POLYNUCLEOTIDES, POLYPEPTIDES AND CANCER
FIELD OF INVENTION
This invention relates to polynucleotides and polypeptides, including the identification of a gene over-expressed in cancer, as well as newly identified polynucleotides, and polypeptides encoded by the polynucleotides. More particularly, the present invention relates to G-protein coupled receptors.
BACKGROUND OF THE INVENTION
It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/ or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991, 351 :353-354). Herein, these proteins are referred to as proteins participating in pathways with G-proteins, or PPG proteins. Some examples of these PPG proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., Proc. Natl,
Acad. Sci., U.S.A., 1987, 84:46-50; Kobilka, B.K., et al, Science, 1987,
238:650-656; Bunzow, J.R, et al, Nature, 1988, 336: 783-787), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M. I., et al, Science, 1991, 252:802-8).
For example, in one form of signal transduction, the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP. GTP also influences hormone binding. A G-protein connects the hormone receptor to adenylate cyclase. G-protein was shown to exchange GTP for bound GDP when activated by a hormone receptor. The GTP-carrying form then binds to the activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-protein itself returns the G-protein to its basal inactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
The G-protein coupled receptor family includes dopamine receptors, which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include, but are not limited to, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorant, and cytomegalovirus receptors.
G-protein coupled receptors are found in numerous sites within a mammalian host. Over the past 15 years, nearly 350 therapeutic agents targeting the seven transmembrane (7 TM) receptors have been successfully introduced onto the market. This success indicates that these receptors have an established, proven history as therapeutic targets. Clearly there is a need for identification and characterization of further receptors which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non-insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; asthma; allergies; benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette's syndrome.
The membrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane alpha - helices connected by extracellular or cytoplasmic loops.
G-protein coupled receptors (otherwise known as 7 TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops forming disulfide bonds that are believed to stabilize functional protein structure. The seven transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.
Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors. Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and /or the carboxy terminus. For several G-protein coupled receptors, such as the beta-adrenoreceptor, phosphorylation by protein kinase A and /or specific receptor kinases mediates receptor desensitization.
For some receptors, the ligand binding sites of G-protein coupled receptors are believed to comprise hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said socket being surrounded by hydrophobic residues of the G-protein coupled receptors. The hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face inward and form polar ligand binding site. TM3 has been implicated in several G-protein coupled receptors as having a ligand binding
site, such as the TM3 aspartate residue. Serine residues in TM5, asparagines residue in TM6 and phenylalanine or tyrosines residues in TM6 or TM7 are also implicated in ligand binding.
G-protein coupled receptors can be intracellularly coupled y heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al, Endoc. Rev., 1989, 10:317-331). Different G-protein alpha- subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors has been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors.
WO 0127158 relates to human olfactory receptors, and provides isolated polynucleotide comprising a sequence encoding a polypeptide which is involved in olfactory sensation. The olfactory receptor polypeptides are to be found within sequences depicted in given polynucleotide sequences SEQ ID NO: l through SEQ ID NO:73 and SEQ ID NOr l l l through SEQ ID NO: 152. Sequences are given for isolated and purified olfactory receptor polypeptides.
SUMMARY OF THE INVENTION
The present invention relates to: the SAN_O787_l polynucleotide which has the nucleotide sequence
set forth in SEQ ID NO: 1 and encodes a SAN_O787_l polypeptide of SEQ ID
NO: 2; the SAN_O399_3 polynucleotide which has the nucleotide sequence set forth in SEQ ID NO: 3 and encodes a SAN_O399_3 polypeptide of SEQ ID NO: 4; the SAN_O437_l polynucleotide which has the nucleotide sequence set forth in SEQ ID NO: 5 and encodes a SAN_O437_l polypeptide of SEQ ID NO: 6; the SAN_O437_4 polynucleotide which has the nucleotide sequence set forth in SEQ ID NO: 7 and encodes a SAN_O437_4 polypeptide of SEQ ID NO: 8; and the SAN_O817_l polynucleotide which has the nucleotide sequence set forth in SEQ ID NO: 9 and encodes a SAN_O817_l polypeptide of SEQ ID NO: 10.
The sequence SAN_0787_1 is known from WO 0127158. We have found that the presence of such a sequence can be a marker for cancer, in that the expression levels of this gene are enhanced in tumor hepatocytes. Accordingly, the present invention provides methods of diagnosing cancer, especially liver cancer.
In one aspect, the present invention comprises detecting cancer by investigating the gene SAN_0787_1. Investigation can involve assessing expression levels of the gene, typically by detecting an increased level of transcription or translation. In a related aspect, the invention involves
detecting variant forms of the gene, or detecting RNA transcribed from such forms, or detecting polypeptides translated from such RNA. Suitable test samples for use in the diagnostic methods can be obtained using cells such as from blood, urine, saliva, sperm, tissue biopsy or autopsy material, and preparations made from such cells, preferably cells from biopsy specimens and prepared RNA samples, more preferably hepatocytes and prepared RNA from mammalian liver.
Acording to the present invention, there is provided in one aspect a method to detect the expression level in a test sample of a target sequence which is an mRNA which hybridizes with a polynucleotide that is complementary to a polynucleotide having the sequence shown in SEQ ID NO: l or complementary to a variant thereof. The test sample is ordinarily obtained from a subject suspected of having liver cancer. A preferred detection method involves detecting hybridization of the target sequence with a probe having a complementary sequence. An alternative preferred detection method involves detecting amplification of the target sequence with primers using a polymerase chain reaction.
Hybridization methods of this invention typically comprise providing a probe complementary to the target sequence, and contacting the sample with the probe under conditions to allow formation of a hybridization complex between the probe and the target sequence. The probe suitably comprises a fragement of a sequence complementary to the target sequence, and ordinarily comprises at least 15, 20, 25, 30, 35 or more nucleotides.
Preferred probes have 30 to 50 nucleotides. The conditions are preferably stringent conditions. The formation of any complex is then qualitatively or quantitatively assessed.
Amplification methods of this invention typically comprise providing forward and reverse primers. The forward primer amplifies at least a part of the target sequence, and hybridizes with at least a part of a polynucleotide sequence complimentary to the target sequence. The reverse primer amplifies at least a part of a polynucleotide sequence complimentary to the target sequence, and hybridizes with at least the target sequence. The sample is contacted with the primers under conditions for a polymerase chain reaction. The primers suitably comprises at least 15, 20, 25, 30, 35 or more nucleotides. Preferred primers have 20 to 30 nucleotides. The formation of any amplified polynucleotide is then qualitatively or quantitatively assessed.
The invention also provides a method to detect a polypeptide which is specifically recognized by a detection agent which specifically recognizes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof, or a fragment of the sequence or variant. A typical detection agent is an antibody, especially an antibody specific for the amino acid sequence shown in SEQ ID NO:2. Such antibodies can be employed in assay methods which include radioimmunoassays, competitive-binding assays, western blot analysis, ELISA assays or protein tip technology, especially an ELISA or western blot method.
A diagnostic method of cancer, especially liver cancer, is also a subject of this invention and comprises the steps of: a) detecting the expression level in a test sample of a target sequence which is an mRNA which hybridizes with a polynucleotide having a sequence complimentary to that of SEQ ID NO: 1 or a variant thereof; and b) comparing the expression level of said mRNA in said test sample with that for a control sample.
The diagnostic method can suitably be carried out by a hybridization method using a probe which hybridizes under stringent conditions with the target sequence. Hybridization assays can be performed on the test sample and the control sample to give data for the expression levels in the test and control samples which can then be compared. Cancer is diagnosed when the expression level of said mRNA in the test sample is indicatively higher than that in the control sample.
The diagnostic method can suitably be carried out by amplification of the target sequence with primers using a polymerase chain reaction. Amplification assays can be performed on the test sample and the control sample to give data for the expression levels in the test and control samples which can then be compared. Cancer is diagnosed when the expression level of the mRNA in the test sample is indicatively higher than that in the control sample.
The present invention further provides a diagnostic method for cancer, especially liver cancer, comprising the steps of: a) detecting the expression level in a test sample of a polypeptide which is specifically recognized by a detection agent which specifically recognizes a polypeptide having the amino acid sequence shown in SEQ ID NO:2 or a variant thereof, or a fragment of the sequence or variant; b) comparing the expression level of said polypeptide in the test sample with that for a control sample.
The diagnostic method can suitably be carried out using a detection agent such as an antibody which binds to the polypeptide. Binding assays can be performed on the test sample and the control sample to give data for the expression levels in the test and control samples which can then be compared. Cancer is diagnosed when the expression level of said polypeptide in the test sample is indicatively higher than that in the control sample.
The invention further provides the polymerase chain reaction primers, which comprise fragment polynucleotides of this invention. Appropriate pairs of forward and reverse primers are provided, either for DNA amplification or more preferably for RNA amplification. Typical primers of this invention include a forward primer which has a length of say 20 - 30 nucleotides, is suited for specifically amplifying the target sequence and hybridizes with a polynucleotide having a sequence complementary to the target sequence. The target sequence is a part or the full length of the SEQ ID NO: l, or a sequence having at least 70% identity thereto. Typical primers of this
invention also include a reverse primer which has a length of say 20 - 30 nucleotides, is suited for specifically amplifying a sequence complimentary to the target sequence and hybridizes with the target sequence. The target sequence is a sequence complimentary to a part or the full length of the SEQ
ID NO: l, or a sequence having at least 70% identity thereto.
Another aspect of this invention resides in the provision of microarray methods and components for such a method. A preferred microarray component comprises a support carrying at least one single stranded polynucleotide which hybridizes under the stringent condition with a target sequence which is mRNA hybridizing with a polynucleotide having a sequence complementary to the sequence shown in SEQ ID NO: l or a varinat thereof.
The invention also provides antibodies which specifically recognize a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof, or a fragment of the sequence or variant. A preferred antibody is specific for the amino acid sequence shown in SEQ ID NO:2.
Kits are also provided by the present invention for diagnosis of liver cancer. Such kits can include appropriate reagents selected from a pair of forward and reverse PCR primers; a hybridization probe which is an oligonucloeotide which hybridizes with a polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant; the microarray component comprising the support carrying the single stranded polynucleotide; or an antibody which specifically recognizes a polypeptide having the amino acid sequence shown in
SEQ ID NO:2 or a variant thereof, or a fragment of the sequence or variant.
Such kits can typically include instructions for using the reagents, along with further components selected from additional reagents and/ or equipment.
A further aspect of this invention resides in a screening method for identifying a test compound as an anti-cancer agent. Such a method can include the steps of: a) detecting the expression level of a gene comprizing polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant thereof in the absence of the test compound; b) detecting the expression level of a gene comprizing polynucleotide having sequence shown in SEQ ID NO: 1 or a variant thereof in the presence of the test compound; and c) comparing the expression levels of said gene in the absence of said test compound with that in presence of said test compound.
With this method, test compounds can be selected for which the expression level of said gene decreased in the presence of the compound. Such compounds are then candidates for investigation as agents against cancer. In particular, the screening method allows identification of compounds of use in treating liver cancer. Pharmaceutical compositions of the compound and a pharmaceutically acceptable carrier are provided by the invention.
An alternative method for detecting cancer comprises assessing the
presence of at least one polymorphism or mutation in a gene selected from the gene SAN_O787_l having the sequence SEQ ID NO: 1 and variants thereof.
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References to variants of the gene SAN_O787_l having the sequence SEQ ID NO: 1 and to the polypeptide having the sequence SEQ ID NO: 2 include allelic forms and other modifications.
The polynucleotide sequence SAN_0399_3 differs from a polynucleotide sequence in WO 0127158 at positions 183, 256 and 716. In SAN_0399_3, the nucleotides are respectively C, T and T, in place of A, C and C at the corresponding positions in the sequence of WO 0127158. Accordingly, in a preferred aspect, this invention provides polynucleotide sequences based on SAN_0399_3, provided that the polynucleotide sequence has one or more of C at position 183, T at position 256 or T at position 716.
The nucleotide differences in SAN_0399_3 compared with the sequence in WO 0127158 lead to changes in the encoded amino acid sequence. The difference at position 183 results in phenylalanine instead of leucine at postion 63. The difference at position 256 results in phenylalanine instead of leucine at position 86. The difference at position 716 results in methionine instead of threonine at position 239. Accordingly, the present invention also provides polypeptide sequences based on SAN_0399_3, provided that the polypeptide has one or more of phenylalanine at postion 63, phenylalanine at position 86 and methionine at position 239.
The polynucleotide sequence SAN_0437_1 differs from a polynucleotide sequence in WO 0127158 by the presence of coding at positions 1 to 63°. Such additional coding will provide an N-terminal region, compared to the polypeptide encoded by the polynucleotide sequence in WO
0127158. The presence of an N-terminal region in a G-protein coupled receptor is often found to be essential for binding of its ligand to the receptor.
Furthermore, the polynucleotide sequence SAN_0437_1 differs from that polynucleotide sequence in WO 0127158 by the presence of coding at positions 999 to 1008. Such additional coding will provide a C-terminal region, compared to the polypeptide encoded by the polynucleotide sequence in WO 0127158. The presence of a C-terminal region in a G-protein coupled receptor is important for signaling through the G-protein. Furthermore, the polynucleotide sequence SAN_0437_1 differs from that polynucleotide sequence in WO 0127158 at positions 360 and 362. In SAN_0437_1, the nucleotides are respectively G and T, in place of A and A at the corresponding position in the sequence of WO 0127158.
The polynucleotide sequence SAN_0437_4 differs from a polynucleotide sequence in WO 0127158 at positions 139 and 720. In SAN_0437_4> the nucleotides are respectively A and T, in place of G and G at the corresponding positions in the sequence of WO 0127158. Accordingly, in a preferred aspect, this invention provides polynucleotide sequences based on SAN_0437_4> provided that the polynucleotide sequence has one or more of A at position 139 or T at position 720.
The nucleotide differences in SAN_0437_4 compared with the sequence in WO 0127158 lead to a change in the encoded amino acid sequence. The difference at position 139 results in isoleucine instead of valine at position 47. Accordingly, the present invention also provides polypeptide sequences based on SAN_0437_4, provided that the polypeptide has one or more of isoleucine at position 47.
The polynucleotide sequence SAN_0817_1 differs from a polynucleotide sequence in WO 0127158 by the absence of coding beyond position 939. Such a change in the coding length will provide a shortened C-terminal region, compared to the polypeptide encoded by the polynucleotide sequence in WO 0127158. The correct C-terminal region in a G-protein coupled receptor is important for signaling through the G-protein.
In yet another aspect, the invention relates to SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_0437_4 and SAN_O817_l polypeptides and recombinant materials and methods for their production.
One aspect of the invention relates to methods for using such SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides and polynucleotides. For SAN_O399_3, SAN_O437_l,
SAN_O437_4 and SAN_O817_l such uses include the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non-insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia;
bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; asthma; allergies; benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette's syndrome, among others.
In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with SAN_0787_1, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l activities or levels.
In a further aspect, the invention exttends to the sequences of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polynucleotides which do not encode a polypeptide which is involved in olfactory sensation, and to SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides which are not olfactory receptor polypeptides.
DESCRIPTION OF THE INVENTION
Definitions
The following definitions are provided to facilitate understanding of certain terms used frequently herein.
"SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_ l" refers, among others, to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, or an allelic or other variant thereof
"Receptor Activity" or "Biological Activity of the Receptor" refers to the metabolic or physiologic function of said SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and immunogenic activities of said SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4, and SAN_O817_l.
"SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l gene" refers to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or allelic or other variants thereof and/or their complements.
"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments,
including the products of a Fab or other immunoglobulin expression library.
"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the . same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.
"Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple- stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
"Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA;
thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
"Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides. "Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques, which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of
flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, PROTEINS-STRUCTURE
AND MOLECULAR PROPERTIES, 2nd Ed., T.E. Creighton, W.H. Freeman and
Company, New York, 1993 and Wold, F., Posttranslational Protein
Modifications: Perspectives and Prospects, pgs 1-12 in POSTTRANSLATIONAL
COVALENT MODIFICATION OF PROTEINS, B.C. Johnson, Ed., Academic
Press, New York, 1983; Seifter et al, "Analysis for protein modifications and non protein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al,
"Protein Synthesis: Posttranslational Modifications and Aging", Ann NY Acad
Sci (1992) 663:48-62.
"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains one or more essential properties, especially a biological function of the parent polynucleotide or polypeptide. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by synthesis.
A variant polynucleotide is typically a polynucleotide with a sequence having at least 80%, 85%, 90% or 95% identity with the parent sequence.
A variant polypeptide is typically a polypeptide with a sequence having at least 80%, 85%, 90% or 95% identity with the parent sequence.
"Identity" is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL
MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York,
1988; BIOCOMPUTING:INFORMATICS AND GENOME PROJECTS, Smith,
D.W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF
SEQUENCE DATA, PART I, Griffin, A.M., and Griffin, H.G., eds., Humana
Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER,
Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991).
While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is known well to skilled artisans (Carillo, H., and Lipton, D., SIAM J Applied Math (1988)
48: 1073). Methods that are commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press,
San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J Applied Math (1988)
48: 1073. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to,
GCS program package (Devereux, J., et at, Nucleic Acids Research (1984) 12
(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al, J. Molec. Biol. (1990)
215: 403).
As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the
polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9.
In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or
3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2, 4, 6, 8, 10. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy
terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
Polypeptides of the Invention
In one aspect, the present invention relates to SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides. The SAN_0787_1, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides include the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, and polypeptides comprising the amino acid sequence which have at least 80% identity to that of SEQ ID NO: 2, 4, 6, 8, 10, 16; over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2, 4, 6, 8, 10. Furthermore, those with at least 97-99% are highly preferred. Also included within SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides are polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2, 4, 6, 8, 10. Furthermore, those with at least 97-99%) are highly preferred. Preferably SAN_O787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides exhibit at least one biological
activity of the receptor. The SAN_O787_l , SAN_0399_3, SAN_O437_l ,
SAN_O437_4 and SAN_O817_l polypeptides may be in the form of the mature protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
Fragments of the SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides are also included in the invention. A fragment is a polypeptide having an amino acid sequence that entirely is the same as part; but not all, of the amino acid sequence of the aforementioned SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides. As with SAN_ O787_l, SAN_O399_3,
SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region.
Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides. In this context "about" includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of SAN_O787_l, SAN_O399_3, SAN_O437_l,
SAN_O437_4 and SAN_O817_l polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Also preferred fragments are characterized by structural or functional attributes comprising alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate receptor activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.
Preferably the polypeptide fragments retain the biological activity of the receptor, including antigenic activity.
Suitably the polypeptide fragments of this invention comprise at least 10, 15, 18, 20, 25, 30, 35, 40 or more amino acids.
Variants of the defined sequence and fragments also form part of the present invention. Preferred variants are those that vary from the referents
by conservative amino acid substitutions, that is, those that substitute a residue for another residue of like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr, among acidic residues
Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.
The SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated Naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
Polynucleotides of the Invention
Another aspect of the invention relates to SAN_O787_l, SAN_O399_3, SAN_O437_l , SAN_O437_4 and SAN_O817_1 polynucleotides. SAN_O787_l , SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polynucleotides include isolated polynucleotides which encode the SAN_O787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides and fragments, and polynucleotides closely related thereto. More specifically, SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polynucleotide of the invention include polynucleotide comprising the nucleotide sequence set
forth in SEQ ID NO: 1, 3, 5, 7, 9 encoding a SAN_O787_l, SAN_O399_3,
SAN_O437_l, SAN_O437_4 and SAN_0817_1, polypeptide of SEQ ID NO: 2, 4,
6, 8, 10 and polynucleotide having the particular sequence of SEQ ID NO: 1, 3,
5, 7, 9. SAN_O787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and
SAN_O817_l polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and
SAN_O817_l polypeptide of SEQ ID NO: 2, 4, 6, 8, 10 over its entire length, and a polynucleotide that is at least 80% identical to that having SEQ ID NO:
1, 3, 5, 7, 9 over its entire length. In this regard, polynucleotides at least 90% identical are particularly preferred, and those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred.
Also included under SAN_O787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO: 1, 3, 5, 7, 9 to hybridize under conditions useable for amplification or for use as a probe or marker. The invention also provides polynucleotides complementary to such SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polynucleotides. SAN_O787_l , SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l of the invention is structurally related to other proteins of the G-protein coupled receptor family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO: 1, 3, 5, 7, 9)
encoding human SAN_O787_l, SAN_O399_3, SAN„O437_l, SAN_O437_4 and SAN_O817_l .
The cDNA sequence of SEQ ID NO: l contains an open reading frame (nucleotide numbers 1 to 927) encoding a polypeptide of 309 amino acids of SEQ ID NO:2.
The cDNA sequence of SEQ ID NO:3 contains an open reading frame (nucleotide numbers 1 to 948) encoding a polypeptide of 316 amino acids of SEQ ID NO:4.
The cDNA sequence of SEQ ID NO: 5 contains an open reading frame (nucleotide numbers 1 to 1005) encoding a polypeptide of 335 amino acids of SEQ ID NO:6.
The cDNA sequence of SEQ ID NO:7 contains an open reading frame (nucleotide numbers 1 to 960) encoding a polypeptide of 320 amino acids of SEQ ID NO:8.
The cDNA sequence of SEQ ID NO:9 contains an open reading frame (nucleotide numbers 1 to 936) encoding a polypeptide of 312 amino acids of SEQ ID NO: 10.
TABLE 1
nucleotide sequences of human SAN_0787_1 , SAN_0399_3 , SAN_0437_1 , SAN_0437_4
and SAN_0817_1 ; SEQ ID NO : 1 , 3 , 5 , 7 , 9 .
SEQ ID NO : 1
1 atgaagagaa agaacttcaσ agaagtgtca gaattcattt tcttgggatt ttctagcttt
61 ggaaagcatσ agataaσcct ctttgtggtt ttcctaactg tctacatttt aactctggtt
121 gctaaσatσa tσattgtgac tatcatctgc attgacσatc atctccacac tσσcatgtat
181 ttcttcctaa gσatgctggc tagttcagag acggtgtaca cactggtcat tgtgccacga
241 atgcttttga gcctcatttt tcataaccaa cctatctcct tggσaggσtg tgctacacaa
301 atgttctttt ttgttatσtt ggccactaat aattgcttcc tgcttaσtgc aatggggtat
361 gaccgctatg tggccatctg σagacccctg agatacactg tcatcatgag caagggaσta
421 tgtgσccagc tggtgtgtgg gtcctttggc attggtctga ctatggσagt tctcσatgtg
481 acagσσatgt tcaatttgcc gttctgtggc aσagtggtag accacttctt ttgtgacatt
541 taccσagtσa tgaaactttσ ttgcattgat aσσactatca atgagataat aaattatggt
601 gtaagttσat ttgtgatttt tgtgcccata ggσctgatat ttatctccta tgtccttgtσ
661 atctcttcca tσcttcaaat tgcσtcagct gagggccgga agaagacctt tgccacctgt
721 gtctcσcacc tcactgtggt tattgtccac tgtggctgtg cctcσattgc ctacctσaag
781 σcgaagtcag aaagttcaat agaaaaagac cttgttctct cagtgacgta σaccatcatc
.841 aσtcccttgc tgaaccctgt tgtttacagt ctgagaaaσa aggaggtaaa ggatgcccta 901 tgσagagttg tgggcagaaa tatttcttaa
SEQ ID NO : 3
1 atgaagatag caaaσaacac agtagtgaca gaatttatcσ tcσttggtσt gactcagtct
61 caagatattc agctcttggt ctttgtgctg atσttaattt tσtacσttat catσctccσt
121 ggaaattttc tcattatttt cacσataagg tcagaccctg ggctcacagσ ccccσtctat
181 ttctttctgg gcaaσttggσ cttcctggat gcatcctact ccttcattgt ggctcccagg
241 atgttggtgg acttσttctσ tgagaagaag gtaatctcct acagaggσtg catcactcag
301 ctctttttct tgcaσttcct tggaggaggg gagggattac tcσttgttgt gatggccttt
361 gacσgctaσa tcgσcatσtg σcggcctctg σaσtgttcaa ctgtcatgaa ccctagagcc
421 tgctatgcaa tgatgttggc tctgtggctt gggggttttg tccactccat tatccaggtg
481 gtσσtσatσc tσcgσttgσc tttttgtggc σcaaaσcagc tggaσaactt cttctgtgat
541 gtccgacagg tcatσaagσt ggσttgcacc gacatgtttg tggtggagσt tctgatggtc
601 ttσaacagtg gcctgatgac aσtcctgtgc tttctggggσ ttctggcttc ctatgcagtc
661 atcctctgcσ atgttcgtag ggσagcttct gaagggaaga acaaggccat gtccatgtgσ
721 accactcgtg tcattattat acttσttatg tttggacctg ctatcttcat ctacatgtgc
781 cctttcaggg ccttaccagc tgacaagatg gtttctctct ttcacacagt gatctttcca
841 ttgatgaatc ctatgattta tacσcttcgc aaccaggaag tgaaaacttc catgaagagg
901 ttattgagtc gacatgtagt ctgtcaagtg gattttataa taagaaactg a
SEQ ID NO: 5
1 atggcaσagg tgagggcgσt gcataaaatc atggcccttt tttctgctaa cagcataggt
61 gctatgaaca actctgacac tcgcatagca ggctgσttcc tcactggcat ccσtgggctg
121 gagcaactac atatctggct gtccatccσc ttctgcatca tgtaσatσgc tgccctggaa
181 ggσaatggca tcctaatttg tgtcatcctc tcccaggcaa tcctgcatga gcccatgtac
241 atattσttat σtatgctggc cagtgctgat gtcttgctct ctaccaccac catgcctaag
301 gcσctggσca atttgtggct aggttatagc σaσatttcct ttgatggctg cctcactσag
361 atgttσttσa ttσacttσσt σttcattcac tctgctgtcc tgctggcσat ggσctttgaσ
421 cgctatgtgg ccatctgctc σcccctgcga tatgtσacaa tcctcacaag caaggtcatt
481 gggaagatcg tσactgcσac cσtgagccgc agσttσatca ttatgtttcσ atccatcttt
541 ctccttgagc acctgcacta ttgccagatc aacatcattg cacacacatt ttgtgagcac
601 atgggcattg cccatctgtσ σtgttσtgat atctcσatσa atgtctggta tgggttggca
661 gctgctcttc tctccacagg cσtggacatc atgσttatta ctgtttσσta catσcacatc
721 ctσcaagσag tcttccgcct cσtttctcaa gatgcσcgct ccaaggσcct gagtacctgt
781 ggatcccata tctgtgtcat cctactcttc tatgtcσctg cccttttttc tgtctttgcc
841 taσaggtttg gtgggagaag catσccatgσ tatgtccata ttσtcctggc cagcctσtac
901 gttgtcattc σtcσtatgct caatcccgtt atttatggag tgaggactaa gccaatactg
961 gaaggggcta agcagatgtt ttcaaatctt gccaaaggat σtaaataa
SEQ ID NO: 7
1 atgσcatctg cctctgccat gatcattttc aacctgagca gttaσaatcσ aggacccttσ 61 attσtggtag ggatσσσagg cctggagcaa ttccatgtgt ggattggaat tσσσttctgt 121 atcatσtaca ttgtagσtat tgtgggaaac tgcatcσttσ tσtacσtσat tgtggtggag 181 σatagtσttc atgaaccσat gttσttcttt ctctσcatgc tggccatgac tgacctcatσ 241 ttgtccaσag σtggtgtgσc taaagσactc agtatctttt ggctaggggc tcgcgaaatc 301 acattcccag gatgccttac acaaatgttc ttccttcaσt ataaσtttgt cctggattσa 361 gσσattσtga tggσσatggc atttgatcac tatgtagcta tctgttctσσ cttgagatat
421 accacσatct tgactcσσaa gacσatσatσ aagagtgcta tgggσatctσ ctttσgaagc 481 ttctgcatσa tσctgccaga tgtattcttg ctgacatgcσ tgσσtttctg caggaσaσgσ 541 atcatacσσc acaσatactg tgagσatata ggtgttgccc agσtcgcctg tgctgatatσ
601 tccatcaact tctggtatgg σttttgtgtt cccatcatga cggtcatctc agatgtgatt 661 ctcattgσtg tttcctacgc aσaσatcctc tgtgctgtct ttggccttcc σtσccaagat
721 gσσtgcσaga aagcσctcgg cacttgtggt tctcatgtσt gtgtσatσσt catgttttat
781 acacctgσct ttttctσcat σctcgcccat cgctttggac aσaatgtctc tcgcaccttσ
841 σaσatcatgt ttgccaatct ctacattgtt atcccacctg cactσaaccc σatggtttac
901 ggagtgaaga σσaagcagat σagagataag gttatacttt tgttttctaa gggtaσagga
961 tga
SEQ ID NO: 9
1 atggaagσag aaaaσσttac agaattatσa aaatttσtσσ tσctgggaσt σtcagatgat 61 cctgaactgσ agσσσgtσσt σtttgggσtg ttcctgtσσa tgtacctggt caσggtgctg 121 gggaacctgσ tσatcattσt ggσcgtcagc tctgactσcσ aσctσcacac σσσσatgtaσ
181 ttcttcctct cσaacctgtc ctttgttgaσ atctgtttca tσtcσaσσac agtσσσσaag 241 atgσtagtga gcatσσaggσ aσggagσaaa gacatσtσσt acatggggtg cσtσaσtσag
301 gtgtattttt taatgatgtt tgσtggaatg gatactttσσ taσtggcσgt gatggσctat 361 gaccggtttg tggccatσtg σσaσσσaσtg σactacacgg tσatcatgaa cσσσtgcσtc 421 tgtggcσtσσ tggttctggc atcttggttσ atσattttσt ggttσtσσct ggttσatatt 481 σtaσtgatga agaggttgaσ cttctσσaσa ggσactgaga ttccgcattt σttσtgtgaa 541 σcggctcagg tcσtσaaggt ggcσtgσtσt aacaccσtσσ tσaataaσat tgtσttgtat
601 gtggσcacgg cactgctggg tgtgtttcσt gtagctggga tcσtσttσtc σtactσtσag 661 attgtσtσct σcttaatggg aatgtcσtcc aσσaagggσa agtaσaaagσ cttttccaσc 721 tgtggatctc acctctgtgt ggtctccttg ttctatggaa caggaσttgg ggtctatctg
781 agttσtgσtg tgaccσattσ ttσσcagagc agσtσσaσσg σσtσagtgat gtaσgσσatg 841 gtσaσσσσσa tgctgaaccσ σttcatctaσ agσσtgagga aσaaggatgt gaagggggσσ 901 ctggaaagac tσσtcagσag ggσcgactσt tgtσcatga
TABLE 2
amino aσid sequenσes of human SAN_0787_1, SAN_0399_3, SAN_0437_1, SAN_0437_4 and SAN_0817_1 SEQ ID NO: 2, 4, 6 , 8, 10.
SEQ ID NO: 2
1 MKRKNFTEVS EFIFLGFSSF GKHQITLFW FLTVYILTLV ANIIIVTIIC IDHHLHTPMY 61 FFLSMLASSE TVYTLVIVPR MLLSLIFHNQ PISLAGCATQ MFFFVILATN NCFLLTAMGY 121. DRYVAICRPL RYTVIMSKGL CAQLVCGSFG IGLTMAVLHV TAMFNLPFCG TWDHFFCDI 181 YPVMKLSCID TTINEIINYG VSSFVIFVPl GLIFISYVLV ISSILQIASA EGRKKTFATC 241 VSHLTWIVH CGCASIAYLK PKSESSIEKD LVLSVTYTII TPLLNPWYS LRNKEVKDAL 301 CRWGRNIS
SEQ ID NO: 4
1 MKIANNTWT EFILLGLTQS QDIQLLVFVL ILIFYLIILP GNFLIIFTIR SDPGLTAPLY 61 FFLGNLAFLD ASYSFIVAPR MLVDFFSEKK VISYRGCITQ LFFLHFLGGG EGLLLWMAF 121 DRYIAICRPL HCSTVMNPRA CYAMMLALWL GGFVHSIIQV VLILRLPFCG PNQLDNFFCD 181 VRQVIKLACT DMFWELLMV FNSGLMTLLC FLGLLASYAV ILCHVRRAAS EGKNKAMSMC 241 TTRVIIILLM FGPAIFIYMC PFRALPADKM VSLFHTVIFP LMNPMIYTLR NQEVKTSMKR 301 LLSRHWCQV DFIIRN
SEQ ID NO : 6
1 MAQVRALHKI MALFSANSIG AMNNSDTRIA GCFLTGIPGL EQLHIWLSIP FCIMYIAALE
61 GNGILICVIL SQAILHEPMY IFLSMLASAD VLLSTTTMPK ALANLWLGYS HISFDGCLTQ
121 MFFIHFLFIH SAVLLAMAFD RYVAICSPLR YVTILTSKVI GKIVTATLSR SFIIMFPSIF 181 LLEHLHYCQI NIIAHTFCEH MGIAHLSCSD ISINV YGLA AALLSTGLDI MLITVSYIHI 241 LQAVFRLLSQ DARSKALSTC GSHICVILLF YVPALFSVFA YRFGGRSIPC YVHILLASLY 301 WIPPMLNPV IYGVRTKPIL EGAKQMFSNL AKGSK
SEQ ID NO: 8
1 MPSASAMIIF NLSSYNPGPF ILVGIPGLEQ FHVWIGIPFC IIYIVAIVGN CILLYLIWE 61 HSLHEPMFFF LSMLAMTDLI LSTAGVPKAL SIF LGAREI TFPGCLTQMF FLHYNFVLDS
121 AILMAMAFDH YVAICSPLRY TTILTPKTII KSAMGISFRS FCIILPDVFL LTCLPFCRTR 181 IIPHTYCEHI GVAQLACADI SINFWYGFCV PIMTVISDVI LIAVSYAHIL CAVFGLPSQD 241 ACQKALGTCG SHVCVILMFY TPAFFSILAH RFGHNVSRTF HIMFANLYIV IPPALNPMVY 301 GVKTKQIRDK VILLFSKGTG
SEQ ID NO: 10
1 MEAENLTELS KFLLLGLSDD PELQPVLFGL FLSMYLVTVL GNLLIILAVS SDSHLHTPMY
61 FFLSNLSFVD ICFISTTVPK M VSIQARSK DISYMGCLTQ VYFLMMFAGM DTFLLAVMAY
121 DRFVAICHPL HYTVIMNPCL CGLLVLAS F IIFWFSLVHI LLMKRLTFST GTEIPHFFCE
181 PAQVLKVACS NTLLNNIVLY VATALLGVFP VAGILFSYSQ IVSSLMGMSS TKGKYKAFST
241 CGSHLCWSL FYGTGLGVYL SSAVTHSSQS SSTASVMYAM VTPMLNPFIY SLRNKDVKGA
301 LERLLSRADS CP
Polynucleotides of the present invention encoding SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides may be obtained using standard cloning and screening from a cDNA library derived from mRNA in cells of human tissues. Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.
The nucleotide sequences encoding SAN_O787_l, SAN_0399_3, SAN_O437_l, SAN_O437_4, and SAN_O817_ polypeptides of SEQ ID NO: 2, 4, 6, 8, 10 may be identical to the polynucleotides encoding sequences contained in Table 1 , or they may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptides of SEQ ID NO:2, 4, 6, 8, 10.
When the polynucleotides of the invention are used for the recombinant productions of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l, polypeptides, the polynucleotides may include the coding sequence for the mature polypeptides or a fragment thereof by itself in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain
preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc Natl Acad Sci USA (1989) 86: 821-824, or is an
HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
Further preferred embodiments are polynucleotides encoding SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l variants comprising the amino acid sequences of the SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l, polypeptides of Table 1 (SEQ ID NO: 2, 4, 6, 8, 10) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted, inserted or added, in any combination.
The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
Polynucleotides of the invention, which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1, 3, 5, 7, 9 or a
fragment thereon may be used as hybridization probes for cDNA and genomic
DNA, to isolate full-length cDNAs and genomic clones encoding SAN_O787_l,
SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l, and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the SAN_p787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and
SAN_O817_1 gene. Such hybridization techniques are known to those of skill in the art. Typically these nucleotide sequences are 80% identical, preferably
90% identical, more preferably 95% identical to that of the referent. The probes generally will comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides.
Particularly preferred probes will range between 30 and 50 nucleotides.
In one embodiment, to obtain polynucleotides encoding the SAN_O787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the SEQ ID NO: 1, 3, 5, 7, 9 or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to those of skill in the art. Stringent hybridization conditions are as defined above or alternatively conditions under overnight incubation at 42 °C in a solution comprising: 50% formamide, δ.times; SSC (150mM NaCl, 15mM sodium citrate), 5. times; Denhardt's solution, and 0.5% SDS, followed by washing the filters in 0.1. times SSC at about 65° C.
The polynucleotides and polypeptides of the present invention may be
employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.
Vectors, Host Cells, Expression
The present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis
cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,
HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
A great variety of expression systems can be used. Such systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression.. Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, Molecular Cloning, A Laboratory Manual (supra).
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
If the SAN_0787_1, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides are to be expressed for use in screening assays, generally, it is preferred that the polypeptide be localized at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If SAN_0787_1, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides are secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
Diagnostic Assays
This invention also relates to the use of SAN_O787_l, SAN_O399_3,
SAN_O437_l, SAN _0437_4 and SAN_O817_l polynucleotides for use as diagnostic reagents. The mutated forms of SAN_0787_1, SAN_O399_3,
SAN_O437_l, SAN_O437_4 and SAN_O817_l gene associated with dysfunction can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and
SAN_O817_l. Individuals carrying mutations in the SAN_O787_l,
SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l genes may be detected by a variety of techniques.
Nucleotides for diagnosis of liver cancer in respect of SAN_O787_l are most suitably obtained from hepatocytes. Nucleotides acids for diagnosis in respect of SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l may be obtained from a subject's cells, such as from blood, urine, saliva, sperm, tissue biopsy or autopsy material. Hepatocytes and liver tissue are preferred sources for use in detecting liver cancer using diagnosis based on SAN_787_ 1. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence
differences may also be detected by alterations in electrophoretic mobility of
DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al, Science (1985) 230: 1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method.
See Cotton et al, Proc Natl Acad Sci USA (1985) 85: 4397-4401. In another embodiment, an array of oligonucleotides probes comprising SAN_O787_l,
SAN_0399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l nucleotide sequences or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M. Chee et al, Science, Vol 274, pp
610-613 (1996)).
The diagnostic assays in respect of SAN_O399_3, SAN_O437_l, SAN_0437_4 and SAN_O817_l offer a process for diagnosing or determining a susceptibility to infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non-insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; asthma; allergies;
benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or
Gilles de la Tourette's syndrome through detection of mutation in the
SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l genes by the methods described.
In addition for SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l, infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non-insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; asthma; allergies; benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette's syndrome, can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of SAN_O399_3, SAN_O437_l, SAN_O437_ 4 and SAN_O817_l polypeptides or SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l mRNAs.
Altered expression of SAN_O787_l, SAN_O399_3, SAN_O437_l,
SAN_O437_4 and SAN_O817_l can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, Rnase protection, Northern blotting, other hybridization methods and DNA microarray technology. Assay techniques that can be used to determine levels of a protein, such as
SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays,
Western Blot analysis, ELISA assays and protein tip technology.
Chromosome Assays
The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of
physically adjacent genes). The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
Antibodies
The polypeptides of the invention or their fragments or analogs thereof or cells expressing them can also be used as immunogens to produce antibodies immuno specific for the SAN_O399_3, SAN_O437_l, SAN_O437_4; SAN_O787_l and SAN_O817_l polypeptides. The term "immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
Antibodies generated against the SAN_0787_1, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides can be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today (1983) 4:72) and the EBV-hybridoma
technique (Cole et al, MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).
Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms including other mammals, may be used to express humanized antibodies.
The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography. Antibodies against SAN_O787_l may be used to treat liver cancer. Antibodies against SAN_O399_3, SAN_O437_l,
SAN_O437_4 and SAN_O817_l polypeptides may be employed to treat infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non- insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; asthma; allergies; benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette's syndrome, among others.
Vaccines
Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l, polypeptides, or a fragments thereof, adequate to produce antibody and/ or T cell immune response to protect said animal. For SAN_O399_3, SAN_O437_l, SAN O437_4 and SAN_O817_l antibodies, the protection can be from infections such as bacterial, fungal, protozoan and viral infections, particular infections caused by HIV-1 or HIV-2; pain; cancers; non- insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary t rombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; asthma; allergies; benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette's syndrome, among others. Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides via a vector directing expression of SAN_O787_l, SAN_O399_3, SAN_O437_l,
SAN_O437_4 and SAN_O817_l polynucleotides in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
Further aspect of the invention relates to an immunological/ or vaccine formulation (or composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to SAN_O787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides wherein the composition comprises SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides or SAN_0787__1, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l genes. The vaccine formulation may further comprise a suitable carrier. Since SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water
systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
Screening Assays
SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides of the present invention may be employed in a screening process for compounds which bind the receptor and which activate (agonists) or inhibit activation of (antagonists) the receptor polypeptide of the present invention. Thus, polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See Coligan et al, Current Protocols in Immunology 1(2): Chapter 5 (1991).
SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4, and SAN_O817_l polypeptides are responsible for many biological functions, including various pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l on the one hand and which can inhibit the function of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_0817_1 on the other hand. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as infections such
as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV- 1 or HIV-2; pain; cancers; non-insulin-dependent diabetes
(NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy ; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; asthma; allergies; benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la
Tourette's syndrome.
Antagonists including inverse agonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non-insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; asthma; allergies; benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias,
such as Huntington's disease or Gilles de la Tourette's syndrome.
In general, such screening procedures involve producing appropriate cells, which express the receptor polypeptide of the present invention on the surface thereof. Such cells include cells from mammals, baculovirus, adenovirus, yeast, Drosophila or E. coli. Cells expressing the receptor (or cell membrane containing the expressed receptor) are then contacted with a test compound to observe binding or stimulation or inhibition of a functional response.
One screening technique includes the use of cells which express receptor of this invention (for example, transfected CHO cells) in a system which measures GTP binding activity of coupled G proteins, extracellular pH or intracellular calcium changes caused by receptor activation. In this technique, compounds may be contacted with cells expressing the receptor polypeptide of the present invention. A second messenger response, e.g., signal transduction, changes of GTP binding activity, pH changes, or changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the receptor.
Another method involves screening for receptor inhibitors by determining inhibition or stimulation of receptor-mediated cAMP and/ or adenylate cyclase accumulation. Such a method involves rransfecting a eukaryotic cell with the receptor of this invention to express the receptor on the cell surface. The cell is then exposed to potential antagonists in the
presence of the receptor of this invention. The amount of cAMP accumulation is then measured. If the potential antagonist binds the receptor, and thus inhibits receptor binding, the levels of receptor-mediated cAMP, or adenylate cyclase, activity will be reduced or increased.
Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast based technology as described in U.S. Pat. No. 5,482,835. The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the receptor is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the receptor, using detection systems appropriate to the cells bearing the receptor at their surfaces. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Standard methods for conducting such screening assays are well understood in the art.
Examples of potential SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligand of the SAN_O787_l, SAN_O399_ 3, SAN_O437_l, SAN_O437_4 and SAN_O817_l e.g., a fragment of the ligand, or small molecules which bind to the receptor but do not elicit a response, so that the activity of the receptor is prevented.
Prophylactic and Therapeutic Methods
This invention provides methods of treating abnormal conditions such as, infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non-insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; asthma; allergies; benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette's syndrome, related to both an excess of and insufficient amounts of SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l activity.
If the activity of SAN_0787_1, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l are in excess, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist including inverse agonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of ligands to the SAN _O787_l,
SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_0817_1 or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of SAN_O787_l, SAN_O399_3, SAN_O437_l,
SAN_O437_4 and SAN_O817_l. Polypeptides still capable of binding the ligand in competition with endogenous SAN_O787_l, SAN_O399_3,
SAN_O437_l, SAN_O437_4 and SAN_O817_l may be administered. Typical embodiments of such competitors comprise fragments of the SAN_O787_l,
SAN_O399_3, SAN_O437_l, SAN_O437_4, and SAN_O817_l polypeptides.
In still another approach, expression of the gene encoding endogenous SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56: 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression CRC Press, Boca Raton, Fla. (1988). Alternatively, oligonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al, Nucleic Acids Res (1979) 6:3073; Cooney et al, Science (1988)241:456; Dervan et al, Science (1991) 251: 1360. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
For treating abnormal conditions related to an under-expression of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l and their activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a
compound which activates SAN_O787_l, SAN_O399_3, SAN_O437_l,
SAN_O437_4 and SAN_O817_l i.e. an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of SAN_O787_l, SAN_O399_3,
SAN_O437_l, SAN_O437_4 and SAN_O817_l by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo. For overview of gene therapy, see
Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic
Approaches, (and references cited therein) in Human Molecular Genetics, T.
Strachan and A. P. Read, BIOS Scientific Publishers Ltd (1996). Another approach is to administer a therapeutic amount of SAN_O787_l,
SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides in combination with a suitable pharmaceutical carrier.
Formulation and Administration
Peptides, such as the soluble form of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides, and agonists and
antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administration, and is well within the skill of the art. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
Preferred forms of systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if properly formulated in enteric or encapsulated formulations, oral administration may also be possible. Administration of these compounds may also be topical and/ or localized, in the form of salves, pastes, gels and the like.
The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection.
Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
Polypeptides used in treatment can also be generated endogenously in the subject in treatment modalities often referred to as "gene therapy" as described above. Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
EXAMPLES OF THE INVENTION
The examples below are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The examples illustrate, but do not limit the invention.
BRIEF DESCRIPTION OF THE DRAWINGS.
Figure 1. Expression of the 5 GPCR-like gene transcripts in human tissues. Reverse transcription (RT) - PCR amplification was performed to detect the GPCR-like gene transcripts. A mixture of the cDNA templates synthesized from 18 human tissue poly(A)+ RNAs was used as a template. Aliquots of the RT PCR products were electrophoresed through a 1.5% agarose gel. Lane 1, SAN_0399_3; lane 2, SAN_0437_1; lane 3, SAN_0437_4; lane 4, SAN _0787_1; lane 5, SAN_0817_1. "M" indicates a 100-bp DNA ladder used as a molecular weight standard. The 600 - bp band appears as a brighter band than the other ladder bands.
Figure 2. The expression pattern of SAN_0399_3 in 24 human tissues. Reverse transcription (RT) - PCR amplification was performed to detect SAN_0399_3 transcripts. 24 human tissue poly(A)+ RNAs were used as a template. Aliquots of the RT-PCR products were electrophoresed through a 1.5% agarose gel. The "marker" indicates a 100-bp DNA ladder used as a molecular weight standard. The 600 - bp band appears as a brighter band than the other ladder bands.
Figure 3. The expression pattern of SAN_0437_1 in 24 human tissues. Reverse transcription (RT) - PCR amplification was performed to detect SAN_0437_1 transcripts. 24 human tissue poly (A) + RNAs were used as a
template. Aliquots of the RT-PCR products were electrophoresed through a
1.5% agarose gel. The "marker" indicates a 100-bp DNA ladder used as a molecular weight standard. The 600- bp band appears as a brighter band than the other ladder bands.
Figure 4. The expression pattern of SAN_0437_4 in 24 human tissues. Reverse transcription (RT) - PCR amplification was performed to detect SAN_0437_4 transcripts. 24 human tissue poly(A)+ RNAs were used as a template. Aliquots of the RT-PCR products were electrophoresed through a 1.5% agarose gel. The "marker" indicates a 100-bp DNA ladder used as a molecular weight standard. The 600- bp band appears as a brighter band than the other ladder bands.
Figure 5. The expression pattern of SAN_0787_1 in 24 human tissues. Reverse transcription (RT) - PCR amplification was performed to detect SAN_0787_1 transcripts. 24 human tissue poly(A)+ RNAs were used as a template. Aliquots of the RT-PCR products were electrophoresed through a 1.5% agarose gel. The "marker" indicates a 100-bp DNA ladder used as a molecular weight standard. The 600- bp band appears as a brighter band than the other ladder bands.
Figure 6. The expression pattern of SAN_0817_1 in 24 human tissues. Reverse transcription (RT) - PCR amplification was performed to detect SAN_0817_1 transcripts. 24 human tissue poly(A)+ RNAs were used as a template. Aliquots of the RT-PCR products were electrophoresed through a
1.5% agarose gel. The "marker" indicates a 100-bp DNA ladder used as a molecular weight standard. The 600- bp band appears as a brighter band than the other ladder bands.
Figure 7. Differential expression of SAN_0787_1 mRNA in normal and tumor liver. SAN_ 0787_1 mRNA in total RNA of human normal and tumor liver was detected by analytical PCR method.
Example 1
In silico Gene Finding
Known 227 human GPCR sequences registered in GPCRDB (Horn, F. et al. Nucleic Acids Res., 26:275-279, 1998) were dissected into fragments of 60 amino acid length overlapping with each other in 30 amino acid lengths. The 3,300 fragments generated were submitted to TBLASN search against EST iuman (downloaded l-Jul-2000). 16,573 ESTs with significant sequence homology to the known GPCR (GPCR-like EST) were obtained and assembled. The GPCR-like ESTs were subjected to BLASTN search against Genbank NT and HTG and those involved in the known genes were eliminated. In this invention, ESTs listed in Table 3 have been left after BLASTN. These ESTs are supposed to be involved in novel GPCR.
The selected ESTs were mapped into the HTG sequences as listed in Table 3. The coding regions embedded in these HTG sequences were obtained
as described below. The individual HTG sequences were dissected into fragments of 5000 bases. The resulted fragments were submitted to BLASTX search against GenPept and GeneSeqp(Derwent). 28 single exon regions encoding novel GPCR-like genes including those given in Table 1 and 2 were identified (Table 3), along with some other gene sequences not listed in the Tables.
Table 3
Preparation of Human Tissue cDNA
In order to detect the GPCR-like gene transcripts, first strand cDNAs were generated by reverse transcription of 18 human tissue poly(A)+ RNAs, and the mixture of the 18 tissue cDNAs was used as a template for polymerase chain reaction (PCR). 13 human adult tissue poly(A)+ RNAs were obtained from Clontech Laboratories, Inc. (Palo Alto, CA): adrenal gland, bone marrow, brain, heart, liver, lung, pancreas, placenta, prostate, skeletal muscle, small intestine, spleen and thyroid. Four human fetal tissue poly(A)+ RNAs were also obtained from Clontech Laboratories: brain, kidney, liver and lung. Human colon poly(A)+ RNA was obtained from BioChain Institute, Inc.
(Hayward, CA).
Reverse transcription reaction was carried out in a nuclease-free microcentrifuge tube for each poly(A)+ RNA of the 18 tissues. Two micrograms of poly(A)+ RNA and 1 μg of oligo(dT)ιs (Roche Diagnostics Corp., Indianapolis, IN) were added to the tube and filled up to 15 μl with water. The mixture was heated to 70°C for 10 minutes and then chilled on ice for 5 minutes. Five microliters of 5 X First-Strand Buffer (Invitrogen Corporation Life Technologies Division, Gaithersburg, MD), 1 μl of 25 mM dNTPs (25 mM each dATP, dCTP, dGTP and dTTP), 2.5 μl of 100 mM DTT, 0.5 μl of RNase inhibitor (40 units/ μl; Toyobo Co., Ltd., Osaka, Japan) and 1 μl of Superscript II reverse transcriptase (200 units/ μl; Invitrogen Corporation Life Technologies Division) were added to the mixture, incubated at 42°C for 90 minutes and then heated to 70°C for 10 minutes. The first strand cDNA was diluted by adding 30 μl of water. 1.25 μl aliquots of each adult tissue cDNA and 2.5 aliquots of each fetal tissue cDNA were mixed and filled up to 1 ml with water. The resultant cDNA mixture was designated as the 18-mix cDNA template solution.
PCR Amplification of Novel GPCR-like cDNA Segments
PCR primers were designed for the GPCR-like genes and 2 known human olfactory receptor genes (OR1A2 and OR3A3 genes), and custom - sythesized by Genset Co., Ltd. (Kyoto, Japan). GenBank accession numbers of OR1A2 and OR3A3 genes are G 7144635 and G 5081803, respectively
(Glusman, G. et al Genomics 63:227-245, 2000). Synthesized DNA was diluted to 10 μM with water. When human genomic DNA was used as a PCR template, all the primer sets gave rise to single bands which corresponded with the expected sizes of the PCR products. Each tube contained a 50 μl reaction mixture which consisted of 67.4 mM Tris-HCI (pH8.8)/ 16.7 mM
(NH4)2S04/2 mM MgCl2/6.74 μM Na2EDTA/ 10% DMSO/0.2 mM dNTPs (0.2 mM each dATP, dCTP, dGTP and dTTP)/ 10 mM 2-mercaptoethanol/ 10 ng/μl human genomic DNA (Clontech Laboratories, Inc.)/0.5 μM forward primer/ 0.5 μM reverse primer/ 0.035 units/ μl Ex Taq DNA polymerase
(Takara Shuzo Co., Ltd., Kyoto, Japan). Forty cycles of PCR was performed under the following conditions: initial denaturation at 94 °C for 5 min; then 40 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min; followed by a final extension of 72°C for 10 minutes. Aliquots of the PCR products were analyzed by agarose gel electrophoresis.
Forty cycles of RT-PCR were next carried out in two microcentrifuge tubes for each gene. Each tube contained a 100 μl reaction mixture which consisted of 67.4 mM Tris-HCI (pH8.8)/ 16.7 mM (NH4)2SO4/2 mM MgCl2/6.74 μM Na2EDTA/ 10% DMSO/0.2 mM dNTPs (0.2 mM each dATP, dCTP, dGTP and dTTP)/ 10 mM 2-mercaptoethanol/0.04% 18-mix cDNA template solution /0.5 μM forward primer/ 0.5 μM reverse primer/ 0.035 units/ μl Ex Taq DNA polymerase (Takara Shuzo Co., Ltd., Kyoto, Japan). PCR was performed under the following conditions: initial denaturation at 94°C for 5 min; then 40 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min; followed by a final extension of 72°C for 10 minutes. RT-PCR products
were purified using Millipore MultiScreen FB 96-Well Filtration Plate
(Millipore Corp., Bedford, MA). Purification was performed according to the manufacturer's instruction. A 200 μl PCR product for each gene was loaded into one well of the MultiScreen plate. At the final step of purification, 57 μl of
10 mM Tris-HCI, pH8.0/0.1 mM Na2EDTA were added to each well of the FB plate, and the plate was centrifuged to elute the purified DNA from the filters.
Five-microliter aliquots of the purified PCR products were electrophoresed through a 1.5% agarose gel and stained with ethidium bromide.
READY- LOAD 100 bp DNA Ladder (Invitrogen Corporation) was used as a molecular weight standard.
Neither of the two primer sets for the known olfactory receptor genes showed any band. Among the GPCR-like genes examined, RT-PCR products derived from five genes showed the bands with expected size, see Figure 1. Nucleotide sequences of the PCR primers for the genes of this invention and for which transcripts were detected in the human tissues are listed in Table 4 and the expected sizes (bp) of the PCR products are also listed. This result demonstrates that the novel GPCR-like genes expressed in the human non-olfactory tissues should have the functions other than olfactory receptors.
Table 4.
PCR primers of GPCR-like genes.
Example 2
1) Tissue Specific Expression of the 5 Genes
In order to examine tissue specific expression of the 5 genes, RT-PCR was performed with 24 human tissue RNAs. Poly(A)+ RNA of human adult adrenal gland, bone marrow, brain, heart, kidney, liver, lung, pancreas, placenta, prostate, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea and uterus and poly(A)+ RNA of human fetal brain , kidney and liver were purchased from Clontech
Laboratories. Human adult colon poly (A) + RNA was obtained from BioChain
Institute, Inc. Reverse transcription was carried out in the same way as described earlier. The first strand cDNA solution prepared from each tissue
RNA was further diluted by 80 times with water. PCR was performed in a 50 μl reaction which consisted of 67.4 mM Tris-HCI (pH8.8)/ 16.7 mM
(NH4)2SO4/2 mM MgCl2/6.74 μM Na2EDTA/ 10% DMSO/0.2 mM dNTPs (0.2 mM each dATP, dCTP, dGTP and dTTP)/ 10 mM 2-mercaptoethanol/4% the diluted first strand cDNA solution/0.5 μM forward primer/0.5 μM reverse primer/0.035 units/ μl Ex Taq DNA polymerase (Takara Shuzo Co., Ltd.,
Kyoto, Japan). Nucleotide sequences of the PCR primers were shown in Table
4. Forty cycles of PCR was performed under the following conditions: initial denaturation at 94°C for 5 min; then 40 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min; followed by a final extension of 72°C for 10 minutes. After the reaction, a 9-μl aliquot of each PCR reaction was electrophoresed through a 1.5% agarose gel and stained with ethidium bromide. READY-LOAD 100 bp
DNA Ladder was used as a molecular weight standard (Figure 2-6).
SAN_0399_3 mRNA was expressed highly in placenta, prostate, skeletal muscle, spinal cord, stomach, testis, thymus and trachea.
SAN_0437_1 mRNA was expressed highly in placenta, prostate, small intestine, spinal cord, spleen, testis, thymus, trachea, uterus and fetal kidney.
SAN_0437_4 mRNA was ubiquitously expressed in the human tissues.
SAN_0787_1 mRNA was expressed highly in colon, kidney, liver and testis.
SAN_0817_1 mRNA was expressed highly in testis.
Example 3
Full Length cDNA Cloning
To obtain a full-length cDNA clone for SAN_0787_1, RT-PCR was performed using human liver cDNA as a template. Human liver poly(A)+RNA was purchased from Clontech Laboratories (Cat. #6510-1, lot 0030614, 1 male Caucasian, 35 yr., sudden death). First strand cDNA template was synthesized by reverse transcription with Superscript π and oligo(dT) primer as described earlier. Nucleotide sequences of PCR primers used were 5'-GCGGCCGCTGTTGAGAATTTACTCCTTGTTG-3' (forward primer) and 5'-GCGGCCGCTAATGGTCTCTTAATGAAATAAT-3' (reverse primer). Both primers contained a linker sequence 5 '-GCGGCC-3' at their 5' ends for cloning into pCR-Blunt IT-TOPO vector (Invitrogen Corp.). PCR was conducted using KOD-plus DNA polymerase (Toyobo Co., Ltd.) and the PCR product was inserted into pCR-Blunt π-TOPO vector using topoisomerase. The resultant plasmid was introduced into Escherichia coli strain TOP 10. Transformants were picked up and the nucleotide sequences of their cDNAs were analyzed
using an ABI 3700 DNA sequencer (Applied Biosystems, Foster City, CA). The nucleotide sequence was identical to the sequence of SEQ ID NO: 1 in the
Table 1 except for one nucleotide substitution at position 697, which led to the amino acid change at the residue 233 from Arg (CGG) to Trp (TGG).
SAN_0787_1 cDNA was also cloned from human kidney poly(A)+RNA (Clontech Laboratories, Cat.#6538-1) and the sequence was identical to that of the cDNA cloned from human liver poly(A)+RNA. To confirm the nucleotide at position 697, SAN_0787_1 DNA was amplified by PCR from human genomic DNA templates that were prepared from two donors, and the PCR products were cloned into pCR-Blunt π-TOPO and the DNA inserts were sequenced. The DNAs were purchased from Roche Diagnostics Corp. and Promega Corp. (Madison, Wl). The nucleotide sequencing analysis revealed that the DNA from Roche Diagnostics (Cat. No.1691112, Lot No.87931520) had a Trp (TGG) codon at the residue 233 and the DNA from Promega (Cat. No.G1471, lot No.A9273001) had an Arg (CGG) codon. These results indicated the presense of single nucleotide polymorphism at nucleotide position 697 of SAN_0787_1 cDNA. The plasmid containing an open reading frame of SAN_0787_1 derived from the Promega's genomic DNA was used in further experiments. An SAN_0787_1 cDNA insert was excised with Notl from the plasmid and subcloned into a Notl site of mammalian expression vector pcDΝA3.1(+) (Invitrogen Corp.). The plasmid having the cDNA insert in the same orientation as CMV promoter of the expression vector was selected. The resultant plasmid can express SAN_0787_1 protein in mammalian cells such as CHO cells, COS cells, 293 cells etc. when the cells are transfected with the
plasmid by conventional methods. Expression of GPCR protein is confirmed by Western blotting of transfected cell lysates using SAN_0787_1 -protein specific antibody which was prepared as described below.
Example 4
Production of Antibodies
Peptide Synthesis
A peptide having an amino acid sequence
(N)-Met-Lys-Arg-Lys-Asn-Phe-Thr-Glu-Val-Ser-Glu-(C) was chosen from the amino-terminal region of SAN_0787_1 protein (SEQ ID NO: 2 in the Table 2) in order to produce a specific antibody for the protein. This peptide with an additional Cys residue at its C-terminal was chemically synthesized using ABI 433A Peptide Synthesizer (PE Applied Biosystems). A Keyhole limpet hemocyanin (KLH), a carrier for the peptide, would bind later to the Cys residue. Boc-amino acid support (0.5 mmol) was used as a starting material and peptide synthesis was performed by conventional methods. Synthesized peptide was released from the support material by treatment with hydrogen fluoride. The peptide was extracted with 0.1%) trifluoroacetic acid and freeze-dried. The peptide was then purified by high-performance liquid chromatography (HPLC) in an acetonitrile-0.1% trifluoroacetic acid solvent system using HPLC Model LC8A (Shimadzu Corp., Kyoto, Japan). Purity of
the peptide obtained was evaluated by HPLC and amino acid composition analysis.
Conjugation of Peptide with Keyhole Limpet Hemocyanin
Purified peptide was coupled with KLH using
N-(6-maleimidocaproyloxy)-succinimide (EMCS, manufactured by Dojindo Laboratories, Kumamoto, Japan). An incorporation ratio of the peptide into the carrier protein was evaluated by amino acid composition analysis.
Immunization of Rabbits with KLH-conjugated Peptide
The peptide-carrier conjugate was suspended in saline solution at a concentration of 1 mg/ml and used to immunize rabbits. The conjugate was mixed well with an equal volume of complete Freund's adjuvant and then injected into the back of two rabbits (body weight 2-2.5 kg) hypodermically or intradermically. For the second or later immunization, incomplete Freund's adjuvant was used. Immunization was performed every two weeks and after the second immunization test-blood collection was performed and the antibody titer in the serum was measured by a solid phase method of an enzyme-linked innmunosorbent assay (ELISA). Antigen peptide was coated on a 96-well plate for ELISA, and a Western horseradish-peroxidase labeled anti-rabbit IgG antibody was used as a second antibody. The exsanguination was conducted 2 months after the first immunization.
Purification of Antibody from Immunized Rabbit Antiserum with Peptide
Affinity Column
The antibody was purified after blood collecting using a peptide affinity column. About 8 mg of the peptide was combined with 5 ml of carrier agarose Affi-Gel 102 (Bio-Rad Laboratories, Inc., Hercules, CA) by conventional methods. About 15 ml of the antiserum was diluted with an equal volume of PBS (containing 0.02 M phosphoric acid buffer solution (pH 7.0), 0.9% sodium chloride) and a precipitate was obtained by the ammonium sulfate precipitation (final concentration 40%) method. This precipitate was dissolved in PBS, and then dialyzed with PBS. The dialyzed antibody solution was used as the semi-purified IgG fraction.
Chromatography operation with an affinity column was carried out in three steps. That is, semi-purified IgG fraction was charged to the affinity column (5 ml), and re-charging a flow- through fraction into the column was repeated 3 times. Next, the column was washed with an excess volume of PBS containing 1 M sodium chloride, and then 15 ml of 4 M magnesium chloride solution, 15 ml of 3.5 M potassium thiocyanate solution, and 10 ml of 0.1 M glycine hydrochloric acid buffer solution (pH 2.3) were poured in the column one by one, and the antibody which had specifically bound to the peptide on the column carrier was eluted as an affinity purified antibody. Since the target antibody was contained in the eluates of 4 M magnesium chloride solution and 3.5 M potassium thiocyanate solution, each eluate was collected. Antibody concentration of the eluate of 4 M magnesium chloride
solution was 61.8 μg/ml for one rabbit and 63.1 μg/ml for the other rabbit.
Antibody concentration of the eluate of 3.5 M potassium thiocyanate solution was 35.0 μg/ml for one rabbit and 40.0 μg/ml for the other rabbit. These antibody cotaining eluates were dialyzed to PBS and used in further experiments as an specific antibody to SAN_0787_1 protein. Titers of the antibody solutions were measured using an ELISA system as described above.
Example 5
Differential Expression of SAN_0787__1 mRNA in Normal and Tumor Liver
Quantitative Analysis of Gene Expression by TaqMan PCR (Real-Time RT-PCR)
An SAN_0787_1 mRNA expression level was analyzed in human normal and tumor liver tissues using ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). This analysis was conducted according to User Bulletin #2 that was provided by the manufacturer. The method involved preparing a standard curve for each gene of interest by assaying a dilution series prepared from a first-strand cDNA in a reverse transcriptase-5'-exonuclease PCR amplification assay. A threshold cycle (ct) for each member of the reverse transcribed-amplification reaction was determined and plotted versus the log of the amount of a first-strand cDNA in dilution series. The plot was used to determine an RNA
equivalent from which the normalized human GAPDH RNA equivalent for
SAN_0787_1 was determined. The assay was used to determine the level of expression of SAN_0787_1 mRNA in sample tissues.
Human liver total RNAs were purchased from Biochain: human normal liver total RNA (Cat. No.61009, Lot.A503276), human tumor liver total RNA (Cat. No.64003, Lot.A505425). The PCR primer pairs and probes were designed using the Primer Express 1.0 program (PE Applied Biosystems). The oligonucleotide hybridization probe and the primer pairs for SAN_0787_1 gene with the following sequences were synthesized: TaqMan probe 5'-FAM-ATATCCTTGGCAGGCTGCGCAA-TAMRA-3', forward primer
5'-ACGAATGCTTTCCAGCCTGA-3', reverse primer
5'-AGCAGTTGTTGGTGGCCAAA-3,. The probe and the primer sequences for human GAPDH gene were as follows: TaqMan probe 5'-JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA-3', foward primer
5'-GAAGGTGAAGGTCGGAGTC-3', reverse primer
5'-GAAGATGGTGATGGGATTTC-3\ The primers were purchased from Amersham Biosciences. The probes were obtained from Applied Biosystems.
First- strand cDNA Synthesis
First-strand cDNA synthesis was performed using TaqMan Reverse Transcription Reagents Kit (PE Applied Biosystems, Cat. No.N808-0234, Lot No.D02956). One microgram total RNA of each sample was dissolved in 20 μl of RNase-free water and denatured for 10 minutes at 65°C and chilled on ice.
First-strand cDNA was synthesized in two steps at 25°C for 10 minutes and at
48°C for 30 minutes in a 100-μl reaction which consisted of 1 x TaqMan RT buffer, 5.5 mM MgCl2, 500 μM each dNTP, 2.5 μM Oligo(dT)ι6, 0.4 units/ μl
RNase inhibitor, 1.25 units/ μl MultiScribe Reverse Transcriptase and 10 ng/ μl denatured total RNA. The reaction was terminated by incubating at
95°C for 5 minutes followed by addition of 2 μl of 0.5 M EDTA to each reaction.
Then 5 μl aliquots of purified cDNA was used for analytical PCR. Human normal liver first-strand cDNA was synthesized in the same way in another tube to prepare reaction standards. The same kind of reaction was carried out in all the samples without reverse transcriptase in order to confirm no genomic DNA contamination in the RNA samples.
Analytical PCR
All of the RT-PCR reactions were performed in duplicates with a final volume of 50 μl. Each RT-PCR reaction includes 25 μl TaqMan Universal PCR Master mix (PE Applied Biosystems), 200 nM forward primer, 200 nM reverse primer, 50 nM probe and 5 μl of first-strand cDNA. RT-PCR conditions were 2 minutes at 50°C, 10 minutes at 95°C, and 50 cycles of 15 seconds at 95°C, 1 minute at 60°C. Standard curves were obtained each time a gene assayed using first-strand cDNA prepared from 7 standard dilution series (designated as quantity 2, 1, 0.4, 0.2, 0.1, 0.04, 0.02). Reactions were run in 96-well plates (MicroAmp Optical 96-well Reaction Plate, PE Applied Biosystems) on ABI PRISM 7700 Sequence Detection System.
Calculation of Relative RNA Equivalents
The equivalent amount of control RNA in each sample was calculated from a plot of Ct versus logιo[cDNA] (Starting Quantity) based on the control cDNA dilution run for each gene. Human GAPDH was used as an internal control to normalize differences in an amount of input sample RNA. mRNA expression of SAN_0787_1 was higher in tumor liver than in normal liver by about 100 fold (Figure 7). No amplification signal was detected in analytical PCR of all the samples when the first strand cDNA was synthesized without reverse transcriptase. This showed no contamination of genomic DNA in the total RNA samples used. Therefore SAN_0787_1 gene can be used as a cancer marker gene especially for liver cancer.
Example 6
Detection of Cancer Marker Genes Using DNA Microarray
Preparation of Probe DNA
Probe DNA of SAN_0787_1 gene was prepared for microarray analysis by RT-PCR using SAN_0787_l-gene specific primers which were listed in Table 4. Forty cycles of RT-PCR were carried out in four microcentrifuge tubes. Each tube contained 100-μl reaction mixture which consisted of 67.4 mM Tris-HCI (pH8.8)/ 16.7 mM (NH4)2SO4/2 mM MgCl2/6.74 μM
Na2EDTA/ 10% DMSO/0.2 mM dNTPs (0.2 mM each dATP, dCTP, dGTP and dTTP)/ 10 mM 2-mercaρtoethanol/0.04% 18-mix cDNA template solution
/0.5 μM forward primer/0.5 μM reverse primer/0.035 units/μl Ex Taq DNA polymerase (Takara Shuzo Co., Ltd.). Preparation of 18-mix cDNA template was described earlier. PCR was performed under the following conditions: initial denaturation at 94°C for 5 min; then 40 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min; followed by a final extension of 72°C for 10 minutes.
RT-PCR products were purified using Millipore MultiScreen FB 96-Well
Filtration Plate (Millipore Corp.). Purification was performed according to the manufacturer's instruction. A 200-μl PCR product was loaded into one well of the MultiScreen plate. At the final step of purification, 57 μl of 10 mM
Tris-HCI, ρH8.0/0.1 mM Na2EDTA were added to each well of the FB plate, and the plate was centrifuged to elute the purified DNA from the filters.
Purified DNA from the two wells were pooled into one microtube and DNA concentration of the purified PCR product was determined by use of
PicoGreen dsDNA Quantitation Reagent Kit (Molecular Probes Inc., Eugene,
OR; Code No. P-7589) according to the manufacturer's instruction. DNA concentration was adjusted to 50 ng/μl, an aliquot of the purified PCR product was electrophoresed through a 1.5% agarose gel and stained with ethidium bromide. READY-LOAD 100 bp DNA Ladder (Invitrogen
Corporation) was used as a molecular weight standard. A single band was detected and its size was about 220 bp as expected. Nucleotide sequence of the PCR product was then determined and confirmed to be identical to that of
SAN_0787_1 gene. Probe DNAs of human house keeping genes β-actin
(GAPDH; GenBank GL28251) and gfyceraldehyde-3-ρhosρhate
dehydrogenase (GenBank GL4503912) were also prepared in the same way and used as positive control spot DNA.
Gene-specific oligonucleotide probes which are about 20 to 80 nucleotides in length can be also used as spot DNAs for detection of expression of SAN_0787_1 gene in normal and tumor tissues.
Spotting Probe DNA onto Microarray Slides
Probe DNAs of human SAN_0787_1 gene, β-actin gene, GAPDH gene and other human genes including GPCR genes were spotted onto microarray slides Type 7 (Amersham Biosciences, Inc., Uppsala, Sweden; Cat. No.RPK0331) using Generation III Array Spotter (Amersham Biosciences, Inc.) according to a manufacturer's instruction. Microarray SocreCard (Amersham Biosciences, Inc., Cat. No. RPK1161) was also spotted onto the same slides. ScoreCard included controls for dynamic range, retio, and positive and negative hybridization controls. For oligonucleotide microarrays spotting methods are optimized for probe DNA to stick well to microarray slides using coated microscope slides such as poly-L lysine slides.
Labeling of Target Nucleotides
Total RNA or poly(A)+RNA is isolated from human normal and diseased tissues such as normal liver and tumor liver tissues by conventional methods. For example RNA is extracted from tissue samples using Trizol reagent
(Invitrogen Corp., Cat. No.15596) according to the manufacturer's protocol.
Poly(A)+RNA is purified from total RNA using commercially available kits such as PolyATtract mRNA Isolation System (Promega Corp., Cat. No. Z5200).
Quality of the isolated RNA is evaluated using Agilent Bioanalyzer 2100 and
LabChip Kits (Agilent Technologies, Inc., Palo Alto, CA).
cDNA is synthesized by reverse transcription from RNA and fluorescent-dye labeled nucleotides, Cy3- and Cy5-dCTP (Amersham Biosciences, Inc., Cat. No. PA53023 and PA55023, respectively), are incorporated into cDNA during its synthesis reaction. In a representative analysis, human normal liver and tumor liver samples are labeled with Cy3- and Cy5-dCTP, respectively. cDNA synthesis, labeling and purification are conducted according to the protocol provided by the manufacturer of microarray slides Type 7. If the amount of total RNA or poly(A)+RNA sample is small, RNA can be amplified using T7 RNA polymerase according to the linear amplification method based on Eberwine protocol (Van Gelder, R. N. et al. Proc. Natl. Acad. Sci. U.S.A. 87(5): 1663-1667, 1990) or using commercially available kits such as RiboAmp RNA Amplification Kit (Takara Shuzo Co., Ltd.). After amplification of RNA, cDNA is synthesized and labeled with Cy3- and Cy-5 dCTP.
Hybridization of DNA Microarray with Labeled Targets
Hybridization of DNA microarray with labeled target cDNAs is performed manually, or automatically using Automated Slide Processor
(Amersham Biosciences, Inc.), in accordance with the protocol provided by the manufacturer of microarray slides Type 7. In a representative analysis,
Cy3-labeled normal liver cDNA and Cy5-labeled tumor liver cDNA are co-hybridized with arrayed probe DNAs containing SAN_0787_1 DNA. After washing the slides, they are processed to fluorescent signal detection steps.
Scanning of Microarray Slides and Analysis of Gene Expression
Scanning of the slides is performed using array scanner GenePix 4000B (Axon Instruments, Inc., Union City, CA) at 532 nm (Cy3) and 635 nm (Cy5). Raw scanned images are processed using GenePix Pro microarray analysis software (Axon Instruments, Inc.). Cy3 and Cy5 scans for each slide are superimposed on to each other and values corresponding to the level of fluorescent intensity for each spot are obtained and exposed to an Excel spreadsheet. Local background is subtracted from the fluorescent value of each spot to obtain a net value. Fluorescence signal intensities of Microarray ScoreCard control spots are used to normalize the input labeled cDNA.
By using DNA microarrays containing SAN_0787_1 gene, diagnosis of liver tumor can be efficiently conducted. DNA microarrays containing SAN_0787_1 gene and other cancer marker genes provide high-throughput diagnosis tools for detecting various tumors in patients' tissues.
Expression of GPCR.
Expression and purification of QPCRs are achieved using bacterial or virus-based expression systems. For expression of GPCRs in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are introduced into suitable bacterial hosts, e.g. , Escherichia coli BL21 (DES) . Antibiotic resistant bacteria express GPCRs upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG) .
Expression of GPCRs in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica California nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of the baculovirus is replaced with cDNA encoding GPCRs by either homologous recombination or bacterial-mediated polyhedrin promoter which drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodopter frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to the baculovirus. (See Engelhard, E.K. et al. (1994) Proc. Natl. Acad, Sci. USA 91: 3224-3227; Sandig, V et al. (1998) Hum. Gene Ther. 7: 1997-1945)
In most expression systems, GPCRs are synthesized as a fusion protein with, e. g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG
or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26 kDa enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech) . Fo 1 lowing purification, the GST moiety can be proteolytically cleaved from GPCRs at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN) . Methods for protein expression and purification are discussed in Ausubel (1995, ch. 10 and 16).
Production of GPCR specific antibodies
GPCRs substantially purified using polyacrylamide gel electrophoresis or other purification techniques, are used to immunize rabbits and to produce antibodies using standard protocols. Rabbits are immunized with the purified GPCRs suspended in complete Freund's adjuvant. Resulting antisera are tested for anti-GPCRs activity by, for example, binding the GPCRs to a substrate, blocking with 1% BSA, reacting with the rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
Information of "SEQ ID NO"
SEQ ID NO: 1 is nucleotide sequence of SAN_O787_l SEQ ID NO: 2 is amino acid sequence of SAN_O787_l
SEQ ID NO: 3 is nucleotide sequence of SAN_O399_3 SEQ ID NO: 4 is amino acid sequence of SAN_O399_3
SEQ ID NO: 5 is nucleotide sequence of SAN_O437_l SEQ ID NO: 6 is amino acid sequence of SAN_O437_l
SEQ ID NO: 7 is nucleotide sequence of SAN_O437_4 SEQ ID NO: 8 is amino acid sequence of SAN_O437_4
SEQ ID NO: 9 is nucleotide sequence of SAN_O817_l SEQ ID NO: 10 is amino acid sequence of SAN_O817_l
SEQ ID NO: 11 is forward primer of SAN_O399_3 SEQ ID NO: 12 is reverse primer of SAN_O399_3 Product size is 453
SEQ ID NO: 13 is forward primer of SAN_O437_l SEQ ID NO: 14 is reverse primer of SAN_O437_l Product size is 495
SEQ ID NO: 15 is forward primer of SAN_O437_4
SEQ ID NO: 16 is reverse primer of SAN_O437_4
Product size is 369
SEQ ID NO: 17 is forward primer of SAN_O787_l SEQ ID NO: 18 is reverse primer of SAN_O787_l Product size is 223
SEQ ID NO: 19 is forward primer of SAN_O817_l SEQ ID NO: 20 is reverse primer of SAN_O817_l Product size is 407.