WO2002102321A2 - Proteines et acides nucleiques les codant - Google Patents

Proteines et acides nucleiques les codant Download PDF

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WO2002102321A2
WO2002102321A2 PCT/US2002/019522 US0219522W WO02102321A2 WO 2002102321 A2 WO2002102321 A2 WO 2002102321A2 US 0219522 W US0219522 W US 0219522W WO 02102321 A2 WO02102321 A2 WO 02102321A2
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polypeptide
amino acid
nucleic acid
seq
acid sequence
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PCT/US2002/019522
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WO2002102321A3 (fr
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David W. Anderson
Xiaojia Guo
Vladimir Y. Gusev
John L. Herrmann
Li Li
Peter S. Mezes
Carol E. A. Pena
Steven K. Spaderna
Mei Zhong
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Curagen Corporation
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Priority to AU2002330857A priority Critical patent/AU2002330857A1/en
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Publication of WO2002102321A3 publication Critical patent/WO2002102321A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides.
  • novel nucleic acids and polypeptides are referred to herein as MOLX, or MOL1, MOL2, MOL3, MOL4, MOL5, MOL6, MOL7, MOL8, MOL9, MOL10, MOL11, MOL12, MOL13, MOL14, MOL15, MOL16, MOL17, MOL 18, MOL19, MOL20, MOL21 or MOL22 nucleic acids and polypeptides.
  • MOLX nucleic acid or polypeptide sequences.
  • the MOLX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a MOLX nucleic acid sequence.
  • the invention also includes an isolated nucleic acid that encodes a MOLX polypeptide, or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 114, 116, 118, 120, 123, 125, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, and 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203 and 205.
  • the invention also features antibodies that immunoselectively bind to MOLX polypeptides, or fragments, homologs, analogs or derivatives thereof.
  • the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically-acceptable carrier.
  • the therapeutic can be, e.g., a MOLX nucleic acid, a MOLX polypeptide, or an antibody specific for a MOLX polypeptide.
  • the invention includes, in one or more containers, a therapeutically- or prophylactically- effective amount of this pharmaceutical composition.
  • the invention also includes methods to identify specific cell or tissue types based on their expression of a MOLX. Also included in the mvention is a method of detecting the presence of a MOLX nucleic acid molecule in a sample by contacting the sample with a MOLX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a MOLX nucleic acid molecule in the sample.
  • compositions of the present invention will have efficacy for treatment of patients suffering from Cancer including pancreatic cancer, adenoma, brain tumor, colon cancer breast cancer, prostate cancer, testis cancer, neurological disorders including age-related disorders, Alzheimer's disease, Stroke, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Behavioral disorders, Addiction, Anxiety, Pain, nephropathy, neurodegenerative disorders, Aneurysms, Fibromuscular dysplasia, metabolic disorders including, failure to thrive, nutritional edema, hypoproteinemia, trypsinogen deficiency disease, chronic and heriditary pancreatitis, enterkinase defieciency, Hypercholesterolemia, Obesity, Diabetes, cardiac disorders comprising tachycardia, erythroderma, long QT syndrome, heart block, Ataxia-telangiectasia, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stea, stea,
  • the method includes contacting a test compound with a MOLX polypeptide and determining if the test compound binds to said MOLX polypeptide. Binding of the test compound to the MOLX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
  • a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., Cancer including pancreatic cancer, adenoma, brain tumor, colon cancer breast cancer, prostate cancer, testis cancer, neurological disorders including age-related disorders, Alzheimer's disease, Stroke, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Behavioral disorders, Addiction, Anxiety, Pain, nephropathy, neurodegenerative disorders, Aneurysms, Fibromuscular dysplasia, metabolic disorders including, failure to thrive, nutritional edema, hypoproteinemia, trypsinogen deficiency disease, chronic and heriditary pancreatitis, enterkinase defieciency, Hypercholesterolemia, Obesity, Diabetes, cardiac disorders comprising tachycardia, erythroderma, long QT syndrome, heart block, Ataxia-telangiectasia, Cardiomyopathy
  • a disclosed encoded MOLli protein has 715 amino acid residues, referred to as the MOLli protein.
  • the disclosed MOLli polypeptide sequence is presented in Table IT using the one-letter amino acid code.
  • a disclosed encoded MOLln protein has 796 amino acid residues, referred to as the MOLln protein.
  • the disclosed MOLln polypeptide sequence is presented in Table 1 AAE using the one-letter amino acid code.
  • MOLlp ORF terminates at a GAG codon at nucleotides 3055-3057. As shown in Table
  • Table 1AAH MOLlp nucleotide sequence (SEQ ID NO: 164).
  • bacterial products are actual ligands for TLRs, or whether they generate ligands via as yet unidentified pattern recognition receptors, has yet to be determined.
  • Signaling pathways activated via the TIR domain trigger the activation of downstream kinases, and transcription factors such as NF-kappaB, and involve the adaptor protein MyD88, which itself contains a TIR domain.
  • nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated, for example but not limited to, in various pathologies /disorders as described below:
  • VHL Von Hippel-Lindau
  • the disclosed MOL3 protein (SEQ ID NO:6) also has good identity with a number of complement compontent proteins, as shown in Table 3C.
  • the disclosed Wnt 8-like protein, MOL4a (also referred to herein as AC004826), is encoded by a nucleic acid, 1064 nucleotides long (SEQ ID NO:7).
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 4-6 and ending with a TGA codon at nucleotides 1057-1059.
  • Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4A, and the start and stop codons are in bold letters.
  • the encoded protein having 351 amino acid residues is presented using the one-letter code in Table 4B (SEQ ID NO:8).
  • the disclosed nucleic acid MOL4a sequence has 881 of 1050 bases (83%) identical to a Mus musculus Wnt 8 mRNA (GENBANK-ID: MMWNT8DPT
  • the disclosed Wnt 8-like protein, MOL4b (also referred to herein as CG53138-05), is encoded by a nucleic acid, 1155 nucleotides long (SEQ ID NO: 140).
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 41-43 and ending with a TGA codon at nucleotides 1148-1150.
  • Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4C, and the start and stop codons are in bold letters.
  • the MOL4b 369 amino acid polypeptide (SEQ ID NO: 141) encoded by SEQ ID NO:140 is presented using the one-letter amino acid code in Table 4D.
  • the Psort profile for MOL4b predicts that this sequence has a signal peptide and is likely to be localized outside the cell with a certainty of 0.4132.
  • NOV4b could also be localized to the lysosome (lumen) with a certainty of 0.1900, to the endoplasmic reticulum (membrane) with a certainty of 0.1000, or to the endoplasmic reticulum (lumen) with a certainty of 0.1000.
  • the most likely cleavage site for a MOL4b peptide is between amino acids 42and 43 (GRS-VN) based on the SignalP result.
  • Expression information was derived from the tissue sources of the sequences that were included in the derivation of the sequence of MOL4b and MOL4c.
  • the sequences are predicted to be expressed in the teratocarcinoma cell line NT2 because of the expression pattern of (GENBANK-ID: gb:GENBANK-ID:AB057725
  • the disclosed Wnt 8-like protein, MOL4d (also referred to herein as CG53138-04), is encoded by a nucleic acid, 1597 nucleotides long (SEQ ID NO: 144).
  • SEQ ID NO: 144 An open reading frame was identified beginning with an ATG initiation codon at nucleotides 41-43 and ending with a TAG codon at nucleotides 1166-1 168.
  • Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4G, and the start and stop codons are in bold letters.
  • the disclosed Wnt 8-like protein, MOL4f (also referred to herein as 250059708), is encoded by a nucleic acid, 952 nucleotides long (SEQ ID NO: 148).
  • An open reading frame was identified beginning with an GGC initiation codon at nucleotides 2-4 and ending with a GGC codon at nucleotides 950-952.
  • Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4K, and the start and stop codons are in bold letters. Because the start and stop codons for MOL4f are not tradistional initiation and termination codons, MOL4f could be a partial reading frame that extends further in the 5' and/or 3' directions.
  • MOL4g (also referred to herein as 246861879), is encoded by a nucleic acid, 726 nucleotides long (SEQ ID NO: 150).
  • An open reading frame was identified beginning with an CGCinitiation codon at nucleotides 1-3 and ending with a GCG codon at nucleotides 724-726.
  • Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4M, and the start and stop codons are in bold letters. Because the start and stop codons for MOL4g are not tradistional initiation and termination codons, MOL4g could be a partial reading frame that extends further in the 5' and/or 3' directions.
  • WISPl cDNA encodes a 367-amino acid protein.
  • Mouse and human WISPl proteins are 84% identical; both have hydrophobic N-terminal signal sequences, 38 conserved cysteine residues, and 4 potential N-linked glycosylation sites. Alignment of the three human WISP proteins showed that WISPl and WISP3 are most similar (42%), whereas WISP2 had 37% identity with WISPl and 32% identity with WISP3.
  • the MOL protein encoded by SEQ ID NO: 9 has 69 amino acid residues and is presented using the one-letter code in Table 5B.
  • the Psort profile for MOL5 predicts that this sequence has a signal peptide and is likely to be localized at the mitochondrial intermembrane space with a certainty of 0.8800.
  • the protein of the invention does not appear to contain a predictable signal peptide.
  • Tbeta4 topically or intraperitoneally increased reepithelialization by 42% over saline controls at 4 d and by as much as 61% at 7 d post- wounding. Treated wounds also contracted at least 11% more than controls by day 7. Increased collagen deposition and angiogenesis were observed in the treated wounds.
  • Tbeta4 stimulated keratinocyte migration in the Boyden chamber assay. After 4-5 h, migration was stimulated 2-3 -fold over migration with medium alone when as little as 10 pg of Tbeta4 was added to the assay.
  • the disclosed novel Trypsin-like MOL6c nucleic acid of 516 nucleotides (also referred to as 246861979) is shown in Table 6E.
  • An open reading begins with an CGC initiation codon at nucleotides 1-3 and ends with a GCGcodon at nucleotides 514-516.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 6E, and the start and stop codons are in bold letters. Because the start and stop codons of MOL6c are notguideoonal initiation and termination codons, MOL6c could be a partial reading frame extending further in the 5' and/or 3' directions.
  • the resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the sequence reported below, which is designated MOL6e, Accession Number GM_87760758_A_da
  • MOL6e Accession Number GM_87760758_A_da
  • Table 6J An open reading frame begins with an ATG initiation codon at nucleotides 8-10 and ends with a TGA codon at nucleotides 713-715.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 61, and the start and stop codons are in bold letters.
  • MOL6e also has high homology to the proteins shown in the BLASTX alignment data in Table 6M.
  • Rowen et al. (1996) mapped the gene corresponding to the third pancreatic trypsinogen cDNA by fluorescence in situ hybridization. They used a cosmid clone containing 3 trypsinogen genes. Strong hybridization to chromosome 7 and weaker hybridization to chromosome 9 were observed. They isolated and partially sequenced 4 cosmid clones from the chromosome 9 region. They found that the region represents a duplication and translocation of a DNA segment from the 3-prime end of the TCRB locus that includes at least seven V(beta) elements and a functional trypsinogen gene denoted T9.
  • MOL7 was found to have 290 of 290 amino acid residues (100 %) identical to the 290 amino acid residue hypothetical 32.6 kD protein from Homo sapiens (human) (ACC: CAB 70765). This protein has similarity to kallikrein.
  • MOL8a A novel human Acetyl LDL Receptor-like nucleic acid was identified by TblastN using CuraGen Corporation's sequence file for MOL probes or homologs and run against the Genomic Daily Files made available by GenBank. The nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein. The disclosed novel MOL8a nucleic acid of 980 nucleotides (also referred to as 11800699-0-16) is shown in Table 8B.
  • the MOL8a protein encoded by SEQ ID NO:16 has 324 amino acid residues, and is presented using the one-letter code in Table 8B (SEQ ID NO:18).
  • the SignalP, Psort and/or Hydropathy profile for MOL8a predict that MOL8a has a signal peptide and is likely to be localized at the plasma membrane with a certainty of0.6000.
  • the SignalP shows a signal sequence with a cleavage site between amino acids 43 and 44. This is typical ofthis type ofmembrane protein. Therefore it is likely that this novel human plasma membrane protein is available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application.
  • the full amino acid sequence of the protein of the invention was found to have 576/729 (79%) identical and 596/729 (81%) similarity to a murine nurse cell receptor amino acid sequence (PatP Accession No. Y85616).
  • the full amino acid sequence of the protein of the invention was also found to have 296 of 741 amino acid residues (39 %) identical and 383 of 741 amino acid residues (51 %) homolog to the 830 amino acid residue acetyl LDL receptor precursor from Homo sapiens (human) (ACC:O43701).
  • SREC scavenger receptor expressed by endothelial cells [Homo sapiens] (SEQ ID NO: 57)
  • Hypercholesterolemia is an autosomal dominant disorder characterized by elevation of serum cholesterol bound to low density lipoprotein (LDL). Mutations in the LDL receptor (LDLR) gene on chromosome 19 cause this disorder. Familial hypercholesterolemia is characterized by elevation of serum cholesterol bound to low density lipoprotein (LDL) and is, hence, one of the conditions producing the hyperlipoproteinemia II phenotype (see OMIM 144400). Heterozygotes develop tendinous xanthomas, corneal arcus, and coronary artery disease; the last usually becomes evident in the fourth or fifth decade. Homozygotes develop these features at a accelerated rate in addition to planar xanthomas, which may be evident at birth in the web between the first two digits.
  • LDL low density lipoprotein
  • HCV Hepatitis C virus
  • BVDV bovine viral diarrheal virus
  • Endocytosis via the LDL receptor was shown to be mediated by complexing of the virus to very low density lipoprotein (VLDL) or LDL, but not high density lipoprotein (HDL).
  • VLDL very low density lipoprotein
  • HDL high density lipoprotein
  • Therapeutic uses of the composition The expression pattern, and protein similarity information for MOL8 that it may function as human LDL Receptor-like protein.
  • nucleic acid and protein of the invention are useful in potential therapeutic applications implicated, for example but not limited to, metabolic disorders, e.g. Hypercholesterolemia, viral diseases, and other diseases and disorders.
  • metabolic disorders e.g. Hypercholesterolemia, viral diseases, and other diseases and disorders.
  • the homology to antigenic secreted and membrane proteins suggests that antibodies directed against the novel genes may be useful in treatment and prevention of metabolic disorders, e.g. Hypercholesterolemia, viral diseases, and other diseases and disorders.
  • Potential therapeutic uses for the invention(s) are, for example but not limited to, the following: (i) Protein therapeutic, (ii) small molecule drug target, (iii) antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) diagnostic and/or prognostic marker, (v) gene therapy (gene delivery/gene ablation), (vi) research tools, and (vii) tissue regeneration in vitro and in vivo (regeneration for all these tissues and cell types composing these tissues and cell types derived from these tissues.
  • the nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in various cancers including those metabolic disorders, e.g. Hypercholesterolemia, viral diseases, and other diseases and disorders.
  • a contemplated MOL8 epitope is from about amino acids 1 to 10.
  • a MOL8 epitope is from about amino acids 50 to 200.
  • a MOL8 epitope contains amino acids 210-400, 475-600, or 625-850.
  • Genomic Daily Files made available by GenBank The nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full- length protein.
  • the disclosed novel MOL9a nucleic acid of 2355 nucleotides (also referred to as 19506719JBJBXT) is shown in Table 9A.
  • An open reading frame begins with an ATG initiation codon at nucleotides 1-3 and ends with a TGA codon at nucleotides 1915-1917.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 9A, and the start and stop codons are in bold letters.
  • MOL9a also has homology to the proteins shown in the BLAST alignments in Table 9C.
  • the cloned open reading frame codes for a 687 amino acid long protein with an overall 95% amino acid identity, to the mature form of the pig neurolysin precursor
  • PCR primers were designed to PCR amplify a DNA segment, representing an ORF, coding for the mature form of 19506719_B EXT.
  • the forward primer includes an, in frame, BamHI restriction site.
  • the reverse primer contains an, in frame, Xhol restriction site.
  • the sequences of the PCR primers are the following:
  • PCR reactions were set up using a total of 5ng cDNA, consisting equal amounts of cDNA derived from human fetal brain, testis, skeletal muscle and mammary, template, 1 microM of each ofthe 19506719_B-EXT Mat-Forw and 19506719_B-EXT FL-Rev primers, 5 micromoles dNTP (Clontech Laboratories, Palo Alto CA) and 1 microliter of 50xAdvantage-HF 2 polymerase (Clontech Laboratories, Palo Alto CA) in 50 microliter volume.
  • the following reaction conditions were used: a) 96°C 3 minutes b) 96°C 30 seconds denaturation c) 60°C 30 seconds annealing d) 72°C 3 minute extension.
  • GGACCATGCTAGACTTTCC 19506719_B-EXT SI; (SEQ ID NO:60) GGAAAGTCTAGCATGGTCC, 19506719_B-EXT S2; (SEQ ID NO:61) GGCTGAACTTGGTGCTCTTCC, 19506719_B-EXT S3; (SEQ ID NO:62) GGAAGAGCACCAAGTTCAGCC, 19506719_B-EXT S4; (SEQ ID NO:63) GGCTTGCTGAACACCTACC, 19506719_B-EXT S7; (SEQ ID NO:64) GGTAGGTGTTCAGCAAGCC, 19506719_B-EXT S8; (SEQ ID NO:65) GCACAGACTGATTTTGCACG, 19506719_B-EXT S9; (SEQ ID NO:66) and CGTGCAAAATCAGTCTGTGC, 19506719_B-EXT S10 (SEQ ID NO:67).
  • MOL9b nucleic acid of 2061 nucleotides (also referred to as MOL9b) is shown in Table 9D. It is thought that MOL9b is an internal fragment of an open reading frame. Therefore its 5' and 3' termini may be extended.
  • the target sequence identified previously, Accession Number 19506719_B_EXT was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species.
  • MOL9c proteins have multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated MOL9c epitope is from about amino acids 25 to 75.
  • a MOL9c epitope is from about amino acids 100 to 200.
  • MOL9a epitopes are found in amino acids 250-400, 450-550, and 650-700.
  • Endopeptidase 24.16 or mitochondrial oligopeptidase is a thiol- and metal-dependent oligopeptidase that is found in multiple intracellular compartments in mammalian cells. From an analysis of the corresponding gene, we found that the distribution of the enzyme to appropriate subcellular locations is achieved by the use of alternative sites for the initiation of transcription.
  • the pig EP 24.16 (MOP) gene spans over 100 kilobases and is organized into 16 exons. The core protein sequence is encoded by exons 5-16 which match perfectly with exons 2-13 of the gene for endopeptidase 24.15, another member of the thimet oligopeptidase family.
  • Neurotensin is a small neuropeptide of 13 amino acids that may function as a neurotransmitter or neuromodulator in the central nervous system. In the CNS, neurotensin is localized to the catecholamine- containing neurons. A catecholamine-producing cell line can also produce NT. Lithium salts, widely used in the treatment of manic-depressive patients, dramatically potentiate
  • Potential therapeutic uses for the invention(s) are, for example but not limited to, the following: (i) Protein therapeutic, (ii) small molecule drug target, (iii) antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) diagnostic and/or prognostic marker, (v) gene therapy (gene delivery/gene ablation), (vi) research tools, and (vii) tissue regeneration in vitro and in vivo (regeneration for all these tissues and cell types composing these tissues and cell types derived from these tissues). These may also function in extracellular matrix remodeling in tissues described above.
  • the MOLlOa protein encoded by SEQ ID NO:27 has 638 amino acid residues, and is presented using the one-letter code in Table 10B (SEQ ID NO:28).
  • PSORT analysis predicts the protein of the invention to be localized in the plasma membrane with a certainty of 0. 6000.
  • SIGNALP analysis it is predicted that the protein of the invention has a signal peptide with most likely cleavage site between positions 57 and 58. Table 10B.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • the nucleic acid sequence has 11625 of 1857 bases (87%) identical to a gb:GENBANK-ID:RNU12425
  • acc:U12425.1 mRNA from Rattus norvegicus (Rattus norvegicus olfactory cyclic nucleotide-gated channel mRNA, complete eds) (Expect 4.0e "316 ).
  • MOLlOb proteins have multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated MOLlOb epitope is from about amino acids 25 to 75.
  • a MOLlOb epitope is from about amino acids 1 to 30.
  • MOLlOb epitopes are found in amino acids 150-250, 275-350, 375-400. and 425-560.
  • Phototransduction is mediated by an enzymatic cascade that ultimately leads to the hydrolysis of cGMP.
  • the photoreceptor cells, rods and cones integrate and respond to cGMP hydrolysis via a cGMP-gated cation channel in the plasma membrane of the outer segment.
  • Kaupp et al. (1989) cloned this channel from bovine retina.
  • Dhallan et al. (1991) used the bovine sequence to isolate cDNA and genomic DNA encompassing the entire protein coding region of the human homolog. Assignment to chromosome 4 was achieved by study of somatic cell hybrids. Pittler et al.
  • the protein similarity information, expression pattern, and map location for MOLl 0 suggest that it may have important structural and/or physiological functions characteristic of the Cyclic-nucleotide gated channel family. Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • MOLl 1 is homologous to members of the chloride channel family of proteins that are important in maintaining physiological ion balance and neuronal signal transduction.
  • the MOLl 1 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in disorders characterized by altered ion regulation and neural signaling, e.g. cystic fibrosis, arrythmia seen in long QT syndrome, Dent's disease, Bartter's syndrome, bronchitis and sinusitis.
  • MOL 12 is homologous to a family of fatty acid-binding proteins important in keratinocyte differentiation.
  • MOL 12 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in disorders characterized by aberrant keratinocyte differentiation, e.g. squamous cell carcinoma and lesional psoriatic skin.
  • MOL 13 is homologous to a family of insulin-like growth factor-binding proteins important in cell proliferation and differentiation.
  • the MOL 13 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in proliferative and apoptotic disorders, e.g. cancer, Alzheimer's disease, and obesity.
  • MOL 15 and MOL22 are homologous to the carboxypeptidase family of proteins important in peptide processing.
  • MOLl 5 and MOL22 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in metabolic disorders of the pancreas, e.g. acute pancreatitis.
  • MOLl 6 is homologous to the mast cell protease-6 family of proteins important in mast cell activation and migration.
  • MOLl 6 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in disorders of the immune system, e.g. infectious inflammatory peritonitis.
  • MOLl 8-19 are homologous to a family of cyto statin-like proteins that are important in modulation of cell shape and motility by controlling cell interactions with the extracellular matrix.
  • MOLl 8-19 nucleic acids and polypeptides, antibodies and related compoimds according to the invention will be useful in therapeutic and diagnostic applications in disorders characterized by altered cell shape, motility, and apoptosis, e.g. cancer and ischemic injury.
  • MOL20-21 are homologous to the chemokine receptor family of proteins that are important in neuronal signal transduction and lymphocyte chemoattraction.
  • a MOLl 1 sequence according to the invention includes a nucleic acid sequence encoding a polypeptide related to the chloride channel family of proteins.
  • a MOLl 1 nucleic acid is found on human chromosome 19.
  • a MOLl 1 nucleic acid and its encoded polypeptide includes the sequences shown in Table 11 A.
  • the disclosed nucleic acid (SEQ ID NO: 183) is 739 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 1-3 and ends with a TAA stop codon at nucleotides 737-739.
  • the representative ORF encodes a 246 amino acid polypeptide (SEQ ID NO: 184) with a predicted molecular weight of 28,017.3 daltons (Da).
  • PSORT analysis of a MOLl 1 polypeptide predicts a plasma membrane protein with a certainty of 0.7900.
  • SIGNALP analysis suggests the presence of a signal peptide with the most likely cleavage site occuring between positions 53 and 54 in SEQ ID NO.: 184.
  • a MOLl 1 nucleic acid has a high degree of homology (92% identity) with a human chloride channel protein P64-like mRNA (CC64; GenBank Accession No.: AK001624), as is shown in Table 1 IB.
  • a MOLl 1 polypeptide also has homology (78% identity, 85%> similarity) with an intracellular human chloride channel polypeptide (ICCP; EMBL Accession No.: AAF19055), as is shown in Table 1 IC.
  • ICCP 1 MALSMPLNGLKEEDKEPLIELFVKAGSDGESIGNCPFSQRLFMILWLKGWFSVTTVDLK 60 MOLli: 61 RKPADLQNKAPGNHPPLIT--STVKS--NKIEEAPEEVLCPPKYLKLSPKHPESNTAGMD 116 l l l lllll III I II II I Ik l llll l lllll l l lll ICCP: 61 RKPADLQNLAPGTHPPFITFNSEVKTDWKIEEFLEEVLCPPKYLKLSP HPESNTAGMD 120
  • ICCP 121 IFAKFSAYIKNSRPEANEALERGLLKTLQKLDEYLNSPLPDEIDENSMEDIKFSTRRFLD 180
  • Chloride channels perform important roles in the regulation of cellular excitability, in transepithelial transport, cell volume regulation, and acidification of intracellular organelles. This variety of functions requires a large number of different chloride channels that are encoded by genes belonging to several unrelated gene families.
  • the CLC family of chloride channels has nine known members in mammals that show a differential tissue distribution and function both in plasma membranes and in intracellular organelles. CLC proteins have about 10-12 transmembrane domains. They probably function as dimers and may have two pores. The functional expression of channels altered by site-directed mutagenesis has led to important insights into their structure-function relationship.
  • the protein encoded by the CF gene functions as a cyclic adenosine monophosphate-regulated chloride channel.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the ability to detect CFTR mutations has led to the recognition of its association with a variety of conditions, including chronic bronchitis, sinusitis with nasal polyps, pancreatitis, and, in men, infertility (Choudari et al, 1999, Gastroenterol. Clin. North Am. 28:543).
  • modulators of CFTR pharmacological agents that interact directly with the
  • CFTR Cl- channel have been identified. Some agents stimulate CFTR by interacting with the nucleotide-binding domains that control channel gating, whereas others inhibit CFTR by binding within the channel pore and preventing Cl- permeation. Knowledge of the molecular pharmacology of CFTR might lead to new treatments for diseases caused by the dysfunction of CFTR.
  • MOLl 1 represents a new member of the chloride channel family. MOLl 1 can be used as a marker for human chromosome 19. MOLl 1 is useful in determining changes in expression of genes contained within the chloride channel protein family. MOLl 1 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of chloride channel-associated proteins.
  • MOLl i nucleic acids, polypeptides, antibodies, and other compositions of the present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving cystic fibrosis, congenital myotonia, Dent disease, an X-linked renal tubular disorder, leukoencephalopathy, malignant hyperthermia, hypertension, arrythmia seen in long QT syndrome, Dent's disease, Bartter's syndrome, bronchitis, sinusitis and other pathologies and disorders.
  • diseases and pathologies including by way of nonlimiting example, those involving cystic fibrosis, congenital myotonia, Dent disease, an X-linked renal tubular disorder, leukoencephalopathy, malignant hyperthermia, hypertension, arrythmia seen in long QT syndrome, Dent's disease, Bartter's syndrome, bronchitis, sinusitis and other pathologies and disorders.
  • a MOL 12 sequence according to the invention includes a nucleic acid sequence encoding a polypeptide related to the fatty acid-binding protein family of proteins.
  • a MOL 12 nucleic acid is found on human chromosome 5.
  • a MOL 12 nucleic acid and its encoded polypeptide includes the sequences shown in Table 12A.
  • the disclosed nucleic acid (SEQ ID NO: 185) is 550 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 27-29 and ends with a TAA stop codon at nucleotides 543-545.
  • a MOL 12 nucleic acid has a high degree of homology (99%> identity) with an uncharacterized region of human chromosome 5, including the clone CTB-139P6 (CHR5; GenBank Accession No.: AC010293), as is shown in Table 12B.
  • a MOL12 polypeptide has homology (71%) identity, 79%) similarity) with a human epidermal fatty acid-binding protein polypeptide (FABP; EMBL Accession No.: Q01469), as is shown in Table 12C.
  • a MOL 12 polypeptide also has homology (71% identity, 79%) similarity) with a human melanogenic inhibitor polypeptide (hMI; PatP Accession No.: R55866) as is shown in Table 12D.
  • MOL12 1 tctgaggacacagccacactcttgtcatgccattgcccttctattctttccttataacat 60
  • MOL12 300 accataaaaatggagagtactttaaaatcatacagttttctcacactcaggggagggaaa 359 ii iiiiiiiiiiii i mi mmimmmmmmmmmmiim
  • MOL12 1 MDTVQQLEERGHLMDSKGFDENKYMKELGVGLALCEKKGAMAKKDCISFFDGKNLTIKME 60 i min i i+iiiiin iiiiiii+ii i IIIII III mum i
  • FABP 1 MATVQQLEGRWRLVDSKGFDE--YMKELGVGIAL-RKMGAMAKPDCIITCDGKNLTIKTE 57 MOL12: 61 STLKSYSFLTLRGGKFKETTGDGRKTQT-CTFTYGTLVRHQKWNGKEGKI-R LKDRKLV 118
  • FABP 118 VECVMNNVTCTRIYEKVE 135 (SEQ ID NO.: 212) Where
  • MOL12 1 MDTVQQLEERGHLMDSKGFDENKYMKELGVGLALCEKKGAMAKKDCISFFDG NLTIKME 60 i mm i i+iiiiiii MIIM M+M I IIIII MI M I I IIII I HMI: 1 MATVQQLEGRWRLVDSKGFDE--YMKELGVGIAL-RKMGAMAKPDCIITCDG NLTIKTE 57 MOL12: 61 STLKSYSFLTLRGGKF ETTGDGR TQT-CTFTYGTLVRHQKWNGKEG I-RKL DRKLV 118
  • HMI 58 STLKTTQFSCTLGEKFEETTADGRKTQTVCNFTDGALVQHQEWDGKESTITRKL DGKLV 117 M0L12: 119 VDCIINNVTCTQIYEKVE 136 (SEQ ID NO.: 211)
  • HMI 118 VECVMNNVTCTRIYEKVE 135 (SEQ ID NO . : 213 )
  • Fatty acid metabolism in mammalian cells depends on a flux of fatty acids, between the plasma membrane and mitochondria or peroxisomes for beta-oxidation, and between other cellular organelles for lipid synthesis.
  • the fatty acid-binding protein (FABP) family consists of small, cytosolic proteins believed to be involved in the uptake, transport, and solubilization of their hydrophobic ligands. Members of this family have highly conserved sequences and tertiary structures.
  • PA-FABP is a cytoplasmic protein, and is expressed in keratinocytes. It is highly up-regulated in psoriatic skin. It shares similarity to other members of the fatty acid-binding proteins and belongs to the fabp/p2/crbp/crabp family of transporter. PA-FABP is believed to have a high specificity for fatty acids, with highest affinity for cl8 chain length. Decreasing the chain length or introducing double bonds reduces the affinity. PA-FABP may be involved in keratinocyte differentiation.
  • E-FABP immunohistochemical localization of the expression of E-FABP in psoriasis, basal and squamous cell carcinomas has been carried out in order to obtain indirect information, at the cellular level, on the transport of the fatty acids (See Masouye et al, 1996, Dermatology 192:208).
  • E-FABP was localized in the upper stratum spinosum and stratum granulosum in normal and non-lesional psoriatic skin.
  • lesional psoriatic epidermis strongly expressed E-FABP in all suprabasal layers, like nonkeratinized oral mucosa.
  • the basal layer did not express E-FABP reactivity in any of these samples.
  • MOL 12 represents a new member of the fatty acid-binding protein family. MOL 12 can be used as a marker for human chromosome 5. MOL 12 is useful in determining changes in expression of genes contained within the fatty acid-binding protein family. MOL 12 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of fatty acid-binding protein associated proteins. MOL 12 nucleic acids, polypeptides, antibodies, and other compositions of the present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving psoriatic skin and cancer, e.g. basal and squamous cell carcinomas.
  • a MOLl 3 sequence according to the invention includes a nucleic acid sequence encoding a polypeptide related to the insulin-like growth factor family of proteins.
  • a MOLl 3 nucleic acid is found on human chromosome 10.
  • a MOL 13 nucleic acid and its encoded polypeptide includes the sequences shown in Table 13 A.
  • the disclosed nucleic acid (SEQ ID NO: 187) is 915 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 1 -3 and ends with a TGA stop codon at nucleotides 913-915.
  • the representative ORF encodes a 304 amino acid polypeptide (SEQ ID NO: 188) with a predicted molecular weight of 32,944.7 Da.
  • MOLl 3 polypeptide is likely to be detected in kidney, spleen, thyroid, brain and salivary gland. PSORT analysis of a MOL 13 polypeptide predicts a secreted protein with a certainty of 0.8200. SIGNALP analysis suggests the presence of a signal peptide with the most likely cleavage site occuring between positions 30 and 31 in SEQ ID NO.: 188.
  • MOL13 121 cgcggctggatgcggctgctagcggagggcgagggctgcgctccctgccggccagaagag 180
  • PSF2 1 MLPPPRPAAALALPVLLLLLWLTPPPTGARPSPGPDYLRRGWMRLLAEGEGCAPCRPEE 60
  • PSF2 61 CAAPRGCLAGRVRDACGCCWECANLEGQLCDLDPSAHFYGHCGEQLECRLDTGGDLSRGE 120 MOL13: 121 VPEPLCACRSQSPLCGSDGHTYSQICRLQEAARARPDANLTVAHPGPCESGPQIVSHPYD 180 PSF2: 121 VlPlElPlLlClAlClRlSlQlSlPlLlClGlSlDlGlHlTlYlSlQlIlClRlLlQlElAlAlRlAlPlDlAlNlLlTlVlAlHlPlGlPlClElSlGlPlQlIlVlSlHlPlYlDl 180 MOL13: 181 TWNVTGQDVIFGCEVFAYPMAS1EWRKDGLDIQLPGDDPHISVQFRGGPQRFEVTGWLQI 240 I
  • MOL13 241 QAVRPSDEGTYRCLGRNALGQVEAPASLTVLTPDQLNSTGIPQLRSLNLVPEEEAESEEN 300
  • PSF2 241 QAVRPSDEGTYRCLARNALGQVEAPASLTVLTPDQLNSTGIPQLRSLNLVPEEEAESEEN 300 MOL13 : 301 DDYY 304 (SEQ ID NO. : 188)
  • MOLl 3 nucleotide sequence showed 99% homology with AAA46731 DNA- encoding sequence for human prostacyclin-stimulating factor-2 (WO 200036105-A1 to Human Genome Sciences, Inc.); 96% homology with AAA46752 Clone HOABR24R, related to human prostacyclin-stimulating factor-2 (WO 200036105-A1 to Human Genome Sciences, Inc.); 87% homology with human FKSG40 mRNA (GenBank ID: AF333487, acc:AF333487.1); 87% homology with human FKSG28 mRNA (GenBank ID: AYO14217, acc:AY)14271.1); 62% homology with Sequence 17 (2036 bp) from U.S. Pat.
  • the insulin-like growth factor binding protein (IGFBP) family comprises six structurally distinct, but highly homologous proteins. They have been identified in serum and other biological fluids, tissue extracts, and cell culture media. cDNAs encoding human IGFBP-4, -5, and -6 have been cloned and expressed these BPs in yeast as ubiquitin (Ub)- IGFBP fusion proteins. Western ligand blotting with 125I-IGF II under non-reducing conditions of recombinant human (rh) IGFBP-containing yeast lysates reveals specific binding bands for IGFBP-4, -5, and -6 at apparent molecular masses of 24-26, 30-32, and 24-26 kDa, respectively, indicating processing of the fusion proteins.
  • IGFBP insulin-like growth factor binding protein
  • Insulin-like growth factor proteins are associated with cancer progression.
  • the down-regulation of Tl A12/mac25 a novel insulin-like growth factor binding-like protein related gene, is associated with disease progression in breast carcinomas.
  • Tl A12/mac25 a novel insulin-like growth factor binding-like protein related gene
  • T1A12 is identical to mac25, an insulin-like growth factor-binding protein related gene.
  • Antibodies generated against the C-terminal region of the Tl A12/mac25 protein were used to investigate its expression in 60 primary breast tissues. Sections of 12 benign, 16 ductal carcinoma in situ and 32 infiltrating ductal carcinoma specimens were examined. Strong immunoperoxidase staining was observed in luminal epithelial cells of normal lobules and ducts, in apocrine cells of cysts and fibroadenomas. Moderate to weak protein expression was found in hyperplastic and DCIS cells, but no specific staining was detected in invasive carcinoma cells.
  • Tl A12/mac25 FISH mapping using a PAC clone localized the Tl A12/mac25 gene to 4ql2-13.
  • Microsatellite length polymorphism was studied using markers for 4q in paired normal and tumor breast tissues. Thirty-three per cent (10/30) of the samples were found to be polymorphic with D4S 189 and D4S231 microsatellite markers and LOH was detected in 50% (5/10) of these informative samples.
  • the data indicate that TlA12/mac25 expression is abrogated during breast cancer progression concomitant with loss of heterozygosity on chromosome 4q.
  • Tl A12/mac25 may therefore have a tumor suppressor-like function and its expression could indicate a disease with a more favorable status, having a better prognosis (See Burger et al, Oncogene 16:2459).
  • Gupta et al. provide evidence from genetic and pharmacologic studies to suggest that cyclooxygenase-2 (COX-2) enzyme plays a role in the development of colorectal cancer (Gupta et al, PNAS 97(24):13275-80, November 21, 2000. However, little is known about the identity or role of the eicosanoid receptor pathways that are activated by COX-derived prostaglandins (PGs). Gupta et al. report that COX-2-derived prostacyclin promotes embryo implantation in mouse uterus via activation of the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) delta.
  • PPAR nuclear hormone receptor peroxisome proliferator-activated receptor
  • MOL 13 represents a new member of the insulin-like growth factor family.
  • MOL 13 can be used as a marker for human chromosome 10.
  • MOLl 3 is useful in determining changes in expression of genes contained within the insulin-like growth factor protein family.
  • MOL 13 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of insulin-like growth factor-like protein associated proteins.
  • MOL 13 nucleic acids, polypeptides, antibodies, and other compositions of the present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving cell proliferative disorders, e.g. cancer.
  • a MOL 14 sequence according to the invention includes a nucleic acid sequence encoding a polypeptide related to the cytokeratin-18 family of proteins.
  • a MOL 14 nucleic acid and its encoded polypeptide includes the sequences shown in Table 14A.
  • the disclosed nucleic acid (SEQ ID NO: 189) is 1,299 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 5-7 and ends with a TAA stop codon at nucleotides 1,286-1,288.
  • the representative ORF encodes a 427 amino acid polypeptide (SEQ ID NO: 190) with a predicted molecular weight of 48,096.8 Da.
  • PSORT analysis of a MOL14 polypeptide predicts localization in the endoplasmic reticulum membrane with a certainty of 0.5500.
  • SIGNALP analysis suggests the lack of a signal peptide. Putative untranslated regions upstream and downstream of the coding sequence are underlined in SEQ ID NO.: 190.
  • a MOL 14 nucleic acid has a high degree of homology (90% identity) with a human keratin-18 mRNA (K-18; GenBank Accession No.: M26326), as is shown in Table 14B.
  • a MOL14 polypeptide has homology (82% identity, 89%) similarity) with a human keratin 18 polypeptide (hKl 8; GenBank Accession No. : S05481), as is shown in Table 14C. TABLE 14B.
  • MOL14 1 CAGCATGAGCTTCACCACTCCCTCCACCTTCTCCACCAACTACCAGTCCCTGGGCTCTGT 60 mmmmiiiiiiii mimmmmimm mimmiim
  • III MM I III III I II K-18 347 GGAGCCTGGAGACCGAGAACCGGAGGCTGGAGAGCAAAATCCGGGAGCACTTGGAGAAGA 406 0L14: 357 -G--ACCCCATGTCAGAGACTGGGGCCATTACTTCAAGACCATCAAGGAACTGAGGGCTC 413 i mm mmimii MMMMMIMM mi mi miiiim
  • K-18 1067 ACGGGATCCTGCTGCACCTTGAGTCAGAGCTGGCACAGACCCGGGCAGAGGGACAGCGCC 1126 M0L14: 1071 AGGTCCAGGAGTACAAGGACTTGCTGAACATCAGGGTCAAGCTGGAGGCTGAGATCGCCA 1130
  • nniiiii m i mmmiiimmimm K-18 1127 AGGCCCAGGAGTATGAGGCCCTGCTGAACATCAAGGTCAAGCTGGAGGCTGAGATCGCCA 1186 MOLl4: 1131 CCTACCGCCGCCTGCTGGAAGACAGCGAGGGCCTCAATCTTGGTGATGCCCTGGACAGCA 1190 ⁇ m ⁇ mm i i miimi
  • M0L14 61 GGLATEMAGGLAEMGGIQNEKETMQSLNDHL-DYLDRVRNLETENWRLESKIQEYLEKR- 118 lllll +IMII I MMM + IMM MMM + I + IM + HK18: 61 GGLATGIAGGLAGMGGIQNEKETMQSLNDRLASYLDRVRSLETENRRLESKIREHLEKKG 120 M0L14: 119 PHVRDWGHYFKTIKELRAQIFANTVDNVHIILQIDNARLAADDFRVKYETELAMRQSVES 178
  • MOL14 358 QEYKDLLNIRVKLEAEIATYRRLLEDSEGLNLGDALDSSNSMQTIQKTTTRQIVDSKWS 317
  • Intermediate filaments are a structurally related family of cellular proteins that appear to be intimately involved with the cytoskeleton.
  • the common structural motif shared by all IFs is a central alpha-helical 'rod domain' flanked by variable N- and C- terminal domains.
  • the rod domain the canonical feature of IFs, has been highly conserved during evolution.
  • the variable terminals have allowed the known IFs to be classified into 6 distinct types by virtue of their differing amino acid sequences (See Steinert and Roop, 1988, Annu. Rev. Biochem. 57:593).
  • Keratins compose types I and II; intermediate filaments desmin, vimentin, GFAP, and peripherin, type III; neurofilaments, type IV, and nuclear lamins, type V.
  • Nestin (600915) has been classed as type VI (See Lendahl et al, 1990, Cell 60:585).
  • the acidic keratins are coded by genes KRT9 to KRTl 9. These genes are located on mouse chromosome 11 and human chromosome 17, except for KRTl 8 which may be located on human chromosome 12 (see later).
  • the basic keratins are coded by genes KRTl to KRT8, which are located on mouse chromosome 15 and human chromosome 12.
  • transgenic mice that express point-mutant K18 and develop chronic hepatitis and hepatocyte fragility in association with disruption of hepatocyte keratin filaments. They showed that transgenic mice expressing mutant Kl 8 are highly susceptible to hepatotoxicity after acute administration of acetaminophen or chronic ingestion of griseofulvin. The authors concluded that the predisposition to hepatotoxicity results directly from the keratin mutation since nontransgenic or transgenic mice that express normal human K18 are more resistant. Hepatotoxicity was manifested by a significant difference in lethality, liver histopathology, and biochemical serum testing.
  • K8/18 is the major keratin pair in epithelia of the type found in liver, pancreas, and intestine.
  • Transgenic mice that express mutant keratin 18, as already noted develop chronic hepatitis, and have an increased susceptibility to drug-induced hepatotoxicity.
  • Ku and colleagues described a hisl271eu (H127L) KRT mutation in a patient with cryptogenic cirrhosis that was germline transmitted.
  • Electron microscopy of in vitro assembled mutant KRTl 8 and wildtype KRT8 showed an assembly defect as compared with normal KRT8/18 assembly.
  • MOL 14 represents a new member of the cytokeratin- 18 family. MOL 14 is useful in determining changes in expression of genes contained within the cytokeratin- 18 protein family. MOL 14 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of cytokeratin- 18-like protein-associated proteins. MOL 14 nucleic acids, polypeptides, antibodies, and other compositions of the present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving hepatic disorders, e.g. cryptogenic cirrhosis.
  • a MOLl 5 sequence according to the invention includes a nucleic acid sequence encoding a polypeptide related to the metallocarboxypeptidase family of proteins.
  • a MOLl 5 nucleic acid maps to human chromosome 20.
  • a MOLl 5 nucleic acid and its encoded polypeptide includes the sequences shown in Table 15 A.
  • a MOL 15 nucleic acid is likely to be expressed in testis, spleen, salivary gland, brain, heart, thyroid, bone marrow, lung, kidney, uterus, ovary and germ cells.
  • the disclosed nucleic acid (SEQ ID NO: 191) is 2,202 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 1-3 and ends with a TGA stop codon at nucleotides 2,200-2,200.
  • the representative ORF encodes a 733 amino acid polypeptide (SEQ ID NO: 192) with a predicted molecular weight of 81 ,573.8 Da.
  • PSORT analysis of a MOL 15 polypeptide predicts a lysosomal localization with a certainty of 0.5487 and a secreted protein with a certainty of 0.5469.
  • SIGNALP analysis suggests the presence of a signal peptide, with the most likely cleavage site between position 20 and 21 of SEQ ID NO.: 192. TABLE 15A.
  • a MOLl 5 polypeptide has homology (84% identity, 89% similarity) with a mouse metallocarboxypeptidase CPX-1 polypeptide (CPXl; EMBL Accession No.: Q9Z100), as is shown in Table 15B. Also, a MOL 15 polypeptide has a high degree of homology with an uncharacterized human protein APG04 (AGP04; PatP Accession No.: B36174), as is shown in Table 15C.
  • MOL15 1 MWGLLLALAGFAPAVGPALGAPRNSVLGLAQPGTTKVPGSTPALHSSPAQPSAETANTSE 60 lll + ll MM II III MM I 111 MM + III CPXl: 1 MWGLLLAVTAFAPSVGLGLGAPSASVPGLA PGSTLAPHSSVAQPSTKANETSE 53
  • MOL15 121 SLRVSDSRLEASSSQSFGLGPHRGRLNIQSGLEDGDLYDGAWCAEEQDADPWFQVDAGHP 180 + MMMMIIMIMIMIIMM + M +11 MM +1
  • CPXl 172 VRFAGIVTQGRNSVWRYDWVTSFKVQFSNDSQTWWKSRN-STGMDIVFPANSDAETPVLN 230 MOL15: 241 LLPEPQVARFIRLLPQTWLQGGAPCLRAEILACPVSDPNDLFLEAPASGSSDPLDFQHHN 300 III II 111+ lll+lll
  • CPXl 231 LLPEPQVARFIRLLPQTWFQGGVPCLRAEILACPVSDPNDLFPEAHTLGSSNSLDFRHHN 290 MOL15: 301 YKAMRKLMKQVQEQCPNITRIYSIGKSYQGLKLYVMEMSDKPGEHELGEPEVRYVAGMHG 360 l ll lllll CPXl: 291 YKAMRKLMKQVNEQCPNITRIYSIGKSHQGLKLYVMEMSDHPGEHELGEPEVRYVAGMHG 350 MOL15: 361 NEALGRELLLLLMQFLCHEFLRGNPRVTRLLSEMRIHLLPSMNPDGYEIAYHRGSELVGW 420
  • MOLl5 421 AEGRWNNQSIDLNHNFADLNTPLWEAQDDGKVPHIVPNHHLPLPTYYTLPNATVAPETRA 480 lllll +1 111111111111 II l + ll! II I n MI: MM mi I
  • CPXl 411 AEGRWTHQGIDLNHNFADLNTQLWYAEDDGLVPDTVPNHHLPLPTYYTLPNATVAPETWA 470 MOL15: 481 VIKWMKRIPFVLSANLHGGELWSYPFDMTRTPWAARELTPTPDDAVFRWLSTVYAGSNL 540 l l llll l + l
  • AGP04 1 MWGLLLALAAFAPAVGPALGAPRNSVLGLAQPGTTKVPGSTPALHSSPAQPPAETANGTS 60 MOL15: 60 EQHVRIRVIKKKKVIMKKRKKLTLTRPTPLVTAGPLVTPTPAGTLDPAEKQEPGCPPLGL 119
  • AGP04 61 EQHVRIRVIKKKKVIMKKRKKLTLTRPTPLVTAGPLVTPTPAGTLDPAEKQETGCPPLGL 120 MOL15: 120 ESLRVSDSRLEASSSQSFGLGPHRGRLNIQSGLEDGDLYDGAWCAEEQDADPWFQVDAGH 179
  • AGP04 181 PTRFSGVITQGRNSVWRYDWVTSYKVQFSNDSRTWWGSRNHSSGMDAVFPANSDPETPVL 240 MOL15 : 240 NLLPEPQVARFIRLLPQTWLQGGAPCLRAEILACPVSDPNDLFLEAPASGSSDPLDFQHH 299 AGP04: 241 NLLPEPQVARFIRLLPQTWLQGGAPCLRAEILACPVSDPNDLFLEAPASGSSDPLDFQHH 300 MOL15 :300 NYKAMRKL KQVQEQCPNITRIYSIGKSYQGLKLYVMEMSDKPGEHELGEPEVRYVAGMH 359 lllll III II
  • AGP04 361 GNEALGRELLLLLMQFLCHEFLRGNPQVTRLLSEMRIHLLPSMNPDGYEIAYHRGSELVG 420 MOL15:420 WAEGRWNNQSIDLNHNFADLNTPLWEAQDDGKVPHIVPNHHLPLPTYYTLPNATVAPETR 479
  • AGP 04 421 WAEGRWNNQSIDLNHNFADLNTPLWEAQDDGKVPHIVPNHHLPLPTYYTLPNATVAPETR 480 MOL15 : 480 AVIKWMKRIPFVLSANLHGGELWSYPFDMTRTPWAARELTPTPDDAVFRWLSTVYAGSN 539 I III 11 II I III III II III 111 II II I II II II II 11 II III 111 II 11 II 11 II 11 II
  • AGP04 481 AVIKWMKRIPFVLSANLHGGELWSYPFDMTRTPWAARELTPTPDDAVFRWLSTVYAGSN 540
  • MOL15 540 LAMQDTSRRPCHSQDFSVHGNIINGADWHTVPGSMNDFSYLHTNCFEVTVELSCDKFPHE 599
  • AGP04 661 WRLLTPGDYMVTASAEGYHSVTRNCRVTFEEGPFPCNFVLTKTPKQRLRELLAAGAKVPP 720 MOL15:720 DLRRRLERLRGQKD 733 (SEQ ID NO.: 192)
  • AGP04 721 DLRRRLERLRGQKD 734 (SEQ ID NO . : 220 ) Where
  • Metallocarboxypeptidases are members of a gene family with broad gene expression patterns and in vivo ftmctions. The recent finding that Cpe(fat)/Cpe(fat) mice, which lack carboxypeptidase E (CPE) activity because of a point mutation, are still capable of a reduced amount of neuroendocrine peptide processing suggested that additional carboxypeptidases (CPs) participate in this processing reaction. Searches for novel members of the CPE gene family led to the discovery of CPD, CPZ, AEBP1, and CPX-2.
  • CPE carboxypeptidase E
  • CPX-1 contains an N-terminal region of 160 amino acids with sequence similarity to the discoidin domain of a variety of proteins.
  • the 410- residue CP-like domain of CPX-1 has 54% to 62%> amino acid sequence identity with AEBP1 and CPX-2 and 33% to 49% amino acid identity with other members of the CPE subfamily.
  • CPX-1 expressed in either the baculovirus system or the mouse AtT-20 cell line does not cleave standard CP substrates.
  • Northern blot analysis shows the highest levels of CPX-1 mRNA in testis and spleen and lower levels in salivary gland, brain, heart, lung, and kidney.
  • In situ hybridization of CPX- 1 mRNA in embryonic and fetal mouse tissue showed expression throughout the head and thorax, with abundance in primordial cartilage and skeletal structures.
  • high levels of CPX-1 mRNA are associated with the nasal mesenchyme, primordial cartilage structures in the ear, and the meninges.
  • CPX-1 mRNA is expressed in multiple developing skeletal structures, including chondrocytes and perichondrial cells of the rib, vertebral, and long-bone primordia.
  • CPX-1 may have a role in development, possibly mediating cell interactions via its discoidin domain. (See Lei et al, 1999, DNA Cell Biology 18:175).
  • MOLl 5 represents a new member of the metallocarboxypeptidase family of proteins.
  • MOL 15 is useful in determining changes in expression of genes contained within the metallocarboxypeptidase protein family.
  • MOL 15 will be useful in identifying testis, spleen, salivary gland, brain, heart, thyroid, bone marrow, lung, kidney, uterus, ovary tissue and germ cells.
  • MOLl 5 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of the metallocarboxypeptidase-associated protein family of proteins.
  • MOL 15 nucleic acids, polypeptides, antibodies, and other compositions of the present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving metabolic disorders of the pancreas, e.g. acute pancreatitis.

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Abstract

L'invention concerne des séquences d'acides nucléiques codant pour des polypeptides associés à un récepteur couplé aux protéines G. Elle concerne aussi des polypeptides codés par ces séquences d'acides nucléiques, des anticorps qui se lient de façon immunospécifique à ces polypeptides, ainsi que des dérivés, variants, mutants ou fragments des ces polypeptides, polynucléotides ou anticorps. Elle concerne enfin des procédés thérapeutiques, de diagnostic, de traitement et de prévention de troubles impliquant l'un de ces acides nucléiques et protéines.
PCT/US2002/019522 2001-06-18 2002-06-18 Proteines et acides nucleiques les codant WO2002102321A2 (fr)

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AU2002330857A AU2002330857A1 (en) 2001-06-18 2002-06-18 Novel proteins and nucleic acids encoding same

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US29913401P 2001-06-18 2001-06-18
US29899401P 2001-06-18 2001-06-18
US60/298,994 2001-06-18
US60/299,134 2001-06-18
US97244601A 2001-10-04 2001-10-04
US09/972,446 2001-10-04
US38636402P 2002-06-06 2002-06-06
US60/386,364 2002-06-06
US38683702P 2002-06-07 2002-06-07
US60/386,837 2002-06-07

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008003925A2 (fr) * 2006-07-06 2008-01-10 Ares Trading S.A. Protéine du complément

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6063594A (en) * 1997-01-31 2000-05-16 Incyte Pharmaceuticals, Inc. DNA encoding a novel human anion channel

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6063594A (en) * 1997-01-31 2000-05-16 Incyte Pharmaceuticals, Inc. DNA encoding a novel human anion channel

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EDWARDS J.C.: 'A novel p64-related C1-channel: subcellular distribution and nephron segment-specific expression' AMERICAN JOURNAL OF PHYSIOLOGY vol. 276, 1999, pages F398 - F408, XP002967889 *
FERNANDEZ-SALAS ET AL.: 'p53 and Tumor necrosis factor alpha regulate the expression of a mitochondrial chloride channel protein' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 274, no. 51, 17 December 1999, pages 36488 - 36497, XP002960760 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008003925A2 (fr) * 2006-07-06 2008-01-10 Ares Trading S.A. Protéine du complément
WO2008003925A3 (fr) * 2006-07-06 2008-02-28 Ares Trading Sa Protéine du complément

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