62 TLDVMMCTASILNLCAISIDRYTAVAMPMLYTSTTRYSSKRRVTVMIAIV VLSFTISCPML 121
NOV3 : 162 NLICD RFCGPNRVNHYFCDMAPVIKLACTDTHVKELALFSLSILVIlYiVPFLLILISYGF 221
GPCR : 122 FGLNNTDQNEC IIANPAFWYSSIVSFYVPFIVTLLVYIK 161
N0V3 : 222 IVNTILKI 229 (SEQ ID NO . 45 ) GPCR : 162 IYIVLRRR 169 (SEQ ID NO . 46 )
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, in one embodiment, the NOV3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. ' Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV3 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
NOV4
A NOV4 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. The NOV3 nucleic acid sequence (SEQ ID NO.: 5) was further analyzed by exon linking and the resulting sequence was identified as NOV4. A NOV4 nucleic acid and its encoded polypeptide includes the sequences shown in Table 16. The disclosed nucleic acid (SEQ ID NO:7) is 1,090 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 15-17 and ends with a TAA stop codon at nucleotides 1,061-1,063. The representative ORF encodes a 314 amino acid polypeptide (SEQ ID NO: 8). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 7. TABLE 16.
AAGAAGTTCTTCAGATGCGAGGTTTCAACAAAACCACTGTGGTTACACAGTTCATCC TGGTGGGTTTCTCCAGCCTGGGGGAGCTCCAGCTGCTACTTTTTGTCATCTTTCTTCT CCTATACTTGACAATCCTGGTGGCCAATGTGACCATCATGGCCGTTATTCGCTTCAG CTGGACTCTCCACACTCCCATGTATGGCTTTCTATTCATCCTTTCATTTTCTGAGTCCT GCTACACTTTTGTCATCATCCCTCAGCTGCTGGTCCACCTGCTCTCAGACACCAAGA CCATCTCCCTCATGGCCTGTGCCACCCAGCTGTTCTTTTTCCTTGGCTTTGCTTGCAC CAACTGCCTCCTCATTGCTGTGATGGGATATGATCGCTATGTAGCAATTTGTCACCCT CTGAGGTACACACTCATCATAAACAAAAGGCTGGGGTTGGAGTTGATTTCTCTCTCA GGGGCCACAGGTTTCTTTATTGCTTTGGTGGCCACCAACCTCATTTGTGACATGCGTT TTTGTGGCCCCAACAGGGTTAACCACTATTTCTGTGACATGGCACCTGTTATCAAGTT AGCCTGCACTGACACCCATGTGAAAGAGCTGGCTTTATTTAGCCTCAGCATCCTGGT AATTATGGTGCCTTTTCTGTTAATTCTCATATCCTATGGCTTCATAGTCAACACCATC CTGAAGATCCCCTCAGCTGAGGGCAAGAAGGCCTTTGTCACCTGTGCCTCACATCTC ACTGTGGTCTTTGTCCACTATGACTGTGCCTCTATCATCTATCTGCGGCCCAAGTCCA AGTCTGCCTCAGACAAGGATCAGTTGGTGGCAGTGACCTACGCAGTGGTTACTCCCT TACTTAATCCTCTTGTCTACAGTCTGAGGAACAAAGAGGTAAAAACTGCATTGAAAA GAGTTCTTGGAATGCCTGTGGCAACCAAGATGAGCTAACAAAAAATAATAATAAAA TTAACTAGGATAGTCACAGAAGAAATCAAAGGCATAAAATTTTCTGACCTTTAATGC ATGTCTCAGACAGTGTTTCCAAGGATTAAGACTACTCTTGCCTTTTTATTTTCTCC (SEQ ID NO.: 7)
MRGFNKTTVVTQFILVGFSSLGELQLLLFVIFLLLYLTILVANVTIMAVIRFSWTLHTPMY GFLFILSFSESCYTFVIIPQLLVHLLSDTKTISLMACATQLFFFLGFACTNCLLIAVMGYDR YVAICHPLRYTLIINKRLGLELISLSGATGFFIALVATNLICDMRFCGPNRVNHYFCDMAP VIKLACTDTHVKELALFSLSILVIMVPFLLILISYGFIVNTILKIPSAEGKKAFVTCASHLTV VFVHYDCASIIYLRPKSKSASDKDQLVAVTYAVVTPLLNPLVYSLRNKEVKTALKRVLG MPVATKMS (SEQ ID NO. : 8)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV4 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. A NOV4 nucleic acid was identified on human chromosome 1.
The NOV4 nucleic acid sequence has a high degree of homology (99% identity) with a human genomic clone corresponding to chromosome 1 (CHRl) (GenBank Accession No.:AL121986), as is shown in Table 17. The NOV4 nucleic acid sequence also has a high degree of homology with the NOV3 sequence (99% identity), as is shown in Table 18. Also, the NOV3 polypeptide has homology (approximately 53% identity, 71% similarity) to the human olfactory receptor 10J1 (OLFR) (GenBank Accession No.: P30954), as is shown in Table 19. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains. NOV4 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 20.
TABLE 17
NOV4 : 1 aagaagttcttcagatgcgaggtttcaacaaaaccactgtggttacacagttcatcctgg 60
CHRl: 145895 aagaagttcttcagatgcgaggtttcaacaaaaccactgtggttacacagttcatcctgg 145836
NOV4 : 61 tgggtttctccagcctgggggagctσcagctgctactttttgtcatctttcttctcctat 120
NNINNININNIIINNNIMNNI 11 II II II II II II I II I II II II I
CHRl: 145835 tgggtttctccagcctgggggagctccagctgctgctttttgtcatctttcttctcctat 145776
NOV4 : 121 acttgacaatcctggtggccaatgtgaccatcatggccgttattcgcttcagctggactc 180
MMMMNNNMMN MINN NNNNNNINNNNINN Nlllll
CHRl: 145775 acttgacaatcctggtggccaatgtgaccatcatggccgttattcgcttcagctggactc 145716
NOV4 : 181 tccacactcccatgtatggctttctattcatcctttcattttctgagtcctgctacactt 240
I ! 111111 ! I II M 1111111111111111111111 II 1111 II 111 IM 11 M 111111
CHRl: 145715 tccacactcccatgtatggctttctattcatcctttcattttctgagtcctgctacactt 145656
NOV4 : 241 ttgtcatcatccctcagctgctggtccacctgctctcagacaccaagaccatctccctca 300
MMMMMMMMMMMIMMMIMMMMIMMMMMIIMMI III
CHRl: 145655 ttgtcatcatccctcagctgctggtccacctgctctcagacaccaagaccatctccttca 145596
NOV4 : 301 tggcctgtgccacccagctgttctttttccttggctttgcttgcaccaactgcctcctca 360 lllll MMMMMMMMMMMMIIMIMM I
CHRl: 145595 tggcctgtgccacccagctgttctttttccttggctttgcttgcaccaactgcctcctca 145536
NOV4 : 361 ttgctgtgatgggatatgatcgctatgtagcaatttgtcaccctctgaggtacacactca 420 llll I llll I II llll II I II llll II llll I I
CHRl: 145535 ttgctgtgatgggatatgatcgctatgtagcaatttgtcaccctctgaggtacacactca 145476
NOV4 : 421 tcataaacaaaaggctggggttggagttgatttctctctcaggggccacaggtttcttta 480
MIMMMMMMMMMMMMMMMMMMMII
CHRl: 145475 tcataaacaaaaggctggggttggagttgatttctctctcaggagccacaggtttcttta 145416 N0V4 : 481 ttgctttggtggccaccaacctcatttgtgacatgcgtttttgtggccccaacagggtta 540
MIMMMIMMMMMMIMMMMMMMMMMMMMMIMMMM
CHRl: 145415 ttgctttggtggccaccaacctcatttgtgacatgcgtttttgtggccccaacagggtta 145356
N0V4 : 541 accactatttctgtgacatggcacctgttatcaagttagcctgcactgacacccatgtga 600
1111 M II M 111 I M 11 M II II llll IM M M 11 II 11 II MM 1111 M MUM !
CHRl: 145355 accactatttctgtgacatggcacctgttatcaagttagcctgcactgacacccatgtga 145296
N0V4 : 601 aagagctggctttatttagcctcagcatcctggtaattatggtgccttttctgttaattc 660
I !! 11111 ! I i 111111 i 111 ) 11111 ) 11111111 ) I II I ) J ) 1111111111 II 1111
CHRl: 145295 aagagctggctttatttagcctcagcatcctggtaattatggtgccttttctgttaattc 145236
N0V4 : 661 tcatatcctatggcttcatagtcaacaccatcctgaagatcccctcagctgagggcaaga 720
I MM 111 Ml M ( I III I III I II I II II I II I II I II II II I II II II II 111 II II
CHRl: 145235 tcatatcctatggcttcatagttaacaccatcctgaagatcccctcagctgagggcaaga 145176
N0V4 : 721 aggcctttgtcacctgtgcctcacatctcactgtggtctttgtccactatgactgtgcct 780 i I i 111111 M 11 ! 11 M 11 M ; ! I i ' 111 M 111111 i 111 i 11 ; i 11 IIIHI
CHRl: 145175 aggcctttgtcacctgtgcctcacatctcactgtggtctttgtccactatggctgtgcct 145116
N0V4 : 781 ctatcatctatctgcggcccaagtccaagtctgcctcagacaaggatcagttggtggcag 840
II II I Mill ! I Ml! MM Mill I! Mill Ml lllll 11 II
CHRl: 145115 ctatcatctatctgcggcccaagtccaagtctgcctcagacaaggatcagttggtggcag 145056
N0V4 : 841 tgacctacgcagtggttactcccttacttaatcctcttgtctacagtctgaggaacaaag 900
IIIMIII MMMMMMMMIMMMMMMMMMMIMMMIMMM
CHRl: 145055 tgacctacacagtggttactcccttacttaatcctcttgtctacagtctgaggaacaaag 144996
N0V4 : 901 aggtaaaaactgcattgaaaagagttcttggaatgcctgtggcaaccaagatgagctaac 960
111 M M M 11 i I II I E I M I M 1111 M 111 II II M I M i 11 M M 11 M I i 1 II i M
CHRl: 144995 aggtaaaaactgcattgaaaagagttcttggaatgcctgtggcaaccaagatgagctaac 144936
N0V4 : 961 aaaaaataataataaaattaactaggatagtcacagaagaaatcaaaggcataaaatttt 1020 llll II IIIMIII II II lllll III lllll lllll II I
CHRl: 144935 aaaaaataataataaaattaactaggatagtcacagaagaaatcaaaggcataaaatttt 144876
N0V4 : 1021 ctgacctttaatgcatgtctcagacagtgtttccaaggattaagactactcttgcctttt 1080
CHRl: 144875 ctgacctttaatgcatgtctcagacagtgtttccaaggattaagactactcttgcctttt 144816
N0V4: 1081 tattttctcc 1090 (SEQ ID NO. 4)
IMIIMMI
CHRl: 144815 tattttctcc 144806 (SEQ ID NO. 42)
TABLE 18
NOV4: 1 AAGAAGTTCTTCAGATGCGAGGTTTCAACAAAACCACTGTGGTTACACAGTTCATCCTGG 60 NOV3: 1 AAGAAGTTCTTCAGATGCGAGGTTTCAACAAAACCACTGTGGTTACACAGTTCATCCTGG 60 N0V4: 61 TGGGTTTCTCCAGCCTGGGGGAGCTCCAGCTGCTACTTTTTGTCATCTTTCTTCTCCTAT 120 NOV3: 61 TGGGT-TTCTCCAGCCTGGGGGAGCTCCAGCTGCTGCTTTTTGTCATCTTTCTTCTCCTAT 120 NOV4: 121 ACTTGACAATCCTGGTGGCCAATGTGACCATCATGGCCGTTATTCGCTTCAGCTGGACTC 180
NOV3: 121 ACTTGACAATCCTGGTGGCCAATGTGACCATCATGGCCGTTATTCGCTTCAGCTGGACTC 180 NOV4: 181 TCCACACTCCCATGTATGGCTTTCTATTCATCCTTTCATTTTCTGAGTCCTGCTACACTT 240
111 i I ' I Ii i 11 ' ! 111 i ! I i ! i i 1111 ! 11 M M 11 i M I II 111 ! ; M ! M 11 ; : ii ;
NOV3: 181 TCCACACTCCCATGTATGGCTTTCTATTCATCCTTTCATTTTCTGAGTCCTGCTACACTT 240 NOV4: 241 TTGTCATCATCCCTCAGCTGCTGGTCCACCTGCTCTCAGACACCAAGACCATCTCCCTCA 300 I I I
N0V3: 241 TTGTCATCATCCCTCAGCTGCTGGTCCACCTGCTCTCAGACACCAAGACCATCTCCTTCA 300 NOV4: 301 TGGCCTGTGCCACCCAGCTGTTCTTTTTCCTTGGCTTTGCTTGCACCAACTGCCTCCTCA 360
N0V3: 301 TGGCCTGTGCCACCCAGCTGTTCTTTTTCCTTGGCTTTGCTTGCACCAACTGCCTCCTCA 360
N0V4: 361 TTGCTGTGATGGGATATGATCGCTATGTAGCAATTTGTCACCCTCTGAGGTACACACTCA 420
N0V3: 361 TTGCTGTGATGGGATATGATCGCTATGTAGCAATTTGTCACCCTCTGAGGTACACACTCA 420
N0V4: 421 TCATAAACAAAAGGCTGGGGTTGGAGTTGATTTCTCTCTCAGGGGCCACAGGTTTCTTTA 480 1 1 1 1 1 1 1 1 I I 1 1 1 1 1 1
N0V3: 421 TCATAAACAAAAGGCTGGGGTTGGAGTTGATTTCTCTCTCAGGAGCCACAGGTTTCTTTA 480 N0V4: 481 TTGCTTTGGTGGCCACCAACCTCATTTGTGACATGCGTTTTTGTGGCCCCAACAGGGTTA 540
N0V3: 481 TTGCTTTGGTGGCCACCAACCTCATTTGTGACATGCGTTTTTGTGGCCCCAACAGGGTTA 540 N0V4: 541 ACCACTATTTCTGTGACATGGCACCTGTTATCAAGTTAGCCTGCACTGACACCCATGTGA 600
; ! i : ; i i ; 111 ' 11111 ' :.11 i ; i ; 1111111 ; i i 11 i ! 1111 : 1111111111 ; ' i i i !
N0V3: 541 ACCACTATTTCTGTGACATGGCACCTGTTATCAAGTTAGCCTGCACTGACACCCATGTGA 600
N0V4: 601 AAGAGCTGGCTTTATTTAGCCTCAGCATCCTGGTAATTATGGTGCCTTTTCTGTTAATTC 660
N0V3: 601 AAGAGCTGGCTTTATTTAGCCTCAGCATCCTGGTAATTATGGTGCCTTTTCTGTTAATTC 660
N0V4: 661 TCATATCCTATGGCTTCATAGTCAACACCATCCTGAAGATCCCCTCAGCTGAGGGCAAGA 720 11 II 111111111111 II 1111111111111111111
N0V3: 661 TCATATCCTATGGCTTCATAGTTAACACCATCCTGAAGATCCCCTCAGCTGAGGGCAAGA 720 N0V4 : 721 AGGCCTTTGTCACCTGTGCCTCACATCTCACTGTGGTCTTTGTCCACTATGACTGTGCCT 780 M I N I M
N0V3: 721 AGGCCTTTGTCACCTGTGCCTCACATCTCACTGTGGTCTTTGTCCACTATGGCTGTGCCT 780 N0V4: 781 CTATCATCTATCTGCGGCCCAAGTCCAAGTCTGCCTCAGACAAGGATCAGTTGGTGGCAG 840
MMMMMMMMMMIMMMMMMIMMIMIMMMMNMMMM
N0V3: 781 CTATCATCTATCTGCGGCCCAAGTCCAAGTCTGCCTCAGACAAGGATCAGTTGGTGGCAG 840
N0V4 : 841 TGACCTACGCAGTGGTTACTCCCTTACTTAATCCTCTTGTCTACAGTCTGAGGAACAAAG 900
N0V3: 841 TGACCTACACAGTGGTTACTCCCTTACTTAATCCTCTTGTCTACAGTCTGAGGAACAAAG 900
N0V4: 901 AGGTAAAAACTGCATTGAAAAGAGTTCTTGGAATGCCTGTGGCAACCAAGATGAGCTAAC 960
) 111 M 11111 M I ) 11111 M ) 111 ) 11 ) 111 ) 1111111111111111 ) 111 ) 11 ) 11
N0V3 : 901 AGGTAAAAACTGCATTGAAAAGAGTTCTTGGAATGCCTGTGGCAACCAAGATGAGCTAAC 960 NOV4 : 961 AAAAAATAATAATAAAATTAACTAGGATAGTCACAGAAGAAATCAAAGGCATAAAATTTT 1020 lllll 11111111 II 111111111 M 111 ! I )! 1111111 N 1111111 lllll
NOV3: 961 AAAAAATAATAATAAAATTAACTAGGATAGTCACAGAAGAAATCAAAGGCATAAAATTTT 1020
NOV4: 1021 CTGACCTTTAATGCATGTCTCAGACAGTGTTTCCAAGGATTAAGACTACTCTTGCCTTTT 1080
[ 111 M I M 1111 ! 1111111111111111111111 M 111 [ I M I M 111 II 11 i 1111
NOV3: 1021 CTGACCTTTAATGCATGTCTCAGACAGTGTTTCCAAGGATTAAGACTACTCTTGCCTTTT 1080 NOV4: 1081 TATTTTCTCC 1090 (SEQ ID NO. 7) NOV3: 1081 TATTTTCTCC 1090 (SEQ ID NO. 5)
TABLE 19
NOV4 : 18 TLITDFVFQGFSSFHEQQITLFGVFLALYILTLAGNIIIVTIIRIDLHLHTPMYFFLSML 77 *4-4.* * - **** * * _ ** 4.** ** * *4- * _ 4** ****** ** 4,* OLFR: 8 TWTQFILVGFSSLGELQLLLFVIFLLLYLTILVANVTI AVIRFSWTLHTPMYGFLFIL 67
NOV4: 78 STSETVYTLVILPRMLSSLVGMSQP SLAGCATQMFFFVTFGITNCFLLTAMGYDRYVAI 137 * * *_ ** ** -*_j._ * *+ 4.4. 4.* * **** *** . * *** * - *********
OLFR: 68 SFSESCYTFVIIPQLLVHLLSDTKTISLMACATQLFFFLGFACTNCLLIAVMGYDRYVAI 127
NOV4: 138 CNPLRYMVIJNKRLRIQLVLGACSIGLIVAITQVTSVFRLPFCA-RKVPHFFCDIRPVMK 196 *4**** 4_* **** 44*4. 4 4 * 4*+ 4 4- ** 4-* *4-***4- **4*
OLFR: 128 CHPLRYTLIINKRLGLELISLSGATGFFIALVATNLICDMRFCGPNRVNHYFCDMAPVIK 187 NOV4: 197 LSCIDTTVNEXXXXXXXXXXXXXPMGLVFISYVLIISTILKIASVEGRKKAFATCASHLT 256
*4>* ** * * * *-J. *** * ._|_***** * ** **** *******
OLFR : 188 LACTDTHVKELALFSLSILVIMVPFLLILISYGFIVNTILKIPSAEG-KKAFVTCASHLT 246
NOV4 : 257 WIVHYSCASIAYLKPKSΞNTREHDQLISVTYTVITPLLNPWYTLRNKEVKDALCRAVG 316 (SEQ ID NO . 49 )
** *** **** ** ***_(__μ 4. ***^-^.*** * .******4-**_(_******* ** * μ*
OLFR: 247 WFVHYDCASIIYLRPKSKSASDKDQLVAVTYAWTPLLNPLVYSLRNKEVKTALKRVLG 306 (SEQ ID NO. 50)
Where * indicates identity and + indicates similarity.
Table 20
NOV4: 43 NVTIMAVIRFS TLHTPMYGFLFILSFSESCYTFVIIPQLLVHLLSDTKTISLMACATQL 102
GPCR: 2 NVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWVVYLEVVGEWKFSRIHCDIFV 61
N0V4: 103 FFFLGFACTNCLLIAVMGYDRYVAICHPLRYTLIIN-KRLGLELISLSGATGFFIALVAT 161
GPCR: 62 TLDVI^CTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIV VLSFTISCPML 121
NOV4: 162 NLICDMRFCGPNRVNHYFCDMAPVIKLACTDTHVKELALFSLSILVIMVPFLLILISYGF 221 GPCR: 122 FGLNNTDQNEC IIANPAFWYSSIVSFYVPFIVTLLVYIK 161
NOV4: 222 IVNTILKI 229 (SEQ ID NO. 51) GPCR: 162 IYIVLRRR 169 (SEQ ID NO. 46) The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV4 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV4 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in treating and/or diagnosing a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
NOV5 A NOV5 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. A NOV5 nucleic acid and its encoded polypeptide includes the sequences shown in Table 21. The disclosed nucleic acid (SEQ ID NO: 9) is 822 nucleotides in length and contains an open reading frame (ORF) that begins at nucleotide 6 and ends with a TGA stop codon at nucleotides 800-802. In addition, C indicates 'G' to 'C substitutions in the sequence to correct stop codons. A representative ORF encodes a 265 amino acid polypeptide (SEQ ID NO: 10). A putative untranslated region downstream ofthe coding sequence is underlined in SEQ ID NO: 9.
TABLE 21
CACACCCCCATGTGCTTCTTCCTCTCCAAACTGTGCTCAGCTGACATCGGTTTCACCT TGGCCATGGTTCCCAAGATGATTGTGAACATGCAGTCGCATAGCAGAGTCATCTCTT ATGAGGGCTGCCTGACACGGATGTCTTTCTTTGTCCTTTTTGCATGTATGGAAGACAT GCTCCTGACTGTGATGGCCTATGACTGCTTTGTAGCCATCTGTCGCCCTCTGCACTAC CCAGTCATCGTGAATCCTCACCTCTGTGTCTTCTTCGTCTTGGTGTCCTTTTTCCTTAG CCCGTTGGATTCCCAGCTGCACAGTTGGATTGTGTTACTATTCACCATCATCAAGAA TGTGGAAATCACTAATTTTGTCTGTGAACCCTCTCAACTTCTCAACCTTGCTTGTTCT GACAGCGTCATCAATAACATATTCATATATTTCGATAGTACTATGTTTGGTTTTCTTC CCATTTCAGGGATCCTTTTGTCTTACTATAAAATTGTCCCCTCCATTCTAAGGATGTC ATCGTCAGATGGGAAGTATAAAGGCTTCTCCACCTGTGGCTCTTACCTGGCAGTTGT TTGCTCATTTGATGGAACAGGCATTGGCATGTACCTGACTTCAGCTGTGTCACCACC CCCCAGGAATGGTGTGGTGGCGTCAGTGATGTATGCTGTGGTCACCCCCATGCTGAA CCTTTTCATCTCAGCCTAGGAAAGAGGGATATACAAAGTGTCCTGCGGAGGCTGTGC AGCAGAACAGTCGAATCTCATGATATGTTCCATCCTTTTTCTTGTGTGGGTGAGAAA GGGCAACCACATTAAA (SEQ ID NO.: 9)
PMCFFLSKLCSADIGFTLAMVPKMIVNMQSHSRVISYEGCLTRMSFFVLFACMEDMLLT VMAYDCFVAICRPLHYPVIVNPHLCVFFVLVSFFLSPLDSQLHSWIVLLFTIIKNVEITNF VCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKGFS TCGSYLAVVCSFDGTGIGMYLTSAVSPPPRNGVVASVMYAVVTPMLNLFIYSLGKRDI QSVLRRLCSRTVESHDMFHPFSCVG (SEQ ID NO.: 10)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV5 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
The NOV5 nucleic acid sequence has a high degree of homology (94% identity) with a human genomic clone cotaining an OR pseudogene (OLFR) (GenBank Accession No.:AF065864), as is shown in Table 22. The NOV5 polypeptide has homology (approximately 67% identity, 79% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No.:043789), as is shown in Table 23. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains. NOV5 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 24.
TABLE 22
NOV5 : 1 CACACCCCCATGTGCTTCTTCCTCTCCAAACTGTGCTCAGCTGACATCGGTTTCACCTTG 60
M M M M M M I M M M M M M M M I I M I I I I I 1 1 I I I I I I I 1 1 1 1 1 1 I I 1 1 OLFR : 136 CACACCCCCATGTGCTTCTTCCTCTCCAACCTGTGCTGGGCTGACATCGGTTTCACCTTG 195 NOV5: 61 GCCATGGTTCCCAAGATGATTGTGAACATGCAGTCGCATAGCAGAGTCATCTCTTATGAG 120
OLFR: 196 GCCACGGTTCCTAAGATGATTGTGGACATGCAGTCTCATACCAGAGTCATCTCTTATGAG 255
NOV5: 121 GGCTGCCTGACACGGATGTCTTTCTTTGTCCTTTTTGCATGTATGGAAGACATGCTCCTG 180
MIMMMMMMM IIIMIII MMMMIMMMM MMIMMMMM
OLFR: 256 GGCTGCCTGACACGGATATCTTTCTTGGTCCTTTTTGCATGTATAGAAGACATGCTCCTG 315
NOV5: 181 ACTGTGATGGCCTATGACTGCTTTGTAGCCATCTGTCGCCCTCTGCACTACCCAGTCATC 240
OLFR: 316 ACTGTGATGGCCTATGACTGCTTTGTAGCCATCTGTCGCCCTCTGCACTACCCAGTCATC 375
NOV5: 241 GTGAATCCTCACCTCTGTGTCTTCTTCGTCTTGGTGTCCTTTTTCCTTAGCCCGTTGGAT 300
1111 M ! 1111 II i I i 11 II I M M 11 I Mill I MIMMMMM MIMM
OLFR: 376 GTGAATCCTCACCTCTGTGTCTTCTTCCTTTTGGTATACTTTTTCCTTAGCTTGTTGGAT 435
NOV5: 301 TCCCAGCTGCACAGTTGGATTGTGTTACTATTCACCATCATCAAGAATGTGGAAATCACT 360
OLFR: 436 TCCCAGCTGCACAGTTGGATTGTGTTACAATTCACCATCATCAAGAATGTGGAAATCTCT 495
NOV5: 361 AATTTTGTCTGTGAACCCTCTCAACTTCTCAACCTTGCTTGTTCTGACAGCGTCATCAAT 420
OLFR: 496 AATTTTGTCTGTGACCCCTCTCAΑCTTCTCAAACTTGCCTGTTCTGACAGCGTCATCAAT 555
NOV5: 421 AACATATTCATATATTTCGATAGTACTATGTTTGGTTTTCTTCCCATTTCAGGGATCCTT 480
I IIMMIII MUM 11 II 11 II II II 111111 II II I II 11 II 111111 II 1111
OLFR: 556 AGCATATTCATGTATTTCCATAGTACTATGTTTGGTTTTCTTCCCATTTCAGGGATCCTT 615
NOV5: 481 TTGTCTTACTATAAAATTGTCCCCTCCATTCTAAGGATGTCATCGTCAGATGGGAAGTAT 540
IMMMMMMMM MMMMMMMMMM Mill MMMMMMIM
OLFR: 616 TTGTCTTACTATAAAATCGTCCCCTCCATTCTAAGGATTTCATCATCAGATGGGAAGTAT 675
NOV5: 541 AAAGGCTTCTCCACCTGTGGCTCTTACCTGGCAGTTGTTTGCTCATTTGATGGAACAGGC 600 llll 1 II M 11 M M I i 111111 II MMMMMMIM llll OLFR 676 AAAGCCTTCTCCACCTGTGGCTCTCACTTGGCAGTTGTTTGCTGATTTTATGGAACAGGC 735
NOV5: 601 ATTGGCATGTACCTGACTTCAGCTGTGTCACCACCCCCCAGGAATGGTGTGGTGGCGTCA 660 MMIMMIIIMIMMMMMMII Mill
OLFR: 736 ATTGGCGTGTACCTGACTTCAGCTGTGTCACCACCCCCCAGGAATGGTGTGGTAGCGTCA 795
NOV5: 661 GTGATGTATGCTGTGGTCACCCCCATGCTGAACCTTTTCATCTACAGCCTAGGAAAGAGG 720
OLFR: 796 GTGATGTACGCTGTGGTCACCCCCATGCTGAACCTTTTCATCTACAGCCTGAGAAACAGG 855
NOV5: 721 GATATACAAAGTGTCCTGCGGAGGCTGTGCAGCAGAACAGTCGAATCTCATGATATGTTC 780
II IIIIMMM 1111 M 111 M 11 111 II 11 II I II 1111 II 11 II II I Mill
OLFR: 856 GACATACAAAGTGCCCTGCGGAGGCTGCTCAGCAGAACAGTCGAATCTCATGATCTGTTC 915 NOV5: 781 CATCCTTT 788 (SEQ ID NO. 53)
IIIMIII
OLFR: 916 CATCCTTT 923 (SEQ ID NO. 54)
TABLE 23
NOV5: 7 PMCFFLSKLCSADIGFTLAMVPKMIVNMQSHSRVISYEGCLTRMSFFVLFACMEDMLLTV 186 ** **** * ****** ****** _L**4.* ********** *4_*** *******4_** * *4>*
OLFR: 1 PiMYFFLSNLSLADIGFTSTTVPKMIVDMQTHSRVISYEGCLTQMSFFVLFACMDDMLLSV 60 NOV5: 187 MAYDCFVAICRPLHYPVIVNPHLCVFFVLVSFFLSPLDSQLHS IVLLFTIIKNVEITNF 366 **** ***** **** 4.*^.** ** * _j_*4-***4-* ******^_ *_j_* * *4>*4.* .**
OLFR: 61 MAYDRFVAICHPLHYRIIMNPRLCGFLILLSFFISLLDSQLHNLIMLQLTCFKDVDISNF 120
NOV5: 367 VCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 546 *4-*****4-* ***4_ ** 4. *** 4.** ******* ****** *** - 4.******
OLFR: 121 FCDPSQLLHLRCSDTFINEMVIYFMGAIFGCLPISGILFSYYKIVSPILRVPTSDGKYKA 180
NOV5: 547 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNGWASV YAWTPMLNLFIYSLGKRDI 726 ******_}.***** * ***4> **_}_*** * ** 4.****** ******* ***** 4.**
OLFR: 181 FSTCGSHLAVVCLFYGTGLVGYLSSAVLPSPRKSMVASVMYTVVTPMLNPFIYSLRNKDI 240
NOV5: 727 QSVLRRLCSRTVESHDMFHPFSCVG 801 (SEQ ID NO. 55) ** * ** * 4.4.** 4. *** 4_*
OLFR: 241 QSALCRLHGRIIKSHHL-HPFCYMG 264 (SEQ ID NO. 56)
Where * indicates identity and + indicates similarity.
TABLE 24 NOV5: 1 PMCFFLSKLCSADIGFTLA VPKMIVNMQSHSRVISYEGCLTRMSFFVLFACMEDMLLTV 60 GPCR: 18 TTNYLIVSLAVADLLVATLVMPWWYLEWGE KFSRIHCDIFVTLDVMMCTASILNLCA 77
NOV5: 61 MAYDCFVAICRPLHYPVIVNPH 82 (SEQ ID NO . 57) GPCR: 78 ISIDRYTAVAMP LYNTRYSSK 99 (SEQ ID NO. 58)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Thus, the NOV5 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV5 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
NOV6 A NOV6 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. A NOV6 nucleic acid and its encoded polypeptide includes the sequences shown in Table 25. The disclosed nucleic acid (SEQ ID NO:l 1) is 930 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 22-24 and ends with a TAA stop codon at nucleotides 907-909. In addition, C indicates 'G' to 'C substitutions in the sequence to correct stop codons. The representative ORF encodes a 294 amino acid polypeptide (SEQ ID NO: 12). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 11.
TABLE 25.
TTGCTGTCCCTGTCCCTGTCCATGTATATGGTCACGGTGCTGAGGAACCTGCT CAGCATCCTGGCTGTCAGCTCTGACTCCCCGCTCCACACCCCCATGTGCTTCT TCCTCTCCAAACTGTGCTCAGCTGACATCGGTTTCACCTTGGCCATGGTTCCC AAGATGATTGTGAACATGCAGTCGCATAGCAGAGTCATCTCTTATGAGGGCT GCCTGACACGGATGTCTTTCTTTGTCCTTTTTGCATGTATGGAAGACATGCTC CTGACTGTGATGGCCTATGACTGCTTTGTAGCCATCTGTCGCCCTCTGCACTA CCCAGTCATCGTGAATCCTCACCTCTGTGTCTTCTTCGTCTTGGTGTCCTTTTT CCTTAGCCCGTTGGATTCCCAGCTGCACAGTTGGATTGTGTTACTATTCACCA TCATCAAGAATGTGGAAATCACTAATTTTGTCTGTGAACCCTCTCAACTTCTC AACCTTGCTTGTTCTGACAGCGTCATCAATAACATATTCATATATTTCGATAG TACTATGTTTGGTTTTCTTCCCATTTCAGGGATCCTTTTGTCTTACTATAAAAT TGTCCCCTCCATTCTAAGGATGTCATCGTCAGATGGGAAGTATAAAGGCTTCT CCACCTGTGGCTCTTACCTGGCAGTTGTTTGCTCATTTGATGGAACAGGCATT GGCATGTACCTGACTTCAGCTGTGTCACCACCCCCCAGGAATGGTGTGGTGG CGTCAGTGATGTATGCTGTGGTCACCCCCATGCTGAACCTTTTCATACTCAGC CTGGGAAAGAGGGATATACAAAGTGTCCTGCGGAGGCTGTGCAGCAGAACA GTCGAATCTCATGATATGTTCCATCCTTTTTCTTGTGTGGGTGAGAAAGGGCA ACCACATTAAATCTCTACATCTGTAAATCCT (SEQ ID NO.: 11)
MYMVTVLRNLLSILAVSSDSPLHTPMCFFLSKLCSADIGFTLAMVPKMIVNMQSHSRVIS YEGCLTRMSFFVLFACMEDMLLTVMAYDCFVAICRPLHYPVIVNPHLCVFFVLVSFFLS PLDSQLHSWIVLLFTIIKNVEITNFVCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILL SYYKIVPSILRMSSSDGKYKGFSTCGSYLAVVCSFDGTGIGMYLTSAVSPPPRNGVASVM YAVVTPMLNLFILSLGKRDIQSVLRRLCSRTVESHDMFHPFSCVGEKGQPH (SEQ ID NO.: 12) The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV6 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. The NOV6 nucleic acid sequence has a high degree of homology (94% identity) with a human genomic clone cotaining an OR pseudogene (OLFR) (GenBank Accession No.:AF065864), as is shown in Table 26. The NOV6 polypeptide has homology (approximately 67% identity, 79% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No.:043789), as is shown in Table 27. As shown in Table 28, the NOV6 polypeptide also has a high degree of homology (99% identity) with the NOV5 polypeptide. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. Thus, NOV5 and NOV6 belong to the same OR subfamily. OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy- terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains. NOV6 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 29.
TABLE 26
NOV6 : 10 ctgtccctgtccatgtatatggtcacggtgctgaggaacctgctcagcatcctggctgtc 69
' M M M M M M M M M I I I I I I I I I I I I I I I I I 1 1 1 1 I I I I 1 1 l l l l l l l l l l l l l OLFR: 58 ctgtccctgtccatgtatctggtcacggtgctgaggaacctgctcatcatcctggctgtc 117
NOV6 : 70 agctctgactccccgctccacacccccatgtgcttcttcctctccaaactgtgctcagct 129 OLFR: 118 agctctgacccccacctccacacccccatgtgcttcttcctctccaacctgtgctgggct 177 NOV6 : 130 gacatcggtttcaccttggccatggttcccaagatgattgtgaacatgcagtcgcatagc 189
111111 II 11111111111111 11111 II! 11 II 111 II 11111 llll 1
OLFR: 178 gacatcggtttcaccttggccacggttcctaagatgattgtggacatgcagtctcatacc 237
NOV6: 190 agagtcatctcttatgagggctgcctgacacggatgtctttctttgtcctttttgcatgt 249 OLFR: 238 agagtcatctcttatgagggctgcctgacacggatatctttcttggtcctttttgcatgt 297
N0V6 : 250 atggaagacatgctcctgactgtgatggcctatgactgctttgtagccatσtgtcgccct 309
II MMMMMMMMMMMMIMMMMMMMMMMMIMIMMM
OLFR: 298 atagaagacatgctcctgactgtgatggcctatgactgctttgtagccatctgtcgccct 357
N0V6 : 310 ctgcactacccagtcatcgtgaatcctcacctctgtgtcttcttcgtcttggtgtccttt 369
I II 111111 i I M I M 111 ! M II I M II 11111 M 1111 II 111 I lllll I llll
OLFR: 358 ctgcactacccagtcatcgtgaatcctcacctctgtgtcttcttccttttggtatacttt 417
NOV6 : 370 ttccttagcccgttggattcccagctgcacagttggattgtgttactattcaccatcatc 429 OLFR: 418 ttccttagcttgttggattcccagctgcacagttggattgtgttacaattcaccatcatc 477
NOV6 : 430 aagaatgtggaaatcactaattttgtctgtgaaccctctcaacttctcaaccttgcttgt 489
MMMMMMIM H 11111 ! i 111111 i I lllll III
OLFR: 478 aagaatgtggaaatctctaattttgtctgtgacccctctcaacttctcaaacttgcctgt 537
NOV6 : 490 tctgacagcgtcatcaataacatattcatatatttcgatagtactatgtttggttttctt 549
MMMMMMIMMM MIIIIIM I II I II 111 II II II II II II II
OLFR: 538 tctgacagcgtcatcaatagcatattcatgtatttccatagtactatgtttggttttctt 597
NOV6 : 550 cccatttcagggatccttttgtcttactataaaattgtcccctσcattctaaggatgtca 609
MMMIIIIMMMIIMMIIIMMIMIII 111 I.I II II II II 11 II 111 III
OLFR: 598 ccσatttcagggatccttttgtcttactataaaatcgtcccctccattctaaggatttca 657
NOV6 : 610 tcgtcagatgggaagtataaaggcttctccacctgtggctσttacctggcagttgtttgc 669
II II IIIMMIMMM
OLFR: 658 tcatcagatgggaagtataaagccttctccacctgtggctctcacttggcagttgtttgc 717
NOV6 : 670 tcatttgatggaacaggcattggcatgtacctgacttcagctgtgtcaccaccccccagg 729 OLFR: 718 tgattttatggaacaggcattggcgtgtacctgacttcagctgtgtcaccaccccccagg 777
NOV6 : 730 aatggtgtggtggcgtcagtgatgtatgctgtggtcacccccatgctgaaccttttcata 789 I Mill lllll IMIMIMIIIMIIIMM
OLFR: 778 aatggtgtggtagcgtcagtgatgtacgctgtggtσacccccatgctgaaccttttcatc 837
NOV6 : 790 ctcagcctgggaaagagggatatacaaagtgtcctgcggaggctgtgcagcagaacagtc 849 OLFR: 838 tacagcctgagaaacagggacatacaaagtgccctgcggaggctgctcagcagaacagtc 897 N0V6: 850 gaatctcatgatatgttccatccttt 875 (SEQ ID NO. 59)
111 E I M 11111 111 II 11111111
OLFR: 898 gaatctcatgatctgttccatccttt 923 (SEQ ID NO. 60)
TABLE 27.
NOV6: 7 P CFFLSKLCSADIGFTI-AMVPK IVNMQSHSRVISYEGCLTRMSFFVLFACMEDMLLTV 186 ** **** * ****** ******4**+************+**********4****+*
OLFR: 1 PMYFFLSNLSLADIGFTSTTVPKMIVDMQTHSRVISYEGCLTQMSFFVLFACMDDMLLSV 60
NOV6: 187 MAYDCFVAICRPLHYPVIWPHLCVFFVLVSFFLSPLDSQLHSWIVLLFTIIKJ EITNF 366 **** ***** **** 4*4** ** * 4*4***4* ******+ * * * *+*4*+**
OLFR: 61 MAYDRFVAICHPLHYRIIM PRLCGFLILLSFFISLLDSQLHNLIMLQLTCFKDVDISNF 120
NOV6: 367 VCEPSQ LNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 546 *4*****4* *** + ** *** 4** ******* ****** ***4 +******
OLFR: 121 FCDPSQLLHLRCSDTFINEMVIYFMGAIFGCLPISGILFSYYKIVSPILRVPTSDGKYKA 180
NOV6: 547 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNGWASVMYAWTPMLNLFIYSLGKRDI 726
******+***** * *** +
OLFR: 181 FSTCGSHLAWCLFYGTG VGYLSSAVLPSPRKS VASVMYTWTPMLNPFIYSLRNKDI 240
NOV6: 727 QSVLRRLCSRTVESHDMFHPFSCVG 801 (SEQ ID NO. 61)
* + * * + ***
OLFR: 241 QSALCR HGRIIKSHHL-HPFCYMG 264 (SEQ ID NO. 62)
Where * indicates identity and + indicates similarity.
TABLE 28
NOV6: 25 PMCFFLSKLCSADIGFTLAMVPKMIVNMQSHSRVISYEGCLTRMSFFVLFACMEDMLLTV 84
************************************************************
NOV5: 1 PMCFFLSKLCSADIGFTLAMVPKMIVNMQSHSRVISYEGCLTRMSFFVLFACMED LLTV 60
NOV6: 85 MAYDCFVAICRPLHYPVIVNPHLCXXXXXXXXXXXXXXXQLHS IVLLFTIIKNVEITNF 144 ************************************************************
NOV5: 61 MAYDCFVAICRPLHYPVIVNPHLCVFFVLVSFFLSPLDSQLHS IVLLFTIIKNVEITNF 120 NOV6: 145 VCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 204
************************************************************
NOV5: 121 VCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 180
NOV6: 205 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNG-VASV YAWTPMLNLFILSLGKRDI 263
********************************** ***************** *******
NOV5: 181 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNGWASVMYAWTPMLNLFIYSLGKRDI 240
NOV6: 264 QSVLRRLCSRTVESHDMFHPFSCVG 288 (SEQ ID NO. 63)
*************************
NOV5: 241 QSVLRRLCSRTVESHDMFHPFSCVG 265 (SEQ ID NO. 10) Where * indicates identity. TABLE 29
NOV6: 9 NLLSILAVSSDSPLHTPMCFFLSKLCSADIGFTLAMVPKMIVNMQSHSRVISYEGCLTRM 68
GPCR: 2 NVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWWYLEWGE KFSRIHCDIFV 61
NOV6: 69 SFFVLFACMEDMLLTVMAYDCFVAICRPLHYPVIVNPHLCVFFVLVSFFLSPLDSQLHSW 128
GPCR: 62 TLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRV TVM 105 NOV6: 129 IVLLFTIIKNVEITNFVCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKI 188 GPCR: 106 IAIV VLSFTISCPMLFG LNNTDQNΞCIIANPAFWYSSIVSFYVPFIVTLLVYIKI 162
NOV6: 189 VPSILRMSSS 198 (SEQ ID NO. 65) GPCR: 163 YIVLRRRRKR 172 (SEQ ID NO. 66)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV6 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV6 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
NOV7
A NOV7 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. A NOV7 nucleic acid and its encoded polypeptide includes the sequences shown in Table 30. The disclosed nucleic acid (SEQ ID NO: 13) is 930 nucleotides in length and contains an open reading frame (ORF) that begins with an ACG initiation codon at nucleotides 10-12 and ends with a TGA stop codon at nucleotides 882-884. In addition, C indicates 'G' to 'C substitutions in the sequence to correct stop codons. The representative ORF encodes a 309 amino acid polypeptide (SEQ ID NO: 12). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 13
TABLE 30. CACAGAGCCACGGAATCTCACAGGTGTCTCAGAATTCCTCCTCCTGGGACTCTCAGA GGATCCAGAACTGCAGCCGGTCCTCGCTTTGCTGTCCCTGTCCCTGTCCATGTATCTG GTCACAGTGCTGAGGAACCTGCTCAGCATCCCGGCTGTCAGCTCTGACTCCCACCTC CACACCCCCACGTACTTCTTCCTCTCCATCCTGTGCTGGGCTGACATCGGTTTCACCT CGGCCACGGTTCCCAAGATGATTGTGGACATGCAGTGGTATAGCAGAGTCATCTCTC ATGCGGGCTGCCTGACACAGATGTCTTTCTTGGTCCTTTTTGCATGTATAGAAGGCAT GCTCCTGACTGTAATGGCCTATGACTGCTTTGTAGGCATCTATCGCCCTCTGCACTAC CCAGTCATCGTGAATCCTCATCTCTGTGTCTTCTTTGTTTTGGTGTCCTTTTTCCTTAG CCTGTTGGATTCCCAGCTGCACAGTTGGATTGTGTTACAATTCACCATCATCAAGAA TGTGGAAATCTCTAATTTTGTCTGTGACCCCTCTCAACTTCTCAAACTTGCCTCTTAT GACAGCGTCATCAATAGCATATTCATATATTTCGATAGTACAATGTTTGGTTTTCTTC CTATTTCAGGGATCCTTTCATCTTACTATAAAATTGTCCCCTCCATTCTAAGGATGTC ATCGTCAGATGGGAAGTATAAAACTTTCTCCACCTATGGCTCTCACCTAGCATTTGTT TGCTCATTTTATGGAACAGGCATTGACATGTACCTGGCTTCAGCTATGTCACCAACC CCCAGGAATGGTGTGGTGGTGTCAGTGATGTAAGCTGTGGTCACCCCCATGCTGAAC CTTTTCATCTACAGCCTGAGAAACAGGGACATACAAAGTGCCCTGCGGAGGCTGCG CAGCAGAAC (SEQ ID NO.: 13)
TEPRNLTGVSEFLLLGLSEDPELQPVLALLSLSLSMYLVTVLRNLLSIPAVSSDSHLHTPT YFFLSILCWADIGFTSATVPKMIVDMQWYSRVISHAGCLTQMSFLVLFACIEGMLLTVM AYDCFVGIYRPLHYPVIVNPHLCVFFVLVSFFLSLLDSQLHSWIVLQFTIIKNVEISNFVCD PSQLLKLASYDSVINSIFIYFDSTMFGFLPISGILSSYYKIVPSILRMSSSDGKYKTFSTYGS HLAFVCSFYGTGIDMYLASAMSPTPRNGVVVSVMXAVVTPMLNLFIYSLRNRDIQSALR RLRSR (SEQ ID NO.: 14)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. The NOV7 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
The NOV7 nucleic acid sequence has a high degree of homology (94% identity) with the human genomic clone pDJ392al7 from chromosome 11 (CHRl 1) (GenBank Accession No.:AC000385), as is shown in Table 31. The NOV7 polypeptide has homology (approximately 68% identity, 78% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No..-043789), as is shown in Table 32. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences.1999, 20:413. OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
NOV7 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 33.
TABLE 31.
NOV7 : 1 cacagagccacggaatctcacaggtgtctcagaattcctcctcctgggactctcagagga 60
NNNNINNNNNNNNNN 11 II II I II II I II II II II II II II 11 II CHRll: 126702 cacagagccacggaatctcacaggtgtctgagaattcctcctcctgggactctcagagga 126643
NOV7 : 61 tccagaactgcagccggtcctcgctttgctgtccctgtccctgtccatgtatctggtcac 120 llll I II II 11 II CHRll: 126642 tccagaactgcagtcggtcctcgctttgctgtccctgtccctgtccctgaatctggtcac 126583
NOV7 : 121 agtgctgaggaacctgctcagcatcccggctgtcagctctgactcccacctccacacccc 180 lllll INN II llll II I Ml I II MUNI MM; I M II 111 II M CHRll: 126582 ggtgctgaggaacctgctcagcatcctggctgtcagctctgactcccccctccacacccc 126523
NOV7 : 181 cacgtacttcttcctctccatcctgtgctgggctgacatcggtttcacctcggccacggt 240
II III lllll II lllll II II I IMIM MM IM I MM II II CHRll: 126522 catgtacttcttcctctccaacctgtgctgggctgacatcggtctcacctcggccacggt 126463
NOV7 : 241 tcccaagatgattgtggacatgcagtggtatagcagagtcatctctcatgcgggctgcct 300
MINN lllll INI I I M 111111 II II M i 111 CHRll: 126462 tcccaaggtgattctggatatgcagtcgcatagcagagtcatctctcatgtgggctgcct 126403
NOV7 : 301 gacacagatgtctttcttggtcctttttgcatgtatagaaggcatgctcctgactgtaat 360 II CHRll: 126402 gacacagatgtctttcttggtcctttttgcatgtatagaaggcatgctcctgactgtgat 126343
NOV7 : 361 ggcctatgactgctttgtaggcatctatcgccctctgcactacccagtcatcgtgaatcc 420 lllll MMMMMMMMMMM IIIMIII CHRll: 126342 ggcctatggctgctttgtagccatctgtcgccctctgcactacccagtcatagtgaatcc 126283
NOV7 : 421 tcatctctgtgtcttctttgttttggtgtcctttttccttagcctgttggattcccagct 480
III lllll I lllll llll CHRll: 126282 tcacctctgtgtcttcttcgttttggtgtcctttttccttaacctgttggattcccagct 126223 N0V7 : 481 gcacagttggattgtgttacaattcaccatcatcaagaatgtggaaatctctaattttgt 540
MMMMMMMIMMMMMMMMMMMMMMMIMMMMMM I
CHRll: 126222 gcacagttggattgtgttacaattcaccatcatcaagaatgtggaaatctctaatttttt 126163
N0V7 : 541 ctgtgacccctctcaacttctcaaacttgcctcttatgacagcgtcatcaatagcatatt 600 II MMMMMMMMMMMM
CHRll: 126162 ctgtgacccctctcagcttctcaaccttgcctgttctgacagcgtcatcaatagcatatt 126103
N0V7 : 601 catatatttcgatagtacaatgtttggttttcttcctatttcagggatcctttcatctta 660 MIIMMMMMM MMMMMMIM lllll
CHRll: 126102 catatatttcgatagtactatgtttggttttcttcccatttcagggatccttttgtctta 126043
N0V7 : 661 ctataaaattgtcccctccattctaaggatgtcatcgtcagatgggaagtataaaacttt 720
II 111 II I Ii 111111 II II 11 M M I M 11 M 1111 II 11 II 111111111111 I II
CHRll: 126042 ctataaaattgtcccctccattctaaggatgtcatcgtcagatgggaagtataaagcctt 125983
N0V7 : 721 ctccacctatggctctcacctagcatttgtttgctcattttatggaacaggcattgacat 780
IMNNIINNIINNNII I IIIIINN INN III
CHRll: 125982 ctccacctatggctctcacctaggagttgtttgctggttttatggaacagtcattggcat 125923
N0V7 : 781 gtacctggcttcagctatgtcaccaacccccaggaatggtgtggtggtgtcagtgatgta 840 MMMMIMMMMMM 111111 ; 1111
CHRll: 125922 gtacctggcttcagccgtgtcaccaccccccaggaatggtgtggtggcatcagtgatgta 125863
N0V7 : 841 agctgtggtcacccccatgctgaaccttttcatctacagcctgagaaacagggacataca 900
.III I III II llll I II llll lllll II II llll II I lllll INN II llll
CHRll: 125862 ggctgtggtcacccccatgctgaaccttttcatctacagcctgagaaacagggacataca 125803
NOW: 901 aagtgccctgcggaggctgcgcagcagaac 930 (SEQ ID NO. 13)
CHRll: 125802 aagtgccctgcggaggctgcgcagcagaac 125773 (SEQ ID NO. 68)
TABLE 32
N0V7: 179 PTYFFLSILCWADIGFTSATVPKMIVDMQWYSRVISHAGCLTQMSFLVLFACIEGMLLTV 358 * ***** * ******* ********** 4*****4 ******** *****4 ***4* OLFR: 1 PMYFFLSNLSLADIGFTSTTVPKMIVDMQTHSRVISYEGCLTQMSFFVLFACMDDMLLSV 60 NOV7: 359 MAYDCFVGIYRPLHYPVIVNPHLCVFFVLVSFFLSLLDSQLHSWIVLQFTIIKNVEISNF 538 **** ** * * * ** _j_* ** ** * 4*4***4********4. * ** * *4* **** OLFR: 61 MAYDRFVAICHPLHYRIIiNPRLCGFLILLSFFISLLDSQLHNLIMLQLTCFKDVDISNF 120 NOV7: 539 VCDPSQLLKLASYDSVINSIFIYFDSTMFGFLPISGILSSYYKIVPSILRMSSSDGKYKT 718
******* * * ** *** 4** ******* ****** *** _j_****** OLFR: 121 FCDPSQLLHLRCSDTFINEMVIYFMGAIFGCLPISGILFSYYKIVSPILRVPTSDGKYKA 180 NOV7: 719 FSTYGSHLAFVCSFYGTGIDMYIιASAMSPTPRNGVWSVM*AWTPMLNLFIYSLRNRDI 898 *** ***** ** *****4 **4** * ** 4* *** ******* *******4** OLFR: 181 FSTCGSHLAWCLFYGTGLVGYLSSAVLPSPRKSMVASVMYTWTPMLNPFIYSLRNKDI 240 NOV7: 899 QSALRRLRSR 928 (SEQ ID NO. 69) **** ** * OLFR: 241 QSALCRLHGR 250 (SEQ ID NO. 70)
Where * indicates identity and + indicates similarity. TABLE 33
NOV7: 44 NLLSIPAVSSDSHLHTPTYFFLSILCWADIGFTSATVPKMIVDMQWYSRVISHAGCLTQM 103 GPCR: 2 NVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMP WYLEWGE KFSRIHCDIFV 61 NOV7: 104 SFLVLFACIEGMLLTVMAYDCFVGIYRPLHYPVIVNPH 141 (SEQ ID NO. 71) GPCR: 62 TLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSK 99 (SEQ ID NO. 72)
The OR family ofthe GPCR superfamily is a group of related proteins that are specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV7 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV7 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
NOV8
A NOV8 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. A NOV8 nucleic acid and its encoded polypeptide includes the sequences shown in Table 34. The disclosed nucleic acid (SEQ ID NO: 15) is 994 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 27-29 and ends with a TGA stop codon at nucleotides 969-971. The representative ORF encodes a 314 amino acid polypeptide (SEQ ID NO: 16). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 15.
TABLE 34.
TGCAGCTAAAGTGCATTGTGTAAAACATGGGGGATGTGAATCAGTCGGTGGCCTCA GACTTCATTCTGGTGGGCCTCTTCAGTCACTCAGGATCACGCCAGCTCCTCTTCTCCC TGGTGGCTGTCATGTTTGTCATAGGCCTTCTGGGCAACACCGTTCTTCTCTTCTTGAT CCGTGTGGACTCCCGGCTCC ATAC ACCCATGTACTTCCTGCTC AGCCAGCTCTCCCTG TTTGACATTGGCTGTCCCATGGTCACCATCCCCAAGATGGCATCAGACTTTCTGCGG GGAGAAGGTGCCACCTCCTATGGAGGTGGTGCAGCTCAAATATTCTTCCTCACACTG ATGGGTGTGGCTGAGGGCGTCCTGTTGGTCCTCATGTCTTATGACCGTTATGTTGCTG TGTGCCAGCCCCTGCAGTATCCTGTACTTATGAGACGCCAGGTATGTCTGCTGATGA TGGGCTCCTCCTGGGTGGTAGGTGTGCTCAACGCCTCCATCCAGACCTCCATCACCC TGCATTTTCCCTACTGTGCCTCCCGTATTGTGGATCACTTCTTCTGTGAGGTGCCAGC CCTACTGAAGCTCTCCTGTGCAGATACCTGTGCCTACGAGATGGCGCTGTCCACCTC AGGGGTGCTGATCCTAATGCTCCCTCTTTCCCTCATCGCCACCTCCTACGGCCACGTG TTGCAGGCTGTTCTAAGCATGCGCTCAGAGGAGGCCAGACACAAGGCTGTCACCAC CTGCTCCTCGCACATCACGGTAGTGGGGCTCTTTTATGGTGCCGCCGTGTTCATGTAC ATGGTGCCTTGCGCCTACCACAGTCCACAGCAGGATAACGTGGTTTCCCTCTTCTAT AGCCTTGTCACCCCTACACTCAACCCCCTTATCTACAGTCTGAGGAATCCGGAGGTG TGGATGGCTTTGGTCAAAGTGCTTAGCAGAGCTGGACTCAGGCAAATGTGCTGACT ACATAGAAACTGCTGGTGAGA (SEQ ID NO.: 15)
MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY FLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLMGVAEGVLLVLMS YDRYVAVCQPLQYPVLMRRQVCLLMMGSSWVVGVLNASIQTSITLHFPYCASRIVDHF FCEVPALLKLSCADTCAYEMALSTSGVLILMLPLSLIATSYGHVLQAVLSMRSEEARHK AVTTCSSHITVVGLFYGAAVFMYMVPCAYHSPQQDNVVSLFYSLVTPTLNPLIYSLRNP EVWMALVKVLSRAGLRQMC (SEQ ID NO. : 16)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. NOV8 nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
The NOV8 polypeptide has homology (approximately 44% identity, 65% similarity) to the human olfactory receptor family 2 subfamily F, member 1 (OLFR) (EMBL Accession No.:NP 036501), as is shown in Table 35. The NOV8 polypeptide also has homology (44% identity, 65% similarity) to the rat olfactor receptor-like protein OLF3 (SwissProt Accession No.: Q13607), as is shown in Table 36. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences. 1999, 20:413. OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains. NOV8 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 37.
TABLE 35
N0V8: 1 MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY 60 ** **4- *4***4-** * 4.* ** * ** .*4- 4*** 4.4.4- ***4*********
OLFR: 1 MGTDNQT VSEFILLGLSSDWDTRVSLFVLFLVMYWTVLGNCLIVLLIRLDSRLHTPMY 60
NOV8: 61 FLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLMGVAEGVLLVLMSY 120 * *4- *** *4- 4.*-_ 4. ** * 4. ***.** _}.* * *** 4-*4_*
OLFR: 61 FFLTNLSLVDVSYATSWPQLLAHFLAEHKAIPFQSCAAQLFFSLALGGIEFVLLAVMAY 120
NOV8: 121 DRYVAVCQPLQYPVLMRRQVCLLMMGSS WGVLNASIQTSITLHFPYCASRIVDHFFCE 180 ******* * + * _|_* 4-* 4. _j.*** * 4-4-4- -|-.* * + ** * * 4.4. 4.** **
OLFR: 121 DRYVAVCDALRYSAIMHGGLCARLAITS VSGFISSPVQTAITFQLPMCRNKFIDHISCE 180
NOV8: 181 VPALLKLSCADTCAYEMALSTSGVLILMLPLSLIATSYGHVLQAVLSMRSEEARHKAVTT 240 4 * _4 .*4.* ** 4. *4 4. * 4,44.** ** *4_ ** 4.4. 4.* 44.* * * ** *
OLFR: 181 LLAWRLACVDTSSNEVTIMVSSIVLLMTPLCLVLLSYIQIISTILKIQSREGRKKAFHT 240 NOV8: 241 CSSHITVVGLFYGAAVFMYMVPCAYHSPQQDNVVSLFYSLVTPTLNPLIYSLRNPEV MA 300
*4-**^.*** * ** * _* *4_ *4- 4. *4_**4-4.4-** ***+****** **
OLFR: 241 CASHLTWALCYGVAIFTYIQPHSSPSVLQEKLFSVFYAILTPMLNPMIYSLRNKEVKGA 300
NOV8: 301 LVKVL 305 (SEQ ID NO. 73) *4-*
OLFR: 301 QKLL 305 (SEQ ID NO. 74)
Where * indicates identity and + indicates similarity. TABLE 36
NOV8: 27 MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY 206 ** ** _ * _***^.** * _* ** * **4-* - ^.*** +4-4- ***4-********* OLFR: 1 MGTDNQT VSΞFILLGLSSD DTRVSLFVLFLVMYWTVLGNCLIVLLIRLDSRLHTPMY 60
NOV8: 207 FLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLMGVAEGVLLVLMSY 386 * *4_ *** *4. 4.*4.4- 4. ** * 4. ***4-** 4.* * *** -* -*
OLFR: 61 FFLTNLSLVDVSYATSWPQLLAHFLAEHKAIPFQSCAAQLFFSLALGGIΞFVLLAVMAY 120
NOV8: 387 DRYVAVCQPLQYPVLMRRQVCLLMMGSS WGVLNASIQTSITLHFPYCASRIVDHFFCE 566 ******* * -* 4.* 4.* 4. +*** * 4-4-4- 4-** -** * * +. 4.** **
OLFR: 121 DRYVAVCDALRYSAIMHGGLCARLAITS VSGFISSPVQTAITFQLPMCRNKFIDHISCE 180 NOV8: 567 VPALLKLSCADTCAYEMALSTSGVLILMLPLSLIATSYGHVLQAVLSMRSEEARHKAVTT 746 4. *4.4. *4.* ** 4. * _ 4. * 4.4.4.** ** * _ ** 44. 4* 4.4* * * ** *
OLFR: 181 LLAWRLACVDTSSNΞVTIMVSSIVLLMTPLCLVLLSYIQIISTILKIQSREGRKKAFHT 240
NOV8: 747 CSSHITVVGLFYGAAVFMYMVPCAYHSPQQDNVVSLFYSLVTPTLNPLIYSLRNPΞVWMA 926 *4_**4_*** * ** * * *4 * 4. * *- 4. *4.**444** ***4****** ** *
OLFR: 241 CASHLTWALCYGVAIFTYIQPHSSPSVLQEKLFSVFYAILTPMLNPMIYSLRNKEVKGA 300 NOV8: 927 LVKVLSR-AGL 956 (SEQ ID NO. 75) *4-* 4. 4.**
OLFR: 301 QKLL KFSGL 311 (SEQ ID NO. 76)
Where * indicates identity and + indicates similarity.
TABLE 37
NOV8: 41 GNTVLLFLIRVDSRLHTPMYFLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQ 100 GPCR: 1 GNVLVCMAVSREKALQTTTIWLIVSIAVADLLVATLVMP VVYLEVVGE KFSRIHCDIF 60
NOV8: 101 IFFLTLMGVAEGVLLVLMSYDRYVAVCQPLQYPVLM-RRQVCLLMMGSSWWGVLNASIQ 159
GPCR: 61 VTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVVLSFTISCPM 120 NOV8: 160 TSITLHFPYCASRIVDHFFCEVPALLKLSCADTCAYEMALSTSGVLILMLPLSLIATSYG 219 GPCR: 121 LFGLNNTDQN ECIIA--NPAFWYSSIVSFYVPFIVTL VYI 160
NOV8: 220 HVLQAVLSMRSEEA 233 (SEQ ID NO. 77) GPCR: 161 KIYIVLRRRRKRVN 174 (SEQ ID NO . 78)
The OR family ofthe GPCR superfamily is a group of related proteins located at the ciliated surface of olfactory sensory neurons in the nasal epithelium. The OR family is involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV8 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV8 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
NOV9 A NOV9 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. A NOV9 nucleic acid and its encoded polypeptide includes the sequences shown in Table 38. The NOV8 nucleic acid sequence (SEQ ID NO.: 15) was further analyzed by exon linking, and the resulting sequence was identified as NOV9. The disclosed nucleic acid (SEQ ID NO: 17) is 994 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 28-30 and ends with a TAG stop codon at nucleotides 979-981. The representative ORF encodes a 317 amino acid polypeptide (SEQ ID NO: 18). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 17.
TABLE 38.
• TGCAGCTAAAGTGCATTGTGTAAAACTATGGGGGATGTGAATCAGTCGGTGGCCTC AGACTTCATTCTGGTGGGCCTCTTCAGTCACTCAGGATCACGCCAGCTCCTCTTCTCC CTGGTGGCTGTCATGTTTGTCATAGGCCTTCTGGGCAACACCGTTCTTCTCTTCTTGA TCCGTGTGGACTCCCGGCTCCACACACCCATGTACTTCCTGCTCAGCCAGCTCTCCCT GTTTGACATTGGCTGTCCCATGGTCACCATCCCCAAGATGGCATCAGACTTTCTGCG GGGAGAAGGTGCCACCTCCTATGGAGGTGGTGCAGCTCAAATATTCTTCCTCACACT GATGGGTGTGGCTGAGGGCGTCCTGTTGGTCCTCATGTCTTATGACCGTTATGTTGCT GTGTGCCAGCCCCTGCAGTATCCTGTACTTATGAGACGCCAGGTATGTCTGCTGATG ATGGGCTCCTCCTGGGTGGTAGGTGTGCTCAACGCCTCCATCCAGACCTCCATCACC CTGCATTTTCCCTACTGTGCCTCCCGTATTGTGGATCACTTCTTCTGTGAGGTGCCAG CCCTACTGAAGCTCTCCTGTGCAGATACCTGTGCCTACGAGATGGCGCTGTCCACCT CAGGGGTGCTGATCCTAATGCTCCCTCTTTCCCTCATCGCCACCTCCTACGGCCACGT GTTGCAGGCTGTTCTAAGCATGCGCTCAGAGGAGGCCAGACACAAGGCTGTCACCA CCTGCTCCTCGCACATCACGGTAGTGGGGCTCTTTTATGGTGCCGCCGTGTTCATGTA CATGGTGCCTTGCGCCTACCACAGTCCACAGCAGGATAACGTGGTTTCCCTCTTCTA TAGCCTTGTCACCCCTACACTCAACCCCCTTATCTACAGTCTGAGGAATCCGGAGGT GTGGATGGCTTTGGTCAAAGTGCTTAGCAGAGCTGGACTCAGGCAAATGTGCATGAC TACATAGAAACTGCTGGTGAGA (SEQ ID NO.: 17)
MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY FLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLMGVAEGVLLVLMS YDRYVAVCQPLQYPVLMRRQVCLLMMGSSWVVGVLNASIQTSITLHFPYCASRIVDHF FCEVPALLKLSCADTCAYEMALSTSGVLILMLPLSLIATSYGHVLQAVLSMRSEEARHK AVTTCSSHITVVGLFYGAAVFMYMVPCAYHSPQQDNVVSLFYSLVTPTLNPLIYSLRNP EVWMALVKVLSRAGLRQMCMTT (SEQ ID NO. : 18)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV9 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
The NOV9 polypeptide has homology (approximately 44% identity, 65% similarity) to the human olfactory receptor family 2 subfamily F, member 1 (OLFR) (EMBL Accession No.:NP 036501), as is shown in Table 39. The NOV9 polypeptide also has a high degree of homology (99% identity) to the NOV8 polypeptide as shown in Table 40. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. Thus NOVS andNOV9 belong to the same subfamily of ORs.
OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy- terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains. NOV9 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 41.
TABLE 39
NOV9 : 1 MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY 60
**4- *+***+** * +* **4-* + 4.*** 4-4-4- ***4-*********
OLFR : 1 MGTDNQTWVSEFILLGLSSD DTRVSLFVLFLVMYWTVLGNCLIVLLIRLDSRLHTPMY 60
NOV9 : 61 FLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLMGVAEGVLLVLMSY 120 * *4- * ** *4- + * + + _j- ** * + ***4«* * 4.* * * * * 4.* >*
OLFR : 61 FFLTNLSLVDVSYATSWPQLLAHFLAEHKAIPFQSCAAQLFFSLALGGIEFVLLAVMAY 120
NOV9 : 121 DRYVAVCQPLQYPVLMRRQVCLLMMGSSWWGVLNASIQTSITLHFPYCASRIVDHFFCE 180 * * * ** * * * + * 4.* + * _ 4.** * * -}-4-+ 4-* * + * * * * 4.+ +* * **
OLFR : 121 DRYVAVCDALRYSAIMHGGLCARLAITS VSGFISSPVQTAITFQLPMCRNKFIDHISCE 180
NOV9 : 181 VPALLKLSCADTCAYEMALSTSGVLILMLPLSLIATSYGHVLQAVLSMRSEEARHKAVTT 240 + * + - -*4-* ** + *+ 4. * 4. + + ** ** *+ ** ++ +* + + * * * ** *
OLFR : 181 LLAWRLACVDTSSNEVTIMVSSIVLLMTPLCLVLLSYIQIISTILKIQSREGRKKAFHT 240
NOV9 : 241 CSSHITWGLFYGAAVFMYMVPCAYHSPQQDNWSLFYSLVTPTLNPLIYSLRNPEV MΑ 300 *4-* * + ** * * * * * + * *+ * + * *+ + * + ** + + + ** * ** + ** * *** * * *
OLFR : 241 CASHLTWALCYGVAIFTYIQPHSSPSVLQEKLFSVFYAILTPMLNPMIYSLRNKEVKGA 300
NOV9: 301 LVKVL 305 (SEQ ID NO. 79) *4-*
OLFR: 301 QKLL 305 (SEQ ID NO. 80)
Where * indicates identity and + indicates similarity.
TABLE 40
NOV9 : 1 MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY 60
************************************************************
NOV8: 1 MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY 60
NOV9: 61 FLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLMGVAEGVLLVLMSY 120 ************************************************************ NOV8: 61 FLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLMGVAEGVLLVLMSY 120
NOV9: 121 DRYVAVCQPLQYPVLMRRQVCLLMMGSSWWGVLNASIQTSITLHFPYCASRIVDHFFCE 180 ************************************************************
NOV8: 121 DRYVAVCQPLQYPVLMRRQVCLLMMGSS WGVLNASIQTSITLHFPYCASRIVDHFFCE 180
N0V9: 181 VPALLKLSCADTCAYΞMALSTSGVLILMLPLSLIATSYGHVLQAVLSMRSEΞARHKAVTT 240 ************************************************************
NOV8: 181 VPALLKLSCADTCAYEMALSTSGVLILMLPLSLIATSYGHVLQAVLSMRSEEARHKAVTT 240
NOV9: 241 CSSHITVVGLFYGAAVFMYMVPCAYHSPQQDNVVSLFYSLVTPTLNPLIYSLRNPEV MA 300 ************************************************************
NOV8 : 241 CSSHITWGLFYGAAVFMYMVPCAYHSPQQDNWSLFYSLVTPTLNPLIYSLRNPEVWMA 300 NOV9 : 301 LVKVLSRAGLRQMCMTT 317 (SEQ ID NO . 17)
**************
NOV8: 301 LVKVLSRAGLRQMC 314 (SEQ ID NO . 15)
Where * indicates identity.
TABLE 41
NOV9: 41 GNTVLLFLIRVDSRLHTPMYFLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQ 100 GPCR: 1 GNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMP WYLΞWGE KFSRIHCDIF 60 NOV9: 101 IFFLTLMGVAEGVLLVLMSYDRYVAVCQPLQYPVLM-RRQVCLLMMGSSWWGVLNASIQ 159 GPCR: 61 VTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVWVLSFTISCPM 120
NOV9: 160 TSITLHFPYCASRIVDHFFCEVPALLKLSCADTCAYEMALSTSGVLILMLPLSLIATSYG 219
GPCR: 121 LFGLNNTDQN ECIIA--NPAFWYSSIVSFYVPFIVTLLVYI 160
NOV9: 220 HVLQAVLSMRSEEA 233 (SEQ ID NO. 83)
GPCR: 161 KIYIVLRRRRKRVN 174 (SEQ ID NO. 84)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV9 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV9 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing. NOV10
A NOV10 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. A NOV10 nucleic acid and its encoded polypeptide includes the sequences shown in Table 42. The disclosed nucleic acid (SEQ ID NO: 19) is 1,077 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 31-33 and ends with a TAG stop codon at nucleotides 1,030- 1,032. The representative ORF encodes a 318 amino acid polypeptide (SEQ ID NO:20). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 19. Exon linking was used to confirm the sequence.
TABLE 42. CAGGTTCATTGACAAGGTCATACCAACCAGATGAATCCAGCAAATCATTCCCAGGT GGCAGGATTTGTTCTACTGGGGCTCTCTCAGGTTTGGGAGCTTCGGTTTGTTTTCTTC ACTGTTTTCTCTGCTGTGTATTTTATGACTGTAGTGGGAAACCTTCTTATTGTGGTCA TAGTGACCTCCGACCCACACCTGCACACAACCATGTATTTTCTCTTGGGCAATCTTTC TTTCCTGGACTTTTGCTACTCTTCCATCACAGCACCTAGGATGCTGGTTGACTTGCTC TCAGGCAACCCTACCATTTCCTTTGGTGGATGCCTGACTCAACTCTTCTTCTTCCACT TCATTGGAGGCATCAAGATCTTCCTGCTGACTGTCATGGCGTATGACCGCTACATTG CCATTTCCCAGCCCCTGCACTACACGCTCATTATGAATCAGACTGTCTGTGCACTCCT TATGGCAGCCTCCTGGGTGGGGGGCTTCATCCACTCCATAGTACAGATTGCATTGAC TATCCAGCTGCCATTCTGTGGGCCTGACAAGCTGGACAACTTTTATTGTGATGTGCCT CAGCTGATCAAATTGGCCTGCACAGATACCTTTGTCTTAGAGCTTTTAATGGTGTCTA ACAATGGCCTGGTGACCCTGATGTGTTTTCTGGTGCTTCTGGGATCGTACACAGCAC TGCTAGTCATGCTCCGAAGCCACTCACGGGAGGGCCGCAGCAAGGCCCTGTCTACCT GTGCCTCTCACATTGCTGTGGTGACCTTAATCTTTGTGCCTTGCATCTACGTCTATAC AAGGCCTTTTCGGACATTCCCCATGGACAAGGCCGTCTCTGTGCTATACACAATTGT CACCCCCATGCTGAATCCTGCCATCTATACCCTGAGAAACAAGGAAGTGATCATGGC CATGAAGAAGCTGTGGAGGAGGAAAAAGGACCCTATTGGTCCCCTGGAGCACAGAC CCTTACATTAGCAGAGGCAGTGACCTGAGAATCTGAAAGATGCTACAGGGTATTAG CAGAGGCAGTGACCTGAGAATCTGAAAGATGCTACAGGGTATTAG (SEQ ID NO.: 19)
MNPANHSQVAGF VLLGLSQVWELRFVFFTVFS AVYFMTVVGNLLIVVIVTSDPHLHTT MYFLLGNLSFLDFCYSSITAPRMLVDLLSGNPTISFGGCLTQLFFFHFIGGIKIFLLTVMAY DRYIAISQPLHYTLIMNQTVCALLMAASWVGGFIHSIVQIALTIQLPFCGPDKLDNFYCD VPQLIKLACTDTFVLELLMVSNNGLVTLMCFLVLLGSYTALLVMLRSHSREGRSKALST CASHIAVVTLIFVPCIYVYTRPFRTFPMDKAVSVLYTIVTPMLNPAIYTLRNKEVIMAMK KLWRRKKDPIGPLEHRPLH (SEQ ID NO. : 20) The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV10 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
The NOV10 polypeptide has homology (approximately 55% identity, 72% similarity) to the olfactory receptor MOR83 (OLFR) (EMBL Accession No. :BAA86125), as is shown in Table 43. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains. NOV10 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 44.
TABLE 43
NOVIO : 79 MNPANHSQVAGFVLLGLSQV ELRFVFFTVFSAVYFMTWGNLLIWIVTSDPHLHTTMY 258
* * ++* *+ * ** * * 4* * *_j_ * 4*44* * *** * 4. * ** **
OLFR: 1 MGALNQTRVTEFIFLGLTDNWVLEILFFVPFTVTYMLTLLGNFLIWTIVFTPRLHNPMY 60 NOVIO: 259 FLLGNLSFLDFCYSSITAPRMLVDLLSGNPTISFGGCLTQLFFFHFIGGIKIFLLTVMAY 438
* * ****4* *4**4* *4** ** **** *4 **** * 4*****4***
OLFR: 61 FFLSNLSFIDICHSSVTVPKMLEGLLLERKTISFDNCIAQLFFLHLFACSEIFLLTIMAY 120
NOVIO: 439 DRYIAISQPLHYTLIiNQTVCALLMAA.SWVGGFIHSIVQIALTIQLPFCGPDKLDNFYCD 618 ***4-** ****4 4** ** *4 * *4** ***_(_** ***4**_}.***4 4*444**
OLFR: 121 DRYVAICIPLHYSNVMNMKVCVQLVFALWLGGTIHSLVQTFLTIRLPYCGPNIIDSYFCD 180
NOVIO: 619 VPQLIKLACTDTFVLELLMVSNNGLVTLMCFLVLLGSYTALLVMLRSHSREGRSKALSTC 798 ** .$.* ******* 4> 4*.j_***.}_* 44*4*** * *** 4* ** * *** ****** OLFR: 181 VPPVIKLACTDTYLTGILIVSNSGTISLVCFLALVTSYTVILFSLRKKSAEGRRKALSTC 240
NOV10: 799 ASHIAWTLIFVPCIYVYTRPFRTFPMDKAVSVLYTIVTPMLNPAIYTLRNKΕVIMAMKK 978 44* **** * ***4+**** 4* _{_** *** ** _***+*** ******_{_** ***
OLFR: 241 SAHFMWTLFFGPCIFLYTRPDSSFSIDIXWSVFYTWTPLLNPLIYTLRNEEVKTAMKH 300
NOV10 : 979 L RRK 993 ( SEQ ID NO . 85 )
* 4*4-
OLFR : 301 LRQRR 305 (SEQ ID NO . 86 )
Where * indicates identity and + indicates similarity. TABLE 44
NOVIO : 41 GNLLIWIVTSDPHLHTTMYFLLGNLSFLDFCYSSITAPRMLVDLLSGNPTISFGGCLTQ 100
GPCR : 1 GNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWWYLEWGEWKFSRIHCDIF 60
NOVIO : 101 LFFFHFIGGIKIFLLTVMAYDRYIAISQPLHYTLIMNQ-TVCALLMAAS VGGFIHSIVQ 159
GPCR : 61 VTLDVMMCTASILNLCAISIDRYTAVA PMLYNTRYSSKRRVTVMIAIVWVLSFT1SCPM 120
NOVIO : 160 IALTIQLPFCGPDKLDNFYCDVPQLI LACTDTFVLELLMVSNNGLVTL CFLVLLGSYT 219 GPCR : 121 LFGLNNTDQNE CIIANPAF Y- -SSIVSFYVPFIVTLLVYI 160
NOVIO : 220 ALLVMLRSHSREGRSKA 236 ( SEQ ID NO . 87 ) GPCR : 161 KIYIVLRRRRKRVNTKR 177 (SEQ ID NO . 88 )
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV10 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV10 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
NOV11
A NOVl 1 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. A NOVl 1 nucleic acid was discovered by exon linking analysis of NOV2 (SEQ ID NO.: 3). A NOVl 1 nucleic acid and its encoded polypeptide includes the sequences shown in Table 45. The disclosed nucleic acid (SEQ ID NO:21) is 1,012 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 54-56 and ends with a TGA stop codon at nucleotides 984-986. The representative ORF encodes a 310 amino acid polypeptide (SEQ ID NO:22). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 21. TABLE 45.
AAACACTTCTCCTAAACCATGAGCATTAACTTGATTTCCTCTGTCATAGGGATATGG GAGACAATATAACATCCATCACAGAGTTCCTCCTACTGGGATTTCCCGTTGGCCCAA GGATTCAGATGCTCCTCTTTGGGCTCTTCTCCCTGTTCTACGTCTTCACCCTGCTGGG GAACGGGACCATACTGGGGCTCATCTCACTGGACTCCAGACTGCACGCCCCCATGTA CTTCTTCCTCTCACACCTGGCGGTCGTCGACATCGCCTACGCCTGCAACACGGTGCC CCGGATGCTGGTGAACCTCCTGCATCCAGCCAAGCCCATCTCCTTTGCGGGCCGCAT GATGCAGACCTTTCTGTTTTCCACTTTTGCTGTCACAGAATGTCTCCTCCTGGTGGTG ATGTCCTATGATCTGTACGTGGCC ATCTGCC ACCCCCTCCGATATTTGGCCATC ATGA CCTGGAGAGTCTGCATCACCCTCGCGGTGACTTCCTGGACCACTGGAGTCCTTTTAT CCTTGATTCATCTTGTGTTACTTCTACCTTTACCCTTCTGTAGGCCCCAGAAAATTTA TCACTTTTTTTGTGAAATCTTGGCTGTTCTCAAACTTGCCTGTGCAGATACCCACATC AATGAGAACATGGTCTTGGCCGGAGCAATTTCTGGGCTGGTGGGACCCTTGTCCACA ATTGTAGTTTCATATATGTGCATCCTCTGTGCTATCCTTCAGATCCAATCAAGGGAAG TTCAGAGGAAAGCCTTCTGCACCTGCTTCTCCCACCTCTGTGTGATTGGACTCTTTTA TGGCACAGCCATTATCATGTATGTTGGACCCAGATATGGGAACCCCAAGGAGCAGA AGAAATATCTCCTGCTGTTTCACAGCCTCTTTAATCCCATGCTCAATCCCCTTATCTG TAGTCTTAGGAACTCAGAAGTGAAGAATACTTTGAAGAGAGTGCTGGGAGTAGAAA GGGCTTTATGAAAAGGATTATGGCATTGTGACTGACA (SEQ ID NO. : 21 )
MGDNITSITEFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYFFL SHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLVVMSYDL YVAICHPLRYLAIMTWRVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEILA VLKLACADTHINENMVLAGAISGLVGPLSTIVVSYMCILCAILQIQSREVQRKAFCTCFSH LCVIGLFYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTLKRV LGVERAL (SEQ ID NO.: 22)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 1 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. The NOVl 1 polypeptide has a high degree of homology (approximately 99% identity) to a human olfactory receptor (OLFR) (EMBL Accession No.:095047), as is shown in Table 46. The NOVl 1 polypeptide also has a high degree of homology (approximately 99% identity) to NOV2, as is shown in Table 47. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences. 1999, 20:413. Therefore, NOVl 1 and NOV2 are two members ofthe same OR subfamily. OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains. NOVl 1 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 48.
TABLE 46 NOVll: 1 MGDNITSITEFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60 ******** ***************************************************
OLFR: 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60 NOVll: 61 FLSHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLVVMSYD 120 ************************************************************
OLFR: 61 FLSHLAVΛ iAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLVVMSYD 120 NOVll: 121 LYVAICHPLRYLAIMT RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCΞI 180
************************************************************ OLFR : 121 LYVAICHPLRYLAIMT RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180
NOVll : 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFCTC 240
********************************************************* **
OLFR: 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFRTC 240 NOVll : 241 FSHLCVIGLFYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300
********* ************************************************** OLFR: 241 FSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300 NOVll : 301 KRVLGVERAL 310 (SEQ ID NO. : 64) ********** OLFR: 301 KRVLGVERAL 310 (SEQ ID NO. : 67)
Where * indicates identity.
TABLE 47
NOVll : 1 MGDNITSITEFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60
******** ***************************************************
NOV2: 1 MGDNITSIRΞFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60
NOVll: 61 FLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLWMSYD 120 ************************************************************
NOV2: 61 FLSHLAWDIAYACNTVPRMLVNLLHPAK ISFAGRMMQTFLFSTFAVTECLLLWMSYD 120 NOVll: 121 LYVAICHPLRYLAIMTWRVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180
************************************************************
NOV2 : 121 LYVAICHPLRYLAIMTWRVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCΞI 180 NOVll : 181 LAVLKLACADTHINΞNMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFCTC 240 ********************************************************* **
NOV2 : 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFRTC 240 NOVll : 241 FSHLCVIGLFYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300 ********* ****** *********** ** *******************************
NOV2: 241 FSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300 NOVll .-301 KRVLGVERAL 310 (SEQ ID NO.: 22)
********** NOV2: 301 KRVLGVERAL 310 (SEQ ID NO.: 4)
Where * indicates identity.
TABLE 48
NOVll : 53 RLHAPMYFFLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECL 112 GPCR : 14 ALQTTTNYLIVSLAVADLLVATLVMP VVYLEVVGEWKFSRIHCDIFVTLDVMMCTASIL 73
NOVll : 113 LLWMSYDLYVAICHPLRYLAIMT -RVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQ 171 GPCR : 74 NLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVWVLSFTISCPMLFGLNNTDQNE- - 131 NOVll : 172 KIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSRE 231 GPCR : 132 - CIIANPAF WYSSIVSFYVPFIVTLLVYIKIYIVLRRRRKRV 173
NOVll : 232 VQRK 235 (SEQ ID NO. : : 81)
GPCR: 174 NTKR 177 (SEQ ID NO. : : 82)
The OR family of the GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 1 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl 1 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
NOV12
A NOVl 2 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. . A NOVl 2 nucleic acid was discovered by exon linking analysis of NOV2 (SEQ ID NO.: 3). A NOV12 nucleic acid and its encoded polypeptide includes the sequences shown in Table 49. The disclosed nucleic acid (SEQ ID NO:23) is 1,014 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 55-57 and ends with a TGA stop codon at nucleotides 985-987. The representative ORF encodes a 310 amino acid polypeptide (SEQ ID NO:24). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 23.
TABLE 49.
TAAACACTTCTCCTAAACCATGAGCATTAACTTGATTTCCTCTGTCATAGGGATATG GGGGACAATATAACATCCATCACAGAGTTCCTCCTACTGGGATTTCCCGTTGGCCCA AGGATTCAGATGCTCCTCTTTGGGCTCTTCTCCCTGTTCTACGTCTTCACCCTGCTGG GGAACGGGACCATACTGGGGCTCATCTCACTGGACTCCAGACTGCACGCCCCCATGT ACTTCTTCCTCTCACACCTGGCGGTCGTCGACATCGCCTACGCCTGCAACACGGTGC CCCGGATGCTGGTGAACCTCCTGCATCCAGCCAAGCCCATCTCCTTTGCGGGCCGCA TGATGCAGACCTTTCTGTTTTCCACTTTTGCTGTCACAGAATGTCTCCTCCTGGTGGT GATGTCCTATGATCTGTACGTGGCCATCTGCCACCCCCTCCGATATTTGGCCATCATG ACCTGGAGAGTCTGCATCACCCTCGCGGTGACTTCCTGGACCACTGGAGTCCTTTTA TCCTTGATTCATCTTGTGTTACTTCTACCTTTACCCTTCTGTAGGCCCCAGAAAATTT ATCACTTTTTTTGTGAAATCTTGGCTGTTCTCAAACTTGCCTGTGCAGATACCCACAT CAATGAGAACATGGTCTTGGCCGGAGCAATTTCTGGGCTGGTGGGACCCTTGTCCAC AATTGTAGTTTCATATATGTGCATCCTCTGTGCTATCCTTCAGATCCAATCAAGGGAA GTTCAGAGGAAAGCCTTCTGCACCTGCTTCTCCCACCTCTGTGTGATTGGACTCTTTT ATGGCACAGCCATTATCATGTATGTTGGACCCAGATATGGGAACCCCAAGGAGCAG AAGAAATATCTCCTGCTGTTTCACAGCCTCTTTAATCCCATGCTCAATCCCCTTATCT GTAGTCTTAGGAACTCAGAAGTGAAGAATACTTTGAAGAGAGTGCTGGGAGTAGAA AGGGCTTTATGAAAAGGATTATGGCATTGTGACTGACAA (SEQ ID NO.: 23)
MGDNITSITEFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYFFL SHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLVVMSYDL YVAICHPLRYLAIMTWRVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEILA VLKLACADTHINENMVLAGAISGLVGPLSTIVVSYMCILCAILQIQSREVQRKAFCTCFSH LCVIGLFYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTLKRV LGVERAL (SEQ ID NO.: 24)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
The NOV12 polypeptide has a high degree of homology (approximately 99% identity) to a human olfactory receptor (OLFR) (EMBL Accession No.:095047), as is shown in Table 50. The NOVl 2 polypeptide also has a high degree of homology (approximately 99% identity) to NOV2, as is shown in Table 51. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. Therefore, NOVl 2 andNOV2 are two members ofthe same OR subfamily. OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains. NOVl 2 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 52.
TABLE 50.
NOV12 : 1 MGDNITSITEFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60 ******** ***************************************************
OLFR: 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60 NOV12: 61 FLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLWMSYD 120
************************************************************ OLFR: 61 FLSHIjAVVDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLVVMSYD 120 NOV12: 121 LYVAICHPLRYLAIMT RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180
************************************************************
OLFR : 121 LYVAICHPLRYLAIMTWRVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180
NOV12 : 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFCTC 240
********************************************************* **
OLFR : 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFRTC 240
NOV12 : 241 FSHLCVIGLFYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300
********* **************************************************
OLFR: 241 FSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300
NOV12: 301 KRVLGVERAL 310 (SEQ ID NO. 24 ) **********
OLFR : 301 KRVLGVERAL 310 (SEQ ID NO . 89 )
Where * indicates identity. TABLE 51.
NOVl2 : 1 MGDNITSITEFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60 ******** *************************************************** NOV2: 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60 NOVl2 : 61 FLSHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGR MQTFLFSTFAVTECLLLVVMSYD 120 ************************************************************ NOV2: 61 FLSHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGR MQTFLFSTFAVTECLLLVVMSYD 120 NOVl2 : 121 LYVAICHPLRYLAIMT RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180 ************************************************************ NOV2: 121 LYVAICHPLRYLAIMT RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCΞI 180 NOVl2 : 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFCTC 240 ********************************************************* ** NOV2: 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFRTC 240 NOVl2 : 241 FSHLCVIGLFYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300
********* ************************************************** NOV2: 241 FSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300 NOVl2 : 301 KRVLGVERAL 310 (SEQ ID NO. : 24)
********** NOV2: 301 KRVLGVERAL 310 (SEQ ID NO . : 4 )
Where * indicates identity.
TABLE 52.
NOV12 : 53 RLHAPMYFFLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECL 112 GPCR : 14 ALQTTTNYLIVSLAVADLLVATLVMP WYLEWGE KFSRIHCDIFVTLDVMMCTASIL 73
NOV12 : 113 LLWMSYDLYVAICHPLRYLAIMTW-RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQ 171
GPCR : 74 NLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIV VLSFTISCPMLFGLNNTDQNE- - 131
NOV12 : 172 KIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSRE 231
GPCR : 132 - CIIANPAF WYSSIVSFYVPFIVTLLVYIKIYIVLRRRRKRV 173
NOV12 : 232 VQRK 235 (SEQ ID NO . : 90 ) GPCR : 174 NTKR 177 ( SEQ ID NO . : 91)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl 2 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
NOV13
A NOVl 3 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. A NOVl 3 nucleic acid and its encoded polypeptide includes the sequences shown in Table 53. The disclosed nucleic acid (SEQ ID NO:25) is 908 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 75-77 and ends with a TAA stop codon at nucleotides 901-903. The representative ORF encodes a 270 amino acid polypeptide (SEQ ID NO:26). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 25.
TABLE 53.
TGTATCTGGTCACGGTGCTGAGGAACCTGCTCAGCATCCTGGCTGTCAGCTCTGACT CCCACCCCCACACACCCATGTACTTCTTCCTCTCCAACCTGTGCTGGGCTGACATCG GTTTCACCTTGGCCACGGTTCCCAAGATGATTGTGGACATGGGGTCGCATAGCAGAG TCATCTCTTATGAGGGCTGCCTGACACAGATGTCTTTCTTTGTCCTTTTTGCATGTAT AGAAGACATGCTCCTGACTGTGATGGCCTATGACCAATTTGTGGCCATCTGTCACCC CCTGCACTACCCAGTCATCATGAATCCTCACCTCTGTGTCTTCTTAGTTTTGGTTTCTT TTTTCCTTAGCCTGTTGGATTCCCAGCTGCACAGTTGGATTGTGTTACAATTCACCTT CTTCAAGAATGTGGAAATCTCTAATTTTTTCTGTGATCCATCTCAACTTCTCAACCTT GCCTGTTCTGACGGCATCATCAATAGCATATTCATATATTTAGATAGTATTCTGTTCA GTTTTCTTCCCATTTCAGGGATCCTTTTGTCTTACTATAAAATTGTCCCCTCCATTCTA AGAATTTCATCGTCAGATGGGAAGTATAAAGCCTTCTCCATCTGTGGCTCTCACCTG GCAGTTGTTTGCTTATTTTATGGAACAGGCATTGGCGTGTACCTAACTTCAGCTGTGT CACCACCCCCCAGGAATGGTGTGGTGGCGTCAGTGATGTATGCTGTGGTCACCCCCA TGCTGAACCCTTTCATCTACAGCCTGAGAAACAGGGATATACAAAGTGTCCTGCGGA GGCTGTGCAGCAGAACAGTCGAATCTCATGATATGTTCCATCCTTTTTCTTGTGTGGG TGAGAAAGGGCAACCACATTAAATCTCTACATCTGTAAATCCT (SEQ ID NO.: 25)
MYFFLSNLCWADIGFTLATVPKMIVDMGSHSRVISYEGCLTQMSFFVLFACIEDMLLTV
MAYDQFVAICHPLHYPVIMNPHLCVFLVLVSFFLSLLDSQLHSWIVLQFTFFKNVEISNFF
CDPSQLLNLACSDGIINSIFIYLDSILFSFLPISGILLSYYKIVPSILRISSSDGKYKAFSICGSH LAVVCLFYGTGIGVYLTSAVSPPPRNGVVASVMYAVVTPMLNPFIYSLRNRDIQSVLRR LCSRTVESHDMFHPFSCVGEKGQPH (SEQ ID NO. : 26)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. The NOV13 polypeptide has homology (approximately 73% identity, 83% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No.:Q9UPJl), as is shown in Table 54. Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. OR proteins have seven transmembrane α-helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains. NOVl 3 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 55.
TABLE 54
NOV12: 1 MYFFLSNLC ADIGFTLATVPKMIVDMGSHSRVISYEGCLTQMSFFVLFACIEDMLLTVM 60 ******** ****** ********* 4.**********************44****4**
OLFR: 1 MYFFLSNLSLADIGFTSTTVPKMIVDMQTHSRVISYEGCLTQMSFFVLFACMDDMLLSVM 60
NOV12: 61 AYDQFVAICHPLHYPVIMNPHLCVFLVLVSFFLSLLDSQLHS IVLQFTFFKNVEISNFF 120 ***+********** 4-**** ** **4-*4_***+********+ *+** * **+*+*****
OLFR: 61 AYDRFVAICHPLHYRIIMNPRLCGFLILLSFFISLLDSQLHNLIMLQLTCFKDVDISNFF 120 NOV12: 121 CDPSQLLNLACSDGIINSIFIYLDSILFSFLPISGILLSYYKIVPSILRISSSDGKYKAF 180
******* + * *** ** . ** +* ******* ****** ***+ +******** OLFR : 121 CDPSQLLHLRCSDTFINEMVIYFMGAIFGCLPISGILFSYYKIVSPILRVPTSDGKYKAF 180
NOV12 : 181 SICGSHLAWCLFYGTGIGVYLTSAVSPPPRNGWASVMYAWTPMLNPFIYSLRNRDIQ 240 * ** ** ** ** * ******+ **4_* ** * * * 4.****** ***** ***** ** *** + * **
OLFR : 181 STCGSHIAWCLFYGTGLVGYLSSAVLPSPRKSMVASVMYTWTPMLNPFIYSLRNKDIQ 240
NOV12: 241 SVLRRLCSRTVESHDMFHPFSCVG 263 (SEQ ID NO.: 24) * * * * * 44.** 4 *** 4.* OLFR : 241 SALCRLHGRIIKSHHL-HPFCYMG 263 ( SEQ ID NO . : 92 )
Where * indicates identity and + indicates similarity.
TABLE 55
NOV13: 1 MYFFLSNLC ADIGFTLATVPKMIVDMGSHSRVISYΞGCLTQMSFFVLFACIEDMLLTVM 60 GPCR: 19 TNYLIVSLAVADLLVATLVMPWWYLEWGE KFSRIHCDIFVTLDVMMCTASILNLCAI 78
NOV13 :61 AYDQFVAICHPLHYPVIMNPHLCVFLVLVSFFLSLLDSQLHS IVLQFTF-FKNVEISNF 119 GPCR: 79 SIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVWVLSF TISCPMLFGLNNTDQNECI 133
NOV13 :120 FCDPSQLLNLACSDGIINSIFIYLDSILFSFLPISGILLSYYKIVPSILRISSS 173 (SEQ ID NO. : 93)
GPCR: 134 IANPAFW YSSIVSFYVPFIVTLLVYIKIYIVLRRRRKR 172 (SEQ ID NO. : 94)
The OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl 3 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
Table 56 shows a multiple sequence alignment of NOVl -13 polypeptides with the known human olfactory receptor 10J1 (GenBank Accession No.: P30954), indicating the homology between the present invention and known members of a protein family.
TABLE 56. NOV4 MRGFNKT--TWTQFILVGFSSLGELQ--LLLFVIFLLLYLTILVANVTIMA
NOV3 MRGFNKT- -TWTQFILVGFSSLGELQ- -LLLFVIFLLLYLTILVANVTIMA
OR_10Jl MLLCFRFGNQSMKRENFTLITDFVFQGFSSFHEQQ- -ITLFGVFLALYILTLAGNIIIVT
NOVl0 MNPANHSQVAGFVLLGLSQVWELR- -FVFFTVFSAVYFMTWGNLLIW
NOV12 MGDNITSIT-EFLLLGFPVGPRIQ- -MLLFGLFSLFYVFTLLGNGTILG NOVll MGDNITSIT-EFLLLGFPVGPRIQ- -MLLFGLFSLFYVFTLLGNGTILG
NOV2 MGDNITSIR-EFLLLGFPVGPRIQ--MLLFGLFSLFYVFTLLGNGTILG NOV9 MGDVNQSVASDFILVGLFSHSGSR--QLLFSLVAVMFVIGLLGNTVLLF
NOV8 MGDVNQSVASDFILVGLFSHSGSR--QLLFSLVAVMFVIGLLGNTVLLF
NOVl_ MEGKNQTNISEFLLLGFSS QQQQ--VLLFALFLCLYLTGLFGNLLILL
NOV6 MYMVTVLRNLLSIL NOV5
NOV13
NOV7 TEPRNLTGVSEFLLLGLSEDPELQPVLALLSLSLSMYLVTVLRNLLSIP
NOV4 VIRFSWTLHTPMYGFLFILSFSESCYTFVIIPQLLVHLLSDTKTISLMACATQLFFFLGF
NOV3 VIRFS TLHTPMYGFLFILSFSESCYTFVIIPQLLVHLLSDTKTISFMACATQLFFFLGF OR_10J1 IIRIDLHLHTPMYFFLSMLSTSETVYTLVILPRMLSSLVGMSQPMSLAGCATQMFFFVTF
NOVIO IVTSDPHLHTTMYFLLGNLSFLDFCYSSITAPRMLVDLLSGNPTISFGGCLTQLFFFHFI
NOV12 LISLDSRLHAPMYFFLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTF
NOVll LISLDSRLHAPMYFFLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTF
NOV2 LISLDSRLHAPMYFFLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTF NOV9 LIRVDSRLHTPMYFLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLM
NOV8 LIRVDSRLHTPMYFLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLM
NOVl AIGSDHCLHTPMYFFLANLSLVDLCLPSATVPKMLLNIQTQTQTISYPGCLAQMYFCMMF
NOV6 AVSSDSPLHTPMCFFLSKLCSADIGFTLAMVPKMIVNMQSHSRVISYEGCLTRMSFFVLF
NOV5 PMCFFLSKLCSADIGFTLAMVPKMIVNMQSHSRVISYEGCLTRMSFFVLF NOV13 MYFFLSNLC ADIGFTLATVPKMIVDMGSHSRVISYEGCLTQMSFFVLF
NOV7 AVSSDSHLHTPTYFFLSILC ADIGFTSATVPKMIVDMQ YSRVISHAGCLTQMSFLVLF
.* * . *.. . * . . .
NOV4 ACTNCLLIAVMGYDRYVAICHPLRYTLIINKRLGLELISLSGATGFFIALVATNLICDMR NOV3 ACTNCLLIAVMGYDRYVAICHPLRYTLIINKRLGLELISLSGATGFFIALVATNLICDMR
OR_10J1 GITNCFLLTAMGYDRYVAICNPLRYMVIMNKRLRIQLVLGACSIGLIVAITQVTSVFRLP
NOVIO GGIKIFLLTVMAYDRYIAISQPLHYTLIMNQTVCALLMAASWVGGFIHSIVQIALTIQLP
NOV12 AVTECLLLWMSYDLYVAICHPLRYLAIMTWRVCITLAVTS TTGVLLSLIHLVLLLPLP
NOVll AVTECLLLWMSYDLYVAICHPLRYLAIMTWRVCITLAVTSWTTGVLLSLIHLVLLLPLP NOV2 AVTECLLLWMSYDLYVAICHPLRYLAIMT RVCITLAVTSWTTGVLLSLIHLVLLLPLP
NOV9 GVAEGVLLVLMSYDRYVAVCQPLQYPVLMRRQVCLLMMGSS WGVLNASIQTSITLHFP
NOV8 GVAEGVLLVLMSYDRYVAVCQPLQYPVLMRRQVCLLMMGSS WGVLNASIQTSITLHFP
NOVl ANMDNFLLTVMAYDRYVAICHPLHYSTIMALRLCASLVAAP VIAILNPLLHTLMMAHLH
NOV6 ACMEDMLLTVMAYDCFVAICRPLHYPVIVNPHLCVFFVLVSFFLSPLDSQLHSWIVLLFT NOV5 ACMEDMLLTVMAYDCFVAICRPLHYPVIVNPHLCVFFVLVSFFLSPLDSQLHSWIVLLFT
NOV13 ACIEDMLLTVMAYDQFVAICHPLHYPVIMNPHLCVFLVLVSFFLSLLDSQLHSWIVLQFT
NOV7 ACIEGMLLTVMAYDCFVGIYRPLHYPVIVNPHLCVFFVLVSFFLSLLDSQLHS IVLQFT
N0V4 FCGPNRVNHYFCDMAPVIKLACTDTHVKELALFSLSILVIMVPFLLILISYGFIVNTILK
NOV3 FCGPNRVNHYFCDMAPVIKLACTDTHVKELALFSLSILVIMVPFLLILISYGFIVNTILK
OR_10J1 FCAR-KVPHFFCDIRPVMKLSCIDTTVNEILTLIISVLVLWPMGLVFISYVLIISTILK
NOVl0 FCGPDKLDNFYCDVPQLIKLACTDTFVLΞLLMVSNNGLVTLMCFLVLLGSYTALL-VMLR
NOV12 FCRPQKIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQ NOVll FCRPQKIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQ
NOV2 FCRPQKIYHFFCEILAVLKLACADTHINΞNMVLAGAISGLVGPLSTIWSYMCILCAILQ
NOV9 YCASRIVDHFFCEVPALLKLSCADTCAYEMALSTSGVLILMLPLSLIATSYGHVLQAVLS
NOV8 YCASRIVDHFFCEVPALLKLSCADTCAYEMALSTSGVLILMLPLSLIATSYGHVLQAVLS
NOVl FCSDNVIHHFFCDINSLLPLSCSDTSLNQLSVLATVGLIFWPSVCILVSYILIVSAVMK NOV6 IIKNVEITNFVCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILR
NOV5 IIKNVEITNFVCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILR
NOV13 FFKNVEISNFFCDPSQLLNLACSDGIINSIFIYLDSILFSFLPISGILLSYYKIVPSILR
NOV7 IIKNVEISNFVCDPSQLLKLASYDSVINSIFIYFDSTMFGFLPISGILSSYYKIVPSILR
NOV4 IPSAEG-KKAFVTCASHLTWFVHYDCASIIYLRPKSKSASDKDQLVAVTYAWTPLLNP NOV3 IPSAEG-KKAFVTCASHLTWFVHYGCASIIYLRPKSKSASDKDQLVAVTYTWTPLLNP OR_10J1 IASVEGRKKAFATCASHLTWIVHYSCASIAYLKPKSENTRΞHDQLISVTYTVITPLLNP NOVIO SHSREGRSKALSTCASHIAWTLIFVPCIYVYTRPFR- -TFPMDKAVSVLYTIVTPMLNP
NOVl2 IQSREVQRKAFCTCFSHLCVIGLFYGTAIIMYVGPRYGNPKΞQKKYLLLFHSLFNPMLNP
NOVl1 IQSREVQRKAFCTCFSHLCVIGLFYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNP
N0V2 IQSREVQRKAFRTCFSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNP
N0V9 RSΞEARHKAVTTCSSHITWGLFYGAAVFMYMVPCAYHSPQQDNVVSLFYSLVTPTLNP
N0V8 MRSEEARHKAVTTCSSHITVVGLFYGAAVFMYMVPCAYHSPQQDNVVSLFYSLVTPTLNP
NOVl VPSAQGKLKAFSTCGSHLALVILFYGAITGVYMSPLSNHSTEKDSAASVIFMWAPVLNP
NOV6 MSSSDGKYKGFSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNG-VASVMYAWTPMLNL
NOV5 MSSSDGKYKGFSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNGWASVMYAWTPMLNL
N0V13 ISSSDGKYKAFSICGSHLAWCLFYGTGIGVYLTSAVSPPPRNGWASVMYAWTPMLNP
NOV7 MSSSDGKYKTFSTYGSHLAFVCSFYGTGIDMYLASAMSPTPRNGVWSVMXAWTPMLNL
NOV4 LVYSLRNKEVKTALKR VLGMPVATKMS (SEQ ID NO. : : 8) NOV3 LVYSLRNKEVKTALKR VLGMPVATKMS (SEQ ID NO. : 6)
R_10Jl WYTLRNKEVKDALCR AVGG KFS (SEQ ID NO. : : 95)
NOVl0 AIYTLRNKEVIMAMKKL RRKKDPIGPLEHRPLH (SEQ ID NO. : : 20)
NOV12 LICSLRNSEVKNTLKR VLG- -VERAL (SEQ ID NO. : : 24)
NOVll LICSLRNSEVKNTLKR VLG- -VERAL (SEQ ID NO. : : 22) NOV2 LICSLRNSEVKNTLKR VLG- -VERAL (SEQ ID NO. : : 4)
NOV9 LIYSLRNPEVWMALVK VLSRAGLRQMCMTT (SEQ ID NO. : : 18)
NOV8 LIYSLRNPEVWMALVK VLSRAGLRQMC (SEQ ID NO. : ; 16)
NOVl FIYSLRNNELKGTLKKTLSRPGAVAHACNPSTLGGRGGWIMRSGDRDHPG (SEQ ID NO. : : 2)
N0V6 FILSLGKRDIQSVLRRLCSRTVESHDMFHPFSCVGEKGQPH (SEQ ID NO. : : 12) NOV5 FIYSLGKRDIQSVLRRLCSRTVESHDMFHPFSCVG (SEQ ID NO. : : 10)
NOV13 FIYSLRNRDIQSVLRRLCSRTVESHDMFHPFSCVGΞKGQPH (SEQ ID NO. : : 26)
NOV7 FIYSLRNRDIQSALRRLRSR (SEQ ID NO. : : 14)
Where "*" indicates a single, fully conserved residue, ":" indicates conservation of strong groups, and "." indicates conservation of weak groups, and OR_10J1 is the known human olfactory receptor 10J1 (GenBank Accession No.: P30954).
The nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in disorders ofthe neuro-olfactory system, such as those induced by trauma, surgery and/or neoplastic disorders. For example, a cDNA encoding the olfactory receptor protein may be useful in gene therapy for treating such disorders, and the olfactory receptor protein may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from disorders ofthe neuro-olfactory system. The novel nucleic acids encoding olfactory receptor protein, and the olfactory receptor protein ofthe invention, or fragments thereof, may further be useful in the treatment of adenocarcinoma; lymphoma; prostate cancer; uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, treatment of Albright hereditary ostoeodystrophy, development of powerful assay system for functional analysis of various human disorders which will help in understanding of pathology of the disease, and development of new drug targets for various disorders. They may also be used in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
NOVX Nucleic Acids
The nucleic acids ofthe invention include those that encode a NOVX polypeptide or protein. As used herein, the terms polypeptide and protein are interchangeable.
In some embodiments, a NOVX nucleic acid encodes a mature NOVX polypeptide. As used herein, a "mature" form of a polypeptide or protein described herein relates to the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the cell in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage ofthe N-terminal methionine residue encoded by the initiation codon of an open reading frame, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal ofthe N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them. Among the NOVX nucleic acids is the nucleic acid whose sequence is provided in SEQ
ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a fragment thereof. Additionally, the invention includes mutant or variant nucleic acids of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a fragment thereof, any of whose bases may be changed from the corresponding bases shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, while still encoding a protein that maintains at least one of its NOVX-like activities and physiological functions (i.e., modulating angiogenesis, neuronal development). The invention further includes the complement ofthe nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, including fragments, derivatives, analogs and homologs thereof. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
One aspect ofthe invention pertains to isolated nucleic acid molecules that encode NOVX proteins or biologically active portions thereof. Also included are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of NOVX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs ofthe DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
"Probes" refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source ofthe nucleic acid. Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule ofthe present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a complement of any of this nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion ofthe nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, as a hybridization probe, NOVX nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook etαl., eds., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et αh, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
A nucleic acid ofthe invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at lease 6 contiguous nucleotides of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a complement thereof Oligonucleotides may be chemically synthesized and may be used as probes. In another embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a portion of this nucleotide sequence. A nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Von der Waals, hydrophobic interactions, etc. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Moreover, the nucleic acid molecule ofthe invention can comprise only a portion ofthe nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, e.g., a fragment that can be used as a probe or primer, or a fragment encoding a biologically active portion of NOVX. Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs ofthe nucleic acids or proteins ofthe invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below. An exemplary program is the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, WI) using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482-489, which is incoφorated herein by reference in its entirety).
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of a NOVX polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the present invention, homologous nucleotide sequences include nucleotide sequences encoding for a NOVX polypeptide of species other than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations ofthe nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, as well as a polypeptide having NOVX activity. Biological activities ofthe NOVX proteins are described below. A homologous amino acid sequence does not encode the amino acid sequence of a human NOVX polypeptide. The nucleotide sequence determined from the cloning ofthe human NOVX gene allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g., from other tissues, as well as NOVX homologues from other mammals. The probe/primer typically comprises a substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 or more consecutive sense strand nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; or an anti-sense strand nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; or of a naturally occurring mutant of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.
Probes based on the human NOVX nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a NOVX protein, such as by measuring a level of a NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
A "polypeptide having a biologically active portion of NOVX" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide ofthe present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically active portion of NOVX" can be prepared by isolating a portion of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 that encodes a polypeptide having a NOVX biological activity (biological activities ofthe NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of NOVX. For example, a nucleic acid fragment encoding a biologically active portion of NOVX can optionally include an ATP-binding domain. In another embodiment, a nucleic acid fragment encoding a biologically active portion of NOVX includes one or more regions.
NOVX Variants
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 due to the degeneracy ofthe genetic code. These nucleic acids thus encode the same NOVX protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 e.g., the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26. In another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26. In addition to the human NOVX nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23 or 25, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of NOVX may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a NOVX protein, preferably a mammalian NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe NOVX gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in NOVX that are the result of natural allelic variation and that do not alter the functional activity of NOVX are intended to be within the scope ofthe invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from the human sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 are intended to be within the scope ofthe invention. Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. For example, a soluble human NOVX cDNA can be isolated based on its homology to human membrane-bound NOVX. Likewise, a membrane-bound human NOVX cDNA can be isolated based on its homology to soluble human NOVX.
Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length. In another embodiment, an isolated nucleic acid molecule ofthe invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
Homologs (/. e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion ofthe particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% ofthe probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C. This hybridization is followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardf s solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C. Other conditions of moderate stringency that may be used are well known in the art. See, e.g., Ausubel et al. feds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g. , as employed for cross-species hybridizations). See, e.g., Ausubel et al. feds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981, Proc Natl Acad Sci USA 78: 6789-6792.
Conservative mutations
In addition to naturally-occurring allelic variants ofthe NOVX sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, thereby leading to changes in the amino acid sequence ofthe encoded NOVX protein, without altering the functional ability ofthe NOVX protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of NOVX without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the NONX proteins ofthe present invention, are predicted to be particularly unamenable to alteration.
Another aspect ofthe invention pertains to nucleic acid molecules encoding ΝOVX proteins that contain changes in amino acid residues that are not essential for activity. Such ΝOVX proteins differ in amino acid sequence from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises. a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8. Preferably, the protein encoded by the nucleic acid is at least about 80% homologous to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, more preferably at least about 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in NOVX is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined.
In one embodiment, a mutant NOVX protein can be assayed for (1) the ability to form proteimprotein interactions with other NOVX proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant NOVX protein and a NOVX receptor; (3) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the ability to bind NOVX protein; or (5) the ability to specifically bind an anti-NOVX protein antibody.
Antisense NOVX Nucleic Acids
Another aspect ofthe invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a NOVX protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding NOVX. The term "coding region" refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of human NOVX corresponds to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding NOVX. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions). Given the coding strand sequences encoding NOVX disclosed herein (e.g., SEQ ID NO:
1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25), antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of NOVX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymemyluracil, 5-methoxyuracil, 2-methylthio-N6-isoρentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NOVX protein to thereby inhibit expression ofthe protein, e.g. , by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) FEBS Lett 215: 327-330).
Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. NOVX Ribozymes and PNA moieties
In still another embodiment, an antisense nucleic acid ofthe invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region. Thus, ribozymes (e. g. , hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for a NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of a NOVX DNA disclosed herein (i.e., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25). For example, a derivative of a Tetrahymena L- 19 IVS RNA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in a NOVX-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, NOVX mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al, (1993) Science 261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe NOVX (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe NOVX gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14: 807-15.
In various embodiments, the nucleic acids of ΝOVX can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein, the terms "peptide nucleic acids" or "PΝAs" refer to nucleic acid mimics, e.g., DΝA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PΝAs has been shown to allow for specific hybridization to DΝA and RΝA under conditions of low ionic strength. The synthesis of PΝA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675. PNAs of NOVX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g. , inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion ' would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl) amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) above). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre etal, 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al, 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
NOVX Polypeptides
A NOVX polypeptide ofthe invention includes the NOVX-like protein whose sequence is provided in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 while still encoding a protein that maintains its NOVX-like activities and physiological functions, or a functional fragment thereof. In some embodiments, up to 20% or more ofthe residues may be so changed in the mutant or variant protein. In some embodiments, the NOVX polypeptide according to the invention is a mature polypeptide.
In general, a NOVX -like variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues ofthe parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect ofthe invention pertains to isolated NOVX proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies. In one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NOVX proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NOVX protein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of NOVX protein having less than about 30% (by dry weight) of non-NOVX protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX protein, still more preferably less than about 10% of non-NOVX protein, and most preferably less than about 5% non-NOVX protein. When the NOVX protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals" includes preparations of NOVX protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis ofthe protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of NOVX protein having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals. Biologically active portions of a NOVX protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence ofthe NOVX protein, e.g., the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 that include fewer amino acids than the full length NOVX proteins, and exhibit at least one activity of a NOVX protein. Typically, biologically active portions comprise a domain or motif with at least one activity ofthe NOVX protein. A biologically active portion of a NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
A biologically active portion of a NOVX protein ofthe present invention may contain at least one ofthe above-identified domains conserved between the NOVX proteins, e.g. TSR modules. Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native NOVX protein. In an embodiment, the NOVX protein has an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 and retains the functional activity ofthe protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 and retains the functional activity ofthe NOVX proteins of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
Determining homology between two or more sequence
To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in either ofthe sequences being compared for optimal alignment between the sequences). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch 1970 J Mol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region ofthe analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part ofthe DNA sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.
The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region. The term "percentage of positive residues" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical and conservative amino acid substitutions, as defined above, occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of positive residues.
Chimeric and fusion proteins
The invention also provides NOVX chimeric or fusion proteins. As used herein, a NOVX "chimeric protein" or "fusion protein" comprises a NOVX polypeptide operatively linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to NOVX, whereas a "non-NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within a NOVX fusion protein the NOVX polypeptide can correspond to all or a portion of a NOVX protein. In one embodiment, a NOVX fusion protein comprises at least one biologically active portion of a NOVX protein. In another embodiment, a NOVX fusion protein comprises at least two biologically active portions of a NOVX protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame to each other. The non-NOVX polypeptide can be fused to the N-terminus or C-terminus ofthe NOVX polypeptide.
For example, in one embodiment a NOVX fusion protein comprises a NOVX polypeptide operably linked to the extracellular domain of a second protein. Such fusion proteins can be further utilized in screening assays for compounds that modulate NOVX activity (such assays are described in detail below).
In another embodiment, the fusion protein is a GST-NO VX fusion protein in which the NOVX sequences are fused to the C-terminus ofthe GST (i.e., glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX.
In another embodiment, the fusion protein is a NOVX-immunoglobulin fusion protein in which the NOVX sequences comprising one or more domains are fused to sequences derived from a member ofthe immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a NOVX ligand and a NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo. In one nonlimiting example, a contemplated NOVX ligand ofthe invention is the NOVX receptor. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of a NOVX cognate ligand. Inhibition ofthe NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival, as well as acute and chronic inflammatory disorders and hyperplastic wound healing, e.g. hypertrophic scars and keloids. Moreover, the NOVX-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with a NOVX ligand. A NOVX chimeric or fusion protein ofthe invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
NOVX agonists and antagonists
The present invention also pertains to variants ofthe NOVX proteins that function as either NOVX agonists (mimetics) or as NOVX antagonists. Variants ofthe NOVX protein can be generated by mutagenesis, e.g., discrete point mutation or truncation ofthe NOVX protein. An agonist ofthe NOVX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form ofthe NOVX protein. An antagonist ofthe NOVX protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe NOVX proteins.
Variants ofthe NOVX protein that function as either NOVX agonists (mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, ofthe NOVX protein for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential NOVX sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.
Polypeptide libraries In addition, libraries of fragments ofthe NOVX protein coding sequence can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of a NOVX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes ofthe NOVX protein. Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of NOVX proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation ofthe vector encoding the gene whose product was detected. Recrusive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
NOVX Antibodies
Also included in the invention are antibodies to NOVX proteins, or fragments of NOVX proteins. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab, and F(ab.)2 fragments, and an Fab expression library. In general, an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature ofthe heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGl5 IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
An isolated NOVX-related protein ofthe invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments ofthe antigen for use as immunogens; An antigenic peptide fragment comprises at least 6 amino acid residues ofthe amino acid sequence ofthe full length protein, such as an amino acid sequence shown in SEQ ID NO: 2, 4, 6 ,8 ,10, 12, 14, 16, 18, or 20, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions ofthe protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments ofthe invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX-related protein that is located on the surface ofthe protein, e.g., a hydrophilic region. A hydrophobicity analysis ofthe human NOVX-related protein sequence will indicate which regions of a NOVX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824- 3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein ofthe invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein ofthe invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative ofthe foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and
Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffϊnity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
Monoclonal Antibodies
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) ofthe monoclonal antibody are identical in all the molecules ofthe population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope ofthe antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells. Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63). The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells can be grown iv vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies ofthe invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells ofthe invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody.
Humanized Antibodies
The antibodies directed against the protein antigens ofthe invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen- binding subsequences of antibodies) that are principally comprised ofthe sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature. 321:522-525 (1986); Riechmann et al, Nature, 332:323-327 (1988); Verhoeyen et al, Science. 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues ofthe human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice o the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol.. 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement ofthe modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No.
5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049. Fab Fragments and Single Chain Antibodies
According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein ofthe invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab.)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab.)2 fragment; (iii) an Fab fragment generated by the treatment ofthe antibody molecule with papain and a reducing agent and (iv) Fv fragments.
Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one ofthe binding specificities is for an antigenic protein ofthe invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because ofthe random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, 1991 EMBOJ., 10:3655-3659.
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzvmology. 121:210 (1986).
According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part ofthe CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield ofthe heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One ofthe Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al, J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen ofthe invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc R), such as Fc RI (CD64), Fc RII (CD32) and Fc RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope ofthe present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
Effector Function Engineering
It can be desirable to modify the antibody ofthe invention with respect to effector function, so as to enhance, e.g., the effectiveness ofthe antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved intemalization capability and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti- tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, ]31I, ,31In, 90Y, and 186Re.
Conjugates ofthe antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
NOVX Recombinant Expression Vectors and Host Cells
Another aspect ofthe invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors ofthe invention comprise a nucleic acid ofthe invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
The recombinant expression vectors ofthe invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein. Such fusion vectors typically serve three purposes: (/) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N. J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et ah, 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the NOVX expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl
(Baldari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif).
Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid ofthe invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBOJ. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987. Genes Dev. 1 : 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al, 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, etal, 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally- regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule ofthe invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription ofthe DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression ofthe antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion ofthe regulation of gene expression using antisense genes see, e.g., Weintraub, et al, "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect ofthe invention pertains to host cells into which a recombinant expression vector ofthe invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope ofthe term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as human, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals. For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell ofthe invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells ofthe invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals The host cells ofthe invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells of the animal includes a fransgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A fransgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome ofthe mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell ofthe animal, prior to development ofthe animal. A transgenic animal ofthe invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinj ection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. Sequences including SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 can be introduced as a fransgene into the genome of a non-human animal. Alternatively, a non-human homologue ofthe human NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a fransgene. Intronic sequences and polyadenylation signals can also be included in the fransgene to increase the efficiency of expression ofthe fransgene. A tissue-specific regulatory sequence(s) can be operably-linked to the NOVX fransgene to direct expression of NOVX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinj ection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence ofthe NOVX fransgene in its genome and/or expression of NOVX mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the fransgene. Moreover, transgenic animals carrying a transgene-encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e.g., the DNA of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25), but more preferably, is a non-human homologue of a human NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression ofthe endogenous NOVX protein). In the homologous recombination vector, the altered portion ofthe NOVX gene is flanked at its 5'- and 3'-termini by additional nucleic acid ofthe NOVX gene to allow for homologous recombination to occur between the exogenous NOVX gene carried by the vector and an endogenous NOVX gene in an embryonic stem cell. The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al, 1987. Cell 51 : 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX gene has homologously- recombined with the endogenous NOVX gene are selected. See, e.g., Li, et al, 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells ofthe animal contain the homologously-recombined DNA by germline transmission ofthe fransgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression ofthe fransgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI . For a description ofthe cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251 :1351-1355. If a cre/loxP recombinase system is used to regulate expression ofthe fransgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a fransgene encoding a selected protein and the other containing a fransgene encoding a recombinase.
Clones ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al, 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter G0 phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring bome of this female foster animal will be a clone ofthe animal from which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions
The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as "active compounds") ofthe invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art.
Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamme (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments ofthe antibody ofthe present invention can be conjugated to the liposomes as described in Martin et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst, 81(19): 1484 (1989).
A pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, infradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants. Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifimgal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a
NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition. The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Coφoration and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. The nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl Acad. Sci. USA 91 : 3054-3057). The pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
Antibodies specifically binding a protein ofthe invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. : 1995; Drug Absoφtion Enhancement : Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York. If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain ofthe target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al, 1993 Proc. Natl. Acad. Sci. USA, 90: 7889-7893. The formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth- inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
The formulations to be used for iv vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly- D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid- glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Screening and Detection Methods The isolated nucleic acid molecules ofthe invention can be used to express NOVX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in a NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein. In addition, the anti-NOVX antibodies ofthe invention can be used to detect and isolate NOVX proteins and modulate NOVX activity. For example, NOVX activity includes growth and differentiation, antibody production, and tumor growth.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
Screening Assays
The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity. The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity ofthe membrane-bound form of a NOVX protein or polypeptide or biologically-active portion thereof. The test compounds ofthe invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997 '. Anticancer Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any ofthe assays ofthe invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al, 1993. Proc. Natl Acad. Sci. U.S.A. 90: 6909; Erb, et al, 1994. Proc. Natl. Acad. Sci. U.S.A. 91 : 11422; Zuckermann, et al, 1994. J. Med. Chem. 37: 2678; Cho, et al, 1993. Science 261: 1303; Carrell, et al, 1994. Angew. Chem. Int. Ed. Engl 33: 2059; Carell, et al, 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al, 1994. J. Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al, 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.). In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NONX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to a ΝOVX protein determined. The cell, for example, can be of mammalian origin or a yeast cell. Determining the ability ofthe test compound to bind to the ΝOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the ΝOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 1251, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of ΝOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds ΝOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with a ΝOVX protein, wherein determining the ability ofthe test compound to interact with a ΝOVX protein comprises determining the ability ofthe test compound to preferentially bind to ΝOVX protein or a biologically-active portion thereof as compared to the known compound. In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of ΝOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity of the ΝOVX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability ofthe NOVX protein to bind to or interact with a NOVX target molecule. As used herein, a "target molecule" is a molecule with which a NOVX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the exfracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A NOVX target molecule can be a non-NOVX molecule or a NOVX protein or polypeptide ofthe invention In one embodiment, a NOVX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound NOVX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.
Determining the ability ofthe NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by one ofthe methods described above for determining direct binding. In one embodiment, determining the ability ofthe NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by determining the activity ofthe target molecule. For example, the activity ofthe target molecule can be determined by detecting induction of a cellular second messenger ofthe target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity ofthe target an appropriate substrate, detecting the induction of a reporter gene (comprising a NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay ofthe invention is a cell-free assay comprising contacting a NOVX protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to bind to the NOVX protein or biologically-active portion thereof. Binding ofthe test compound to the NOVX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with a NOVX protein, wherein determining the ability ofthe test compound to interact with a NOVX protein comprises determining the ability ofthe test compound to preferentially bind to NOVX or biologically- active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity ofthe NOVX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability ofthe NOVX protein to bind to a NOVX target molecule by one ofthe methods described above for determining direct binding. In an alternative embodiment, determining the ability ofthe test compound to modulate the activity of NO VX protein can be accomplished by determining the ability ofthe NOVX protein further modulate a NOVX target molecule. For example, the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined as described above.
In yet another embodiment, the cell-free assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with a NOVX protein, wherein determining the ability ofthe test compound to interact with a NOVX protein comprises determining the ability ofthe NOVX protein to preferentially bind to or modulate the activity of a NOVX target molecule. The cell-free assays ofthe invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubihzing agent such that the membrane-bound form of NOVX protein is maintained in solution. Examples of such solubihzing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, N-dodecyl-- N,N-dimethyl-3-ammonio-l -propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol- 1 -propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy- 1 -propane sulfonate (CHAPSO). In more than one embodiment ofthe above assay methods ofthe invention, it may be desirable to immobilize either NOVX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both ofthe proteins, as well as to accommodate automation ofthe assay. Binding of a test compound to NOVX protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both ofthe proteins to be bound to a matrix. For example, GST-NO VX fusion proteins or GST-target fusion proteins can be • adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays ofthe invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding ofthe NOVX protein to its target molecule, can be derivatized to the wells ofthe plate, and unbound target or NOVX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX mRNA or protein in the cell is determined. The level of expression of NOVX mRNA or protein in the presence ofthe candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence ofthe candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression. Alternatively, when expression of NOVX mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression. The level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.
In yet another aspect ofthe invention, the NOVX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos, et al., 1993. Cell 72: 223-232; Madura, et al, 1993. J. Biol. Chem. 268: 12046-12054; Bartel, et al, 1993. Biotechniques 14: 920-924; Iwabuchi, et al, 1993. Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins" or "NOVX-bp") and modulate NOVX activity. Such NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements ofthe NOVX pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain ofthe known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a NOVX-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX. The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
Detection Assays
Portions or fragments ofthe cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) identify an individual from a minute biological sample (tissue typing); and (ii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.
Tissue Typing
The NOVX sequences ofthe invention can be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences ofthe invention are useful as additional DNA markers for RFLP ("restriction fragment length polymoφhisms," described in U.S. Patent No. 5,272,057).
Furthermore, the sequences ofthe invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR primers from the 5'- and 3 '-termini ofthe sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences ofthe invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences ofthe invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much ofthe allelic variation is due to single nucleotide polymoφhisms (SNPs), which include restriction fragment length polymoφhisms (RFLPs).
Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymoφhisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 are used, amore appropriate number of primers for positive individual identification would be 500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic
(predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX expression or activity.
Disorders associated with aberrant NOVX expression of activity include, for example, disorders of olfactory loss, e.g. trauma, HIV illness, neoplastic growth, and neurological disorders, e.g. Parkinson's disease and Alzheimer's disease.
The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in a NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive puφose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity. Another aspect ofthe invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype ofthe individual (e.g., the genotype ofthe individual examined to determine the ability ofthe individual to respond to a particular agent.) Yet another aspect ofthe invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials. These and other agents are described in further detail in the following sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein. One agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with a detectable label. Antibodies directed against a protein of the invention may be used in methods known within the art relating to the localization and/or quantitation ofthe protein (e.g., for use in measuring levels ofthe protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies against the proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antigen binding domain, are utilized as pharmacologically-active compounds.
An antibody specific for a protein ofthe invention can be used to isolate the protein by standard techniques, such as immunoaffinity chromatography or immunoprecipitation. Such an antibody can facilitate the purification ofthe natural protein antigen from cells and of recombinantly produced antigen expressed in host cells. Moreover, such an antibody can be used to detect the antigenic protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe antigenic protein. Antibodies directed against the protein can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 1251, 1 iI, 35S or 3H.
Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method ofthe invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of NOVX genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. In one embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect NOVX protein or nucleic acid.
Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. Such disorders include for example, disorders of olfactory loss, e.g. trauma, HIV illness, neoplastic growth, and neurological disorders, e.g. Parkinson's disease and Alzheimer's disease.
Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant NOVX expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant NOVX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g., wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant NOVX expression or activity).
The methods ofthe invention can also be used to detect genetic lesions in a NOVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a NOVX-protein, or the misexpression ofthe NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from a NOVX gene; (ii) an addition of one or more nucleotides to a NOVX gene; (iii) a substitution of one or more nucleotides of a NOVX gene, (iv) a chromosomal rearrangement of a NOVX gene; (v) an alteration in the level of a messenger RNA transcript of a NOVX gene, (vi) aberrant modification of a NOVX gene, such as ofthe methylation pattern of the genomic DNA, (vii) the presence of a non- wild-type splicing pattern of a messenger RNA transcript of a NOVX gene, (viii) a non-wild-type level of a NOVX protein, (ix) allelic loss of a NOVX gene, and (x) inappropriate post-translational modification of a NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a NOVX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells. In certain embodiments, detection ofthe lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et αl, 1988. Science 241 : 1077-1080; and Nakazawa, et αl, 1994. Proc. Nαtl. Acαd. Sci. USA 91 : 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et αl, 1995. Nucl. Acids Res. 23: 675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to a NONX gene under conditions such that hybridization and amplification ofthe ΝONX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size ofthe amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al, 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), franscriptional amplification system (see, Kwoh, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qβ Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in a ΝOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DΝA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DΝA indicates mutations in the sample DΝA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al, 1996. Human Mutation 7: 244-255; Kozal, et al, 1996. Nat. Med. 2: 753-759. For example, genetic mutations in NOVX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al, supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and confrol to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the NONX gene and detect mutations by comparing the sequence of the sample ΝOVX with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Νaeve, et al, 1995. Biotechniques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen, et al, 1996. Adv. Chromatography 36: 127-162; and Griffin, et al, 1993. Appl. Biochem. Biotechnol 38: 147-159).
Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g., Myers, et al, 1985. Science 230: 1242. In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions ofthe duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with Sj nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion ofthe mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, etal, 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al, 1992. Methods Enzymol 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection. In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et al, 1994. Carcinogenesis 15: 1657-1662. According to an exemplary embodiment, a probe based on a NOVX sequence, e.g., a wild-type NOVX sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymoφhism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al, 1989. Proc. Natl. Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl 9: 73-79. Single-stranded DNA fragments of sample and confrol NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al, 1991. Trends Genet. 7: 5.
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al, 1985. Nature 313: 495. When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al, 1986. Nature 324: 163; Saiki, et al, 1989. Proc. Nat Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA. Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center ofthe molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al, 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11 : 238). In addition it may be desirable to introduce a novel restriction site in the region ofthe mutation to create cleavage-based detection. See, e.g., Gasparini, et al, 1992. Mol Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3'-terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a NONX gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which ΝONX is expressed may be utilized in the prognostic assays described herein. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on ΝONX activity (e.g., ΝONX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g. disorders of olfactory loss, e.g. trauma, HIN illness, neoplastic growth, and neurological disorders, e.g. Parkinson's disease and Alzheimer's disease). In conjunction with such treatment, the pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drug) ofthe individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concenfration ofthe pharmacologically active drug. Thus, the pharmacogenomics ofthe individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration ofthe individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NONX protein, expression of ΝONX nucleic acid, or mutation content of ΝONX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol, 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymoφhisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nifrofurans) and consumption of fava beans .
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymoφhisms of drug metabolizing enzymes (e.g., Ν-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymoφhisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymoφhic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of C YP2D6 and C YP2C 19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite moφhine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification. Thus, the activity of NONX protein, expression of ΝOVX nucleic acid, or mutation content of ΝOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymoφhic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a ΝOVX modulator, such as a modulator identified by one ofthe exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of ΝOVX (e.g., the ability to modulate aberrant cell proliferation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase ΝOVX gene expression, protein levels, or upregulate ΝOVX activity, can be monitored in clinical trails of subjects exhibiting decreased ΝOVX gene expression, protein levels, or downregulated ΝOVX activity.
Alternatively, the effectiveness of an agent determined by a screening assay to decrease ΝOVX gene expression, protein levels, or downregulate ΝOVX activity, can be monitored in clinical trails of subjects exhibiting increased ΝOVX gene expression, protein levels, or upregulated ΝOVX activity. In such clinical trials, the expression or activity of ΝOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers ofthe immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including ΝOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates ΝOVX activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RΝA prepared and analyzed for the levels of expression of ΝOVX and other genes implicated in the disorder. The levels of gene expression (t'.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one ofthe methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative ofthe physiological response ofthe cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment ofthe individual with the agent.
In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-adminisfration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a NOVX protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the pre-adminisfration sample with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration ofthe agent to the subject accordingly. For example, increased administration ofthe agent may be desirable to increase the expression or activity of NOVX to higher levels than detected, i.e., to increase the effectiveness ofthe agent. Alternatively, decreased administration ofthe agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i.e., to decrease the effectiveness ofthe agent. Methods of Treatment
The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant
NOVX expression or activity. Disorders associated with aberrant NOVX expression include, for example, disorders of olfactory loss, e.g. trauma, HIV illness, neoplastic growth, and neurological disorders, e.g. Parkinson's disease and Alzheimer's disease. These methods of treatment will be discussed more fully, below. Disease and Disorders
Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (fv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and antagonists, including additional peptide mimetic ofthe invention or antibodies specific to a peptide ofthe invention) that alter the interaction between an aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity ofthe expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like). Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX activity. Subjects at risk for a disease that is caused or contributed to by aberrant NOVX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe NOVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of NOVX aberrancy, for example, a NOVX agonist or NOVX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods ofthe invention are further discussed in the following subsections.
Therapeutic Methods
Another aspect ofthe invention pertains to methods of modulating NOVX expression or activity for therapeutic purposes. The modulatory method ofthe invention involves contacting a cell with an agent that modulates one or more ofthe activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NOVX protein, a peptide, a NOVX peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NOVX protein activity. Examples of such stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell. In another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NOVX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering a NOVX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX expression or activity.
Stimulation of NOVX activity is desirable in situations in which NOVX is abnormally downregulated and/or in which increased NOVX activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated ). Another example of such a situation is where the subject has an immunodeficiency disease (e.g., AIDS).
Antibodies ofthe invention, including polyclonal, monoclonal, humanized and fully human antibodies, may used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Such an effect may be one of two kinds, depending on the specific nature ofthe interaction between the given antibody molecule and the target antigen in question. In the first instance, administration ofthe antibody may abrogate or inhibit the binding ofthe target with an endogenous ligand to which it naturally binds. In this case, the antibody binds to the target and masks a binding site ofthe naturally occurring ligand, wherein the ligand serves as an effector molecule. Thus the receptor mediates a signal transduction pathway for which ligand is responsible.
Alternatively, the effect may be one in which the antibody elicits a physiological result by virtue of binding to an effector binding site on the target molecule. In this case the target, a receptor having an endogenous ligand which may be absent or defective in the disease or pathology, binds the antibody as a surrogate effector ligand, initiating a receptor-based signal transduction event by the receptor.
A therapeutically effective amount of an antibody ofthe invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning ofthe target, and in other cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity ofthe antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody, or antibody fragment ofthe invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.
Determination ofthe Biological Effect ofthe Therapeutic In various embodiments ofthe invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment ofthe affected tissue.
In various specific embodiments, in vitro assays may be performed with representative cells ofthe type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any ofthe animal model system known in the art may be used prior to administration to human subjects.
The invention will be further described in the following examples, which do not limit the scope ofthe invention described in the claims.
EXAMPLES
Example 1.: Method of Identifying the Nucleic Acids Encoding the G-Protein Coupled Receptors.
Novel nucleic acid sequences were identified by TblastN using CuraGen Corporation's sequence file run against the Genomic Daily Files made available by GenBank. The nucleic acids were further predicted by the program GenScan™, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
OTHER EMBODIMENTS
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope ofthe invention, which is defined by the scope ofthe appended claims. Other aspects, advantages, and modifications are within the scope ofthe following claims.

Claims

What is claimed is:
1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: a) a mature form ofthe amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26; b) a variant of a mature form ofthe amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15% ofthe amino acid residues in the sequence ofthe mature form are so changed; c) the amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26; d) a variant ofthe amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% ofthe amino acid residues in the sequence are so changed; and e) a fragment of any of a) through d).
2. The polypeptide of claim 1 that is a naturally occurring allelic variant ofthe sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
3. The polypeptide of claim 2, wherein the variant is the translation of a single nucleotide polymoφhism.
4. The polypeptide of claim 1 that is a variant polypeptide described therein, wherein any amino acid specified in the chosen sequence is changed to provide a conservative substitution.
5. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: a) a mature form ofthe amino acid sequence given SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26; b) a variant of a mature form ofthe amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 wherein any amino acid in the mature form ofthe chosen sequence is changed to a different amino acid, provided that no more than 15% ofthe amino acid residues in the sequence of the mature form are so changed; c) the amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26; d) a variant ofthe amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% ofthe amino acid residues in the sequence are so changed; e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 or any variant of said polypeptide wherein any amino acid ofthe chosen sequence is changed to a different amino acid, provided that no more than 10% ofthe amino acid residues in the sequence are so changed; and f) the complement of any of said nucleic acid molecules.
6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant.
7. The nucleic acid molecule of claim 5 that encodes a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant.
8. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises a single nucleotide polymoφhism encoding said variant polypeptide.
9. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of a) the nucleotide sequence selected from the group consisting of SEQ JJD NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; b) a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 is changed from that selected from the group consisting ofthe chosen sequence to a different nucleotide provided that no more than 15% ofthe nucleotides are so changed; c) a nucleic acid fragment ofthe sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; and d) a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 is changed from that selected from the group consisting ofthe chosen sequence to a different nucleotide provided that no more than 15% ofthe nucleotides are so changed.
10. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a complement of said nucleotide sequence.
11. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises a nucleotide sequence in which any nucleotide specified in the coding sequence ofthe chosen nucleotide sequence is changed from that selected from the group consisting ofthe chosen sequence to a different nucleotide provided that no more than 15% ofthe nucleotides in the chosen coding sequence are so changed, an isolated second polynucleotide that is a complement ofthe first polynucleotide, or a fragment of any of them.
12. A vector comprising the nucleic acid molecule of claim 11.
13. The vector of claim 12, further comprising a promoter operably linked to said nucleic acid molecule.
14. A cell comprising the vector of claim 12.
15. An antibody that binds immunospecifically to the polypeptide of claim 1.
16. The antibody of claim 15, wherein said antibody is a monoclonal antibody.
17. The antibody of claim 15, wherein the antibody is a humanized antibody.
18. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising:
(a) providing said sample;
(b) introducing said sample to an antibody that binds immunospecifically to the polypeptide; and
(c) deterrriining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.
19. A method for determining the presence or amount ofthe nucleic acid molecule of claim 5 in a sample, the method comprising:
(a) providing said sample;
(b) infroducmg said sample to a probe that binds to said nucleic acid molecule; and
(c) determining the presence or amount of said probe bound to said nucleic acid molecule, thereby determining the presence or amount ofthe nucleic acid molecule in said sample.
20. A method of identifying an agent that binds to the polypeptide of claim 1, the method comprising: (a) introducing said polypeptide to said agent; and
(b) determining whether said agent binds to said polypeptide.
21. A method for identifying a potential therapeutic agent for use in treatment of a pathology, wherein the pathology is related to aberrant expression or aberrant physiological interactions ofthe polypeptide of claim 1, the method comprising:
(a) providing a cell expressing the polypeptide of claim 1 and having a property or function ascribable to the polypeptide;
(b) contacting the cell with a composition comprising a candidate substance; and
(c) determining whether the substance alters the property or function ascribable to the polypeptide; whereby, if an alteration observed in the presence ofthe substance is not observed when the. cell is contacted with a composition devoid ofthe substance, the substance is identified as a potential therapeutic agent.
22. A method for modulating the activity ofthe polypeptide of claim 1 , the method comprising introducing a cell sample expressing the polypeptide of said claim with a compound that binds to said polypeptide in an amount sufficient to modulate the activity ofthe polypeptide.
23. A method of treating or preventing a pathology associated with the polypeptide of claim 1, said method comprising administering the polypeptide of claim 1 to a subject in which such treatment or prevention is desired in an amount sufficient to freat or prevent said pathology in said subject.
24. The method of claim 23, wherein said subject is a human.
25. A method of treating or preventing a pathology associated with the polypeptide of claim 1, said method comprising administering to a subject in which such treatment or prevention is desired a NOVX nucleic acid in an amount sufficient to treat or prevent said pathology in said subject.
26. The method of claim 25, wherein said subject is a human.
27. A method of treating or preventing a pathology associated with the polypeptide of claim 1, said method comprising administering to a subject in which such treatment or prevention is desired a NOVX antibody in an amount sufficient to treat or prevent said pathology in said subject.
28. The method of claim 27, wherein the subject is a human.
29. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.
30. A pharmaceutical composition comprising the nucleic acid molecule of claim 5 and a pharmaceutically acceptable carrier.
31. A pharmaceutical composition comprising the antibody of claim 15 and a pharmaceutically acceptable carrier.
32. A kit comprising in one or more containers, the pharmaceutical composition of claim 29.
33. A kit comprising in one or more containers, the pharmaceutical composition of claim 30.
34. A kit comprising in one or more containers, the pharmaceutical composition of claim 31.
35. The use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease selected from a pathology associated with the polypeptide of claim 1 , wherein said therapeutic is the polypeptide of claim 1.
36. The use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease selected from a pathology associated with the polypeptide of claim 1, wherein said therapeutic is a NOVX nucleic acid.
37. The use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease selected from a pathology associated with the polypeptide of claim 1, wherein said therapeutic is a NOVX antibody.
38. A method for screening for a modulator of activity or of latency or predisposition to a pathology associated with the polypeptide of claim 1, said method comprising: a) administering a test compound to a test animal at increased risk for a pathology associated with the polypeptide of claim 1, wherein said test animal recombinantly expresses the polypeptide of claim 1; b) measuring the activity of said polypeptide in said test animal after administering the compound of step (a); and c) comparing the activity of said protein in said test animal with the activity of said polypeptide in a control animal not administered said polypeptide, wherein a change in the activity of said polypeptide in said test animal relative to said control animal indicates the test compound is a modulator of latency of, or predisposition to, a pathology associated with the polypeptide of claim 1.
39. The method of claim 38 , wherein said test animal is a recombinant test animal that expresses a test protein fransgene or expresses said fransgene under the confrol of a promoter at an increased level relative to a wild-type test animal, and wherein said promoter is not the native gene promoter of said fransgene.
40. A method for determining the presence of or predisposition to a disease associated with altered levels ofthe polypeptide of claim 1 in a first mammalian subject, the method comprising: a) measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and b) comparing the amount of said polypeptide in the sample of step (a) to the amount ofthe polypeptide present in a confrol sample from a second mammalian subject known not to have, or not to be predisposed to, said disease, wherein an alteration in the expression level ofthe polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to said disease.
41. A method for determining the presence of or predisposition to a disease associated with altered levels ofthe nucleic acid molecule of claim 5 in a first mammalian subject, the method comprising: a) measuring the amount ofthe nucleic acid in a sample from the first mammalian subject; and b) comparing the amount of said nucleic acid in the sample of step (a) to the amount ofthe nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level ofthe nucleic acid in the first subject as compared to the confrol sample indicates the presence of or predisposition to the disease.
42. A method of treating a pathological state in a mammal, the method comprising administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 or a biologically active fragment thereof.
43. A method of freating a pathological state in a mammal, the method comprising administering to the mammal the antibody of claim 15 in an amount sufficient to alleviate the pathological state.
PCT/US2001/001513 2000-01-13 2001-01-16 Odorant receptor polypeptides and nucleic acids encoding same WO2001051632A2 (en)

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US17819100P 2000-01-26 2000-01-26
US17822700P 2000-01-26 2000-01-26
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US21832400P 2000-07-14 2000-07-14
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