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US20030166067A1 - Islet cell antigen 1851 - Google Patents

Islet cell antigen 1851 Download PDF

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Publication number
US20030166067A1
US20030166067A1 US10/124,089 US12408902A US2003166067A1 US 20030166067 A1 US20030166067 A1 US 20030166067A1 US 12408902 A US12408902 A US 12408902A US 2003166067 A1 US2003166067 A1 US 2003166067A1
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Prior art keywords
amino acid
acid residue
seq
islet cell
cell antigen
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US10/124,089
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Wayne Kindsvogel
Laura Jelinek
Paul Sheppard
William Hagopian
James LaGasse
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Zymogenetics Inc
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Zymogenetics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Insulin-dependent diabetes mellitus is a disease resulting from the autoimmune destruction of the insulin-producing ⁇ -cells of the pancreas.
  • Studies directed at identifying the autoantigen(s) responsible for ⁇ -cell destruction have generated several candidates, including poorly characterized islet cell antigens (ICA) (Bottazzo et al., Lancet 2: 1279-83, 1974), insulin (Palmer et al., Science 222: 1337-39, 1983), glutamic acid decarboxylase (GAD) (Baekkeskov et al., Nature 298: 167-69, 1982; Baekkeskov et al., Nature 347: 151-56, 1990), and a 64 kD islet cell antigen that is distinct from GAD and that which yields 37 kD and 40 kD fragments upon trypsin-digestion (Christie et al., Diabetes 41: 782-87, 1992).
  • ICA islet cell antigens
  • Antibodies to the 40 kD, and more particularly the 37 kD, ICA fragments are detected when clinical onset of IDDM is imminent and are found to be closely associated with IDDM development (Christie et al., Diabetes 41: 782-87, 1992).
  • Diabetic sera containing antibodies specific to the 40 kD fragment were recently found to bind to the intracellular domain of the protein tyrosine phosphatase, IA-2/ICA512 (Lu et al., Biochem. Biophys. Res. Comm. 204: 930-36, 1994; Lan et al., DNA Cell Biol. 13: 505-14, 1994; Rabin et al., J. Immunol.
  • Antibodies specific to the 37 kD fragment are thought to bind either to a posttranslational in vivo modification of IA-2/ICA512 or a different, but probably related, protein precursor (Passini et al., ibid.).
  • ICA 512 was initially isolated as an autoantigen from an islet cell cDNA library, and was subsequently shown to be related to the receptor-linked protein tyrosine phosphatase family (Rabin et al., ibid.). ICA 512 was later found to be identical to a mouse and human protein tyrosine phosphatase, IA-2, isolated from brain and insulinoma cDNA libraries (Lu et al., ibid.; and Lan et al., ibid.).
  • diabetes-associated autoantigens especially combinations of autoantigens, genotypes, such as HLA DR and HLA DQ, and loci, such as the polymorphic region in the 5′ flanking region of the insulin gene; in prediabetic individuals have been shown to be useful predictive markers of IDDM, see for example, Bell et al., ( Diabetes 33:176-83, 1984); Sheehy et al., ( J. Clin. Invest. 83:830-35, 1989); and Bingley et al., ( Diabetes 43: 1304-10, 1994).
  • the present invention fulfills this need by providing novel autoantigens as well as related compositions and methods.
  • the autoantigens of the present invention represent a new ⁇ -cell antigen.
  • the present invention also provides other, related advantages.
  • the present invention provides an isolated polynucleotide which forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM, comprising a DNA segment encoding a mammalian islet cell antigen polypeptide of SEQ ID NO:16 from Leu, amino acid residue 636 to Gln, amino acid residue 1012.
  • the invention also provides a mammalian islet cell antigen polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818.
  • the invention also provides allelic variants of these polypeptides.
  • the isolated polynucleotide encodes a mammalian islet cell antigen polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818.
  • the isolated polynucleotide encodes a mammalian islet cell antigen polypeptide of SEQ ID NO:16 from Phe, amino acid residue 612, to Gln, amino acid residue 1012.
  • the invention further provides allelic variants of these polypeptides.
  • the isolated polynucleotide encoding a polypeptide of SEQ ID NO:16 from Ala, amino acid residue 1, to Gln, amino acid residue 1012.
  • the isolated polynucleotide encoding a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818.
  • the invention further provides allelic variants of these polypeptides.
  • the isolated polynucleotide is a DNA molecule comprising a coding sequence corresponding to SEQ ID NO:21 from nucleotide 1325 to nucleotide 2455.
  • the DNA molecule comprises a coding sequence corresponding to SEQ ID NO:15 from nucleotide 1909 to nucleotide 3039.
  • the invention also provides allelic variants of these molecules.
  • the invention further provides complements of polynucleotide molecules which specifically hybridize to these molecules.
  • the isolated polynucleotide is a DNA molecule comprising a coding sequence corresponding to SEQ ID NO:21 from nucleotide 1254 to nucleotide 2455.
  • the isolated polynucleotide is a DNA molecule comprising a coding sequence corresponding to SEQ ID NO:15 from nucleotide 1837 to nucleotide 3039.
  • the invention also provides allelic variants of these molecules.
  • the invention further provides complements of polynucleotide molecules which specifically hybridize to these molecules.
  • the DNA molecule comprises a coding sequence corresponding to SEQ ID NO:15 from nucleotide 4 to nucleotide 3039.
  • the DNA molecule comprises a coding sequence corresponding to SEQ ID NO:21 from nucleotide 2 to nucleotide 2455.
  • the invention also provides allelic variants of these molecules.
  • the invention further provides complements of polynucleotide molecules which specifically hybridize to these molecules.
  • the invention also provides an isolated polynucleotide molecule which encodes a complete coding sequence of a mammalian islet cell antigen polypeptide comprising the sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
  • the invention also provides mammalian islet cell antigens that are primate islet cell antigens.
  • the invention also provides DNA constructs comprising a first DNA segment encoding a human islet cell antigen polypeptide operably linked to additional DNA segments required for the expression of the first DNA segment.
  • the invention further provides a first DNA segment that is an isolated polynucleotide molecule encoding a human islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818.
  • the invention also provides a first DNA segment that is an isolated polynucleotide molecule encoding a human islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818.
  • the invention provides a first DNA segment that is an isolated polynucleotide molecule encoding a human islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818.
  • the invention further provides host cells containing such DNA constructs, as well as methods for producing human islet cell antigen polypeptides comprising the steps of culturing such host cell and isolating the human islet cell antigen polypeptide.
  • the invention further provides isolated mammalian islet cell antigen polypeptides, wherein said isolated mammalian islet cell antigen polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818.
  • IDDM comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818.
  • the invention further provides isolated mammalian islet cell antigen polypeptides comprising the amino acid sequence of SEQ ID NO:16 from Leu, amino acid residue 636 to Gln, amino acid residue 1012.
  • the invention also provides isolated polypeptides of SEQ ID NO:16 from Phe, amino acid residue 612 to Gln, amino acid residue 1012.
  • the invention also provides isolated polypeptides of SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818.
  • the invention further provides isolated polypeptides of SEQ ID NO:16 from Ala, amino acid residue 1 to Gln, amino acid residue 1012.
  • the invention also provides isolated polypeptides of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818.
  • the invention further provides allelic variants of these polypeptides.
  • the invention still further provides an isolated polypeptide which is a full length mammalian islet cell antigen protein comprising the sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
  • the invention also provides mammalian islet cell antigens that are primate islet cell antigens.
  • a method for determining the presence of an autoantibody to a human islet cell antigen polypeptide in a biological sample comprising the steps of contacting the biological sample with the human islet cell antigen polypeptide, which comprises an amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof, under conditions conducive to immune complex formation, and detecting the presence of immune complex formation between the human islet cell antigen polypeptide and the autoantibody to a human islet cell antigen, thereby determining the presence of autoantibodies to the human islet cell antigen in the biological sample.
  • the invention further provides human islet cell antigen polypeptide, which comprises an amino
  • the invention provides a method for predicting the clinical course of diabetes in a patient, comprising testing a biological sample from a patient for the presence of human islet cell antigen polypeptides comprising the amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof, wherein the polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM, and classifying the patient for clinical course of diabetes based on the presence or absence of human islet cell antigens in the sample.
  • the invention further provides a method of predicting the clinical course of IDDM by testing one or more additional predictive markers associated with risk of or protection from IDDM.
  • the invention provides methods of predicting the clinical course where the predictive marker is an autoantibody to an antigen selected from the group consisting of GAD65, IA-2/ICA512 or insulin.
  • the invention also provides methods wherein the predictive marker is a genotype selected from the group consisting of HLA DR and HLA DQ.
  • the invention also provides methods wherein the predictive marker is a polymorphic region in the 5′ flanking region of a human insulin gene.
  • the invention also provides a method for treating a patient to prevent an autoimmune response to a human islet cell antigen polypeptide comprising inducing immunological tolerance in the patient by administering a mammalian islet cell antigen polypeptide comprising the amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof, that specifically binds a human islet cell antigen receptor on immature or mature T or B lymphocytes.
  • the invention also provides oligonucleotide probes of at least about 16 nucleotides, wherein which the oligonucleotide is at least 85% homologous to a sequence of the mammalian islet cell antigen DNA sequence of SEQ ID Nos:15 or 21.
  • the invention further provides isolated antibodies which specifically bind to human islet cell antigen polypeptides which comprise the amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof.
  • the invention provides monoclonal antibodies.
  • the invention provides a hybridoma which produces the monoclonal antibody.
  • the invention also provides a diagnostic kit for use in detecting autoantibodies to pancreatic ⁇ -islet cells, comprising a container containing an islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof, wherein the polypeptide forms an immune complex with autoantibodies from a patient at risk of or predisposed to develop IDDM, and one or more containers containing additional reagents.
  • an islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue
  • a pharmaceutical composition comprising an islet cell antigen comprising an amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof, in combination with a pharamceutically acceptable carrier or vehicle.
  • a method for monitoring the disease state in a patient comprising testing a biological sample from a patient for the presence of human islet cell antigen post-translationally modified polypeptides, determining the concentration of the peptides and correlating the peptide levels in the sample with the disease state in the patient.
  • the invention provides that the human islet cell antigen post-translationally modified polypeptide comprises the sequence of SEQ ID NO:22 from His, amino acid residue 1 to Glu, amino acid residue 227.
  • the biological sample is plasma or serum.
  • the invention provides a method for monitoring the disease state in a patient comprising exposing T cells to islet cell antigen 1851 peptides, detecting T and correlating T cell reactivity with disease state.
  • the invention provides that the T cells are from peripheral blood mononuclear cells from a prediabetic patient.
  • the invention further provides that the disease state is conversion from prediabetes to diabetes.
  • Allelic variant Any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • Biological sample A sample that is derived from or contains cells, cell components or cell products, including, but not limited to, cell culture supernatants, cell lysates, cleared cell lysates, cell extracts, tissue extracts, blood plasma, serum, and fractions thereof, from a patient.
  • polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence.
  • sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.
  • Immune Complex Formation A noncovalently bound molecule formed between an antigen and an antibody specific for that antigen, resulting in an extensively cross-linked mass. Conditions conducive to complex formation are known in the art and easily adaptable by those skilled in art, for example, the degree of complex formation is in proportion to the relative amounts of available antigen and antibody. Such complexes can be used, for example, to identify and/or quantify the presence of either antigen or antibody in a biological sample, identify and characterize particular antibodies in tissues and cells, or to stimulate an immune response.
  • Isolated When applied to a protein the term “isolated” indicates that the protein is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated protein is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the proteins in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When applied to a polynucleotide molecule the term “isolated” indicates that the molecule is removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated and may include naturally occurring 5′ and 3′ untranslated regions such as promoters and terminators, the identification of such will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985).
  • Operably linked Indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • the DNA sequences encoding the polypeptides of the present invention were unexpectedly identified during screening of a primate islet cell cDNA library, and human insulinoma cDNA, for autoantigens toward human diabetic sera.
  • Analysis of the macaque cDNA clones revealed a unique, previously unknown islet cell antigen which contained regions of homology to the protein tyrosine phosphatase family, especially the protein tyrosine phosphatase IA2/ICA512. This novel islet cell antigen has been designated 1851 or ICA512 ⁇ .
  • the present invention provides islet cell antigen polypeptides which are ⁇ -cell autoantigens. These autoantigens were reactive with human prediabetic and diabetic sera.
  • the invention also provides methods for using the islet cell antigen polypeptides for the detection, diagnosis, and treatment of IDDM.
  • Representative islet cell antigen polypeptides of the present invention comprise the amino acid sequences in SEQ ID NOs:4, 16 or 22 and/or are encoded by polynucleotide sequences comprising the sequences of SEQ ID NOs:3, 15 and 21 and form an immune complex with autoantibodies from a patient at risk of or predisposed to develop IDDM.
  • the islet cell antigen polypeptides of the present invention are preferably from mammals, especially primates including humans.
  • Preferred polypeptides of the present invention include isolated polypeptides selected from the group consisting of a polypeptide of SEQ ID NO:2 from Leu, amino acid residue 265, to Gln amino acid residue 641.
  • the invention also provides polypeptides of SEQ ID NO:2 from Glu, amino acid residue 1, to Gln, amino acid residue 641.
  • the invention further provides macaque polypeptides of SEQ ID NO:16 from Ala, amino acid residue 1 to Gln, amino acid residue 1012 and human polypeptides of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818 and SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818.
  • the invention further provides allelic variants and isolated sequences that are substantially identical to the representative polypeptide sequences of SEQ ID NOs:2, 16 and 22 and their species homologs.
  • substantially identical is used herein to denote proteins having 50%, preferably 60%, more preferably 70%, and most preferably at least 80%, sequence identity to the representative sequences shown in SEQ ID NO:2, 16 or 22 or its species homologs. Within preferred embodiments, such proteins will be at least 90% identical, and most preferably 95% or more identical, to SEQ ID NO:2, 16 or 22 or their species homologs.
  • Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids are indicated by the standard one-letter codes).
  • Total ⁇ ⁇ number ⁇ ⁇ of ⁇ ⁇ identical ⁇ ⁇ matches [ length ⁇ ⁇ of ⁇ ⁇ the ⁇ ⁇ longer ⁇ ⁇ sequence ⁇ ⁇ plus ⁇ ⁇ the ⁇ number ⁇ ⁇ of ⁇ ⁇ gaps ⁇ ⁇ introduced ⁇ ⁇ into ⁇ ⁇ the ⁇ ⁇ longer sequence ⁇ ⁇ in ⁇ ⁇ order ⁇ ⁇ to ⁇ ⁇ align ⁇ ⁇ the ⁇ ⁇ two ⁇ ⁇ sequences ] ⁇ 100
  • Substantially identical proteins are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the folding or activity of the protein; small deletions, typically of one to about 30 amino acids; amidation of the amino- or carboxyl-terminal; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-85, 1989). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g. protein tyrosine phosphatase activity, Strueli et al., EMBO J. 9: 2399-407, 1990, or binding to autoantibodies in prediabetic or diabetic sera) to identify amino acid residues that are critical to the activity of the molecule.
  • biological activity e.g. protein tyrosine phosphatase activity, Strueli et al., EMBO J. 9: 2399-407, 1990, or binding to autoantibodies in prediabetic or diabetic sera
  • Sites of ligand-receptor interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.
  • the present invention further provides isolated polynucleotide molecules encoding islet cell antigen polypeptides which form immune complexes with autoantibodies from a patient at risk of or predisposed to develop IDDM.
  • Useful polynucleotide molecules in this regard include mRNA, genomic DNA, cDNA and synthetic DNA.
  • cDNA is preferred.
  • the invention provides an isolated polynucleotide molecule wherein the molecule is a DNA molecule comprising a coding sequence corresponding to SEQ ID NO:1 from nucleotide 795 to nucleotide 1922.
  • the invention also provides a DNA molecule comprising a coding sequence corresponding to SEQ ID NO:1 from nucleotide 1 to nucleotide 2168.
  • the invention also provides a DNA molecule comprising a coding sequence corresponding to nucleotide 4 to nucleotide 3039 of SEQ ID NO: 15.
  • the invention also provides DNA molecules from nucleotide 1325 to nucleotide 2455, from nucleotide 1254 to nucleotide 2455 and from nucleotide 2 to nucleotide 2544 of SEQ ID NO:21.
  • the invention also provides allelic variants of the sequences shown in SEQ ID NOs:1, 15 or 21, and polynucleotide molecules that specifically hybridize to allelic variants.
  • polynucleotide molecules will hybridize to the representative DNA sequences of SEQ ID NOs:1, 15, 21 or their allelic variants under stringent conditions (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989).
  • stringent conditions refers to hybridizing conditions that employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% polyvinylpyrrolidone/50 mM sodium citrate at 42° C.; or employ 50% formamide, 5 ⁇ SSC (0.75 M NaCl, 0.075M sodium pyrophosphate, 5 ⁇ Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C.
  • a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% polyvinylpyrrolidone/50 mM sodium citrate at 42° C.
  • formamide for example, 50% (vol/vol) formamide with 0.
  • hybridizable polynucleotide molecules would include genetically engineered or synthetic variants of the representative islet cell antigen polynucleotide sequence, SEQ ID NO: 1, and polynucleotide molecules that encode one or more amino acid substitutions, deletions or additions, preferably of a minor nature, as discussed above.
  • Genetically engineered variants may be obtained by using oligonucleotide-directed site-specific mutagenesis, by use of restriction endonuclease digestion and adapter ligation, polymerase chain reaction (PCR), or other methods well established in the literature (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989, and Smith et al., Genetic Engineering: Principles and Methods, Plenum Press, 1981; which are incorporated herein by reference).
  • PCR polymerase chain reaction
  • hybridizable polynucleotide molecules may encompass sequences containing degeneracies in the DNA code wherein host-preferred codons are substituted for the analogous codons in the representative sequences of SEQ ID NOs: 1, 15 and 21.
  • cDNA libraries from different tissues can be screened to obtain a full length cDNA, which encodes a full length mammalian islet cell antigen polypeptides.
  • Another option for obtaining the complete coding sequence comprises using 5′ RACE (Rapid Amplification cDNA Ends) PCR.
  • RACE Rapid Amplification cDNA Ends
  • PCR is an art recognized PCR-based method for amplifying the 5′ ends of incomplete cDNAs, a frequent occurrence in cDNA cloning.
  • PCR is carried out on specially prepared cDNA which contains unique anchor sequences, using anchor primers provided with the 5′ RACE reagents available from, for example, Clontech, Palo Alto, Calif.
  • the 5′-RACE-Ready cDNA can be purchased commercially (Clontech), or prepared according to known methods.
  • a secondary PCR reaction can then be carried out using the anchor primer and a nested 3′ primer, according to known methods.
  • Transmembrane, or receptor-linked, protein tyrosine phosphatases consist of a conserved cytoplasmic domain which may have one or two (tandemly duplicated) catalytic regions, a single transmembrane domain, a highly variable extracellular domain and a signal peptide. These structural features suggest that receptor-linked protein tyrosine phosphatases would be capable of binding ligand and transducing external signal, but no ligands as of yet have been identified.
  • the macaque 1851 polypeptide has an approximately 611 amino acid extracellular domain, from Ala, amino acid residue 1 to Lys, amino acid residue 611 of SEQ ID NO:16, containing a post translational modification dibasic site, at amino acid residue 423-424, or a tribasic site at amino acid residues 422-424; a 24 amino acid transmembrane domain comprising amino acid residue 241 to amino acid residue 265 of SEQ ID NO:2 or Phe, amino acid residue 612 to Cys, amino acid residue 635 of SEQ ID NO:16 and an approximately 375 amino acid cytoplasmic domain comprising the amino acid residue 265 to amino acid residue 640 of SEQ ID NO:2 or Leu, amino acid residue 636 to Gln, amino acid residue 1012 of SEQ ID NO:16.
  • the representative amino acid sequence of the human islet cell antigen 1851 (SEQ ID NO:22) has 417 amino acids of an extracellular domain, from His, amino acid residue 1 to Lys, amino acid residue 417 of SEQ ID NO:22; a 24 amino acid residue transmembrane domain, from Phe, amino acid residue 418 to Cys, amino acid residue 441, of SEQ ID NO:22; and a 376 amino acid cytoplasmic domain, from Leu, amino acid residue 442 to Gln, amino acid residue 818 of SEQ ID NO:22.
  • the cytoplasmic domain of 1851 contains many regions that are conserved between members of the protein tyrosine phosphatase family. Within the cytoplasmic domain of protein tyrosine phosphatases is a catalytic region of about 230 amino acids, which contains a highly conserved catalytic core segment of approximately 11 amino acid residues (VHCXAGXXRXG SEQ ID NO:13) where the first three X's are any amino acid, the fourth X is S or T, and the cysteine appears to be essential to the catalytic mechanism (Fischer et al., Science 253: 401-06).
  • the catalytic core sequence of the representative macaque 1851 polypeptide sequences of SEQ ID Nos:2 and 16 and human 1851 polypeptide sequence represented by SEQ ID NO:22 differs from other members of the protein tyrosine phosphatase family in that alanine has been replaced by aspartic acid and the second variable amino acid (X) is alanine.
  • 1851 like IA-2/ICA512, has a single catalytic region. Deletion of C-terminal amino acids from the intracellular domain of human islet cell antigen 1851 reduced reactivity with new onset IDDM sera, suggesting this region may play a role in defining an autoantibody epitope.
  • a comparison between the overall human and macaque islet cell antigen 1851 nucleotide and amino acid sequences shows a 96.2% nucleotide identity and a 94.6% amino acid identity, in particular there was 97% identity within the nucleotide sequence and 98.9% identity within the amino acid sequence of the corresponding cytoplasmic domains, 100% identity within the transmembrane domain.
  • the tissue distribution of human islet cell antigen 1851 is generally neuroendocrine.
  • Northern analysis showed strong hybridization to human mRNA from brain and pancreas and weaker hybridization in spinal cord, thyroid, adrenal and GI tract. In situ hybridization using macaque tissues further localized pancreatic and adrenal expression to islets and adrenal medulla, respectively.
  • Northern blot analysis of rat phogrin showed expression in brain, pancreas and ⁇ and ⁇ cell tumor lines (Wasmeier and Hutton, ibid.); mouse IA-2 ⁇ in brain, pancreas, stomach and in insulinoma and glucagomoma cell lines (Lu et al., Proc. Natl. Acad. Sci.
  • IA-2/ICA512 and human islet cell antigen 1851 yielded a 40 kD IA-2/ICA512 fragment and a 37 kD islet cell antigen 1851 fragment. These correspond to the 37 kD and 40 kD tryptic fragments described by Christie et al. ( J. Exp. Med. 172:789-94, 1990), Payton et al. ( J. Clin. Invest. 96:1506-11, 1995), Bonifacio et al. ( J. Immunol. 155:5419-26, 1995), Lu et al. ( Proc. Natl. Acad. Sci. USA 93:2307-11, 1996) and Wasmeier and Hutton (ibid.).
  • the invention provides isolated DNA molecules that are useful in producing recombinant islet cell antigens.
  • each individual domain or combinations of the domains may be prepared synthetically or by recombinant DNA techniques for use in the present invention.
  • the present invention provides the advantage that islet cell antigens are produced in high quantities that may be readily purified using methods known in the art (see generally; Scopes, Protein Purification, Springer-Verlag, NY, 1982).
  • the proteins of the present invention may be synthesized following conventional synthesis methods, such as the solid-phase synthesis method of Barany and Merrifield (in The Peptides. Analysis, Synthesis, Biology Vol. 2, Gross and Meienhofer, eds, Academic Press, NY, pp. 1-284, 1980), by partial solid-phase techniques, by fragment condensation or by classical solution addition.
  • DNA molecules of the present invention can be isolated using standard cloning methods such as those described by Maniatis et al. ( Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982; which is incorporated herein by reference), Sambrook et al., ( Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989), or Mullis et al. (U.S. Pat. No. 4,683,195) which are incorporated herein by reference.
  • the coding sequences of the present invention can be synthesized using standard techniques that are well known in the art, such as by synthesis on an automated DNA synthesizer.
  • SEQ ID NOs: 1, 15 and 21 The sequence of a polynucleotide molecule encoding a representative islet cell antigen polypeptide is shown in SEQ ID NOs: 1, 15 and 21 and the corresponding amino acid sequences are shown in SEQ ID NOs: 2, 16 and 22.
  • SEQ ID NOs: 1, 15 and 21 The sequence of a polynucleotide molecule encoding a representative islet cell antigen polypeptide is shown in SEQ ID NOs: 1, 15 and 21 and the corresponding amino acid sequences are shown in SEQ ID NOs: 2, 16 and 22.
  • allelic variants of the DNA sequence shown in SEQ ID NO: 1, 15 and 21 including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO: 2, 16 and 22.
  • the macaque sequence disclosed herein is useful for isolating polynucleotide molecules encoding islet cell antigen polypeptides from other species (“species homologs”).
  • the macaque cDNA was used to conduct a sequence search for a human homolog. A match was found as an expressed sequence tag (EST) from a human fetal brain library submitted to the Genbank database (GenBank ID: TO361, clone ID: HFBCV88).
  • Other preferred species homologs include mammalian homologs such as bovine, canine, porcine, ovine, and equine proteins. Methods for using sequence information from a first species to clone a corresponding polynucleotide sequence from a second species are well known in the art. See, for example, Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.
  • DNA molecules of the present invention or portions thereof may be used as probes, for example, to directly detect 1851 sequences in cells or biological samples.
  • DNA molecules are generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences and will generally comprise at least about 16 nucleotides, more often from about 17 nucleotides to about 25 or more nucleotides, sometimes 40 to 60 nucleotides, and in some instances a substantial portion or even the entire 1851 gene or cDNA.
  • the synthetic oligonucleotides of the present invention have at least 85% identity to a representative macaque or human 1851 DNA sequence (SEQ ID Nos:1, 15 and 21) or their complements.
  • the molecules are labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle, etc., according to methods known in the art.
  • Probes of the present invention may also be used in diagnostic methods to detect autoantibodies in diabetic and prediabetic sera.
  • DNA molecules used within the present invention may be labeled and used in a hybridization procedure similar to the Southern or dot blot.
  • conditions that allow the DNA molecules of the present invention to hybridize to the representative DNA sequence of SEQ ID NO:1, 15 or 21 or their allelic variants may be determined by methods well known in the art (reviewed, for example, by Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; which is incorporated herein by reference).
  • Those skilled in the art will be capable of varying hybridization conditions (i.e.
  • allelic variants may be identified using DNA molecules of the present invention and, for example, the polymerase chain reaction (PCR) (disclosed by Saiki et al., Science 239: 487, 1987; Mullis et al., U.S. Pat. No. 4,686,195; and Mullis et al., U.S. Pat. No. 4,683,202) to amplify DNA sequences, which are subsequently detected by their characteristic size on agarose gels or which may be sequenced to detect sequence abnormalities.
  • PCR polymerase chain reaction
  • DNA molecules encoding the islet cell antigen polypeptides of the present invention may be inserted into DNA constructs.
  • a DNA construct is understood to refer to a DNA molecule, or a clone of such a molecule, either single- or double-stranded, which has been modified through human intervention to contain segments of DNA combined and juxtaposed in a manner that would not otherwise exist in nature.
  • DNA constructs of the present invention comprise a first DNA segment encoding an islet cell antigen polypeptide operably linked to additional DNA segments required for the expression of the first DNA segment.
  • additional DNA segments will generally include promoters and transcription terminators, and may further include enhancers and other elements.
  • One or more selectable markers may also be included.
  • DNA constructs useful for expressing cloned DNA segments in a variety of prokaryotic and eukaryotic host cells can be prepared from readily available components or purchase from commercial suppliers.
  • a DNA sequence encoding a protein of the present invention is operably linked to a transcription promoter and terminator within a DNA construct.
  • the construct will commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • the first DNA segment is an isolated polynucleotide molecule encoding a mammalian islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:4, wherein the polypeptide forms an immune complex with autoantibodies from a patient at risk of or predisposed to IDDM.
  • the first DNA segment is an isolated polynucleotide encoding a polypeptide of SEQ ID NO:2 from Leu, amino acid residue 265 to Gln, amino acid residue 641.
  • the first DNA segment is an isolated polynucleotide encoding a polypeptide of SEQ ID NO:2 from Ser, amino acid residue 1, to Gln, amino acid residue 641.
  • the first DNA segment is an isolated polynucleotide encoding a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818.
  • the first DNA segment is an isolated polynucleotide encoding a polypeptide of SEQ ID NO:16 from Ala, amino acid residue 1 to Gln, amino acid residue 1012.
  • the proteins of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells.
  • Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., ibid., which are incorporated herein by reference.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence is joined to the DNA sequence encoding a protein of the present invention in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5′ to the DNA sequence encoding the protein of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
  • the secretory signal sequence may be that normally associated with a protein of the present invention, or may be from a gene encoding another secreted protein.
  • a preferred vector system for use in the present invention is the pZCEP vector system as disclosed by Jelineck et al., Science, 259: 1615-16, 1993.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
  • cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No.
  • CRL 1573 Graham et al., J. Gen. Virol. 36:59-72, 1977
  • Chinese hamster ovary e.g. CHO-K1; ATCC No. CCL 61
  • Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Md.
  • strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus.
  • Prokaryotic cells can also serve as host cells for use in carrying out the present invention. Particularly preferred are strains of the bacteria Escherichia coli, although Bacillus and other genera are also useful. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.).
  • the protein When expressing the proteins in bacteria such as E. coli, the protein may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate. The denatured protein is then refolded by diluting the denaturant. In the latter case, the protein can be recovered from the periplasmic space in a soluble form.
  • Fungal cells are also suitable as host cells.
  • Saccharomyces ssp. Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia guillermondii, Pichia methanolica, and Candida maltosa transformation systems are known in the art. See, for example, Kawasaki, U.S. Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No.
  • Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign proteins therein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and Bang et al., U.S. Pat. No. 4,775,624, which are incorporated herein by reference.
  • the use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
  • a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
  • Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as “amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
  • the recombinant islet cell antigen polypeptides expressed using the methods described herein are isolated and purified by conventional procedures, including separating the cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulfate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography or affinity chromatography, or the like.
  • Methods of protein purification are known in the art (see generally, Scopes, R., Protein Purification, Springer-Verlag, NY (1982), which is incorporated herein by reference) and may be applied to the purification of the recombinant proteins of the present invention.
  • Substantially pure recombinant islet cell antigen polypeptides of at least about 50% is preferred, at least about 70-80% more preferred, and 95-99% or more homogeneity most preferred, particularly for pharmaceutical uses.
  • the recombinant islet cell antigen polypeptides may then be used diagnostically, therapeutically, etc. as further described below.
  • Recombinant 1851 polypeptides can also be produced by expressing islet cell antigen DNA fragments, such as fragments generated by digesting an islet cell antigen cDNA at convenient restriction sites.
  • the isolated recombinant polypeptides or cell-conditioned media are then assayed for activity as described in the examples below.
  • the proteins of the present invention may be synthesized following conventional synthesis methods such as the solid-phase synthesis using the method of Barany and Merrifield (in The Peptides. Analysis, Synthesis, Biology Vol. 2, Gross and Meienhofer, eds, Academic Press, NY, pp. 1-284, 1980, which are incorporated herein by reference), by partial solid-phase techniques, by fragment condensation or by classical solution addition.
  • Short polypeptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 amino acids, which correspond to selected islet cell antigen polypeptide regions can be readily synthesized and then screened in screening assays designed to identify peptides having a desired activity, such as domains which are responsible for or contribute to binding activity, immunodominant epitopes (particularly those recognized by autoantibodies), and the like.
  • 1851 polypeptides may also be prepared from cells that naturally produce 1851 protein (such as islet cells).
  • 1851 polypeptides may be prepared from islet cells by isolation of a membrane fraction. This 1851-enriched fraction is then used to detect autoantibodies to 1851 in prediabetic and diabetic sera.
  • Islet cell antigen polypeptides produced according to the present invention can be used diagnostically, in the detection and quantitation of autoantibodies in a biological sample, that is, any sample derived from or containing cells, cell components or cell products, including, but not limited to, cell culture supernatants, cell lysates, cleared cell lysates, cell extracts, tissue extracts, blood plasma, serum, and fractions thereof.
  • a biological sample that is, any sample derived from or containing cells, cell components or cell products, including, but not limited to, cell culture supernatants, cell lysates, cleared cell lysates, cell extracts, tissue extracts, blood plasma, serum, and fractions thereof.
  • This information can then be used to monitor the progression or regression of the potentially harmful autoantibodies in individuals at risk of, or with a predisposition to develop IDDM, and would be useful for predicting the clinical course of the disease in a patient.
  • the assay results can also find use in monitoring the effectiveness of therapeutic measures for treatment of IDDM or related diseases.
  • autoantibodies directed to the polypeptides of the present invention are quantified directly by measuring the binding of autoantibodies in a biological sample to recombinant or synthetic islet cell antigen polypeptides.
  • the biological sample is contacted with at least one islet cell antigen polypeptide of the invention under conditions conducive to immune complex formation.
  • the immune complexes formed between the islet cell antigen polypeptide and the antibodies are then detected, and the presence and quantity of autoantibodies can then be used to diagnose or direct treatment of IDDM.
  • the immune complexes can be detected by means of antibodies that bind to the islet cell antigen of the present invention or by labeling the polypeptide as described below.
  • the serum level of a patient's autoantibodies to the islet cell antigen polypeptides in serum can be measured by competitive binding with labeled or unlabeled antibodies to the islet cell antigen polypeptides of the present invention.
  • Unlabeled 1851 polypeptides can be used in combination with labeled antibodies that bind to human antibodies or to islet cell antigens.
  • the islet cell antigen polypeptide can be directly labeled.
  • labels can be employed, such as radionuclides, particles (e.g., gold, ferritin, magnetic particles, red blood cells), fluors, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), chemiluminescers, biotin and other compounds that provide for the detection of the labeled polypeptide or protein.
  • an 1851 polypeptide can be radiolabeled using conventional methods such as in vitro transcription and translation. Radiolabeled 1851 polypeptide is combined with patient serum under conditions suitable for immune complex formation. Immune complexes are then separated, such as by binding to protein A.
  • Precipitated 1851 polypeptides are then quantitated by conventional methods, such as gel electrophoresis, fluorography, densitometry or by direct counting of immunoprecipitated, radiolabeled antigen.
  • the amount of 1851 polypeptide precipitated by test sera can be statistically compared to mean counts precipitated by healthy control sera, each measured separately.
  • an 1851 polypeptide antigen, labeled with biotin is combined with patient serum under conditions suitable for immune complex formation.
  • the serum is then transferred to a protein A-coated container, such as a well of an assay plate, and the container is allowed to stand so that immune complexes can form.
  • the container is then washed, and streptavidin, conjugated to a suitable enzyme (e.g. alkaline phosphatase), is added.
  • streptavidin conjugated to a suitable enzyme (e.g. alkaline phosphatase)
  • a chromogenic substrate is then added, and the presence of 1851 polypeptide auto
  • autoantibodies to islet cell antigen polypeptides can be identified and, if desired, extracted from a patient's serum by binding to 1851 polypeptides of the present invention.
  • the islet cell antigen polypeptides may be attached, e.g., by adsorption, to an insoluble or solid support, such as ELISA microtiter well, microbead, filter membrane, insoluble or precipitable soluble polymer, etc. to function as an affinity resin.
  • the captured autoantibodies can then be identified by several methods. For example, antisera or monoclonal antibodies to the antibodies can be used. These antisera or monoclonal antibodies are typically non-human in origin, such as rabbit, goat, mouse, etc. These anti-antibodies can be detected directly if attached to a label such as 125 I, enzyme, biotin, etc., or can be detected indirectly by a labeled secondary antibody made to specifically detect the anti-antibody.
  • the diagnostic methods of the present invention can be used in conjunction with other known assays and diagnostic techniques (see for example, WO 95/07464, incorporated herein by reference in its entirety).
  • Such other assays and techniques include measurement of body mass index (BMI), defined as the quotient of the patient's weight in kg divided by the square of height in meters; C-peptide level (Heding, Diabetologia 11: 541-548 (1975); Landin-Olsson et al., Diabetologia 33: 561-568 (1990)); or one or more additional diabetes-associated autoantibodies, genotypes or loci.
  • BMI body mass index
  • a low BMI i.e. less than about 25
  • other indicators is suggestive of type I diabetes.
  • BMI is thus a useful indicator for distinguishing type I from type II diabetes.
  • C-peptide level can be measured using standard methods, such as that of Heding (ibid.), in which insulin and proinsulin are removed from serum and C-peptide is measured in the resulting insulin-free fraction radioimmunologically.
  • the islet cell antigen polypeptides of the current invention can also be used to assess T cell reactivity, as a method for monitoring the disease state in a patient.
  • Mammalian islet cell antigen 1851 peptides will generally comprise at least about 12 amino acids, and more often from about 15 amino acids to about 20 or more amino acids. In some instances, a substantial portion or domain or even the entire 1851 protein, can be used to assess T cell reactivity in peripheral blood mononuclear cells (PBMNCs) from prediabetics.
  • PBMNCs peripheral blood mononuclear cells
  • Methods for detecting such in vitro activity are known in the art, including a proliferation assay measuring 3 H-thymidine incorporation, analysis of activation markers, such as CD69, or measuring cytokine production, such as IL-2.
  • Mammalian cells such as COS cells or L cells, may also be transfected with appropriate Class I or Class II alleles specific for the islet cell antigen of the present invention.
  • MHC molecules may be soluble or membrane bound, and the 1851 antigenic polypeptide may be recombinantly tethered to the N-terminal region of the ⁇ or ⁇ chain using a flexible linker containing, for example, repeating glycine residues separated by a serine residue, such that the antigenic peptide binds to the MHC molecule and is properly presented to the T cell.
  • the antigenic peptide may be exogenously loaded into the MHC peptide binding grove.
  • the MHC-antigenic peptide complex can then be used to assess the reactivity of peripheral blood T cells derived from prediabetic or diabetic patients. This reactivity may be assessed by methods known in the art, such as 3 H thymidine incorporation, cytokine production or cytolysis.
  • islet cell antigen expressed in microorganisms can be “fed” to peripheral blood mononuclear cells (PBMN). The antigen-fed cells can then be used to stimulate peripheral blood T cells derived from diabetics or prediabetics.
  • the islet cell antigen polypeptides are also contemplated to be advantageous for use as immunotherapeutics to induce immunological tolerance or nonresponsiveness (anergy) to 1851 polypeptide autoantigens in patients predisposed or already mounting an immune response to 1851 polypeptide autoantigens of the islet ⁇ -cells.
  • This therapy can take the form of autoantigenic 1851 peptides bound to an appropriate MHC Class I or Class II molecule as described above.
  • the therapy can also be in the form of oral tolerance (Weiner et al., Nature 376: 177-80, 1995), or IV tolerance, for example.
  • polypeptide antigens in suppression of autoimmune disease is disclosed by Wraith, et al., ( Cell 59: 247-55, 1989).
  • Tolerance can be induced in patients, although conditions for inducing such tolerance will vary according to a variety of factors.
  • tolerance can be induced by parenteral injection of an islet cell antigenic polypeptide, either with recombinant polypeptide or synthetic antigen, or more conveniently by oral administration in an appropriate formulation. The precise amount of administration, its mode and frequency of dosages will vary.
  • the precise amounts and frequency of administration will also vary, for adults about 1 to 1,000 mg/kg can be administered by a variety of routes, such as parenterally, orally, by aerosols, intradermal injection, etc. For neonates the doses will generally be higher than those administered to adults; e.g. 100 to 1,000 mg/kg.
  • the islet cell antigen 1851 polypeptides will typically be more tolerogenic when administered in a soluble form rather than an aggregrated or particulate form. Persistence of an islet cell antigen polypeptide of the invention is generally needed to maintain tolerance in an adult, and thus may require more frequent administration of the antigen, or its administration in a form which extends the half-life of the islet cell antigen. See for example, Sun et al. ( Proc. Natl. Acad. Sci. USA 91: 10795-99, 1994).
  • the islet cell antigen polypeptides described herein are also contemplated to be advantageous for use as immunotherapeutics in treating longer term IDDM patients that have been identified by autoantibody testing at the time of clinical non-insulin dependent diabetes mellitus (NIDDM) diagnosis. Intervention in these patients may be especially effective, perhaps due to the slowly progressive nature of their ⁇ cell destruction. Since the numbers of such patients is nearly the same as those with classical childhood IDDM, there is a need for such therapeutic intervention (Hagopian et al., J. Clin. Invest. 91:368-74; 1993; Harris and Robbins, Diabetes Care 17:1337-40, 1994; and Kobayashi et al., Diabetes 45:622-26, 1996).
  • the N-terminal domain of islet cell antigen 1851 is expected to be inside the insulin secretory granule.
  • the islet cell antigen polypeptides of the current invention contain post translational modification sites within the N-terminal domain.
  • a dibasic site or tribasic site at amino acid residues 228-230 (Arg-Lys-Lys) in SEQ ID NO:22 and amino acid residues 422-424 (Arg-Lys-Lys) in SEQ ID NO:16 could result in cleavage of a 420 amino acid post-translationally modified mammalian islet cell antigen polypeptide from the islet cell antigen 1851 polypeptide.
  • All or part of this cleaved polypeptide may be released from the ⁇ cell via either the constitutive secretory pathway for granule halo components, or via the regulated pathway involved in insulin release.
  • Detection and quantitation of post translationally modified polypeptides in a biological sample can be used diagnostically to monitor disease state in a patient.
  • the presence or absence of such polypeptides in prediabetic and diabetic sera can be determined, for example by radioimmunoassay, and the concentration of such polypeptides in such an individual serum sample can be measured. This information can then be used, for example, to monitor insulin secretory activity, such as ⁇ cell insulin secretory rates; or to indicate altered ⁇ cell physiology associated with cellular stress as in an immune attack. Peptide levels could be an indicator of ⁇ cell distress or ⁇ cell death, and would be useful for predicting the disease state in a patient.
  • the peptides herein function serve in paracrine or endocrine signaling to other islet cells or remote cells in other organs.
  • a post-translationally modified mammalian islet cell antigen polypeptide comprises the sequence of SEQ ID NO:22 from His, amino acid residue 1 to Glu, amino acid residue 227.
  • the biological sample is blood.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an islet cell antigen polypeptide of the present invention, together with a pharmaceutically acceptable carrier or vehicle, such as saline, buffered saline, water or the like.
  • a pharmaceutically acceptable carrier or vehicle such as saline, buffered saline, water or the like.
  • Formulations may further include one or more excipients, preservatives, solubilizers, etc.
  • Methods of formulation are well known in the art and are disclosed, for example, in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton Pa., 1990, which is incorporated herein by reference.
  • Therapeutic doses will generally be in the range of 0.1 to 100 ⁇ g/kg of patient weight, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art.
  • a therapeutically effective amount of an islet cell antigen polypeptide of the present invention is an amount sufficient to produce a clinically significant reduction in ⁇ -cell loss or a delay of clinical onset of IDDM.
  • the present invention provides diagnostic kits for use with the recombinant or synthetic islet cell antigen polypeptides of the present invention, in detecting autoantibodies to pancreatic ⁇ -islet cells.
  • 1851 polypeptides may be provided, usually in lyophilized form, in a container, either alone or in conjunction with additional reagents, such as 1851-specific antibodies, labels, and/or anti-human antibodies and the like.
  • the 1851 polypeptides and antibodies which may be conjugated to a label or unconjugated, are included in the kits with buffers, such as Tris phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum albumin, and the like. Frequently it will be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1 to 99% of the total composition. Where an antibody capable of binding to the islet cell antigen polypeptide autoantibody or to the recombinant or synthetic 1851 polypeptide is employed in an assay, this will typically be present in a separate vial.
  • islet cell antigen polypeptides including derivatives thereof, as well as portions or fragments of these polypeptides, are utilized to prepare antibodies for diagnostic or therapeutic uses which specifically bind to islet cell antigen polypeptides.
  • antibodies includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab′) 2 and Fab fragments, as well as recombinantly produced binding partners. These binding partners incorporate the variable regions from a gene which encodes a specifically binding monoclonal antibody.
  • Antibodies are defined to be specifically binding if they bind to the islet cell antigen polypeptides with a K a of greater than or equal to 10 7 /M.
  • K a of greater than or equal to 10 7 /M.
  • the affinity of a monoclonal antibody or binding partner may be readily determined by one of ordinary skill in the art (see, Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949).
  • polyclonal antibodies may be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats, for example.
  • the immunogenicity of the islet cell antigen polypeptide may be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant.
  • an adjuvant such as Freund's complete or incomplete adjuvant.
  • assays known to those skilled in the art may be utilized to detect antibodies which specifically bind to an islet cell antigen. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations, enzyme-linked immuno-sorbent assays, dot blot assays, inhibition or competition assays, and sandwich assays.
  • mRNA is isolated from a B cell population and used to create heavy and light chain immunoglobulin cDNA expression libraries in a suitable vector such as the ⁇ IMMUNOZAP(H) and ⁇ IMMUNOZAP(L) vectors, which may be obtained from Stratogene Cloning Systems (La Jolla, Calif.). These vectors are then screened individually or are co-expressed to form Fab fragments or antibodies (Huse et al., Science 246: 1275-81, 1989; Sastry et al., Proc. Natl. Acad. Sci. USA 86: 5728-32, 1989). Positive plaques are subsequently converted to a non-lytic plasmid which allows high level expression of monoclonal antibody fragments in E. coli.
  • Binding partners such as those described above may also be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene which encodes a specifically binding antibody.
  • the construction of these proteins may be readily accomplished by one of ordinary skill in the art (see for example, Larrick et al., Biotechnology 7: 934-38, 1989; Reichmann et al., Nature 322: 323-27, 1988 and Roberts et al. Nature 328: 731-34, 1987). Once suitable antibodies or binding partners have been obtained, they may be isolated or purified by many techniques well described in the literature (see for example, Antibodies: A Laboratory Manual, ibid.).
  • Suitable techniques include protein or peptide affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns or any combination of these techniques.
  • isolated as used to define antibodies or binding partners means “substantially free of other blood components.”
  • Antibodies of the present invention may be produced by immunizing an animal, a wide variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats can be used, with a recombinant or synthetic islet cell antigen polypeptide or a selected portion thereof (e.g., a peptide). For example, by selected screening one can identify a region of the islet cell antigen polypeptide such as that predominantly responsible for recognition by anti-islet cell antigen polypeptide antibodies, or a portion which comprises an epitope of a islet cell antigen polypeptide variable region, which may thus serve as a islet cell antigen polypeptide-specific marker.
  • Antibody producing cells obtained from the immunized animals are immortalized and screened, or screened first for, e.g., the production of antibody which inhibits the interaction of the anti-islet cell antigen polypeptide autoantibody with the islet cell antigen polypeptide and then immortalized.
  • a human antigen such as an 1851 polypeptide
  • Such methods are generally known in the art and are described in, for example, U.S. Pat. No. 4,816,397, and EP publications 173,494 and 239,400, which are incorporated herein by reference.
  • DNA sequences which encode a human monoclonal antibody or portions thereof that specifically bind to islet cell antigen polypeptides by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246: 1275-81, 1989, incorporated herein by reference, and then cloning and amplifying the sequences which encode the antibody (or binding fragment) of the desired specificity.
  • the mammalian islet cell antigen polypeptides can be used to clone T cells which have specific receptors for the islet cell antigen polypeptide.
  • the T cells or membrane preparations thereof can be used to immunize animals to produce antibodies to the islet cell antigen polypeptide receptors on T cells.
  • the antibodies can be polyclonal or monoclonal. If polyclonal, the antibodies can be murine, lagomorph, equine, ovine, or from a variety of other mammals.
  • Monoclonal antibodies will typically be murine in origin, produced according to known techniques, or human, as described above, or combinations thereof, as in chimeric or humanized antibodies.
  • the anti-islet cell antigen polypeptide receptor antibodies thus obtained can then be administered to patients to reduce or eliminate T cell subpopulations which recognize and participate in the immunological destruction of islet cell antigen polypeptide bearing cells in an individual predisposed to or already suffering from a disease, such as IDDM.
  • the islet cell antigen polypeptide T cell receptors can thus be identified, cloned and sequenced, and receptor polypeptides synthesized which bind to the islet cell antigen polypeptides and block recognition of the islet cell antigen polypeptide-bearing cells, thereby impeding the autoimmune response against host islet cells. Howell et al. ( Science 246: 668-70, 1989) have demonstrated that T cell receptor peptides can block the formation of the tri-molecular complex between T cells, autoantigen and major histocompatibilty complex in an autoimmune disease model.
  • Antibodies and binding partners of the present invention may be used in a variety of ways.
  • the tissue distribution of the islet cell antigen may be determined by incubating tissue slices with a labeled monoclonal antibody which specifically binds to the islet cell antigen polypeptides, followed by detection of the presence of the bound antibody.
  • Labels suitable for use within the present invention are well known in the art and include, among others, fluorescein, isothiocyanate, phycoerythrin, horseradish peroxidase, and colloidal gold.
  • the antibodies of the present invention may also be used for the purification of the islet cell antigen polypeptides of the present invention.
  • Antibodies of the present invention may be used as a marker reagent to detect the presence of islet cell antigen polypeptides on cells or in solution. Such antibodies are also useful for western analysis or immunoblotting, particularly of purified cell secreted material. Polyclonal, affinity purified polyclonal, monoclonal and single chain antibodies are suitable for use in this regard. In addition, proteolytic and recombinant fragments and epitope binding domains can be used herein. Chimeric, humanized, veneered, CDR-replaced, reshaped or other recombinant whole or partial antibodies are also suitable.
  • RNA from the islets was isolated according to the method of Chirgwin et al., Biochemistry 18: 52-94, 1994, incorporated herein by reference, using polytron homogenization in guanidinium thiocynate and LiCl centrifugation. Poly(A)+ RNA was isolated using oligo d(T) cellulose chromatography (Aviv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972).
  • First strand cDNA was synthesized from two-time poly d(T)-selected liver poly(A) + RNA.
  • Ten microliters of a solution containing 10 ⁇ g of liver poly(A) + RNA was mixed with 2 ⁇ l of 20 pmole/ ⁇ l first strand primer ZC3747 (SEQ ID NO:8) and 4 ⁇ l of diethylpyrocarbonate-treated water. The mixture was heated at 65° C. for 4 minutes and cooled by chilling on ice.
  • the first strand cDNA synthesis was initiated by the addition of 8 ⁇ l of 5 ⁇ SUPERSCRIPT buffer (GIBCO BRL, Gaithersburg, Md.), 4 ⁇ l of 100 mM dithiothreitol, and 2.0 ⁇ l of a deoxynucleotide triphosphatate solution containing 10 mM each of dATP, dGTP, dTTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology Inc., Piscataway, N.J.) to the RNA-primer mixture. The reaction mixture was incubated at 45° C. for 4 minutes.
  • the unlabeled cDNA was resuspended in 50 ⁇ l water and used for the second strand synthesis.
  • the length of first strand cDNA was assessed by resuspending the labeled cDNA in 20 ⁇ l water and determining the cDNA size by agarose gel electrophoresis.
  • Second strand synthesis was performed on the RNA-DNA hybrid from the first strand synthesis reaction under conditions that promoted first strand priming of second strand synthesis resulting in DNA hairpin formation.
  • a reaction mixture was prepared containing 20.0 ⁇ l of 5 ⁇ polymerase I buffer (100 mM Tris, pH 7.4, 500 mM KCl, 25 mM MgCl 2, 50 MM (NH 4 ) 2 SO 4 ), 1.0 ⁇ l of 100 mM dithiothreitol, 2.0 ⁇ l of a solution containing 10 mM of each deoxynucleotide triphosphate, 3.0 ⁇ l 5 mM ⁇ -NAD, 1.0 ⁇ l of 3 U/ ⁇ l E.
  • the reactions were each terminated by serial phenol/chloroform and chloroform/isoamylalcohol extractions.
  • the DNA from each reaction was precipitated in the presence of ethanol and 2.5 M ammonium acetate.
  • the DNA from the unlabeled reaction was resuspended in 100 ⁇ l water.
  • the labeled DNA was resuspended and electrophoresed as described above.
  • the single-stranded DNA in the hairpin structure was cleaved using mung bean nuclease.
  • the reaction mixture contained 20 ⁇ l of 10 ⁇ Mung Bean Nuclease Buffer (Stratagene Cloning Systems, La Jolla, Calif.), 16 ⁇ l of 100 mM dithiothreitol, 54 ⁇ l water, 100 ⁇ l of the second strand cDNA, and 10 ⁇ l of a 1:10 dilution of Mung Bean Nuclease, final concentration 10.5 U/ ⁇ l (Promega Corp., Madison, Wis.) in Stratagene MB dilution Buffer (Stratagene Cloning Systems). The reaction was incubated at 37° C.
  • the resuspended cDNA was blunt-ended with T4 DNA polymerase.
  • the cDNA which was resuspended in a volume of 50 ⁇ l of water, was mixed with 50 ⁇ l of 5 ⁇ T4 DNA polymerase buffer. (250 mM Tris-HCl, pH 8.0, 250 mM KCl, 25 MM MgCl 2 ), 3 ⁇ l of 100 mM dithiothreitol, 3 ⁇ l of a solution containing 10 mM of each deoxynucleotide triphosphate, and 4 ⁇ l of 1.0 U/ ⁇ l T4 DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.). After an incubation at 10° C.
  • the reaction was terminated by serial phenol/chloroform and chloroform/isoamylalcohol extractions.
  • the cDNA fragments less than 400 bp in length were removed by chromatography on a Clontech TE400 spin column (Clontech, Palo Alto, Calif.).
  • the DNA was ethanol precipitated and resuspended in 9 ⁇ l of water. Based on the incorporation of 32 P-dCTP, the yield of cDNA was estimated to be 4 ⁇ g from a starting mRNA template of 10 ⁇ g.
  • Eco RI adapters (Pharmacia LKB Biotechnology Inc., Piscataway, N.J.) were added to the cDNA prepared above to facilitate the cloning of the cDNA into a mammalian expression vector.
  • the reaction was terminated by the addition of 185 ⁇ l water, 25 ⁇ l REACT 2 buffer (Gibco BRL) followed by an incubation at 65° C. for between 30 and 60 minutes. After incubation, the reaction was terminated by serial phenol/chloroform and chloroform/isoamylalcohol extractions and ethanol precipitation as described above. Following centrifugation, the DNA pellet was washed with 70% ethanol and was air dried. The pellet was resuspended in 89 ⁇ l of water.
  • the cDNA was digested with Xho I, resulting in a cDNA having a 5′ Eco RI adhesive end and a 3′ Xho I adhesive end.
  • the Xho I restriction site at the 3′ end of the cDNA was introduced through the ZC3747 primer (SEQ ID NO:8).
  • the restriction digestion was terminated by serial phenol/chloroform and chloroform/isoamylalcohol extractions.
  • the cDNA was ethanol precipitated, and the resulting pellet was washed with 70% ethanol and air-dried. The pellet was resuspended in 1 ⁇ loading buffer (10 mM phosphate buffer, pH 8.8, 5% glycerol, 0.125% bromphenol blue).
  • the resuspended cDNA was heated to 65° C. for 10 minutes, cooled on ice and electrophoresed on a 0.8% low melt agarose gel (Seaplaque GTG Low Melt Agarose, FMC Corp., Rockland, Me.) using a 1 Kb ladder (Gibco BRL) as size markers.
  • the contaminating adapters and by-product fragments below 600 bp in size were excised from the gel.
  • the electrodes were reversed, and the cDNA was electrophoresed until concentrated near the lane origin. The area of the gel containing the concentrated DNA was excised, placed in a microfuge tube, and the approximate volume of the gel slice was determined.
  • TE Tris HCl pH 7.4, 1 mM disodium ethylenediaminetetraacetate.2 H 2 O (EDTA)
  • EDTA disodium ethylenediaminetetraacetate.2 H 2 O
  • the sample was centrifuged at 14,000 ⁇ g for 10 minutes to remove the undigested agarose.
  • the cDNA in the supernatant was ethanol precipitated.
  • the cDNA pellet was washed with 70% ethanol, air dried and resuspended in 37 ⁇ l of water.
  • the cDNA recovered from the agarose gel was phosphorylated using T4 polynucleotide kinase.
  • the reaction consisted of 37 ⁇ l cDNA, 5 ⁇ l 10 ⁇ Stratagene Ligase Buffer (Stratagene Cloning Systems).
  • the reaction was cooled to room temperature where 5 ⁇ l 10 mM ATP (Pharmacia) and 3 ⁇ l T4 DNA polymerase (10 U/ ⁇ l, Stratagene) were added. The reaction was incubated at 37° C. for 45 minutes and at 65° C. for 10 minutes. The reaction was terminated by serial phenol/chloroform extractions. The samples were chromatographed through a Clontech TE400 spin column and were precipitated in the presence of 2.5 M ammonium acetate. The cDNA was resuspended in 15 ⁇ l of 2.5 mM Tris-HCl, pH 8.0, 0.25 mM EDTA.
  • the resulting Eco RI-Xho I cDNA library was cloned into the E. coli vector pZCEP (Jelinek et al., Science 259: 1614-16, 1993).
  • Eco RI-Xho I linearized pZCEP was ligated with the Eco RI-Xho I cDNA library.
  • the resulting plasmids were electroporated into the E. coli strain DH10B ELECTROMAX ⁇ (Gibco BRL).
  • the library was plated to obtain >5 ⁇ 10 5 independent colonies and aliquoted into 120 pools to give approximately 5,000 colonies per pool. An aliquot of the cells from each pool was removed for use in preparing plasmid DNA.
  • Plasmid DNA was prepared from each pool and the resulting plasmid DNA was digested with RNAse (Boehringer Mannheim) according to the manufacturer's instructions. The RNAse reaction was terminated by a phenol/chloroform/isoamylalcohol (24:24:1) extraction, and the DNA was ethanol precipitated. The pools were systematically screened as described in the examples below.
  • Macaque DNA from each pool was transfected into COS-7 cells (African Green Monkey Kidney cells, ATCC CRL 1651) using the method essentially described by McMahan et al. ( EMBO J. 10: 2821-32, 1991; which is incorporated by reference herein in its entirety).
  • 1-2 ⁇ g of macaque islet cell library pooled DNA was added into 100 ⁇ l of serum free medium (SFM, F/DV medium, 10 mg/l transferrin, 2 ⁇ g/l selenium, 10 mg/l fetuin, 5 mg/l insulin, 1 L-glutamine, 25 mM Hepes, 1 mM NaPyruvate, and 0.1 mM NEAA).
  • SFM serum free medium
  • F/DV medium serum free medium
  • transferrin 10 mg/l transferrin
  • 2 ⁇ g/l selenium 10 mg/l fetuin
  • 5 mg/l insulin 1 L-glutamine
  • 25 mM Hepes 25 mM Hepes
  • 1 mM NaPyruvate 1 mM NaPyruvate
  • 0.1 mM NEAA 0.1 mM NEAA
  • Recombinant radiolabeled GAD was expressed in the presence of 35 S Methionine (Amersham Corp., Arlington Heights, Ill.) using the Sp6 bacteriophage promoter and the TNT reticulocyte lysate kit (Promega), according to manufacturer's direction. 35 S Methionine incorporation was determined by precipitation using trichloroacetic acid (TCA), and 25% or more incorporation was considered acceptable. Radiolabeled antigen was stored at ⁇ 80° C. until use.
  • Radiolabeled antigen was diluted 1:10 in immunoprecipitation buffer (150 mM NaCl, 1% v/v Triton X-114 (Sigma Chemical Co., St. Louis, Mo.), 0.05% Bovine serum albumin (Sigma), 10 mM benzamidine (Sigma), and 10 mM HEPES pH 7.4).
  • the antigen was incubated for preclearing for 4 hours at 4° C. with 50 ⁇ l normal human serum. Immunoglobulin was removed using 200 ⁇ l Protein A Sepharose beads (Pharmacia LKB Biotechnology Inc.) for 45 minutes.
  • the cleared supernatant was diluted to 50,000 TCA-precipitable counts per minute (cpm) per 400 ⁇ l immunoprecipitation buffer.
  • cpm TCA-precipitable counts per minute
  • 4° C. 4° C. overnight with mixing by gentle rotation.
  • Antigen-antibody complexes were precipitated by 16 ⁇ l Protein A Sepharose, and the pellet was washed 5 times in ice-cold wash buffer which consisted of 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% BSA, and 0.25% Triton X-114.
  • Antigen was dissociated from the pellet by boiling in the presence of 2% SDS and 5% ⁇ -mercaptoethanol, and counted by scintillation counting in scintillation fluid. Counts per minute reflect the level of autoantibodies present in the sera to capture the antigen.
  • First strand cDNA was synthesized using a Superscript ⁇ Preamplification System (GIBCO BRL) according to the manufacturer's directions.
  • GEBCO BRL Superscript ⁇ Preamplification System
  • One and one half microliters of a solution containing 5 ⁇ g total U87MG RNA was mixed with 1 ⁇ l oligo dT solution and 11.5 ⁇ l diethylpyrocarbonate-treated water. The mixture was heated at 70° C. for 10 minutes and cooled by chilling on ice.
  • First strand cDNA synthesis was initiated by the addition of 2 ⁇ l Superscript ⁇ II buffer, 2 ⁇ l 0.1 M dithiothreitol, 1 ⁇ l deoxynucleotide triphosphate solution containing 10 mM each of dATP, dGTP, dTTP, and dCTP, and 1 ⁇ l of 200 U/ ⁇ l Superscript ⁇ II reverse transcriptase to the RNA-primer mixture.
  • the reaction was incubated at room temperature for 10 minutes followed by an incubation at 42° C. for 50 minutes, then 70° C. for 15 minutes, then cooled on ice.
  • the reaction was terminated by addition of 1 ⁇ l RNase H which was incubated at 37° C. for 20 minutes, then cooled on ice.
  • a 100 ⁇ l PCR reaction mixture was then prepared containing 20 ⁇ l of first strand template, 8 ⁇ l 10 ⁇ synthesis buffer, 3.3 ⁇ M ZC8802 (SEQ ID NO:9, contains 5′ Xho I site and ATG), 5.4 ⁇ M ZC8803 (SEQ ID NO:10, contains Eco RI site following stop codon), 65 ⁇ l dH 2 O and 1 wax bead (AmpliWax ⁇ , Perkin-Elmer Cetus, Norwalk, Conn.). Following an initial cycle of 95° C. for 2 minutes, 4° C. for 10 minutes, 5 U Taq polymerase was added, and the reaction was amplified for 30 cycles of 1 minute at 95° C., 2 minutes at 55° C.
  • EmWi Twenty milliliters of EmWi was diluted 1:1 in 0.1 M NaPO 4 buffer, pH 8.0 and incubated with an equal volume of Protein A covalently linked to Sepharose beads (Zymed, South San Francisco, Calif.) for affinity purification. After gentle mixing for 45 minutes at 4° C., the slurry was loaded onto a column and washed with 10 column volumes of 0.1 M NaPO 4 buffer, pH 8.0 and one column volume of 0.01 M NaPO 4 buffer, pH 8.0, before elution of immunoglobulins with 0.05 M Na citrate buffer, pH 3.5. Eluted immunoglobulins were immediately neutralized to pH 7.0 with 2 M Tris, pH 8.0.
  • Eluted fractions were evaluated by spectrophotometric absorption at 280 nM, and peak fractions were pooled, aliquoted and flash frozen for storage at ⁇ 80° C. Typically the concentration was 4 mg/ml IgG.
  • COS-7 cells were grown to confluence in 150 ml T-flasks, washed with PBS, fixed in 4% paraformaldehyde, and permeabilized by freeze/thaw. The pooled sera were diluted to 1 mg/ml in PBS and incubated with the permeabilized COS-7 cell lysate overnight at 4° C. Supernatant was cleared at 100,000 ⁇ g and aliquotted for storage at ⁇ 80° C. for use in the binding assay.
  • the cells were fixed with 1 ml 50% ETOH/50% acetone for 5 minutes at room temperature followed by two washes in PBS and two washes in 1% bovine serum albumin (BSA) in PBS.
  • BSA bovine serum albumin
  • the precleared serum (EmWi) was diluted to 0.2 mg/ml in a 5% BSA in PBS solution, and 500 ⁇ l was added to each of the slides which were then covered, wrapped in plastic wrap, and rocked gently on a rocker overnight at room temperature.
  • the slides were then washed three times in a 1% BSA/PBS solution, three minutes for each wash. Following the final wash, the slides were blocked for 10 minutes with 1 ml 5% BSA/4% normal goat serum (Sigma) in PBS at room temperature. The blocking buffer was removed, and 500 ⁇ l of 0.02 mg/ml biotinylated Protein A (Amersham Corp., Arlington Heights, Ill.) in 5% BSA/4% normal goat serum/PBS was added, followed by a 30 minute incubation at room temperature.
  • the slides were washed three times with 1% BSA/PBS, three minutes for each wash, then 500 ⁇ l streptavidin-gold (Amersham) diluted 1:50 in 5% BSA/4% normal goat serum/PBS was added to each slide. Following a 60 minute incubation at room temperature the slides were washed three times in 1% BSA/PBS and one final time in PBS. The slides were then fixed by adding 0.5 ml of 9% formaldehyde/45% acetone in PBS for 30 seconds followed by three, 3 minute washes in dH 2 O.
  • a 50 ⁇ l reaction mixture was prepared containing 0.05 ⁇ g of the plasmid DNA from pool #18; 20 pmole of ZC 8802 and ZC 8803 (SEQ ID NOS:9 and 10, respectively); 10 nmoles of each deoxynucleotide triphosphate (Pharmacia); 4 ⁇ l 10 ⁇ synthesis buffer (Boehringer Mannheim), and 2.5 U Taq polymerase (Boehringer Mannheim).
  • the PCR reaction was run for 24 cycles (1 minute at 94° C., 1 minute at 56° C., and 1 minute at 72° C.). An approximately 1.1 Kb band was isolated on a low melt agarose gel electrophoresis and random primed using the MEGAPRIME ⁇ Kit (Amersham) according to the manufacturer's instructions.
  • the filter was hybridized in a solution containing 6 ⁇ SSC, 0.1% SDS, 5 ⁇ Denhardt's, 200 ⁇ g/ml denatured, sheared salmon sperm DNA, and 1 ⁇ 10 5 cpm/ml of 32 P-labeled PCR fragment.
  • the filter was hybridized overnight at 65° C.
  • the excess label was removed by two, 15 minute washes with 2 ⁇ SSC, 0.1% SDS at 65° C.
  • the filter was exposed to film overnight at ⁇ 80° C. with two screens.
  • M1.18.5.1 One clone, designated M1.18.5.1, was sequenced, revealing a 2,170 bp coding region which contained regions of homology to the protein tyrosine phosphatase family, especially IA-2/ICA512. Comparison of the full length human protein tyrosine phosphatase IA-2/ICA512 with M1.18.5.1 suggests that the coding region of M1.18.5.1 is missing amino terminal sequence corresponding to approximately 400 amino acids.
  • the partial nucleic acid sequence and deduced amino acid sequence of M1.18.5.1 is shown in SEQ ID NO 1 and SEQ ID NO: 2.
  • M1.18.5.1 was re-transfected into COS-7 cells and assayed as described above.
  • the JoGr sera which had a high titer to IA-2, was added to the screen and both detected M1.18.5.1.
  • the 2,170 nucleotide sequence from M1.18.5.1 was used to conduct a sequence search for a human homolog. A match was found in the GenBank database (GenBank ID: TO361, clone ID: HFBCV88) submitted by The Institute of Genomic Research, Gaithersburg, Md., as an expressed sequence tag (EST) from a human fetal brain library (Stratagene Cloning Systems). HFBCV88 (EST24415.seq), a 127 amino acid polypeptide, SEQ ID NO:5, had homology to a region of the cytoplasmic domain of M1.18.5.1. The closest human DNA sequence to HFBCV88 is HSICA512, islet cell antigen ICA-512.
  • An oligonucleotide primer (ZC10,011 SEQ ID NO:11) was made to a conserved region between 1851 and HFBCV88 which differed from the corresponding sequence of mouse and human IA-2/ICA512 in that an arginine was substituted for a methionine.
  • a portion of the human homologue of M1.18.5.1 was identified in human insulinoma cDNA by PCR.
  • a PCR reaction was performed in a 100 ⁇ l final volume using 12.5 ng Marathon-ready human insulinoma cDNA prepared according to manufacturer's instruction (Marathon ⁇ cDNA Amplification Kit, Clontech), 20 pmoles each of primers ZC 10,011 (SEQ ID NO:11) and ZC 10,019 (SEQ ID NO:14), and the reagents provided in the Marathon ⁇ PCR kit (Clontech) according to the manufacturer's instructions.
  • the reaction was amplified for 30 cycles (1 minute at 94° C., 30 seconds at 60° C., 5 minutes at 68° C.) followed by a 10 minute extension at 72° C.
  • An 800 bp (WK11111, SEQ ID NO:32) and a 1,200 bp (WK121315, SEQ ID NO:34) fragment were isolated by low melt agarose gel electrophoresis.
  • a 3′RACE Marathon PCR was also performed in a 50 ⁇ l final volume using 12.5 ng Marathon-ready human insulinoma cDNA, 10 pmoles each of primers ZC 10,177 (SEQ ID NO:12) the complement to ZC 10,011, and AP-1 (adaptor primer, supplied with kit), and the reagents provided in the 3′RACE Marathon ⁇ PCR kit (Clontech), according to the manufacturer's instructions.
  • the reaction was amplified for 30 cycles (30 seconds at 94° C., 30 seconds at 68° C.).
  • a 900 bp and a 2,000 bp (WK121111, SEQ ID NO:33) fragment were isolated by low melt agarose electrophoresis.
  • Additional immunoprecipitation assays were performed with a spectrum of serum samples, including 91 healthy control sera (median age 22 years, range 1-49 years, 49% males and 51% females); 183 newly diagnosed IDDM patients sampled at onset (median age 11 years, 51% males and 49% females); and 60 first degree relatives of type I diabetic patients sampled a mean of 2.0 years before onset (median age 12 years, 58% males and 42% females).
  • Parallel autoantibody assays used the intracellular domain of IA-2/ICA512. Immunoprecipitation assays were as described above.
  • Antigen-antibody complexes were precipitated using 20 ⁇ l Protein A Sepharose, and the pellet was washed 3 times in ice-cold wash buffer (which consisted of 10 mM HEPES pH 7.4, 150 mM NaCl, 0.25% BSA, and 0.25% Triton X-114) and one cold water wash. Antigen was dissociated from the pellet by boiling in the presence of 2% SDS and 5% ⁇ -mercaptoethanol, counted by scintillation counting in scintillation fluid, and the results expressed as islet cell antigen 1851 index (Hagopian et al., Diabetes 42:631-36, 1993). Counts per minute reflect the level of autoantibodies present in the sera that can capture the antigen. Assay cutoff was an index of 0.04, determined as the mean +3 standard deviations of 91 control sera. Assay sensitivity, specificity, and positive predictive value were calculated (Hagopian et al., ibid., 1995).
  • IA-2/ICA512 autoantibodies recognized only epitopes shared with islet cell antigen 1851
  • the intracellular domain of IA-2/ICA512 was expressed in baby hamster kidney cells (BHK cells).
  • the 1.2 kb IA-2/ICA512 intracellular fragment (SEQ ID NO:30) from Example 3 was ligated into pZEM219b under the SV40 promoter (Busby et al., J. Biol. Chem. 266:15286-92, 1991) and cellular expression was determined by immunocytochemistry using rabbit polyclonal antiserum to IA-2/ICA512 (Rabin et al., J. Immunol. 152:3183-88, 1994).
  • IA-2/ICA512-transfected BHK cells were homogenized in homogenization buffer (0.25% Triton X-114, 10 mM benzamidine). Using Western blotting, the concentration of recombinant intracellular IA-2/ICA512 was estimated at 7 ⁇ g/ml of cell extract.
  • Immunoprecipitation assays were done using radiolabeled islet cell antigen 1851 in the presence of 0.5 ⁇ g of unlabeled IA-2/ICA512 per microliter of islet cell antigen 1851 positive sera, as a competitor. Islet cell antigen 1851 autoantibodies not fully blocked by this amount of IA-2/ICA512 were subjected to repeated immunoprecipitation assays using a 2.5 fold increase of unlabeled IA-2/ICA512 as a competitor. As a control, extracts from non-transfected BHK cells were used.
  • the filters were prehybridized in 20 ml hybridization buffer (6 ⁇ SSC, 0.5% SDS, 5 ⁇ Denhardts and 0.2 mg/ml boiled salmon sperm DNA) overnight at 65° C.
  • the filters were then hybridized in 20 ml hybridization buffer containing 1 ⁇ 10 6 cpm/ml ⁇ 32 P-ATP labeled hybridization probe (ZC10504 SEQ ID NO:18) overnight at 65° C.
  • the labeled hybridization probe was prepared by adding to a 5 ⁇ l final volume 30 pmol oligo ZC10504 (SEQ ID NO:18), T4 polynucleotide kinase buffer, 37.5 pmol ⁇ 32 P-ATP and 10 U T4 polynucleotide kinase The reaction was incubated for 1 hour at room temperature and unincorporated ATP was removed using a Stratagene push column according the manufacturer's instructions (Stratagene Cloning Systems, La Jolla, Calif.).
  • 5′ RACE PCR was used to generate the remaining 5′ cDNA fragments of macaque islet cell antigen 1851.
  • a vector-specific oligonucleotide primer ZC11197, SEQ ID NO:29
  • a macaque specific primer ZC11654, SEQ ID NO:28
  • 1 ng macaque islet cell cDNA library from Example 1, 40 mM dNTPs, TAQ Polymerase buffer and 1.25 U TAQ Polymerase.
  • a one minute denaturation at 94° C. was followed by 30 amplification cycles (30 seconds at 94° C., 1 minute at 60° C., 2 minutes at 72° C.) followed by a 6 minute extension at 72° C.).
  • the fragments were isolated by agarose electrophoresis, excised and separated from the agarose using the Qiagen Qiaquick Gel Extraction System (Qiagen, Inc., Chatsworth, Calif.) according to manufacturer's instruction.
  • the fragments were subcloned into pGEM-T (Promega Corp., Madison, Wis.), using the TA Cloning Kit (Promega Corp.) according to the manufacturer's instructions.
  • the resulting plasmids pJML8, 7, 9 and 10 respectfully, were used to transform E. coli DH10B cells. Transformants were screened for presence of insert, followed by sequencing of the insert.
  • the 5′ RACE fragments (SEQ ID Nos:23, 24, 25, 26 and 27) contain overlapping segments and were aligned with the macaque islet cell antigen 1851 sequence of SEQ ID NO:1 to give a full length macaque islet cell antigen 1851 DNA sequence as represented in SEQ ID NO:15.
  • Comparison of the human protein tyrosine phosphatase IA-2/ICA512 cDNA and amino acid sequences with those of the macaque islet cell antigen 1851 cDNA and amino acid sequences suggests that the coding region is missing the start methionine.
  • a vector containing the full length macaque sequence can be created using PCR.
  • the macaque 5′ RACE fragments (SEQ ID NOs: 23, 24, 25, 26 and 27) can be joined using PCR.
  • a clone shown to possess the complete coding sequence can then be digested with convenient restriction sites and subcloned into a vector of choice. Clones can be screened for correct insertion of the full length sequence and subjected to DNA sequence analysis.
  • PCR using macaque derived primers was done to identify remaining 5′ cDNA sequence for the human islet cell antigen 1851 (SEQ ID NO:6).
  • To a 50 ⁇ l final volume was added 5 pmol each of two gene-specific oligonucleotide primers ZC10504, SEQ ID NO:18 and ZC11653, SEQ ID NO:17, 1 ng Marathon-ready insulinoma cDNA, prepared according to manufacturer's instruction (Marathon ⁇ cDNA Amplification Kit, Clontech), 40 mM dNTPs, TAQ Polymerase buffer and 1.25 U TAQ Polymerase.
  • the reaction was denatured at 94° C. for one minute, amplified for 30 cycles (30 seconds at 94° C., 1 minute at 63° C., 2 minutes at 72° C.), followed by a 6 minute extension at 72° C.).
  • a 1263 bp fragment (SEQ ID NO:31) was isolated by agarose electrophoresis. The isolated fragment was then excised and subcloned into pGEM-T using the TA Cloning Kit (Promega, Corp.), as described above. The clones were then analyzed for the presence of insert, and those containing insert were subjected to DNA sequence analysis. The human islet cell antigen 1851 fragments can be joined using PCR to give the human sequence as represented in SEQ ID NO:21. Clones can be screened for correct insertion of the fragments and subjected to DNA sequence analysis.
  • ExpressHyb ⁇ (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 37° C. using 5 ⁇ 10 6 cpm/ml of labeled probe. The blots were then washed three times at room temperature, once at 50° C. for 30 minutes, once at 60° C., in 6 ⁇ SSC, 0.1% SDS. A final wash at 68° C. with 2 ⁇ SSC, 0.05% SDS for 20 minutes was done prior to autoradiography. Two transcript sizes were detected. A strong 5.5 kb band and a weaker 3.3 kb band were detected in brain, pancreas and prostate, with lesser signals in spinal cord, thyroid, adrenal and GI tract. With the exception of prostate, this represents the expected neuroendocrine distribution.
  • in situ hybridization was performed on macaque pancreas, adrenal gland and muscle.
  • the 38 nucleotide islet cell antigen 1851 oligonucleotide (SEQ ID NO:18), a 38 bp IC-2/ICA512 oligonucleotide (SEQ ID NO:19) and a 30 bp insulin ⁇ -chain probe for pancreatic islets (Petersen et al., Diabetes 42:484-95, 1993) (SEQ ID NO:20) were end-labeled with 33 P-dATP (New England Nuclear, Boston, Mass.) using terminal deoxytransferase (GIBCO BRL) according to manufacturer's instructions.
  • Frozen sections (14 ⁇ m) from macaque pancreas, adrenal, pituitary and muscle were fixed in 4% paraformaldehyde, followed by acetylation with acetic anhydride and then delipidated in chloroform prior to use. Labeled probes (2 pmol/ml) were incubated on the sections overnight and then washed in two changes of 1 ⁇ SSC at 60° C. for 30 minutes, followed by dehydration in ethanol and apposition to autoradiography film (Hyperfilm Betamax, Amersham Corp., Arlington Heights, Ill.) for 2 to 6 days.
  • the slides were then coated with NTB2 Track emulsion (Eastman Kodak, Rochester, N.Y.) and exposed for 12-18 days before development and counterstain with cresyl violet. Images were captured using a Dage 72 CCD camera and a MCID M2 imaging system (Imaging Research, Ontario, Canada). Strong hybridization was detected in pancreatic islets and adrenal medulla but not in muscle. The IA-2/ICA512 and the insulin ⁇ chain probes hybridized to islets.

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Abstract

A mammalian islet cell antigen polypeptide involved in the development of insulin-dependent diabetes mellitus (IDDM) is disclosed. This islet cell antigen polypeptide, 1851, was found to contain regions of homology to the protein tyrosine phosphatase family. Methods for diagnosis and treatment, including use in immunoprecipitation assays and the induction of immune tolerance using the recombinant mammalian polypeptides and antibodies specific to mammalian islet cell antigen 1851 polypeptides are presented.

Description

    BACKGROUND OF THE INVENTION
  • Insulin-dependent diabetes mellitus (IDDM) is a disease resulting from the autoimmune destruction of the insulin-producing β-cells of the pancreas. Studies directed at identifying the autoantigen(s) responsible for β-cell destruction have generated several candidates, including poorly characterized islet cell antigens (ICA) (Bottazzo et al., [0001] Lancet 2: 1279-83, 1974), insulin (Palmer et al., Science 222: 1337-39, 1983), glutamic acid decarboxylase (GAD) (Baekkeskov et al., Nature 298: 167-69, 1982; Baekkeskov et al., Nature 347: 151-56, 1990), and a 64 kD islet cell antigen that is distinct from GAD and that which yields 37 kD and 40 kD fragments upon trypsin-digestion (Christie et al., Diabetes 41: 782-87, 1992).
  • Detection of specific autoantigens in prediabetic individuals has been used as a predictive marker to identify, before clinical onset and significant β-cell loss has occurred, those at greater risk of developing IDDM (Gorsuch et al., [0002] Lancet 2: 1363-65, 1981; Baekkeskov et al., J. Clin. Invest. 79: 926-34, 1987; Johnstone et al., Diabetologia 32: 382-86, 1989; Ziegler et al., Diabetes 38: 1320-25, 1989; Baekkeskov et al., Nature (Lond) 347: 151-56, 1990; Bonifacio et al., Lancet 335: 147-49, 1990; and Bingley et al. Diabetes 43: 1304-10, 1994).
  • Antibodies to the 40 kD, and more particularly the 37 kD, ICA fragments are detected when clinical onset of IDDM is imminent and are found to be closely associated with IDDM development (Christie et al., [0003] Diabetes 41: 782-87, 1992). Diabetic sera containing antibodies specific to the 40 kD fragment were recently found to bind to the intracellular domain of the protein tyrosine phosphatase, IA-2/ICA512 (Lu et al., Biochem. Biophys. Res. Comm. 204: 930-36, 1994; Lan et al., DNA Cell Biol. 13: 505-14, 1994; Rabin et al., J. Immunol. 152: 3183-88, 1994; Payton et al., J. Clinc. Invest. 96: 1506-11, 1995; and Passini et al., Proc. Natl. Acad. Sci. USA 92: 9412-16, 1995). Antibodies specific to the 37 kD fragment are thought to bind either to a posttranslational in vivo modification of IA-2/ICA512 or a different, but probably related, protein precursor (Passini et al., ibid.).
  • ICA 512 was initially isolated as an autoantigen from an islet cell cDNA library, and was subsequently shown to be related to the receptor-linked protein tyrosine phosphatase family (Rabin et al., ibid.). ICA 512 was later found to be identical to a mouse and human protein tyrosine phosphatase, IA-2, isolated from brain and insulinoma cDNA libraries (Lu et al., ibid.; and Lan et al., ibid.). [0004]
  • Detection of diabetes-associated autoantigens, especially combinations of autoantigens, genotypes, such as HLA DR and HLA DQ, and loci, such as the polymorphic region in the 5′ flanking region of the insulin gene; in prediabetic individuals have been shown to be useful predictive markers of IDDM, see for example, Bell et al., ([0005] Diabetes 33:176-83, 1984); Sheehy et al., (J. Clin. Invest. 83:830-35, 1989); and Bingley et al., (Diabetes 43: 1304-10, 1994). There is therefore a need in the art for autoantigens that would serve to improve detection and diagnosis of IDDM. The present invention fulfills this need by providing novel autoantigens as well as related compositions and methods. The autoantigens of the present invention represent a new β-cell antigen. The present invention also provides other, related advantages.
    • SUMMARY OF THE INVENTION
  • The present invention provides an isolated polynucleotide which forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM, comprising a DNA segment encoding a mammalian islet cell antigen polypeptide of SEQ ID NO:16 from Leu, amino acid residue 636 to Gln, amino acid residue 1012. The invention also provides a mammalian islet cell antigen polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818. The invention also provides allelic variants of these polypeptides. Within one aspect of the invention, the isolated polynucleotide encodes a mammalian islet cell antigen polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818. Within another aspect of the invention, the isolated polynucleotide encodes a mammalian islet cell antigen polypeptide of SEQ ID NO:16 from Phe, amino acid residue 612, to Gln, amino acid residue 1012. The invention further provides allelic variants of these polypeptides. Within another aspect, the isolated polynucleotide encoding a polypeptide of SEQ ID NO:16 from Ala, amino acid residue 1, to Gln, amino acid residue 1012. Within another aspect, the isolated polynucleotide encoding a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818. The invention further provides allelic variants of these polypeptides. Within another aspect, the isolated polynucleotide is a DNA molecule comprising a coding sequence corresponding to SEQ ID NO:21 from nucleotide 1325 to nucleotide 2455. In still another aspect, the DNA molecule comprises a coding sequence corresponding to SEQ ID NO:15 from nucleotide 1909 to nucleotide 3039. The invention also provides allelic variants of these molecules. The invention further provides complements of polynucleotide molecules which specifically hybridize to these molecules. In yet another aspect, the isolated polynucleotide is a DNA molecule comprising a coding sequence corresponding to SEQ ID NO:21 from nucleotide 1254 to nucleotide 2455. Within another aspect, the isolated polynucleotide is a DNA molecule comprising a coding sequence corresponding to SEQ ID NO:15 from nucleotide 1837 to nucleotide 3039. The invention also provides allelic variants of these molecules. The invention further provides complements of polynucleotide molecules which specifically hybridize to these molecules. In still another aspect, the DNA molecule comprises a coding sequence corresponding to SEQ ID NO:15 from nucleotide 4 to nucleotide 3039. In still another aspect, the DNA molecule comprises a coding sequence corresponding to SEQ ID NO:21 from nucleotide 2 to nucleotide 2455. The invention also provides allelic variants of these molecules. The invention further provides complements of polynucleotide molecules which specifically hybridize to these molecules. The invention also provides an isolated polynucleotide molecule which encodes a complete coding sequence of a mammalian islet cell antigen polypeptide comprising the sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738. The invention also provides mammalian islet cell antigens that are primate islet cell antigens. [0006]
  • The invention also provides DNA constructs comprising a first DNA segment encoding a human islet cell antigen polypeptide operably linked to additional DNA segments required for the expression of the first DNA segment. The invention further provides a first DNA segment that is an isolated polynucleotide molecule encoding a human islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818. The invention also provides a first DNA segment that is an isolated polynucleotide molecule encoding a human islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818. Within another aspect, the invention provides a first DNA segment that is an isolated polynucleotide molecule encoding a human islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818. The invention further provides host cells containing such DNA constructs, as well as methods for producing human islet cell antigen polypeptides comprising the steps of culturing such host cell and isolating the human islet cell antigen polypeptide. [0007]
  • The invention further provides isolated mammalian islet cell antigen polypeptides, wherein said isolated mammalian islet cell antigen polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818. The invention further provides isolated mammalian islet cell antigen polypeptides comprising the amino acid sequence of SEQ ID NO:16 from Leu, amino acid residue 636 to Gln, amino acid residue 1012. [0008]
  • The invention also provides isolated polypeptides of SEQ ID NO:16 from Phe, amino acid residue 612 to Gln, amino acid residue 1012. The invention also provides isolated polypeptides of SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818. The invention further provides isolated polypeptides of SEQ ID NO:16 from Ala, amino acid residue 1 to Gln, amino acid residue 1012. The invention also provides isolated polypeptides of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818. The invention further provides allelic variants of these polypeptides. The invention still further provides an isolated polypeptide which is a full length mammalian islet cell antigen protein comprising the sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738. The invention also provides mammalian islet cell antigens that are primate islet cell antigens. [0009]
  • Within yet another aspect of the invention is provided a method for determining the presence of an autoantibody to a human islet cell antigen polypeptide in a biological sample, comprising the steps of contacting the biological sample with the human islet cell antigen polypeptide, which comprises an amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof, under conditions conducive to immune complex formation, and detecting the presence of immune complex formation between the human islet cell antigen polypeptide and the autoantibody to a human islet cell antigen, thereby determining the presence of autoantibodies to the human islet cell antigen in the biological sample. The invention further provides human islet cell antigen polypeptides that are detectably labeled. [0010]
  • Within a further embodiment the invention provides a method for predicting the clinical course of diabetes in a patient, comprising testing a biological sample from a patient for the presence of human islet cell antigen polypeptides comprising the amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof, wherein the polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM, and classifying the patient for clinical course of diabetes based on the presence or absence of human islet cell antigens in the sample. The invention further provides a method of predicting the clinical course of IDDM by testing one or more additional predictive markers associated with risk of or protection from IDDM. The invention provides methods of predicting the clinical course where the predictive marker is an autoantibody to an antigen selected from the group consisting of GAD65, IA-2/ICA512 or insulin. The invention also provides methods wherein the predictive marker is a genotype selected from the group consisting of HLA DR and HLA DQ. The invention also provides methods wherein the predictive marker is a polymorphic region in the 5′ flanking region of a human insulin gene. [0011]
  • The invention also provides a method for treating a patient to prevent an autoimmune response to a human islet cell antigen polypeptide comprising inducing immunological tolerance in the patient by administering a mammalian islet cell antigen polypeptide comprising the amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof, that specifically binds a human islet cell antigen receptor on immature or mature T or B lymphocytes. [0012]
  • The invention also provides oligonucleotide probes of at least about 16 nucleotides, wherein which the oligonucleotide is at least 85% homologous to a sequence of the mammalian islet cell antigen DNA sequence of SEQ ID Nos:15 or 21. [0013]
  • The invention further provides isolated antibodies which specifically bind to human islet cell antigen polypeptides which comprise the amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof. Within another aspect, the invention provides monoclonal antibodies. Within yet another aspect, the invention provides a hybridoma which produces the monoclonal antibody. [0014]
  • The invention also provides a diagnostic kit for use in detecting autoantibodies to pancreatic β-islet cells, comprising a container containing an islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof, wherein the polypeptide forms an immune complex with autoantibodies from a patient at risk of or predisposed to develop IDDM, and one or more containers containing additional reagents. [0015]
  • Within another embodiment of the invention is provided a pharmaceutical composition comprising an islet cell antigen comprising an amino acid sequence selected from the group consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, and allelic variants thereof, in combination with a pharamceutically acceptable carrier or vehicle. [0016]
  • Within a further embodiment of the invention is provided a method for monitoring the disease state in a patient comprising testing a biological sample from a patient for the presence of human islet cell antigen post-translationally modified polypeptides, determining the concentration of the peptides and correlating the peptide levels in the sample with the disease state in the patient. The invention provides that the human islet cell antigen post-translationally modified polypeptide comprises the sequence of SEQ ID NO:22 from His, amino acid residue 1 to Glu, amino acid residue 227. The invention further provides that the biological sample is plasma or serum. [0017]
  • Within yet a further embodiment, the invention provides a method for monitoring the disease state in a patient comprising exposing T cells to islet cell antigen 1851 peptides, detecting T and correlating T cell reactivity with disease state. The invention provides that the T cells are from peripheral blood mononuclear cells from a prediabetic patient. The invention further provides that the disease state is conversion from prediabetes to diabetes. [0018]
    • DETAILED DESCRIPTION OF THE INVENTION
  • Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter: [0019]
  • Allelic variant—Any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. [0020]
  • Biological sample—A sample that is derived from or contains cells, cell components or cell products, including, but not limited to, cell culture supernatants, cell lysates, cleared cell lysates, cell extracts, tissue extracts, blood plasma, serum, and fractions thereof, from a patient. [0021]
  • Complements of polynucleotide molecules—Polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′. [0022]
  • Immune Complex Formation—A noncovalently bound molecule formed between an antigen and an antibody specific for that antigen, resulting in an extensively cross-linked mass. Conditions conducive to complex formation are known in the art and easily adaptable by those skilled in art, for example, the degree of complex formation is in proportion to the relative amounts of available antigen and antibody. Such complexes can be used, for example, to identify and/or quantify the presence of either antigen or antibody in a biological sample, identify and characterize particular antibodies in tissues and cells, or to stimulate an immune response. [0023]
  • Isolated—When applied to a protein the term “isolated” indicates that the protein is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated protein is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the proteins in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When applied to a polynucleotide molecule the term “isolated” indicates that the molecule is removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated and may include naturally occurring 5′ and 3′ untranslated regions such as promoters and terminators, the identification of such will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, [0024] Nature 316: 774-78, 1985).
  • Operably linked—Indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator. [0025]
  • The DNA sequences encoding the polypeptides of the present invention were unexpectedly identified during screening of a primate islet cell cDNA library, and human insulinoma cDNA, for autoantigens toward human diabetic sera. Analysis of the macaque cDNA clones revealed a unique, previously unknown islet cell antigen which contained regions of homology to the protein tyrosine phosphatase family, especially the protein tyrosine phosphatase IA2/ICA512. This novel islet cell antigen has been designated 1851 or ICA512β. [0026]
  • The present invention provides islet cell antigen polypeptides which are β-cell autoantigens. These autoantigens were reactive with human prediabetic and diabetic sera. The invention also provides methods for using the islet cell antigen polypeptides for the detection, diagnosis, and treatment of IDDM. [0027]
  • Representative islet cell antigen polypeptides of the present invention comprise the amino acid sequences in SEQ ID NOs:4, 16 or 22 and/or are encoded by polynucleotide sequences comprising the sequences of SEQ ID NOs:3, 15 and 21 and form an immune complex with autoantibodies from a patient at risk of or predisposed to develop IDDM. The islet cell antigen polypeptides of the present invention are preferably from mammals, especially primates including humans. Preferred polypeptides of the present invention include isolated polypeptides selected from the group consisting of a polypeptide of SEQ ID NO:2 from Leu, amino acid residue 265, to Gln amino acid residue 641. The invention also provides polypeptides of SEQ ID NO:2 from Glu, amino acid residue 1, to Gln, amino acid residue 641. The invention further provides macaque polypeptides of SEQ ID NO:16 from Ala, amino acid residue 1 to Gln, amino acid residue 1012 and human polypeptides of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818 and SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818. The invention further provides allelic variants and isolated sequences that are substantially identical to the representative polypeptide sequences of SEQ ID NOs:2, 16 and 22 and their species homologs. The term “substantially identical” is used herein to denote proteins having 50%, preferably 60%, more preferably 70%, and most preferably at least 80%, sequence identity to the representative sequences shown in SEQ ID NO:2, 16 or 22 or its species homologs. Within preferred embodiments, such proteins will be at least 90% identical, and most preferably 95% or more identical, to SEQ ID NO:2, 16 or 22 or their species homologs. [0028]
  • Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., [0029] Bull. Math. Bio. 48: 603-616, 1986; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids are indicated by the standard one-letter codes). The percent identity of the optimum alignment is then calculated as:
    TABLE 1
    A R N D C Q E G H I L K M F P S T W Y V
    A 4
    R −1 5
    N −2 0 6
    D −2 2 1 6
    C 0 −3 −3 −3 9
    Q −1 1 0 0 −3 5
    E −2 0 0 2 −4 2 5
    G 0 −2 0 −1 −3 −2 −2 6
    H −2 0 1 −1 −3 0 0 −2 8
    I −1 −3 −3 −3 −1 −3 −3 −4 −3 4
    L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4
    K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5
    M −1 −1 −2 −3 −1 0 −2 −3 −2 1 −2 −1 5
    F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6
    P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7
    S 1 −1 2 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4
    T 0 −1 0 −1 −1 −1 −1 −2 −2 −2 −1 −1 −1 −2 −1 1 5
    W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11
    Y −2 −2 −2 −3 −2 −2 −2 −3 2 −1 −1 −2 −2 3 −3 −2 −2 2 7
    V 0 −3 −3 −3 −2 −2 −2 −3 −3 3 2 −2 1 −1 −2 −2 0 −3 −1 4
  • [0030] Total   number   of   identical   matches [ length   of   the   longer   sequence   plus   the   number   of   gaps   introduced   into   the   longer sequence   in   order   to   align   the   two   sequences ] × 100
    Figure US20030166067A1-20030904-M00001
  • Substantially identical proteins are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the folding or activity of the protein; small deletions, typically of one to about 30 amino acids; amidation of the amino- or carboxyl-terminal; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain. See, in general, Ford et al., [0031] Protein Expression and Purification 2: 95-107, 1991, which is incorporated herein by reference.
    TABLE 2
    Conservative amino acid substitutions
    Basic: arginine
    lysine
    histidine
    Acidic: glutamic acid
    aspartic acid
    Polar: glutamine
    asparagine
    Hydrophobic: leucine
    isoleucine
    valine
    Aromatic: phenylalanine
    tryptophan
    tyrosine
    Small: glycine
    alanine
    serine
    threonine
    methionine
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, [0032] Science 244, 1081-85, 1989). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g. protein tyrosine phosphatase activity, Strueli et al., EMBO J. 9: 2399-407, 1990, or binding to autoantibodies in prediabetic or diabetic sera) to identify amino acid residues that are critical to the activity of the molecule. Sites of ligand-receptor interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.
  • Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer ([0033] Science 241:53-57, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-156, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a protein, selecting for functional protein, and then sequencing the mutagenized proteins to determine the spectrum of allowable substitutions at each position. These methods allow the rapid determination of the importance of individual amino acid residues in a protein of interest, and can be applied to proteins of unknown structure.
  • The present invention further provides isolated polynucleotide molecules encoding islet cell antigen polypeptides which form immune complexes with autoantibodies from a patient at risk of or predisposed to develop IDDM. Useful polynucleotide molecules in this regard include mRNA, genomic DNA, cDNA and synthetic DNA. For production of recombinant islet cell antigen polypeptides, cDNA is preferred. The invention provides an isolated polynucleotide molecule wherein the molecule is a DNA molecule comprising a coding sequence corresponding to SEQ ID NO:1 from nucleotide 795 to nucleotide 1922. The invention also provides a DNA molecule comprising a coding sequence corresponding to SEQ ID NO:1 from nucleotide 1 to nucleotide 2168. The invention also provides a DNA molecule comprising a coding sequence corresponding to nucleotide 4 to nucleotide 3039 of SEQ ID NO: 15. The invention also provides DNA molecules from nucleotide 1325 to nucleotide 2455, from nucleotide 1254 to nucleotide 2455 and from nucleotide 2 to nucleotide 2544 of SEQ ID NO:21. The invention also provides allelic variants of the sequences shown in SEQ ID NOs:1, 15 or 21, and polynucleotide molecules that specifically hybridize to allelic variants. Such polynucleotide molecules will hybridize to the representative DNA sequences of SEQ ID NOs:1, 15, 21 or their allelic variants under stringent conditions (Sambrook et al., [0034] Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989). As used herein, the term “stringent conditions” refers to hybridizing conditions that employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% polyvinylpyrrolidone/50 mM sodium citrate at 42° C.; or employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075M sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS. Such hybridizable polynucleotide molecules would include genetically engineered or synthetic variants of the representative islet cell antigen polynucleotide sequence, SEQ ID NO: 1, and polynucleotide molecules that encode one or more amino acid substitutions, deletions or additions, preferably of a minor nature, as discussed above. Genetically engineered variants may be obtained by using oligonucleotide-directed site-specific mutagenesis, by use of restriction endonuclease digestion and adapter ligation, polymerase chain reaction (PCR), or other methods well established in the literature (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989, and Smith et al., Genetic Engineering: Principles and Methods, Plenum Press, 1981; which are incorporated herein by reference). In addition, hybridizable polynucleotide molecules may encompass sequences containing degeneracies in the DNA code wherein host-preferred codons are substituted for the analogous codons in the representative sequences of SEQ ID NOs: 1, 15 and 21.
  • Analysis of the representative cDNA sequences of SEQ ID NO:1, 15 and 21 and their representative polypeptide sequences of SEQ ID NO:2, 16 and 22, show that they contain regions of homology to transmembrane protein tyrosine phosphatases. Comparison of the human protein tyrosine phosphatase IA-2/ICA512 cDNA and amino acid sequences with those of 1851 suggests that the coding region of macaque 1851 is missing amino-terminal sequence corresponding to approximately 1 amino acid and human 1851 is missing approximately 200 amino acid residues of the amino terminus. To recover the 5′ region, cDNA libraries from different tissues can be screened to obtain a full length cDNA, which encodes a full length mammalian islet cell antigen polypeptides. Another option for obtaining the complete coding sequence comprises using 5′ RACE (Rapid Amplification cDNA Ends) PCR. RACE is an art recognized PCR-based method for amplifying the 5′ ends of incomplete cDNAs, a frequent occurrence in cDNA cloning. To obtain the 5′ portion of a cDNA, PCR is carried out on specially prepared cDNA which contains unique anchor sequences, using anchor primers provided with the 5′ RACE reagents available from, for example, Clontech, Palo Alto, Calif. and a 3′ primer based on known sequence. The 5′-RACE-Ready cDNA can be purchased commercially (Clontech), or prepared according to known methods. A secondary PCR reaction can then be carried out using the anchor primer and a nested 3′ primer, according to known methods. Once a full-length cDNA is obtained, it is expressed and analyzed for overall structural similarity to known protein tyrosine phosphatases, and examined for features such as a continuous open reading frame flanked by translation initiation and termination sites and a potential signal sequence. [0035]
  • Transmembrane, or receptor-linked, protein tyrosine phosphatases consist of a conserved cytoplasmic domain which may have one or two (tandemly duplicated) catalytic regions, a single transmembrane domain, a highly variable extracellular domain and a signal peptide. These structural features suggest that receptor-linked protein tyrosine phosphatases would be capable of binding ligand and transducing external signal, but no ligands as of yet have been identified. Based on the representative amino acid sequence of SEQ ID NOs:2 and 15, the macaque 1851 polypeptide has an approximately 611 amino acid extracellular domain, from Ala, amino acid residue 1 to Lys, amino acid residue 611 of SEQ ID NO:16, containing a post translational modification dibasic site, at amino acid residue 423-424, or a tribasic site at amino acid residues 422-424; a 24 amino acid transmembrane domain comprising amino acid residue 241 to amino acid residue 265 of SEQ ID NO:2 or Phe, amino acid residue 612 to Cys, amino acid residue 635 of SEQ ID NO:16 and an approximately 375 amino acid cytoplasmic domain comprising the amino acid residue 265 to amino acid residue 640 of SEQ ID NO:2 or Leu, amino acid residue 636 to Gln, amino acid residue 1012 of SEQ ID NO:16. The representative amino acid sequence of the human islet cell antigen 1851 (SEQ ID NO:22) has 417 amino acids of an extracellular domain, from His, amino acid residue 1 to Lys, amino acid residue 417 of SEQ ID NO:22; a 24 amino acid residue transmembrane domain, from Phe, amino acid residue 418 to Cys, amino acid residue 441, of SEQ ID NO:22; and a 376 amino acid cytoplasmic domain, from Leu, amino acid residue 442 to Gln, amino acid residue 818 of SEQ ID NO:22. [0036]
  • The cytoplasmic domain of 1851 contains many regions that are conserved between members of the protein tyrosine phosphatase family. Within the cytoplasmic domain of protein tyrosine phosphatases is a catalytic region of about 230 amino acids, which contains a highly conserved catalytic core segment of approximately 11 amino acid residues (VHCXAGXXRXG SEQ ID NO:13) where the first three X's are any amino acid, the fourth X is S or T, and the cysteine appears to be essential to the catalytic mechanism (Fischer et al., [0037] Science 253: 401-06). The catalytic core sequence of the representative macaque 1851 polypeptide sequences of SEQ ID Nos:2 and 16 and human 1851 polypeptide sequence represented by SEQ ID NO:22 differs from other members of the protein tyrosine phosphatase family in that alanine has been replaced by aspartic acid and the second variable amino acid (X) is alanine. 1851, like IA-2/ICA512, has a single catalytic region. Deletion of C-terminal amino acids from the intracellular domain of human islet cell antigen 1851 reduced reactivity with new onset IDDM sera, suggesting this region may play a role in defining an autoantibody epitope. Removal of the C-terminal 27 amino acids decreased reactivity from 19/53 sera (36%) to 10/53 sera (19%), a 47% decrease. Removal of the C-terminal 80 amino acids decreased reactivity further to 9/53 sera (17%), a 53% decrease, and removal of the C-terminal 160 amino acids abolished all recognition by all 53 new onset IDDM sera. This is similar to the reports of one of two described intracellular IA-2/ICA512 autoantibody epitopes (Bonifacio et al., J. Immunol. 155:5419-426, 1995). That human islet cell antigens 1851 and human IA-2/ICA512 are each precipitated by sera that do not precipitate the other suggests that each antigen has unique autoantibody epitopes, which is consistent with previous findings regarding the 37 kD and 40 kD tryptic fragments (Payton et al., J. Clin. Invest. 96:1506-11, 1995). A comparison between the overall human and macaque islet cell antigen 1851 nucleotide and amino acid sequences shows a 96.2% nucleotide identity and a 94.6% amino acid identity, in particular there was 97% identity within the nucleotide sequence and 98.9% identity within the amino acid sequence of the corresponding cytoplasmic domains, 100% identity within the transmembrane domain. There is 77% amino acid identity within the cytoplasmic domain between the claimed human (SEQ ID NO:22) and macaque (SEQ ID NO:16) islet cell antigen 1851 sequences and the reported human IA-2/ICA512 sequences (Lan et al., ibid.; and Rabin et al., ibid.). Between the full length macaque islet cell antigen 1851 sequence (as represented in SEQ ID Nos:15 and 16) and rat phogrin sequences (Wasmeier and Hutton, J. Biol. Chem. 271:18161-70, 1996) there was less homology, 75.5% identity within the nucleotide sequence and 69.9% identity within the amino acid sequence.
  • In contrast, there is little homology in the extracellular regions of transmembrane protein tyrosine phosphatases. Some contain Ig-like and/or fibronectin type III repeats (Streuli et al., [0038] J. Exp. Med. 168: 1523, 1988; Hariharan et al., Proc. Natl. Acad. Aci. USA 88: 11266, 1991); others have glycosylated segments (Sap et al., Proc. Natl. Acad. Sci. USA 87:6112, 1990; and Krueger et al., EMBO J. 9: 3241, 1990) and a conserved cysteine-rich region (Tonks et al., J. Biol. Chem. 265: 10674-80, 1990) (Lan et al. ibid.). There is 31% identity between macaque islet cell antigen 1851 (as represented by SEQ ID NO:15) and IA-2/ICA512 (Lan et al., ibid.; and Rabin et al., ibid.) within the extracellular domain.
  • The tissue distribution of human islet cell antigen 1851 is generally neuroendocrine. Northern analysis showed strong hybridization to human mRNA from brain and pancreas and weaker hybridization in spinal cord, thyroid, adrenal and GI tract. In situ hybridization using macaque tissues further localized pancreatic and adrenal expression to islets and adrenal medulla, respectively. Northern blot analysis of rat phogrin showed expression in brain, pancreas and α and βcell tumor lines (Wasmeier and Hutton, ibid.); mouse IA-2β in brain, pancreas, stomach and in insulinoma and glucagomoma cell lines (Lu et al., [0039] Proc. Natl. Acad. Sci. USA 93:2307-11, 1996); human IA-2 in brain, pituitary and pancreas, four insulinoma cell lines and a glioblastoma cell line (Lan et al., ibid.); and human ICA512, brain and pancreas (Rabin et al., ibid.).
  • Limited trypsinization of IA-2/ICA512 and human islet cell antigen 1851 yielded a 40 kD IA-2/ICA512 fragment and a 37 kD islet cell antigen 1851 fragment. These correspond to the 37 kD and 40 kD tryptic fragments described by Christie et al. ([0040] J. Exp. Med. 172:789-94, 1990), Payton et al. (J. Clin. Invest. 96:1506-11, 1995), Bonifacio et al. (J. Immunol. 155:5419-26, 1995), Lu et al. (Proc. Natl. Acad. Sci. USA 93:2307-11, 1996) and Wasmeier and Hutton (ibid.).
  • Members of the protein tyrosine phosphatase family have been shown to display alternative mRNA splicing (Moeller et al., WO 94/21800; Hall et al., [0041] J. Immunol. 141: 2781-87, 1988; Johnson et al., J. Biol. Chem. 264: 6220-29, 1989; Streuli and Saito, EMBO J. 8: 787-96, 1989; Matthews et al., Proc. Natl. Acad. Sci. USA 87: 4444-48, 1990; Walton and Dixon, Ann. Rev. Biochem. 62: 101-20, 1993; and Pan et al., J. Biol. Chem. 268: 19284-91, 1993). Alternative splicing may be important in autoantibody recognition; “inappropriate” splicing could lead to autoimmunity by activating T cells, for example.
  • The invention provides isolated DNA molecules that are useful in producing recombinant islet cell antigens. As will be evident to one skilled in the art, each individual domain or combinations of the domains may be prepared synthetically or by recombinant DNA techniques for use in the present invention. Thus, the present invention provides the advantage that islet cell antigens are produced in high quantities that may be readily purified using methods known in the art (see generally; Scopes, [0042] Protein Purification, Springer-Verlag, NY, 1982). Alternatively, the proteins of the present invention may be synthesized following conventional synthesis methods, such as the solid-phase synthesis method of Barany and Merrifield (in The Peptides. Analysis, Synthesis, Biology Vol. 2, Gross and Meienhofer, eds, Academic Press, NY, pp. 1-284, 1980), by partial solid-phase techniques, by fragment condensation or by classical solution addition.
  • DNA molecules of the present invention can be isolated using standard cloning methods such as those described by Maniatis et al. ([0043] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982; which is incorporated herein by reference), Sambrook et al., (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989), or Mullis et al. (U.S. Pat. No. 4,683,195) which are incorporated herein by reference. Alternatively, the coding sequences of the present invention can be synthesized using standard techniques that are well known in the art, such as by synthesis on an automated DNA synthesizer.
  • The sequence of a polynucleotide molecule encoding a representative islet cell antigen polypeptide is shown in SEQ ID NOs: 1, 15 and 21 and the corresponding amino acid sequences are shown in SEQ ID NOs: 2, 16 and 22. Those skilled in the art will recognize that these sequences correspond to one allele of either the macaque or human gene, and that allelic variation is expected to exist. Allelic variants of the DNA sequence shown in SEQ ID NO: 1, 15 and 21 including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO: 2, 16 and 22. [0044]
  • The macaque sequence disclosed herein is useful for isolating polynucleotide molecules encoding islet cell antigen polypeptides from other species (“species homologs”). In particular, the macaque cDNA was used to conduct a sequence search for a human homolog. A match was found as an expressed sequence tag (EST) from a human fetal brain library submitted to the Genbank database (GenBank ID: TO361, clone ID: HFBCV88). This 127 amino acid polypeptide, SEQ ID NO:5, had homology to a region of the cytoplasmic domain of M1.18.5.1 (SEQ ID NO:2) and was used to design PCR primers to clone a 1.1 kD cytoplasmic portion (SEQ ID NOs:6 and 7) of the human 1851 sequence, as described in the examples below. Other preferred species homologs include mammalian homologs such as bovine, canine, porcine, ovine, and equine proteins. Methods for using sequence information from a first species to clone a corresponding polynucleotide sequence from a second species are well known in the art. See, for example, Ausubel et al., eds., [0045] Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.
  • DNA molecules of the present invention or portions thereof may be used as probes, for example, to directly detect 1851 sequences in cells or biological samples. Such DNA molecules are generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences and will generally comprise at least about 16 nucleotides, more often from about 17 nucleotides to about 25 or more nucleotides, sometimes 40 to 60 nucleotides, and in some instances a substantial portion or even the entire 1851 gene or cDNA. The synthetic oligonucleotides of the present invention have at least 85% identity to a representative macaque or human 1851 DNA sequence (SEQ ID Nos:1, 15 and 21) or their complements. For use as probes, the molecules are labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle, etc., according to methods known in the art. Probes of the present invention may also be used in diagnostic methods to detect autoantibodies in diabetic and prediabetic sera. [0046]
  • DNA molecules used within the present invention may be labeled and used in a hybridization procedure similar to the Southern or dot blot. As will be understood by those skilled in the art, conditions that allow the DNA molecules of the present invention to hybridize to the representative DNA sequence of SEQ ID NO:1, 15 or 21 or their allelic variants may be determined by methods well known in the art (reviewed, for example, by Sambrook et al. [0047] Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; which is incorporated herein by reference). Those skilled in the art will be capable of varying hybridization conditions (i.e. stringency of hybridization) of the DNA molecules as appropriate for use in the various procedures by methods well known in the literature (see, for example, Sambrook et al., ibid., pages 11.45-11.53). The higher the stringency of hybridization, the lower the number of mismatched sequences detected. Alternatively, lower stringency will allow related sequences to be identified.
  • Alternatively, allelic variants may be identified using DNA molecules of the present invention and, for example, the polymerase chain reaction (PCR) (disclosed by Saiki et al., [0048] Science 239: 487, 1987; Mullis et al., U.S. Pat. No. 4,686,195; and Mullis et al., U.S. Pat. No. 4,683,202) to amplify DNA sequences, which are subsequently detected by their characteristic size on agarose gels or which may be sequenced to detect sequence abnormalities.
  • DNA molecules encoding the islet cell antigen polypeptides of the present invention may be inserted into DNA constructs. As used within the context of the present invention a DNA construct is understood to refer to a DNA molecule, or a clone of such a molecule, either single- or double-stranded, which has been modified through human intervention to contain segments of DNA combined and juxtaposed in a manner that would not otherwise exist in nature. DNA constructs of the present invention comprise a first DNA segment encoding an islet cell antigen polypeptide operably linked to additional DNA segments required for the expression of the first DNA segment. Within the context of the present invention, additional DNA segments will generally include promoters and transcription terminators, and may further include enhancers and other elements. One or more selectable markers may also be included. DNA constructs useful for expressing cloned DNA segments in a variety of prokaryotic and eukaryotic host cells can be prepared from readily available components or purchase from commercial suppliers. [0049]
  • In general, a DNA sequence encoding a protein of the present invention is operably linked to a transcription promoter and terminator within a DNA construct. The construct will commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. [0050]
  • In one embodiment the first DNA segment is an isolated polynucleotide molecule encoding a mammalian islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:4, wherein the polypeptide forms an immune complex with autoantibodies from a patient at risk of or predisposed to IDDM. In another embodiment, the first DNA segment is an isolated polynucleotide encoding a polypeptide of SEQ ID NO:2 from Leu, amino acid residue 265 to Gln, amino acid residue 641. In another embodiment, the first DNA segment is an isolated polynucleotide encoding a polypeptide of SEQ ID NO:2 from Ser, amino acid residue 1, to Gln, amino acid residue 641. [0051]
  • Within yet another embodiment, the first DNA segment is an isolated polynucleotide encoding a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818. In another embodiment, the first DNA segment is an isolated polynucleotide encoding a polypeptide of SEQ ID NO:16 from Ala, amino acid residue 1 to Gln, amino acid residue 1012. [0052]
  • The proteins of the present invention can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., [0053] Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., ibid., which are incorporated herein by reference.
  • To direct a protein of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence is joined to the DNA sequence encoding a protein of the present invention in the correct reading frame. Secretory signal sequences are commonly positioned 5′ to the DNA sequence encoding the protein of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830). The secretory signal sequence may be that normally associated with a protein of the present invention, or may be from a gene encoding another secreted protein. [0054]
  • Cultured mammalian cells are also preferred hosts within the present invention. A preferred vector system for use in the present invention is the pZCEP vector system as disclosed by Jelineck et al., [0055] Science, 259: 1615-16, 1993. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987), and cationic lipid transfection using commercially available reagents including the Boehringer Mannheim Transfection-Reagent (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl ammoniummethylsulfate; Boehringer Mannheim, Indianapolis, Ind.) or LIPOFECTIN˜ reagent (N-[1-(2,3-Dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioeleoyl phosphatidylethanolamine; GIBCO-BRL, Gaithersburg, Md.) using the manufacturer-supplied directions, which are incorporated herein by reference. The production of recombinant proteins in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134, which are incorporated herein by reference. Preferred cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Md. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus.
  • Prokaryotic cells can also serve as host cells for use in carrying out the present invention. Particularly preferred are strains of the bacteria [0056] Escherichia coli, although Bacillus and other genera are also useful. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing the proteins in bacteria such as E. coli, the protein may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate. The denatured protein is then refolded by diluting the denaturant. In the latter case, the protein can be recovered from the periplasmic space in a soluble form.
  • Fungal cells are also suitable as host cells. For example, Saccharomyces ssp., [0057] Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia guillermondii, Pichia methanolica, and Candida maltosa transformation systems are known in the art. See, for example, Kawasaki, U.S. Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075, Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Pat. No. 4,935,349, which is incorporated herein by reference. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228, which is incorporated herein by reference.
  • Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign proteins therein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and Bang et al., U.S. Pat. No. 4,775,624, which are incorporated herein by reference. The use of [0058] Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.” A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as “amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. [0059]
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. [0060]
  • The recombinant islet cell antigen polypeptides expressed using the methods described herein are isolated and purified by conventional procedures, including separating the cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulfate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography or affinity chromatography, or the like. Methods of protein purification are known in the art (see generally, Scopes, R., [0061] Protein Purification, Springer-Verlag, NY (1982), which is incorporated herein by reference) and may be applied to the purification of the recombinant proteins of the present invention. Substantially pure recombinant islet cell antigen polypeptides of at least about 50% is preferred, at least about 70-80% more preferred, and 95-99% or more homogeneity most preferred, particularly for pharmaceutical uses. Once purified, partially or to homogeneity, as desired, the recombinant islet cell antigen polypeptides may then be used diagnostically, therapeutically, etc. as further described below.
  • Recombinant 1851 polypeptides can also be produced by expressing islet cell antigen DNA fragments, such as fragments generated by digesting an islet cell antigen cDNA at convenient restriction sites. The isolated recombinant polypeptides or cell-conditioned media are then assayed for activity as described in the examples below. Alternatively, the proteins of the present invention may be synthesized following conventional synthesis methods such as the solid-phase synthesis using the method of Barany and Merrifield (in [0062] The Peptides. Analysis, Synthesis, Biology Vol. 2, Gross and Meienhofer, eds, Academic Press, NY, pp. 1-284, 1980, which are incorporated herein by reference), by partial solid-phase techniques, by fragment condensation or by classical solution addition. Short polypeptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 amino acids, which correspond to selected islet cell antigen polypeptide regions can be readily synthesized and then screened in screening assays designed to identify peptides having a desired activity, such as domains which are responsible for or contribute to binding activity, immunodominant epitopes (particularly those recognized by autoantibodies), and the like.
  • Although the use of recombinant 1851 polypeptides is preferred within the methods of the present invention, 1851 polypeptides may also be prepared from cells that naturally produce 1851 protein (such as islet cells). For example, 1851 polypeptides may be prepared from islet cells by isolation of a membrane fraction. This 1851-enriched fraction is then used to detect autoantibodies to 1851 in prediabetic and diabetic sera. [0063]
  • Islet cell antigen polypeptides produced according to the present invention can be used diagnostically, in the detection and quantitation of autoantibodies in a biological sample, that is, any sample derived from or containing cells, cell components or cell products, including, but not limited to, cell culture supernatants, cell lysates, cleared cell lysates, cell extracts, tissue extracts, blood plasma, serum, and fractions thereof. By means of having islet cell antigen polypeptides which specifically bind to autoantibodies in prediabetic and diabetic sera, the presence or absence of such autoantibodies can be determined, and the concentration of such autoantibodies in an individual can be measured. This information can then be used to monitor the progression or regression of the potentially harmful autoantibodies in individuals at risk of, or with a predisposition to develop IDDM, and would be useful for predicting the clinical course of the disease in a patient. The assay results can also find use in monitoring the effectiveness of therapeutic measures for treatment of IDDM or related diseases. [0064]
  • As will be recognized by those skilled in the art, numerous types of immunoassays are available for use in determining the presence of autoantibodies. For instance, direct and indirect binding assays, competitive assays, sandwich assays, and the like, as are generally described in, e.g., U.S. Pat. Nos. 4,642,285; 4,376,110; 4,016,043; 3,879,262; 3,852,157; 3,850,752; 3,839,153; 3,791,932; and Harlow and Lane, [0065] Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, N.Y., 1988, each incorporated herein by reference. In one assay format, autoantibodies directed to the polypeptides of the present invention are quantified directly by measuring the binding of autoantibodies in a biological sample to recombinant or synthetic islet cell antigen polypeptides. The biological sample is contacted with at least one islet cell antigen polypeptide of the invention under conditions conducive to immune complex formation. The immune complexes formed between the islet cell antigen polypeptide and the antibodies are then detected, and the presence and quantity of autoantibodies can then be used to diagnose or direct treatment of IDDM. The immune complexes can be detected by means of antibodies that bind to the islet cell antigen of the present invention or by labeling the polypeptide as described below. Separation steps (e.g., washes) may be necessary in some cases to distinguish specific binding over background. In another format, the serum level of a patient's autoantibodies to the islet cell antigen polypeptides in serum can be measured by competitive binding with labeled or unlabeled antibodies to the islet cell antigen polypeptides of the present invention. Unlabeled 1851 polypeptides can be used in combination with labeled antibodies that bind to human antibodies or to islet cell antigens. Alternatively, the islet cell antigen polypeptide can be directly labeled. A wide variety of labels can be employed, such as radionuclides, particles (e.g., gold, ferritin, magnetic particles, red blood cells), fluors, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), chemiluminescers, biotin and other compounds that provide for the detection of the labeled polypeptide or protein. For example, an 1851 polypeptide can be radiolabeled using conventional methods such as in vitro transcription and translation. Radiolabeled 1851 polypeptide is combined with patient serum under conditions suitable for immune complex formation. Immune complexes are then separated, such as by binding to protein A. Precipitated 1851 polypeptides are then quantitated by conventional methods, such as gel electrophoresis, fluorography, densitometry or by direct counting of immunoprecipitated, radiolabeled antigen. The amount of 1851 polypeptide precipitated by test sera can be statistically compared to mean counts precipitated by healthy control sera, each measured separately. In an alternative format, an 1851 polypeptide antigen, labeled with biotin, is combined with patient serum under conditions suitable for immune complex formation. The serum is then transferred to a protein A-coated container, such as a well of an assay plate, and the container is allowed to stand so that immune complexes can form. The container is then washed, and streptavidin, conjugated to a suitable enzyme (e.g. alkaline phosphatase), is added. A chromogenic substrate is then added, and the presence of 1851 polypeptide autoantibodies in the sample is indicated by a color change. Additional assay formats will be evident to those skilled in the art.
  • Thus, autoantibodies to islet cell antigen polypeptides can be identified and, if desired, extracted from a patient's serum by binding to 1851 polypeptides of the present invention. The islet cell antigen polypeptides may be attached, e.g., by adsorption, to an insoluble or solid support, such as ELISA microtiter well, microbead, filter membrane, insoluble or precipitable soluble polymer, etc. to function as an affinity resin. The captured autoantibodies can then be identified by several methods. For example, antisera or monoclonal antibodies to the antibodies can be used. These antisera or monoclonal antibodies are typically non-human in origin, such as rabbit, goat, mouse, etc. These anti-antibodies can be detected directly if attached to a label such as [0066] 125I, enzyme, biotin, etc., or can be detected indirectly by a labeled secondary antibody made to specifically detect the anti-antibody.
  • The diagnostic methods of the present invention can be used in conjunction with other known assays and diagnostic techniques (see for example, WO 95/07464, incorporated herein by reference in its entirety). Such other assays and techniques include measurement of body mass index (BMI), defined as the quotient of the patient's weight in kg divided by the square of height in meters; C-peptide level (Heding, [0067] Diabetologia 11: 541-548 (1975); Landin-Olsson et al., Diabetologia 33: 561-568 (1990)); or one or more additional diabetes-associated autoantibodies, genotypes or loci. A low BMI (i.e. less than about 25) in combination with other indicators is suggestive of type I diabetes. BMI is thus a useful indicator for distinguishing type I from type II diabetes. C-peptide level can be measured using standard methods, such as that of Heding (ibid.), in which insulin and proinsulin are removed from serum and C-peptide is measured in the resulting insulin-free fraction radioimmunologically.
  • The islet cell antigen polypeptides of the current invention can also be used to assess T cell reactivity, as a method for monitoring the disease state in a patient. Mammalian islet cell antigen 1851 peptides will generally comprise at least about 12 amino acids, and more often from about 15 amino acids to about 20 or more amino acids. In some instances, a substantial portion or domain or even the entire 1851 protein, can be used to assess T cell reactivity in peripheral blood mononuclear cells (PBMNCs) from prediabetics. Methods for detecting such in vitro activity are known in the art, including a proliferation assay measuring [0068] 3H-thymidine incorporation, analysis of activation markers, such as CD69, or measuring cytokine production, such as IL-2. Correlations can be drawn between T cell reactivity to islet cell antigen 1851 and conversion from prediabetes to diabetes. This correlation would be consistent with the appearance of autoantibodies to islet cell antigen peptides late in prediabetes (Christie et al., Diabetes 43:1254-59, 1994).
  • Mammalian cells, such as COS cells or L cells, may also be transfected with appropriate Class I or Class II alleles specific for the islet cell antigen of the present invention. Such MHC molecules may be soluble or membrane bound, and the 1851 antigenic polypeptide may be recombinantly tethered to the N-terminal region of the α or β chain using a flexible linker containing, for example, repeating glycine residues separated by a serine residue, such that the antigenic peptide binds to the MHC molecule and is properly presented to the T cell. Alternatively, the antigenic peptide may be exogenously loaded into the MHC peptide binding grove. The MHC-antigenic peptide complex can then be used to assess the reactivity of peripheral blood T cells derived from prediabetic or diabetic patients. This reactivity may be assessed by methods known in the art, such as [0069] 3H thymidine incorporation, cytokine production or cytolysis. Alternatively, islet cell antigen expressed in microorganisms can be “fed” to peripheral blood mononuclear cells (PBMN). The antigen-fed cells can then be used to stimulate peripheral blood T cells derived from diabetics or prediabetics.
  • The islet cell antigen polypeptides are also contemplated to be advantageous for use as immunotherapeutics to induce immunological tolerance or nonresponsiveness (anergy) to 1851 polypeptide autoantigens in patients predisposed or already mounting an immune response to 1851 polypeptide autoantigens of the islet β-cells. This therapy can take the form of autoantigenic 1851 peptides bound to an appropriate MHC Class I or Class II molecule as described above. The therapy can also be in the form of oral tolerance (Weiner et al., [0070] Nature 376: 177-80, 1995), or IV tolerance, for example. The use of polypeptide antigens in suppression of autoimmune disease is disclosed by Wraith, et al., (Cell 59: 247-55, 1989). Tolerance can be induced in patients, although conditions for inducing such tolerance will vary according to a variety of factors. In a neonate, tolerance can be induced by parenteral injection of an islet cell antigenic polypeptide, either with recombinant polypeptide or synthetic antigen, or more conveniently by oral administration in an appropriate formulation. The precise amount of administration, its mode and frequency of dosages will vary.
  • To induce immunological tolerance to the islet cell autoantigens in an adult susceptible to or already suffering from a islet cell antigen related disease such as IDDM, the precise amounts and frequency of administration will also vary, for adults about 1 to 1,000 mg/kg can be administered by a variety of routes, such as parenterally, orally, by aerosols, intradermal injection, etc. For neonates the doses will generally be higher than those administered to adults; e.g. 100 to 1,000 mg/kg. [0071]
  • The islet cell antigen 1851 polypeptides will typically be more tolerogenic when administered in a soluble form rather than an aggregrated or particulate form. Persistence of an islet cell antigen polypeptide of the invention is generally needed to maintain tolerance in an adult, and thus may require more frequent administration of the antigen, or its administration in a form which extends the half-life of the islet cell antigen. See for example, Sun et al. ([0072] Proc. Natl. Acad. Sci. USA 91: 10795-99, 1994).
  • The islet cell antigen polypeptides described herein are also contemplated to be advantageous for use as immunotherapeutics in treating longer term IDDM patients that have been identified by autoantibody testing at the time of clinical non-insulin dependent diabetes mellitus (NIDDM) diagnosis. Intervention in these patients may be especially effective, perhaps due to the slowly progressive nature of their β cell destruction. Since the numbers of such patients is nearly the same as those with classical childhood IDDM, there is a need for such therapeutic intervention (Hagopian et al., [0073] J. Clin. Invest. 91:368-74; 1993; Harris and Robbins, Diabetes Care 17:1337-40, 1994; and Kobayashi et al., Diabetes 45:622-26, 1996).
  • The N-terminal domain of islet cell antigen 1851 is expected to be inside the insulin secretory granule. The islet cell antigen polypeptides of the current invention contain post translational modification sites within the N-terminal domain. A dibasic site or tribasic site at amino acid residues 228-230 (Arg-Lys-Lys) in SEQ ID NO:22 and amino acid residues 422-424 (Arg-Lys-Lys) in SEQ ID NO:16 could result in cleavage of a 420 amino acid post-translationally modified mammalian islet cell antigen polypeptide from the islet cell antigen 1851 polypeptide. All or part of this cleaved polypeptide may be released from the β cell via either the constitutive secretory pathway for granule halo components, or via the regulated pathway involved in insulin release. Detection and quantitation of post translationally modified polypeptides in a biological sample (that is, any sample derived from or containing cells, cell components or cell products, including, but not limited to, cell culture supernatants, cell lysates, cleared cell lysates, cell extracts, tissue extracts, blood plasma, serum, and fractions thereof) can be used diagnostically to monitor disease state in a patient. The presence or absence of such polypeptides in prediabetic and diabetic sera can be determined, for example by radioimmunoassay, and the concentration of such polypeptides in such an individual serum sample can be measured. This information can then be used, for example, to monitor insulin secretory activity, such as β cell insulin secretory rates; or to indicate altered β cell physiology associated with cellular stress as in an immune attack. Peptide levels could be an indicator of β cell distress or β cell death, and would be useful for predicting the disease state in a patient. Alternatively, the peptides herein function serve in paracrine or endocrine signaling to other islet cells or remote cells in other organs. The assay results can also find use in monitoring the effectiveness of therapeutic measures for treatment of IDDM or related diseases. In a preferred embodiment, a post-translationally modified mammalian islet cell antigen polypeptide comprises the sequence of SEQ ID NO:22 from His, amino acid residue 1 to Glu, amino acid residue 227. In another preferred embodiment the biological sample is blood. [0074]
  • The present invention also relates to a pharmaceutical composition comprising an islet cell antigen polypeptide of the present invention, together with a pharmaceutically acceptable carrier or vehicle, such as saline, buffered saline, water or the like. Formulations may further include one or more excipients, preservatives, solubilizers, etc. Methods of formulation are well known in the art and are disclosed, for example, in [0075] Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton Pa., 1990, which is incorporated herein by reference. Therapeutic doses will generally be in the range of 0.1 to 100 μg/kg of patient weight, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. In general, a therapeutically effective amount of an islet cell antigen polypeptide of the present invention is an amount sufficient to produce a clinically significant reduction in β-cell loss or a delay of clinical onset of IDDM.
  • In a related aspect, the present invention provides diagnostic kits for use with the recombinant or synthetic islet cell antigen polypeptides of the present invention, in detecting autoantibodies to pancreatic β-islet cells. Thus, 1851 polypeptides may be provided, usually in lyophilized form, in a container, either alone or in conjunction with additional reagents, such as 1851-specific antibodies, labels, and/or anti-human antibodies and the like. The 1851 polypeptides and antibodies, which may be conjugated to a label or unconjugated, are included in the kits with buffers, such as Tris phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum albumin, and the like. Frequently it will be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1 to 99% of the total composition. Where an antibody capable of binding to the islet cell antigen polypeptide autoantibody or to the recombinant or synthetic 1851 polypeptide is employed in an assay, this will typically be present in a separate vial. [0076]
  • Within one aspect of the present invention, islet cell antigen polypeptides, including derivatives thereof, as well as portions or fragments of these polypeptides, are utilized to prepare antibodies for diagnostic or therapeutic uses which specifically bind to islet cell antigen polypeptides. As used herein, the term “antibodies” includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab′)[0077] 2 and Fab fragments, as well as recombinantly produced binding partners. These binding partners incorporate the variable regions from a gene which encodes a specifically binding monoclonal antibody. Antibodies are defined to be specifically binding if they bind to the islet cell antigen polypeptides with a Ka of greater than or equal to 107/M. The affinity of a monoclonal antibody or binding partner may be readily determined by one of ordinary skill in the art (see, Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949).
  • Methods for preparing polyclonal and monoclonal antibodies have been well described in the literature (see for example, Sambrook et al., [0078] Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, Fla., 1982, which is incorporated herein by reference) As would be evident to one of ordinary skill in the art, polyclonal antibodies may be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats, for example. The immunogenicity of the islet cell antigen polypeptide may be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant. A variety of assays known to those skilled in the art may be utilized to detect antibodies which specifically bind to an islet cell antigen. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations, enzyme-linked immuno-sorbent assays, dot blot assays, inhibition or competition assays, and sandwich assays.
  • Additional techniques for the preparation of monoclonal antibodies may be utilized to construct and express recombinant monoclonal antibodies. Briefly, mRNA is isolated from a B cell population and used to create heavy and light chain immunoglobulin cDNA expression libraries in a suitable vector such as the λIMMUNOZAP(H) and λIMMUNOZAP(L) vectors, which may be obtained from Stratogene Cloning Systems (La Jolla, Calif.). These vectors are then screened individually or are co-expressed to form Fab fragments or antibodies (Huse et al., [0079] Science 246: 1275-81, 1989; Sastry et al., Proc. Natl. Acad. Sci. USA 86: 5728-32, 1989). Positive plaques are subsequently converted to a non-lytic plasmid which allows high level expression of monoclonal antibody fragments in E. coli.
  • Binding partners such as those described above may also be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene which encodes a specifically binding antibody. The construction of these proteins may be readily accomplished by one of ordinary skill in the art (see for example, Larrick et al., [0080] Biotechnology 7: 934-38, 1989; Reichmann et al., Nature 322: 323-27, 1988 and Roberts et al. Nature 328: 731-34, 1987). Once suitable antibodies or binding partners have been obtained, they may be isolated or purified by many techniques well described in the literature (see for example, Antibodies: A Laboratory Manual, ibid.). Suitable techniques include protein or peptide affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns or any combination of these techniques. Within the context of the present invention, the term “isolated” as used to define antibodies or binding partners means “substantially free of other blood components.”
  • Antibodies of the present invention may be produced by immunizing an animal, a wide variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats can be used, with a recombinant or synthetic islet cell antigen polypeptide or a selected portion thereof (e.g., a peptide). For example, by selected screening one can identify a region of the islet cell antigen polypeptide such as that predominantly responsible for recognition by anti-islet cell antigen polypeptide antibodies, or a portion which comprises an epitope of a islet cell antigen polypeptide variable region, which may thus serve as a islet cell antigen polypeptide-specific marker. Antibody producing cells obtained from the immunized animals are immortalized and screened, or screened first for, e.g., the production of antibody which inhibits the interaction of the anti-islet cell antigen polypeptide autoantibody with the islet cell antigen polypeptide and then immortalized. As the generation of human monoclonal antibodies to a human antigen, such as an 1851 polypeptide, may be difficult with conventional immortalization techniques, it may be desirable to first make non-human antibodies and then transfer via recombinant DNA techniques the antigen binding regions of the non-human antibodies, e.g. the F(ab′)2 or hypervariable regions, to human constant regions (Fc) or framework regions to produce substantially human molecules. Such methods are generally known in the art and are described in, for example, U.S. Pat. No. 4,816,397, and EP publications 173,494 and 239,400, which are incorporated herein by reference. [0081]
  • Alternatively, one may isolate DNA sequences which encode a human monoclonal antibody or portions thereof that specifically bind to islet cell antigen polypeptides by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., [0082] Science 246: 1275-81, 1989, incorporated herein by reference, and then cloning and amplifying the sequences which encode the antibody (or binding fragment) of the desired specificity.
  • In another aspect of the invention, the mammalian islet cell antigen polypeptides can be used to clone T cells which have specific receptors for the islet cell antigen polypeptide. Once the islet cell antigen polypeptide specific T cells are isolated and cloned using techniques generally available to the skilled artisan, the T cells or membrane preparations thereof can be used to immunize animals to produce antibodies to the islet cell antigen polypeptide receptors on T cells. The antibodies can be polyclonal or monoclonal. If polyclonal, the antibodies can be murine, lagomorph, equine, ovine, or from a variety of other mammals. Monoclonal antibodies will typically be murine in origin, produced according to known techniques, or human, as described above, or combinations thereof, as in chimeric or humanized antibodies. The anti-islet cell antigen polypeptide receptor antibodies thus obtained can then be administered to patients to reduce or eliminate T cell subpopulations which recognize and participate in the immunological destruction of islet cell antigen polypeptide bearing cells in an individual predisposed to or already suffering from a disease, such as IDDM. Further, the islet cell antigen polypeptide T cell receptors can thus be identified, cloned and sequenced, and receptor polypeptides synthesized which bind to the islet cell antigen polypeptides and block recognition of the islet cell antigen polypeptide-bearing cells, thereby impeding the autoimmune response against host islet cells. Howell et al. ([0083] Science 246: 668-70, 1989) have demonstrated that T cell receptor peptides can block the formation of the tri-molecular complex between T cells, autoantigen and major histocompatibilty complex in an autoimmune disease model.
  • Antibodies and binding partners of the present invention may be used in a variety of ways. The tissue distribution of the islet cell antigen, for example, may be determined by incubating tissue slices with a labeled monoclonal antibody which specifically binds to the islet cell antigen polypeptides, followed by detection of the presence of the bound antibody. Labels suitable for use within the present invention are well known in the art and include, among others, fluorescein, isothiocyanate, phycoerythrin, horseradish peroxidase, and colloidal gold. The antibodies of the present invention may also be used for the purification of the islet cell antigen polypeptides of the present invention. The coupling of antibodies to solid supports and their use in purification of proteins is well known in the literature (see for example, [0084] Methods in Molecular Biology, Vol. 1, Walker (Ed.), Humana Press, New Jersey, 1984, which is incorporated by reference herein in its entirety). Antibodies of the present invention may be used as a marker reagent to detect the presence of islet cell antigen polypeptides on cells or in solution. Such antibodies are also useful for western analysis or immunoblotting, particularly of purified cell secreted material. Polyclonal, affinity purified polyclonal, monoclonal and single chain antibodies are suitable for use in this regard. In addition, proteolytic and recombinant fragments and epitope binding domains can be used herein. Chimeric, humanized, veneered, CDR-replaced, reshaped or other recombinant whole or partial antibodies are also suitable.
  • The following examples are offered by way of illustration, not by way of limitation.[0085]
    • EXAMPLES Example 1 Synthesis of Macaque Islet Cell cDNA and Preparation of a Macaque Islet Cell cDNA Library
  • Islets of Langerhans (˜100,000) were isolated by collagenase digestion and Ficoll density gradient centrifugation from pancreas of [0086] Macaca nemestrina (obtained from the University of Washington Primate Center, Seattle, Wash.). These cells were then flash frozen in liquid nitrogen and stored at −80° C. until use. Total RNA from the islets was isolated according to the method of Chirgwin et al., Biochemistry 18: 52-94, 1994, incorporated herein by reference, using polytron homogenization in guanidinium thiocynate and LiCl centrifugation. Poly(A)+ RNA was isolated using oligo d(T) cellulose chromatography (Aviv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972).
  • First strand cDNA was synthesized from two-time poly d(T)-selected liver poly(A)[0087] +RNA. Ten microliters of a solution containing 10 μg of liver poly(A)+RNA was mixed with 2 μl of 20 pmole/μl first strand primer ZC3747 (SEQ ID NO:8) and 4 μl of diethylpyrocarbonate-treated water. The mixture was heated at 65° C. for 4 minutes and cooled by chilling on ice.
  • The first strand cDNA synthesis was initiated by the addition of 8 μl of 5× SUPERSCRIPT buffer (GIBCO BRL, Gaithersburg, Md.), 4 μl of 100 mM dithiothreitol, and 2.0 μl of a deoxynucleotide triphosphatate solution containing 10 mM each of dATP, dGTP, dTTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology Inc., Piscataway, N.J.) to the RNA-primer mixture. The reaction mixture was incubated at 45° C. for 4 minutes. After incubation, 10.0 μl of 200 U/μl SUPERSCRIPT reverse transcriptase (GIBCO BRL) was added. The efficiency of the first strand synthesis was analyzed in a parallel reaction by the addition of 10 μCi of [0088] 32P-αdCTP to a 5 μl aliquot of the reaction mixture to label the reaction products. The first strand synthesis reaction mixtures were incubated at 45° C. for 45 minutes followed by a 15 minute incubation at 50° C. Unincorporated nucleotides were removed from each reaction by precipitating the cDNA in the presence of 8 μg of glycogen carrier, 2.5 M ammonium acetate and 2.5 volume ethanol. The unlabeled cDNA was resuspended in 50 μl water and used for the second strand synthesis. The length of first strand cDNA was assessed by resuspending the labeled cDNA in 20 μl water and determining the cDNA size by agarose gel electrophoresis.
  • Second strand synthesis was performed on the RNA-DNA hybrid from the first strand synthesis reaction under conditions that promoted first strand priming of second strand synthesis resulting in DNA hairpin formation. A reaction mixture was prepared containing 20.0 μl of 5× polymerase I buffer (100 mM Tris, pH 7.4, 500 mM KCl, 25 mM MgCl[0089] 2, 50 MM (NH4)2SO4), 1.0 μl of 100 mM dithiothreitol, 2.0 μl of a solution containing 10 mM of each deoxynucleotide triphosphate, 3.0 μl 5 mM α-NAD, 1.0 μl of 3 U/μl E. coli DNA ligase (New England Biolabs, Inc., Beverly, Mass.), 5.0 μl of 10 U/μl E. coli DNA polymerase (Gibco BRL) and 50.0 μl of the unlabeled first strand DNA. A parallel reaction in which a 10 μl aliquot of the second strand synthesis was labeled by the addition of 10 μCi of 32P-αdCTP was used to monitor the efficiency of second strand synthesis. The reaction mixtures were incubated at room temperature for 5 minutes followed by the addition of 1.5 μl of 2 U/μl RNase H (Gibco BRL) to each reaction mixture. The reactions were incubated at 15° C. for 2 hours and 15 minutes, followed by a 15 minute incubation at room temperature. The reactions were each terminated by serial phenol/chloroform and chloroform/isoamylalcohol extractions. The DNA from each reaction was precipitated in the presence of ethanol and 2.5 M ammonium acetate. The DNA from the unlabeled reaction was resuspended in 100 μl water. The labeled DNA was resuspended and electrophoresed as described above.
  • The single-stranded DNA in the hairpin structure was cleaved using mung bean nuclease. The reaction mixture contained 20 μl of 10× Mung Bean Nuclease Buffer (Stratagene Cloning Systems, La Jolla, Calif.), 16 μl of 100 mM dithiothreitol, 54 μl water, 100 μl of the second strand cDNA, and 10 μl of a 1:10 dilution of Mung Bean Nuclease, final concentration 10.5 U/μl (Promega Corp., Madison, Wis.) in Stratagene MB dilution Buffer (Stratagene Cloning Systems). The reaction was incubated at 37° C. for 15 minutes, and the reaction was terminated by the addition of 20 μl of Tris-HCl, pH 8.0 followed by sequential extractions with phenol/chloroform and chloroform/isoamylalcohol. Following the extractions, the DNA was precipitated in ethanol and resuspended in water. [0090]
  • The resuspended cDNA was blunt-ended with T4 DNA polymerase. The cDNA, which was resuspended in a volume of 50 μl of water, was mixed with 50 μl of 5× T4 DNA polymerase buffer. (250 mM Tris-HCl, pH 8.0, 250 mM KCl, 25 MM MgCl[0091] 2), 3 μl of 100 mM dithiothreitol, 3 μl of a solution containing 10 mM of each deoxynucleotide triphosphate, and 4 μl of 1.0 U/μl T4 DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.). After an incubation at 10° C. for 60 minutes, the reaction was terminated by serial phenol/chloroform and chloroform/isoamylalcohol extractions. The cDNA fragments less than 400 bp in length were removed by chromatography on a Clontech TE400 spin column (Clontech, Palo Alto, Calif.). The DNA was ethanol precipitated and resuspended in 9 μl of water. Based on the incorporation of 32P-dCTP, the yield of cDNA was estimated to be 4 μg from a starting mRNA template of 10 μg.
  • Eco RI adapters (Pharmacia LKB Biotechnology Inc., Piscataway, N.J.) were added to the cDNA prepared above to facilitate the cloning of the cDNA into a mammalian expression vector. A 9 μl aliquot of the cDNA and 975 pmole of the adapter (15 μl) were mixed with 3 μl 10× ligase buffer (Promega Corp.), 1 μl 10 mM ATP, and 20 Units (2 μl), of T4 DNA ligase. (Promega Corp.). The reaction was incubated for 16 hours at a temperature gradient of 4° C. to 15° C. The reaction was terminated by the addition of 185 μl water, 25 μl REACT 2 buffer (Gibco BRL) followed by an incubation at 65° C. for between 30 and 60 minutes. After incubation, the reaction was terminated by serial phenol/chloroform and chloroform/isoamylalcohol extractions and ethanol precipitation as described above. Following centrifugation, the DNA pellet was washed with 70% ethanol and was air dried. The pellet was resuspended in 89 μl of water. [0092]
  • To facilitate the directional insertion of the cDNA into a mammalian expression vector, the cDNA was digested with Xho I, resulting in a cDNA having a 5′ Eco RI adhesive end and a 3′ Xho I adhesive end. The Xho I restriction site at the 3′ end of the cDNA was introduced through the ZC3747 primer (SEQ ID NO:8). The restriction digestion was terminated by serial phenol/chloroform and chloroform/isoamylalcohol extractions. The cDNA was ethanol precipitated, and the resulting pellet was washed with 70% ethanol and air-dried. The pellet was resuspended in 1× loading buffer (10 mM phosphate buffer, pH 8.8, 5% glycerol, 0.125% bromphenol blue). [0093]
  • The resuspended cDNA was heated to 65° C. for 10 minutes, cooled on ice and electrophoresed on a 0.8% low melt agarose gel (Seaplaque GTG Low Melt Agarose, FMC Corp., Rockland, Me.) using a 1 Kb ladder (Gibco BRL) as size markers. The contaminating adapters and by-product fragments below 600 bp in size were excised from the gel. The electrodes were reversed, and the cDNA was electrophoresed until concentrated near the lane origin. The area of the gel containing the concentrated DNA was excised, placed in a microfuge tube, and the approximate volume of the gel slice was determined. An aliquot of TE (10 mM Tris HCl pH 7.4, 1 mM disodium ethylenediaminetetraacetate.2 H[0094] 2O (EDTA)) equivalent to half the volume of the gel slice was added to the tube, and the agarose was melted by heating to 65° C. for fifteen minutes. Following equilibration of the sample to 42° C., approximately 5 units of β-Agarase I (New England Biolabs, Inc.) was added. The sample was incubated for 2 hours to digest the agarose. After incubation, a 0.1× volume of 3M sodium acetate was added to the sample, and the mixture was incubated on ice for fifteen minutes. After incubation, the sample was centrifuged at 14,000×g for 10 minutes to remove the undigested agarose. The cDNA in the supernatant was ethanol precipitated. The cDNA pellet was washed with 70% ethanol, air dried and resuspended in 37 μl of water. The cDNA recovered from the agarose gel was phosphorylated using T4 polynucleotide kinase. The reaction consisted of 37 μl cDNA, 5 μl 10× Stratagene Ligase Buffer (Stratagene Cloning Systems). Following a 5 minute incubation at 65° C., the reaction was cooled to room temperature where 5 μl 10 mM ATP (Pharmacia) and 3 μl T4 DNA polymerase (10 U/μl, Stratagene) were added. The reaction was incubated at 37° C. for 45 minutes and at 65° C. for 10 minutes. The reaction was terminated by serial phenol/chloroform extractions. The samples were chromatographed through a Clontech TE400 spin column and were precipitated in the presence of 2.5 M ammonium acetate. The cDNA was resuspended in 15 μl of 2.5 mM Tris-HCl, pH 8.0, 0.25 mM EDTA.
  • The resulting Eco RI-Xho I cDNA library was cloned into the [0095] E. coli vector pZCEP (Jelinek et al., Science 259: 1614-16, 1993). Eco RI-Xho I linearized pZCEP was ligated with the Eco RI-Xho I cDNA library. The resulting plasmids were electroporated into the E. coli strain DH10B ELECTROMAX˜ (Gibco BRL). The library was plated to obtain >5×105 independent colonies and aliquoted into 120 pools to give approximately 5,000 colonies per pool. An aliquot of the cells from each pool was removed for use in preparing plasmid DNA. The remaining cell mixtures were brought to a final concentration of 15% glycerol, aliquoted and frozen at −80° C. Plasmid DNA was prepared from each pool and the resulting plasmid DNA was digested with RNAse (Boehringer Mannheim) according to the manufacturer's instructions. The RNAse reaction was terminated by a phenol/chloroform/isoamylalcohol (24:24:1) extraction, and the DNA was ethanol precipitated. The pools were systematically screened as described in the examples below.
    • Example 2 Transfection of Macaque DNA into COS-7 Cells
  • Macaque DNA from each pool was transfected into COS-7 cells (African Green Monkey Kidney cells, ATCC CRL 1651) using the method essentially described by McMahan et al. ([0096] EMBO J. 10: 2821-32, 1991; which is incorporated by reference herein in its entirety). Briefly, one day prior to transfection approximately 2×105 COS-7 cells in 2 ml growth medium containing 10% fetal bovine serum (Dulbecco's modified Eagle's medium (DMEM), 1% L-glutamine, 1% PNS antibiotic mix (Gibco BRL), 25 mM Hepes, and 1 mM NaPyruvate) were plated on sterile, single-chamber slides (Nunc AS, Roskilde, Denmark) that had been coated with 10 μg/ml of human fibronectin in PBS for 30 minutes at 37° C. and washed with phosphate buffered saline (PBS). For each pool to be tested, 1-2 μg of macaque islet cell library pooled DNA was added into 100 μl of serum free medium (SFM, F/DV medium, 10 mg/l transferrin, 2 μg/l selenium, 10 mg/l fetuin, 5 mg/l insulin, 1 L-glutamine, 25 mM Hepes, 1 mM NaPyruvate, and 0.1 mM NEAA). To each DNA sample was added 100 μl SFM containing 12 μl LipofectAMINE˜ (Gibco BRL). The transfection solution was mixed by pipetting up and down and kept at room temperature for 15 to 45 minutes. To each mix was added 0.8 ml SFM which was then gently added to the COS-7 cells which had been washed once with SFM. The cells were incubated at 37° C., 5% CO2 for 4-5 hours. One milliliter of growth medium containing 20% FBS was added to each slide. Slides were incubated overnight at 37° C., 5% CO2. The spent medium was removed and replaced with 2 ml growth medium containing 10% FBS and the cells incubated for 24 to 48 hours, preferably 48 hours, at 37° C., 5% CO2.
    • Example 3 Diabetic Sera
  • Sera from two prediabetic subjects, EmWi and JoGr, were selected for screening the islet cell cDNA library. Sera from both subjects were characterized for autoantibodies to known β-cell antigens using techniques known in the art. The sera were tested for GAD65 autoantibodies using an in vitro transcription/translation assay (Grubin et al., [0097] Diabetologia 37: 344-50, 1994) followed by immunoprecipitation using radiolabeled recombinant human GAD65 according to Hagopian et al., J. Clin. Invest. 91: 368-74, 1993.
  • Recombinant radiolabeled GAD was expressed in the presence of [0098] 35S Methionine (Amersham Corp., Arlington Heights, Ill.) using the Sp6 bacteriophage promoter and the TNT reticulocyte lysate kit (Promega), according to manufacturer's direction. 35S Methionine incorporation was determined by precipitation using trichloroacetic acid (TCA), and 25% or more incorporation was considered acceptable. Radiolabeled antigen was stored at −80° C. until use.
  • Radiolabeled antigen was diluted 1:10 in immunoprecipitation buffer (150 mM NaCl, 1% v/v Triton X-114 (Sigma Chemical Co., St. Louis, Mo.), 0.05% Bovine serum albumin (Sigma), 10 mM benzamidine (Sigma), and 10 mM HEPES pH 7.4). The antigen was incubated for preclearing for 4 hours at 4° C. with 50 μl normal human serum. Immunoglobulin was removed using 200 μl Protein A Sepharose beads (Pharmacia LKB Biotechnology Inc.) for 45 minutes. The cleared supernatant was diluted to 50,000 TCA-precipitable counts per minute (cpm) per 400 μl immunoprecipitation buffer. Four microliters of serum from diabetic or control patients was separately incubated in duplicate with 400 μl diluted antigen at 4° C. overnight with mixing by gentle rotation. Antigen-antibody complexes were precipitated by 16 μl Protein A Sepharose, and the pellet was washed 5 times in ice-cold wash buffer which consisted of 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% BSA, and 0.25% Triton X-114. Antigen was dissociated from the pellet by boiling in the presence of 2% SDS and 5% β-mercaptoethanol, and counted by scintillation counting in scintillation fluid. Counts per minute reflect the level of autoantibodies present in the sera to capture the antigen. [0099]
  • Autoantibodies to the protein tyrosine phosphatase IA-2/ICA512 were detected as above using a radiolabeled cytoplasmic domain of human IA-2/ICA512 (Lan et al., [0100] DNA Cell Biology 13: 505-14, 1994; and Hagopian et al., Autoimmunity 21: 61, 1995). The complete cytoplasmic domain of human IA-2 was isolated by RT-PCR from U87MG glioblastoma cells (ATCC M85). Briefly, total RNA was prepared from 5×107 glioblastoma cells which were homogenized in 3.5 ml guanidine/LiCl followed by CsCl centrifugation. First strand cDNA was synthesized using a Superscript˜ Preamplification System (GIBCO BRL) according to the manufacturer's directions. One and one half microliters of a solution containing 5 μg total U87MG RNA was mixed with 1 μl oligo dT solution and 11.5 μl diethylpyrocarbonate-treated water. The mixture was heated at 70° C. for 10 minutes and cooled by chilling on ice.
  • First strand cDNA synthesis was initiated by the addition of 2 μl Superscript˜ II buffer, 2 μl 0.1 M dithiothreitol, 1 μl deoxynucleotide triphosphate solution containing 10 mM each of dATP, dGTP, dTTP, and dCTP, and 1 μl of 200 U/μl Superscript˜ II reverse transcriptase to the RNA-primer mixture. The reaction was incubated at room temperature for 10 minutes followed by an incubation at 42° C. for 50 minutes, then 70° C. for 15 minutes, then cooled on ice. The reaction was terminated by addition of 1 μl RNase H which was incubated at 37° C. for 20 minutes, then cooled on ice. [0101]
  • A 100 μl PCR reaction mixture was then prepared containing 20 μl of first strand template, 8 μl 10× synthesis buffer, 3.3 μM ZC8802 (SEQ ID NO:9, contains 5′ Xho I site and ATG), 5.4 μM ZC8803 (SEQ ID NO:10, contains Eco RI site following stop codon), 65 μl dH[0102] 2O and 1 wax bead (AmpliWax˜, Perkin-Elmer Cetus, Norwalk, Conn.). Following an initial cycle of 95° C. for 2 minutes, 4° C. for 10 minutes, 5 U Taq polymerase was added, and the reaction was amplified for 30 cycles of 1 minute at 95° C., 2 minutes at 55° C. and 3 minutes at 72° C. The reaction mixture was then stored at 4° C. The resulting 1.2 kb fragment (SEQ. ID. No.30) was digested with Eco RI-Xho I, treated with RNAse, then isolated by low melt agarose gel electrophoresis and ligated into Eco RI-Xho I linearized pZCEP. Sera were screened for IA-2/ICA512 autoantibodies as described above for GAD autoantibodies.
  • Both EmWi and JoGr sera showed reactivity to IA-2/ICA512. The sera were titered for IA-2/ICA512 reactivity on vector only transfected COS-7 cells using techniques known in the art, see for example, Greenbaum et al. ([0103] Diabetes 41: 1570-1574, 1992). The sera were separately adsorbed with porcine insulin (Hoechst, 10 mg/ml) and GAD (1 mg/ml) until reactivity was abolished in the respective antibody assays. These sera were then retitered for IA-2/ICA512 as above. JoGr had IA-2/ICA512 reactivity of 280 JDFU (Juvenile Diabetes Foundation Units) which persisted at >130 JDFU after adsorption. EmWi had IA-2/ICA512 reactivity of 140 JDFU which persisted at >130 JDFU after adsorption. EmWi had the lowest background staining and was therefore used for primary screening.
  • Twenty milliliters of EmWi was diluted 1:1 in 0.1 M NaPO[0104] 4 buffer, pH 8.0 and incubated with an equal volume of Protein A covalently linked to Sepharose beads (Zymed, South San Francisco, Calif.) for affinity purification. After gentle mixing for 45 minutes at 4° C., the slurry was loaded onto a column and washed with 10 column volumes of 0.1 M NaPO4 buffer, pH 8.0 and one column volume of 0.01 M NaPO4 buffer, pH 8.0, before elution of immunoglobulins with 0.05 M Na citrate buffer, pH 3.5. Eluted immunoglobulins were immediately neutralized to pH 7.0 with 2 M Tris, pH 8.0. Eluted fractions were evaluated by spectrophotometric absorption at 280 nM, and peak fractions were pooled, aliquoted and flash frozen for storage at −80° C. Typically the concentration was 4 mg/ml IgG. COS-7 cells were grown to confluence in 150 ml T-flasks, washed with PBS, fixed in 4% paraformaldehyde, and permeabilized by freeze/thaw. The pooled sera were diluted to 1 mg/ml in PBS and incubated with the permeabilized COS-7 cell lysate overnight at 4° C. Supernatant was cleared at 100,000×g and aliquotted for storage at −80° C. for use in the binding assay.
    • Example 4 Binding Assay
  • The macaque DNA transformed COS-7 cells on single chamber slides, from Example 2, were prepared for assay by removing spent medium from the slides and washing the cells 3 times in PBS at room temperature. The cells were fixed with 1 ml 50% ETOH/50% acetone for 5 minutes at room temperature followed by two washes in PBS and two washes in 1% bovine serum albumin (BSA) in PBS. The precleared serum (EmWi) was diluted to 0.2 mg/ml in a 5% BSA in PBS solution, and 500 μl was added to each of the slides which were then covered, wrapped in plastic wrap, and rocked gently on a rocker overnight at room temperature. [0105]
  • The slides were then washed three times in a 1% BSA/PBS solution, three minutes for each wash. Following the final wash, the slides were blocked for 10 minutes with 1 ml 5% BSA/4% normal goat serum (Sigma) in PBS at room temperature. The blocking buffer was removed, and 500 μl of 0.02 mg/ml biotinylated Protein A (Amersham Corp., Arlington Heights, Ill.) in 5% BSA/4% normal goat serum/PBS was added, followed by a 30 minute incubation at room temperature. The slides were washed three times with 1% BSA/PBS, three minutes for each wash, then 500 μl streptavidin-gold (Amersham) diluted 1:50 in 5% BSA/4% normal goat serum/PBS was added to each slide. Following a 60 minute incubation at room temperature the slides were washed three times in 1% BSA/PBS and one final time in PBS. The slides were then fixed by adding 0.5 ml of 9% formaldehyde/45% acetone in PBS for 30 seconds followed by three, 3 minute washes in dH[0106] 2O.
  • An equal volume of silver enhancement solution and initiator (IntenSE˜ M Silver Enhancement Kit, Amersham) were mixed in a 15 ml conical tube, and 0.5 ml was added to each slide. The slides were allowed to develop for 20 minutes or until the desired color intensity was achieved. The slides were then rinsed twice for five minutes in dH[0107] 2O and air dried. A single positive pool (#18) containing approximately 5,000 clones was found out of approximately 50 pools screened using EmWi sera.
  • To isolate the positive clone(s) from pool #18, one 150 mm plate was plated to give approximately 10,000 colonies from the #18 pool. Filter lifts were prepared using the methods essentially described by Hanahan and Meselson ([0108] Gene 10: 63, 1980) and Maniatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982), which are incorporated herein by reference in their entirety. The hybridization probe was obtained by PCR amplification of plasmid DNA from pool #18. Briefly, an aliquot of the plasmid DNA from pool #18 was subjected to PCR amplification using oligonucleotides ZC8802 and ZC8803 (SEQ ID NOS:9 and 10, respectively). A 50 μl reaction mixture was prepared containing 0.05 μg of the plasmid DNA from pool #18; 20 pmole of ZC 8802 and ZC 8803 (SEQ ID NOS:9 and 10, respectively); 10 nmoles of each deoxynucleotide triphosphate (Pharmacia); 4 μl 10× synthesis buffer (Boehringer Mannheim), and 2.5 U Taq polymerase (Boehringer Mannheim). The PCR reaction was run for 24 cycles (1 minute at 94° C., 1 minute at 56° C., and 1 minute at 72° C.). An approximately 1.1 Kb band was isolated on a low melt agarose gel electrophoresis and random primed using the MEGAPRIME˜ Kit (Amersham) according to the manufacturer's instructions.
  • The filter was hybridized in a solution containing 6× SSC, 0.1% SDS, 5× Denhardt's, 200 μg/ml denatured, sheared salmon sperm DNA, and 1×10[0109] 5 cpm/ml of 32P-labeled PCR fragment. The filter was hybridized overnight at 65° C. The excess label was removed by two, 15 minute washes with 2× SSC, 0.1% SDS at 65° C. The filter was exposed to film overnight at −80° C. with two screens.
  • Eighteen positive colonies were detected. Six of these colonies were cultured and subjected to a second round of filter lifts as described above, and from this two positive clones were identified. Restriction endonuclease analysis showed that both contained an approximate 2 Kb insert. One clone, designated M1.18.5.1, was sequenced, revealing a 2,170 bp coding region which contained regions of homology to the protein tyrosine phosphatase family, especially IA-2/ICA512. Comparison of the full length human protein tyrosine phosphatase IA-2/ICA512 with M1.18.5.1 suggests that the coding region of M1.18.5.1 is missing amino terminal sequence corresponding to approximately 400 amino acids. The partial nucleic acid sequence and deduced amino acid sequence of M1.18.5.1 is shown in SEQ ID NO 1 and SEQ ID NO: 2. [0110]
  • M1.18.5.1 was re-transfected into COS-7 cells and assayed as described above. In addition to the EmWi sera, the JoGr sera, which had a high titer to IA-2, was added to the screen and both detected M1.18.5.1. [0111]
    • Example 5 Isolation of Human Islet Cell Antigen 1851
  • The 2,170 nucleotide sequence from M1.18.5.1 (SEQ ID NO 1) was used to conduct a sequence search for a human homolog. A match was found in the GenBank database (GenBank ID: TO361, clone ID: HFBCV88) submitted by The Institute of Genomic Research, Gaithersburg, Md., as an expressed sequence tag (EST) from a human fetal brain library (Stratagene Cloning Systems). HFBCV88 (EST24415.seq), a 127 amino acid polypeptide, SEQ ID NO:5, had homology to a region of the cytoplasmic domain of M1.18.5.1. The closest human DNA sequence to HFBCV88 is HSICA512, islet cell antigen ICA-512. [0112]
  • An oligonucleotide primer (ZC10,011 SEQ ID NO:11) was made to a conserved region between 1851 and HFBCV88 which differed from the corresponding sequence of mouse and human IA-2/ICA512 in that an arginine was substituted for a methionine. Combined with a 128 fold degenerate primer (ZC10,019 SEQ ID NO:14, AARGCNACNGTNGAYAAY, wherein R is A or G, N is A, C, T, or G, and Y is C or T) which lies just upstream of the transmembrane domain, in the extracellular domain, a portion of the human homologue of M1.18.5.1 was identified in human insulinoma cDNA by PCR. Briefly, a PCR reaction was performed in a 100 μl final volume using 12.5 ng Marathon-ready human insulinoma cDNA prepared according to manufacturer's instruction (Marathon˜ cDNA Amplification Kit, Clontech), 20 pmoles each of primers ZC 10,011 (SEQ ID NO:11) and ZC 10,019 (SEQ ID NO:14), and the reagents provided in the Marathon˜ PCR kit (Clontech) according to the manufacturer's instructions. The reaction was amplified for 30 cycles (1 minute at 94° C., 30 seconds at 60° C., 5 minutes at 68° C.) followed by a 10 minute extension at 72° C. An 800 bp (WK11111, SEQ ID NO:32) and a 1,200 bp (WK121315, SEQ ID NO:34) fragment were isolated by low melt agarose gel electrophoresis. [0113]
  • A 3′RACE Marathon PCR was also performed in a 50 μl final volume using 12.5 ng Marathon-ready human insulinoma cDNA, 10 pmoles each of primers ZC 10,177 (SEQ ID NO:12) the complement to ZC 10,011, and AP-1 (adaptor primer, supplied with kit), and the reagents provided in the 3′RACE Marathon˜ PCR kit (Clontech), according to the manufacturer's instructions. The reaction was amplified for 30 cycles (30 seconds at 94° C., 30 seconds at 68° C.). A 900 bp and a 2,000 bp (WK121111, SEQ ID NO:33) fragment were isolated by low melt agarose electrophoresis. [0114]
  • The 800 bp, (SEQ ID NO:32) 1,200 bp, (SEQ ID NO:34) and 2,000 bp (SEQ ID NO:33) PCR fragments were independently subcloned into pCR1 (Invitrogen Inc., San Diego, Calif.), using the TA Cloning Kit (Invitrogen Inc.) according to the manufacturer's instructions. The resulting plasmids (11.1.1, 11.1.2, and 11.1.3, respectfully) were used to transform [0115] E. coli XL-1 cells. Transformants were screened for presence of insert, followed by sequencing of the insert.
    • Example 6 Detection of Human Islet Cell Antigen Autoantibodies
  • An approximately 1.1 kb (SEQ ID NO:6) Eco RI-Hind III cytoplasmic fragment of human islet cell antigen 1851 cDNA was inserted into the vector pcDNAII (Invitrogen, San Diego, Calif.), and designated IL1851-3. The resultant polypeptide was transcribed and translated in vitro using a TNT Coupled. Reticulocyte Lysate System (Promega), according the manufacturer's instructions. [0116]
  • The labeled, synthesized cytoplasmic portion of human islet cell antigen 1851 was used to screen diabetic sera from six patients, for the presence of autoantibodies. Protein A-Sepharose immunoprecipitation, as described above, showed that sera from all six reacted positively with the in vitro synthesized, human islet cell antigen, and indicated that the major autoepitope is likely present on this polypeptide. [0117]
  • Additional immunoprecipitation assays were performed with a spectrum of serum samples, including 91 healthy control sera (median age 22 years, range 1-49 years, 49% males and 51% females); 183 newly diagnosed IDDM patients sampled at onset (median age 11 years, 51% males and 49% females); and 60 first degree relatives of type I diabetic patients sampled a mean of 2.0 years before onset (median age 12 years, 58% males and 42% females). Parallel autoantibody assays used the intracellular domain of IA-2/ICA512. Immunoprecipitation assays were as described above. Briefly, 4 μl of serum from diabetic or control patients were separately incubated in duplicate with 400 μl [0118] 35S radiolabeled antigen (cytoplasmic portion of human islet cell antigen 1851, SEQ ID NO: 6, in immunoprecipitation buffer (10 mM Hepes, 0.05% BSA, 150 mM NaCl, 10 mM benzamidine, and 1% Triton X114)) at 4° C. overnight with mixing by gentle rotation (Hagopian et al., J. Clinc. Invest. 91:368-74, 1995). Antigen-antibody complexes were precipitated using 20 μl Protein A Sepharose, and the pellet was washed 3 times in ice-cold wash buffer (which consisted of 10 mM HEPES pH 7.4, 150 mM NaCl, 0.25% BSA, and 0.25% Triton X-114) and one cold water wash. Antigen was dissociated from the pellet by boiling in the presence of 2% SDS and 5% β-mercaptoethanol, counted by scintillation counting in scintillation fluid, and the results expressed as islet cell antigen 1851 index (Hagopian et al., Diabetes 42:631-36, 1993). Counts per minute reflect the level of autoantibodies present in the sera that can capture the antigen. Assay cutoff was an index of 0.04, determined as the mean +3 standard deviations of 91 control sera. Assay sensitivity, specificity, and positive predictive value were calculated (Hagopian et al., ibid., 1995).
  • Immunoprecipitation assays revealed autoantibodies in 56/183 (30.6%) newly diagnosed IDDM patients, 28/60 (46.7%) first degree relatives later progressing to clinical diabetes, but only 1/91 (1.1%) healthy control subject groups. For first degree relatives, this represents a positive predictive value of 58% and a sensitivity of 48%. [0119]
  • Of sera from 153 newly diagnosed patients, 83 (54%) recognized IC-2/ICA512 and 48 (31%) recognized islet cell antigen 1851. Only 1/48 (2%) from the sera recognizing islet cell antigen 1851 did not precipitate IA-2/ICA512, but 35/83 (42%) from the sera reactive with IA-2/ICA512 did not bind islet cell antigen 1851. Of those positive for both antigens, reactivity to IA-2/ICA512 was generally stronger than that to islet cell antigen 1851. [0120]
  • The intracellular domains of human islet cell antigen 1851 and IA-2/ICA512 were expressed and radiolabeled by in vitro transcription and translation using a TNT Coupled Reticulocyte Lysate System (Promega), according the manufacturer's instructions, as described above. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of the resulting radiolabeled polypeptide revealed, for human islet cell antigen 1851, a major band of 46 kD and a minor band at 33 kD, both immunoprecipitated by IDDM sera. Limited trypsin digest of the radiolabeled immunoprecipitated intracellular fragment of macaque and human islet cell antigen 1851 and IA-2/ICA512 was done using the method of Christie et al. ([0121] J. Exp. Med. 172:789-94, 1990), followed by SDS-PAGE and autoradiography, which revealed a 37 kD product from both macaque and human islet cell antigen 1851. This product was distinct from the 40 kD product produced by limited trypinization of the intracellular domain of IA-2/ICA512.
  • In order to test whether IA-2/ICA512 autoantibodies recognized only epitopes shared with islet cell antigen 1851, the intracellular domain of IA-2/ICA512 was expressed in baby hamster kidney cells (BHK cells). The 1.2 kb IA-2/ICA512 intracellular fragment (SEQ ID NO:30) from Example 3 was ligated into pZEM219b under the SV40 promoter (Busby et al., [0122] J. Biol. Chem. 266:15286-92, 1991) and cellular expression was determined by immunocytochemistry using rabbit polyclonal antiserum to IA-2/ICA512 (Rabin et al., J. Immunol. 152:3183-88, 1994). IA-2/ICA512-transfected BHK cells were homogenized in homogenization buffer (0.25% Triton X-114, 10 mM benzamidine). Using Western blotting, the concentration of recombinant intracellular IA-2/ICA512 was estimated at 7 μg/ml of cell extract.
  • Immunoprecipitation assays, as described above, were done using radiolabeled islet cell antigen 1851 in the presence of 0.5 μg of unlabeled IA-2/ICA512 per microliter of islet cell antigen 1851 positive sera, as a competitor. Islet cell antigen 1851 autoantibodies not fully blocked by this amount of IA-2/ICA512 were subjected to repeated immunoprecipitation assays using a 2.5 fold increase of unlabeled IA-2/ICA512 as a competitor. As a control, extracts from non-transfected BHK cells were used. Recombinant intracellular IA-2/ICA512 fully blocked islet cell antigen 1851 reactivity in 29/53 islet cell antigen 1851 positive sera, while a median of 21.4% (range 3%-55%) of original immunoreactivity was retained in 24/53 sera. Increasing the IA-2/ICA512 concentration did not reduce this residual immunoreactivity, suggesting that unique islet cell antigen 1851 epitopes are being recognized in certain sera. [0123]
    • Example 7 Cloning the Remaining 5′ Sequence of Macaque and Human Islet Cell Antigen 1851 cDNA
  • To obtain the remaining 5′ macaque cDNA sequence one pool (#12) from the macaque library described in Example 1 was plated at 10,000 colonies/150 mm plate. Filter lifts were prepared (Maniatis et al. ([0124] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982) and denatured with 0.5 M NaOH for four minutes, neutralized with 1 M Tris pH 8.0 for 2 minutes followed by renaturation with 1 M Tris pH 8.0/1.5 M NaCl for 2 minutes. Filters were cross linked in a UV Stratalinker (1200 μJ) (Stratagene Cloning Systems, La Jolla, Calif.). The filters were prehybridized in 20 ml hybridization buffer (6× SSC, 0.5% SDS, 5× Denhardts and 0.2 mg/ml boiled salmon sperm DNA) overnight at 65° C. The filters were then hybridized in 20 ml hybridization buffer containing 1×106 cpm/ml γ32P-ATP labeled hybridization probe (ZC10504 SEQ ID NO:18) overnight at 65° C. The labeled hybridization probe was prepared by adding to a 5 μl final volume 30 pmol oligo ZC10504 (SEQ ID NO:18), T4 polynucleotide kinase buffer, 37.5 pmol γ32P-ATP and 10 U T4 polynucleotide kinase The reaction was incubated for 1 hour at room temperature and unincorporated ATP was removed using a Stratagene push column according the manufacturer's instructions (Stratagene Cloning Systems, La Jolla, Calif.). Following the hybridization, excess unbound label was removed from the filters with eight washes in 2× SSC/0.1 SDS (2 times with 20 ml, 5 times with 30 ml and a final wash in 100 ml) for 5 to 10 minutes at 65° C. The filters were exposed to film overnight at −80° C.
  • Several positive colonies were detected. One of these colonies was cultured from a replica plated colony and subjected to sequence analysis. The clone, 12.10504.1, contained 2,736 bp coding region (SEQ ID NO:23), containing the cytoplasmic and transmembrane domains and extending the 5′ end of the macaque extracellular domain sequence (SEQ ID NO:1) by 609 bp. [0125]
  • 5′ RACE PCR was used to generate the remaining 5′ cDNA fragments of macaque islet cell antigen 1851. To a 50 μl final volume was added 5 pmol of a vector-specific oligonucleotide primer (ZC11197, SEQ ID NO:29), 5 pmol of a macaque specific primer (ZC11654, SEQ ID NO:28), 1 ng macaque islet cell cDNA library from Example 1, 40 mM dNTPs, TAQ Polymerase buffer and 1.25 U TAQ Polymerase. A one minute denaturation at 94° C. was followed by 30 amplification cycles (30 seconds at 94° C., 1 minute at 60° C., 2 minutes at 72° C.) followed by a 6 minute extension at 72° C.). [0126]
  • Four independent 5′RACE PCR reactions were run, each using a different pool from the macaque library as template. Four fragments were obtained, a 738 bp fragment (SEQ. ID. No. 24) extending the 5′ end by 246 bp; a 932 bp fragment (SEQ ID NO:25) extending the 5′ end by 193 bp; a 999 bp fragment (SEQ ID NO:26) extending the 5′ end by 68 bp and a 1011 bp fragment (SEQ ID NO:27) which contained the remaining 5′ sequence with the exception of the start methionine. The fragments were isolated by agarose electrophoresis, excised and separated from the agarose using the Qiagen Qiaquick Gel Extraction System (Qiagen, Inc., Chatsworth, Calif.) according to manufacturer's instruction. The fragments were subcloned into pGEM-T (Promega Corp., Madison, Wis.), using the TA Cloning Kit (Promega Corp.) according to the manufacturer's instructions. The resulting plasmids pJML8, 7, 9 and 10 respectfully, were used to transform [0127] E. coli DH10B cells. Transformants were screened for presence of insert, followed by sequencing of the insert.
  • The 5′ RACE fragments (SEQ ID Nos:23, 24, 25, 26 and 27) contain overlapping segments and were aligned with the macaque islet cell antigen 1851 sequence of SEQ ID NO:1 to give a full length macaque islet cell antigen 1851 DNA sequence as represented in SEQ ID NO:15. Comparison of the human protein tyrosine phosphatase IA-2/ICA512 cDNA and amino acid sequences with those of the macaque islet cell antigen 1851 cDNA and amino acid sequences (SEQ ID NOs: 15 and 16) suggests that the coding region is missing the start methionine. [0128]
  • A vector containing the full length macaque sequence can be created using PCR. The macaque 5′ RACE fragments (SEQ ID NOs: 23, 24, 25, 26 and 27) can be joined using PCR. A clone shown to possess the complete coding sequence can then be digested with convenient restriction sites and subcloned into a vector of choice. Clones can be screened for correct insertion of the full length sequence and subjected to DNA sequence analysis. [0129]
  • PCR using macaque derived primers was done to identify remaining 5′ cDNA sequence for the human islet cell antigen 1851 (SEQ ID NO:6). To a 50 μl final volume was added 5 pmol each of two gene-specific oligonucleotide primers ZC10504, SEQ ID NO:18 and ZC11653, SEQ ID NO:17, 1 ng Marathon-ready insulinoma cDNA, prepared according to manufacturer's instruction (Marathon˜ cDNA Amplification Kit, Clontech), 40 mM dNTPs, TAQ Polymerase buffer and 1.25 U TAQ Polymerase. The reaction was denatured at 94° C. for one minute, amplified for 30 cycles (30 seconds at 94° C., 1 minute at 63° C., 2 minutes at 72° C.), followed by a 6 minute extension at 72° C.). [0130]
  • A 1263 bp fragment (SEQ ID NO:31) was isolated by agarose electrophoresis. The isolated fragment was then excised and subcloned into pGEM-T using the TA Cloning Kit (Promega, Corp.), as described above. The clones were then analyzed for the presence of insert, and those containing insert were subjected to DNA sequence analysis. The human islet cell antigen 1851 fragments can be joined using PCR to give the human sequence as represented in SEQ ID NO:21. Clones can be screened for correct insertion of the fragments and subjected to DNA sequence analysis. Comparison of the human protein tyrosine phosphatase IA-2/ICA512 cDNA with that of the human islet cell antigen 1851 sequences (SEQ ID NO:21 and 22) suggests that the coding region is missing 5′ sequence corresponding to approximately 600 bp. Including the 3′ untranslated region, but not the 5′ untranslated region, the estimated mRNA size for the human sequence is 5 kb, which is consistent with the 5.5 kb mRNA observed in Northern blots discussed below. To obtain the remaining 5′ human islet cell antigen 1851 cDNA sequence, additional PCR or 5′ RACE PCR reactions can be performed as described above. [0131]
    • Example 8 Tissue Distribution
  • Human Multiple Tissue Northern Blots (MTN I, MTN II, and MTN III; Clontech, Palo Alto, Calif.) were probed to determine the tissue distribution of human islet cell antigen 1851 expression. A 38 nucleotide oligonucleotide sequence just external to the transmembrane region of human islet cell antigen 1851, which is distinct from the corresponding sequence of IA-2/ICA512 (SEQ ID NO:18) was radioactively labeled with γ[0132] 32P using a T4 nucleotide kinase (GIBCO BRL, Gaithersburg, Md.) according to the manufacturer's specifications. ExpressHyb˜ (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 37° C. using 5×106 cpm/ml of labeled probe. The blots were then washed three times at room temperature, once at 50° C. for 30 minutes, once at 60° C., in 6× SSC, 0.1% SDS. A final wash at 68° C. with 2× SSC, 0.05% SDS for 20 minutes was done prior to autoradiography. Two transcript sizes were detected. A strong 5.5 kb band and a weaker 3.3 kb band were detected in brain, pancreas and prostate, with lesser signals in spinal cord, thyroid, adrenal and GI tract. With the exception of prostate, this represents the expected neuroendocrine distribution.
  • In order to define tissue localization further, in situ hybridization was performed on macaque pancreas, adrenal gland and muscle. The 38 nucleotide islet cell antigen 1851 oligonucleotide (SEQ ID NO:18), a 38 bp IC-2/ICA512 oligonucleotide (SEQ ID NO:19) and a 30 bp insulin β-chain probe for pancreatic islets (Petersen et al., [0133] Diabetes 42:484-95, 1993) (SEQ ID NO:20) were end-labeled with 33P-dATP (New England Nuclear, Boston, Mass.) using terminal deoxytransferase (GIBCO BRL) according to manufacturer's instructions. Frozen sections (14 μm) from macaque pancreas, adrenal, pituitary and muscle were fixed in 4% paraformaldehyde, followed by acetylation with acetic anhydride and then delipidated in chloroform prior to use. Labeled probes (2 pmol/ml) were incubated on the sections overnight and then washed in two changes of 1× SSC at 60° C. for 30 minutes, followed by dehydration in ethanol and apposition to autoradiography film (Hyperfilm Betamax, Amersham Corp., Arlington Heights, Ill.) for 2 to 6 days. The slides were then coated with NTB2 Track emulsion (Eastman Kodak, Rochester, N.Y.) and exposed for 12-18 days before development and counterstain with cresyl violet. Images were captured using a Dage 72 CCD camera and a MCID M2 imaging system (Imaging Research, Ontario, Canada). Strong hybridization was detected in pancreatic islets and adrenal medulla but not in muscle. The IA-2/ICA512 and the insulin β chain probes hybridized to islets.
  • From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. [0134]
  • 0
    SEQUENCE LISTING
    (1) GENERAL INFORMATION:
    (iii) NUMBER OF SEQUENCES: 34
    (2) INFORMATION FOR SEQ ID NO: 1:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 2171 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (ix) FEATURE:
    (A) NAME/KEY: Coding Sequence
    (B) LOCATION: 1...1923
    (D) OTHER INFORMATION:
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
    GAA TTC GGC ACG AGC GGA GTT CAG GAC GAC GAT GAC AGA CTT TAC CAA 48
    Glu Phe Gly Thr Ser Gly Val Gln Asp Asp Asp Asp Arg Leu Tyr Gln
    1 5 10 15
    GAG GTC CAT CGT CTG AGT GCC ACA CTC GGG GGC CTC CTG CAG GAC CAC 96
    Glu Val His Arg Leu Ser Ala Thr Leu Gly Gly Leu Leu Gln Asp His
    20 25 30
    GGG TCT CGA CTC TCG CCT GGA GCC CTC CCC TTT GCA AAG CCC CTC AAA 144
    Gly Ser Arg Leu Ser Pro Gly Ala Leu Pro Phe Ala Lys Pro Leu Lys
    35 40 45
    ATG GAG AGG AAG AAA TCC GAG CGC CCT GAG GCT TCC CTG TCT TCA GAA 192
    Met Glu Arg Lys Lys Ser Glu Arg Pro Glu Ala Ser Leu Ser Ser Glu
    50 55 60
    GAG GAG ACT GCC GGA GTG GAG AAC GTC AAG AGC CAG ACG TAT TCC AAA 240
    Glu Glu Thr Ala Gly Val Glu Asn Val Lys Ser Gln Thr Tyr Ser Lys
    65 70 75 80
    GAC CTG CTG GGG CAG CAG CCG CAT TCG GAG CCC GGG GCA GGC GCG TTT 288
    Asp Leu Leu Gly Gln Gln Pro His Ser Glu Pro Gly Ala Gly Ala Phe
    85 90 95
    GGG GAG CTC CAA AAC CAG ATG CCT GGG CCC TCG GAG GAG GAG CAG AGC 336
    Gly Glu Leu Gln Asn Gln Met Pro Gly Pro Ser Glu Glu Glu Gln Ser
    100 105 110
    CTT CCA GCG GGT GCT CAG GAG GCC CTC GGC GAC GGC CTG CAA TTG GAA 384
    Leu Pro Ala Gly Ala Gln Glu Ala Leu Gly Asp Gly Leu Gln Leu Glu
    115 120 125
    GTC AAG CCT TCC GAG GAA GAG GCA CGG TGC TAC ATC GTG ACA GAC AGA 432
    Val Lys Pro Ser Glu Glu Glu Ala Arg Cys Tyr Ile Val Thr Asp Arg
    130 135 140
    GAC CCC CTG CGC CCC GAG GAA GGA AGG CAG CTG GTG GAG GAC GTC GCC 480
    Asp Pro Leu Arg Pro Glu Glu Gly Arg Gln Leu Val Glu Asp Val Ala
    145 150 155 160
    CGC CTC CTG CAG ATG CCC AGC AGC ACA TTC GCC GAC GTG GAG GTT CTC 528
    Arg Leu Leu Gln Met Pro Ser Ser Thr Phe Ala Asp Val Glu Val Leu
    165 170 175
    GGA CCA GCA GTG ACC TTC AAA GTG GGC GCC AAT GTC CAG AAC GTG ACC 576
    Gly Pro Ala Val Thr Phe Lys Val Gly Ala Asn Val Gln Asn Val Thr
    180 185 190
    ACT GCG GAT GTG GAG AAG GCC ACA GTT GAC AAC AAA GAC AAA CTG GAG 624
    Thr Ala Asp Val Glu Lys Ala Thr Val Asp Asn Lys Asp Lys Leu Glu
    195 200 205
    GAA ACC TCT GGA CTG AAA ATT CTT CAA ACC GGA GTC GGG TCG AAA AGC 672
    Glu Thr Ser Gly Leu Lys Ile Leu Gln Thr Gly Val Gly Ser Lys Ser
    210 215 220
    AAA CTC AAG TTC CTG CCT CCT CAG GCG GAG CAA GAA GAC TCA ACC AAG 720
    Lys Leu Lys Phe Leu Pro Pro Gln Ala Glu Gln Glu Asp Ser Thr Lys
    225 230 235 240
    TTC ATC GCG CTC ACC CTG GTC TCC CTC GCC TGC ATC CTG GGC GTC CTC 768
    Phe Ile Ala Leu Thr Leu Val Ser Leu Ala Cys Ile Leu Gly Val Leu
    245 250 255
    CTG GCC TCT GGC CTC ATC TAC TGC CTA CGC CAT AGC TCT CAG CAC AGG 816
    Leu Ala Ser Gly Leu Ile Tyr Cys Leu Arg His Ser Ser Gln His Arg
    260 265 270
    CTG AAG GAG AAG CTC TCG GGA CTA GGG CGC GAC CCA GGT GCA GAT GCC 864
    Leu Lys Glu Lys Leu Ser Gly Leu Gly Arg Asp Pro Gly Ala Asp Ala
    275 280 285
    ACC GCC GCC TAC CAG GAG CTG TGC CGC CAG CGT ATG GCC ACG CGG CCA 912
    Thr Ala Ala Tyr Gln Glu Leu Cys Arg Gln Arg Met Ala Thr Arg Pro
    290 295 300
    CCA GAC CGG CCC GAG GGC CCG CAC ACA TCC CGC ATC AGC AGC GTC TCG 960
    Pro Asp Arg Pro Glu Gly Pro His Thr Ser Arg Ile Ser Ser Val Ser
    305 310 315 320
    TCC CAG TTC AGC GAC GGG CCG ATG CCC AGC CCC TCC GCA CGC AGC AGC 1008
    Ser Gln Phe Ser Asp Gly Pro Met Pro Ser Pro Ser Ala Arg Ser Ser
    325 330 335
    GCC TCG TCC TGG TCC GAG GAG CCC GTG CAG TCC AAC ATG GAC ATC TCC 1056
    Ala Ser Ser Trp Ser Glu Glu Pro Val Gln Ser Asn Met Asp Ile Ser
    340 345 350
    ACC GGC CAC ATG ATC CTG TCC TAC ATG GAG GAC CAC CTG AAG AAC AAG 1104
    Thr Gly His Met Ile Leu Ser Tyr Met Glu Asp His Leu Lys Asn Lys
    355 360 365
    AAC CGG CTG GAG AAG GAG TGG GAG GCG CTG TGT GCC TAC CAG GCG GAG 1152
    Asn Arg Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala Tyr Gln Ala Glu
    370 375 380
    CCC AAC AGC TCA CTT GTG GCC CAG AAG GAG GAG AAT GTG CCC AAG AAC 1200
    Pro Asn Ser Ser Leu Val Ala Gln Lys Glu Glu Asn Val Pro Lys Asn
    385 390 395 400
    CGC TCC CTG GCC GTG CTG ACC TAT GAC CAC TCC CGG GTC CTA CTG AAG 1248
    Arg Ser Leu Ala Val Leu Thr Tyr Asp His Ser Arg Val Leu Leu Lys
    405 410 415
    GCG GAG AAC AGC CAC AGC CAC TCG GAC TAC ATC AAC GCC AGC CCC ATC 1296
    Ala Glu Asn Ser His Ser His Ser Asp Tyr Ile Asn Ala Ser Pro Ile
    420 425 430
    ATG GAT CAC GAC CCG AGG AAC CCC GCG TAC ATC GCC ACC CAG GGA CCG 1344
    Met Asp His Asp Pro Arg Asn Pro Ala Tyr Ile Ala Thr Gln Gly Pro
    435 440 445
    CTG CCC GCC ACC GTG GCC GAC TTT TGG CAG ATG GTG TGG GAG AGC GGC 1392
    Leu Pro Ala Thr Val Ala Asp Phe Trp Gln Met Val Trp Glu Ser Gly
    450 455 460
    TGC GTG GTG ATC GTC ATG CTG ACA CCC CTC ACA GAG AAC GGC GTC CGG 1440
    Cys Val Val Ile Val Met Leu Thr Pro Leu Thr Glu Asn Gly Val Arg
    465 470 475 480
    CAG TGC TAC CAC TAC TGG CCA GAT GAA GGC TCC AAC CTC TAC CAC ATC 1488
    Gln Cys Tyr His Tyr Trp Pro Asp Glu Gly Ser Asn Leu Tyr His Ile
    485 490 495
    TAT GAG GTG AAC CTG GTC TCC GAG CAC ATC TGG TGC GAG GAC TTT CTG 1536
    Tyr Glu Val Asn Leu Val Ser Glu His Ile Trp Cys Glu Asp Phe Leu
    500 505 510
    GTG AGG AGC TTC TAT CTG AAG AAC CTG CAG ACC AAC GAG ACG CGC ACC 1584
    Val Arg Ser Phe Tyr Leu Lys Asn Leu Gln Thr Asn Glu Thr Arg Thr
    515 520 525
    GTG ACC CAG TTC CAC TTC CTG AGT TGG TAT GAC CGA GGA GTC CCC TCC 1632
    Val Thr Gln Phe His Phe Leu Ser Trp Tyr Asp Arg Gly Val Pro Ser
    530 535 540
    TCC TCA AGA TCC CTC CTG GAC TTC CGC AGA AAA GTA AAC AAG TGC TAC 1680
    Ser Ser Arg Ser Leu Leu Asp Phe Arg Arg Lys Val Asn Lys Cys Tyr
    545 550 555 560
    AGG GGC CGT TCT TGT CCA ATA ATT GTT CAT TGC AGT GAC GGT GCA GGC 1728
    Arg Gly Arg Ser Cys Pro Ile Ile Val His Cys Ser Asp Gly Ala Gly
    565 570 575
    CGG AGC GGC ACC TAC GTC CTG ATC GAC ATG GTT CTC AAC AAG ATG GCC 1776
    Arg Ser Gly Thr Tyr Val Leu Ile Asp Met Val Leu Asn Lys Met Ala
    580 585 590
    AAA GGT GCT AAA GAG ATT GAT ATC GCA GCA ACC CTG GAG CAC TTG AGG 1824
    Lys Gly Ala Lys Glu Ile Asp Ile Ala Ala Thr Leu Glu His Leu Arg
    595 600 605
    GAC CAG AGA CCC GGC ATG GTC CAG ACG AAG GAG CAG TTT GAG TTC GCG 1872
    Asp Gln Arg Pro Gly Met Val Gln Thr Lys Glu Gln Phe Glu Phe Ala
    610 615 620
    CTG ACA GCC GTG GCT GAA GAG GTG AAT GCC ATC CTC AAG GCC CTT CCC 1920
    Leu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys Ala Leu Pro
    625 630 635 640
    CAG TGAGCAGCGG CCTCGGGGCC TCGGGGGAGC CCCCACCCCC CGGATGTCGT CAGGAA 1979
    Gln
    TCGTGATCTG ACTTTAATTG TGTGTCTTCT ATTATAACTG CATAGTAATA GGGCCCTTAG 2039
    CTCTCCCGTA GTCAGCGCAG TTTAGCAGTT AAGCAGTTAA AATGTGTATT TTTGTTTAAT 2099
    CCAACAATAA TAAAGAGAGA TTTGTGGAAA AATCCCAAAA AAAAAAAAAA AAAAAAAAAA 2159
    AAAAAACTCG AG 2171
    (2) INFORMATION FOR SEQ ID NO: 2:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 641 amino acids
    (B) TYPE: amino acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (v) FRAGMENT TYPE: internal
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
    Glu Phe Gly Thr Ser Gly Val Gln Asp Asp Asp Asp Arg Leu Tyr Gln
    1 5 10 15
    Glu Val His Arg Leu Ser Ala Thr Leu Gly Gly Leu Leu Gln Asp His
    20 25 30
    Gly Ser Arg Leu Ser Pro Gly Ala Leu Pro Phe Ala Lys Pro Leu Lys
    35 40 45
    Met Glu Arg Lys Lys Ser Glu Arg Pro Glu Ala Ser Leu Ser Ser Glu
    50 55 60
    Glu Glu Thr Ala Gly Val Glu Asn Val Lys Ser Gln Thr Tyr Ser Lys
    65 70 75 80
    Asp Leu Leu Gly Gln Gln Pro His Ser Glu Pro Gly Ala Gly Ala Phe
    85 90 95
    Gly Glu Leu Gln Asn Gln Met Pro Gly Pro Ser Glu Glu Glu Gln Ser
    100 105 110
    Leu Pro Ala Gly Ala Gln Glu Ala Leu Gly Asp Gly Leu Gln Leu Glu
    115 120 125
    Val Lys Pro Ser Glu Glu Glu Ala Arg Cys Tyr Ile Val Thr Asp Arg
    130 135 140
    Asp Pro Leu Arg Pro Glu Glu Gly Arg Gln Leu Val Glu Asp Val Ala
    145 150 155 160
    Arg Leu Leu Gln Met Pro Ser Ser Thr Phe Ala Asp Val Glu Val Leu
    165 170 175
    Gly Pro Ala Val Thr Phe Lys Val Gly Ala Asn Val Gln Asn Val Thr
    180 185 190
    Thr Ala Asp Val Glu Lys Ala Thr Val Asp Asn Lys Asp Lys Leu Glu
    195 200 205
    Glu Thr Ser Gly Leu Lys Ile Leu Gln Thr Gly Val Gly Ser Lys Ser
    210 215 220
    Lys Leu Lys Phe Leu Pro Pro Gln Ala Glu Gln Glu Asp Ser Thr Lys
    225 230 235 240
    Phe Ile Ala Leu Thr Leu Val Ser Leu Ala Cys Ile Leu Gly Val Leu
    245 250 255
    Leu Ala Ser Gly Leu Ile Tyr Cys Leu Arg His Ser Ser Gln His Arg
    260 265 270
    Leu Lys Glu Lys Leu Ser Gly Leu Gly Arg Asp Pro Gly Ala Asp Ala
    275 280 285
    Thr Ala Ala Tyr Gln Glu Leu Cys Arg Gln Arg Met Ala Thr Arg Pro
    290 295 300
    Pro Asp Arg Pro Glu Gly Pro His Thr Ser Arg Ile Ser Ser Val Ser
    305 310 315 320
    Ser Gln Phe Ser Asp Gly Pro Met Pro Ser Pro Ser Ala Arg Ser Ser
    325 330 335
    Ala Ser Ser Trp Ser Glu Glu Pro Val Gln Ser Asn Met Asp Ile Ser
    340 345 350
    Thr Gly His Met Ile Leu Ser Tyr Met Glu Asp His Leu Lys Asn Lys
    355 360 365
    Asn Arg Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala Tyr Gln Ala Glu
    370 375 380
    Pro Asn Ser Ser Leu Val Ala Gln Lys Glu Glu Asn Val Pro Lys Asn
    385 390 395 400
    Arg Ser Leu Ala Val Leu Thr Tyr Asp His Ser Arg Val Leu Leu Lys
    405 410 415
    Ala Glu Asn Ser His Ser His Ser Asp Tyr Ile Asn Ala Ser Pro Ile
    420 425 430
    Met Asp His Asp Pro Arg Asn Pro Ala Tyr Ile Ala Thr Gln Gly Pro
    435 440 445
    Leu Pro Ala Thr Val Ala Asp Phe Trp Gln Met Val Trp Glu Ser Gly
    450 455 460
    Cys Val Val Ile Val Met Leu Thr Pro Leu Thr Glu Asn Gly Val Arg
    465 470 475 480
    Gln Cys Tyr His Tyr Trp Pro Asp Glu Gly Ser Asn Leu Tyr His Ile
    485 490 495
    Tyr Glu Val Asn Leu Val Ser Glu His Ile Trp Cys Glu Asp Phe Leu
    500 505 510
    Val Arg Ser Phe Tyr Leu Lys Asn Leu Gln Thr Asn Glu Thr Arg Thr
    515 520 525
    Val Thr Gln Phe His Phe Leu Ser Trp Tyr Asp Arg Gly Val Pro Ser
    530 535 540
    Ser Ser Arg Ser Leu Leu Asp Phe Arg Arg Lys Val Asn Lys Cys Tyr
    545 550 555 560
    Arg Gly Arg Ser Cys Pro Ile Ile Val His Cys Ser Asp Gly Ala Gly
    565 570 575
    Arg Ser Gly Thr Tyr Val Leu Ile Asp Met Val Leu Asn Lys Met Ala
    580 585 590
    Lys Gly Ala Lys Glu Ile Asp Ile Ala Ala Thr Leu Glu His Leu Arg
    595 600 605
    Asp Gln Arg Pro Gly Met Val Gln Thr Lys Glu Gln Phe Glu Phe Ala
    610 615 620
    Leu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys Ala Leu Pro
    625 630 635 640
    Gln
    (2) INFORMATION FOR SEQ ID NO: 3:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 894 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (ix) FEATURE:
    (A) NAME/KEY: Coding Sequence
    (B) LOCATION: 1...894
    (D) OTHER INFORMATION:
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
    CGC CAT AGC TCT CAG CAC AGG CTG AAG GAG AAG CTC TCG GGA CTA GGG 48
    Arg His Ser Ser Gln His Arg Leu Lys Glu Lys Leu Ser Gly Leu Gly
    1 5 10 15
    GGC GAC CCA GGT GCA GAT GCC ACT GCC GCC TAC CAG GAG CTG TGC CGC 96
    Gly Asp Pro Gly Ala Asp Ala Thr Ala Ala Tyr Gln Glu Leu Cys Arg
    20 25 30
    CAG CGT ATG GCC ACG CGG CCA CCA GAC CGA CCT GAG GGC CCG CAC ACG 144
    Gln Arg Met Ala Thr Arg Pro Pro Asp Arg Pro Glu Gly Pro His Thr
    35 40 45
    TCA CGC ATC AGC AGC GTC TCA TCC CAG TTC AGC GAC GGG CCG ATC CCC 192
    Ser Arg Ile Ser Ser Val Ser Ser Gln Phe Ser Asp Gly Pro Ile Pro
    50 55 60
    AGC CCC TCC GCA CGC AGC AGC GCC TCA TCC TGG TCC GAG GAG CCT GTG 240
    Ser Pro Ser Ala Arg Ser Ser Ala Ser Ser Trp Ser Glu Glu Pro Val
    65 70 75 80
    CAG TCC AAC ATG GAC ATC TCC ACC GGC CAC ATG ATC CTG TCC TAC ATG 288
    Gln Ser Asn Met Asp Ile Ser Thr Gly His Met Ile Leu Ser Tyr Met
    85 90 95
    GAG GAC CAC CTG AAG AAC AAG AAC CGG CTG GAG AAG GAG TGG GAA GCG 336
    Glu Asp His Leu Lys Asn Lys Asn Arg Leu Glu Lys Glu Trp Glu Ala
    100 105 110
    CTG TGC GCC TAC CAG GCG GAG CCC AAC AGC TCG TTC GTG GCC CAG AGG 384
    Leu Cys Ala Tyr Gln Ala Glu Pro Asn Ser Ser Phe Val Ala Gln Arg
    115 120 125
    GAG GAG AAC GTG CCC AAG AAC CGC TCC CTG GCC GTG CTG ACC TAT GAC 432
    Glu Glu Asn Val Pro Lys Asn Arg Ser Leu Ala Val Leu Thr Tyr Asp
    130 135 140
    CAC TCC CGG GTC CTG CTG AAG GCG GAG AAC AGC CAC AGC CAC TCA GAC 480
    His Ser Arg Val Leu Leu Lys Ala Glu Asn Ser His Ser His Ser Asp
    145 150 155 160
    TAC ATC AAC GCT AGC CCC ATC ATG GAT CAC GAC CCG AGG AAC CCC GCG 528
    Tyr Ile Asn Ala Ser Pro Ile Met Asp His Asp Pro Arg Asn Pro Ala
    165 170 175
    TAC ATC GCC ACC CAG GGA CCG CTG CCC GCC ACC GTG GCT GAC TTT TGG 576
    Tyr Ile Ala Thr Gln Gly Pro Leu Pro Ala Thr Val Ala Asp Phe Trp
    180 185 190
    CAG ATG GTG TGG GAG AGC GGC TGC GTG GTG ATC GTC ATG CTG ACA CCC 624
    Gln Met Val Trp Glu Ser Gly Cys Val Val Ile Val Met Leu Thr Pro
    195 200 205
    CTC GCG GAG AAC GGC GTC CGG CAG TGC TAC CAC TAC TGG CCG GAT GAA 672
    Leu Ala Glu Asn Gly Val Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu
    210 215 220
    GGC TCC AAT CTC TAC CAC ATC TAT GAG GTG AAC CTG GTC TCC GAG CAC 720
    Gly Ser Asn Leu Tyr His Ile Tyr Glu Val Asn Leu Val Ser Glu His
    225 230 235 240
    ATC TGG TGT GAG GAC TTC CTG GTG AGG AGC TTC TAT CTG AAG AAC CTG 768
    Ile Trp Cys Glu Asp Phe Leu Val Arg Ser Phe Tyr Leu Lys Asn Leu
    245 250 255
    CAG ACC AAC GAG ACG CGC ACC GTG ACG CAG TTC CAC TTC CTG AGT TGG 816
    Gln Thr Asn Glu Thr Arg Thr Val Thr Gln Phe His Phe Leu Ser Trp
    260 265 270
    TAT GAC CGA GGA GTC CCT TCC TCC TCA AGG TCC CTC CTG GAC TTC CGC 864
    Tyr Asp Arg Gly Val Pro Ser Ser Ser Arg Ser Leu Leu Asp Phe Arg
    275 280 285
    AGA AAA GTA AAC AAG TGC TAC AGG GGC CGT 894
    Arg Lys Val Asn Lys Cys Tyr Arg Gly Arg
    290 295
    (2) INFORMATION FOR SEQ ID NO: 4:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 298 amino acids
    (B) TYPE: amino acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (v) FRAGMENT TYPE: internal
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
    Arg His Ser Ser Gln His Arg Leu Lys Glu Lys Leu Ser Gly Leu Gly
    1 5 10 15
    Gly Asp Pro Gly Ala Asp Ala Thr Ala Ala Tyr Gln Glu Leu Cys Arg
    20 25 30
    Gln Arg Met Ala Thr Arg Pro Pro Asp Arg Pro Glu Gly Pro His Thr
    35 40 45
    Ser Arg Ile Ser Ser Val Ser Ser Gln Phe Ser Asp Gly Pro Ile Pro
    50 55 60
    Ser Pro Ser Ala Arg Ser Ser Ala Ser Ser Trp Ser Glu Glu Pro Val
    65 70 75 80
    Gln Ser Asn Met Asp Ile Ser Thr Gly His Met Ile Leu Ser Tyr Met
    85 90 95
    Glu Asp His Leu Lys Asn Lys Asn Arg Leu Glu Lys Glu Trp Glu Ala
    100 105 110
    Leu Cys Ala Tyr Gln Ala Glu Pro Asn Ser Ser Phe Val Ala Gln Arg
    115 120 125
    Glu Glu Asn Val Pro Lys Asn Arg Ser Leu Ala Val Leu Thr Tyr Asp
    130 135 140
    His Ser Arg Val Leu Leu Lys Ala Glu Asn Ser His Ser His Ser Asp
    145 150 155 160
    Tyr Ile Asn Ala Ser Pro Ile Met Asp His Asp Pro Arg Asn Pro Ala
    165 170 175
    Tyr Ile Ala Thr Gln Gly Pro Leu Pro Ala Thr Val Ala Asp Phe Trp
    180 185 190
    Gln Met Val Trp Glu Ser Gly Cys Val Val Ile Val Met Leu Thr Pro
    195 200 205
    Leu Ala Glu Asn Gly Val Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu
    210 215 220
    Gly Ser Asn Leu Tyr His Ile Tyr Glu Val Asn Leu Val Ser Glu His
    225 230 235 240
    Ile Trp Cys Glu Asp Phe Leu Val Arg Ser Phe Tyr Leu Lys Asn Leu
    245 250 255
    Gln Thr Asn Glu Thr Arg Thr Val Thr Gln Phe His Phe Leu Ser Trp
    260 265 270
    Tyr Asp Arg Gly Val Pro Ser Ser Ser Arg Ser Leu Leu Asp Phe Arg
    275 280 285
    Arg Lys Val Asn Lys Cys Tyr Arg Gly Arg
    290 295
    (2) INFORMATION FOR SEQ ID NO: 5:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 127 amino acids
    (B) TYPE: amino acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
    Ala Ser Pro Ile Met Asp His Asp Pro Arg Asn Pro Ala Tyr Ile Ala
    1 5 10 15
    Thr Gln Gly Pro Leu Pro Ala Thr Val Ala Asp Phe Trp Gln Met Val
    20 25 30
    Trp Glu Ser Gly Cys Val Val Ile Val Met Leu Thr Pro Leu Ala Glu
    35 40 45
    Asn Gly Val Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu Gly Ser Asn
    50 55 60
    Leu Tyr His Ile Tyr Glu Val Asn Leu Val Ser Glu His Ile Trp Cys
    65 70 75 80
    Glu Asp Phe Leu Val Arg Ser Phe Tyr Leu Lys Asn Leu Gln Thr Asn
    85 90 95
    Glu Thr Arg Thr Val Thr Gln Phe Pro Leu Ser Xaa Trp Tyr Asp Arg
    100 105 110
    Xaa Val Pro Ser Phe Leu Lys Val Pro Xaa Trp Thr Ser Ala Glu
    115 120 125
    (2) INFORMATION FOR SEQ ID NO: 6:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 1163 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (ix) FEATURE:
    (A) NAME/KEY: Coding Sequence
    (B) LOCATION: 27...1154
    (D) OTHER INFORMATION:
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
    AAGCTATGCA TCAAGCTTCC ACCATG CGC CAT AGC TCT CAG CAC AGG CTG AAG 53
    Arg His Ser Ser Gln His Arg Leu Lys
    1 5
    GAG AAG CTC TCG GGA CTA GGG GGC GAC CCA GGT GCA GAT GCC ACT GCC 101
    Glu Lys Leu Ser Gly Leu Gly Gly Asp Pro Gly Ala Asp Ala Thr Ala
    10 15 20 25
    GCC TAC CAG GAG CTG TGC CGC CAG CGT ATG GCC ACG CGG CCA CCA GAC 149
    Ala Tyr Gln Glu Leu Cys Arg Gln Arg Met Ala Thr Arg Pro Pro Asp
    30 35 40
    CGA CCT GAG GGC CCG CAC ACG TCA CGC ATC AGC AGC GTC TCA TCC CAG 197
    Arg Pro Glu Gly Pro His Thr Ser Arg Ile Ser Ser Val Ser Ser Gln
    45 50 55
    TTC AGC GAC GGG CCG ATC CCC AGC CCC TCC GCA CGC AGC AGC GCC TCA 245
    Phe Ser Asp Gly Pro Ile Pro Ser Pro Ser Ala Arg Ser Ser Ala Ser
    60 65 70
    TCC TGG TCC GAG GAG CCT GTG CAG TCC AAC ATG GAC ATC TCC ACC GGC 293
    Ser Trp Ser Glu Glu Pro Val Gln Ser Asn Met Asp Ile Ser Thr Gly
    75 80 85
    CAC ATG ATC CTG TCC TAC ATG GAG GAC CAC CTG AAG AAC AAG AAC CGG 341
    His Met Ile Leu Ser Tyr Met Glu Asp His Leu Lys Asn Lys Asn Arg
    90 95 100 105
    CTG GAG AAG GAG TGG GAA GCG CTG TGC GCC TAC CAG GCG GAG CCC AAC 389
    Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala Tyr Gln Ala Glu Pro Asn
    110 115 120
    AGC TCG TTC GTG GCC CAG AGG GAG GAG AAC GTG CCC AAG AAC CGC TCC 437
    Ser Ser Phe Val Ala Gln Arg Glu Glu Asn Val Pro Lys Asn Arg Ser
    125 130 135
    CTG GCC GTG CTG ACC TAT GAC CAC TCC CGG GTC CTG CTG AAG GCG GAG 485
    Leu Ala Val Leu Thr Tyr Asp His Ser Arg Val Leu Leu Lys Ala Glu
    140 145 150
    AAC AGC CAC AGC CAC TCA GAC TAC ATC AAC GCT AGC CCC ATC ATG GAT 533
    Asn Ser His Ser His Ser Asp Tyr Ile Asn Ala Ser Pro Ile Met Asp
    155 160 165
    CAC GAC CCG AGG AAC CCC GCG TAC ATC GCC ACC CAG GGA CCG CTG CCC 581
    His Asp Pro Arg Asn Pro Ala Tyr Ile Ala Thr Gln Gly Pro Leu Pro
    170 175 180 185
    GCC ACC GTG GCT GAC TTT TGG CAG ATG GTG TGG GAG AGC GGC TGC GTG 629
    Ala Thr Val Ala Asp Phe Trp Gln Met Val Trp Glu Ser Gly Cys Val
    190 195 200
    GTG ATC GTC ATG CTG ACA CCC CTC GCG GAG AAC GGC GTC CGG CAG TGC 677
    Val Ile Val Met Leu Thr Pro Leu Ala Glu Asn Gly Val Arg Gln Cys
    205 210 215
    TAC CAC TAC TGG CCG GAT GAA GGC TCC AAT CTC TAC CAC ATC TAT GAG 725
    Tyr His Tyr Trp Pro Asp Glu Gly Ser Asn Leu Tyr His Ile Tyr Glu
    220 225 230
    GTG AAC CTG GTC TCC GAG CAC ATC TGG TGT GAG GAC TTC CTG GTG AGG 773
    Val Asn Leu Val Ser Glu His Ile Trp Cys Glu Asp Phe Leu Val Arg
    235 240 245
    AGC TTC TAT CTG AAG AAC CTG CAG ACC AAC GAG ACG CGC ACC GTG ACG 821
    Ser Phe Tyr Leu Lys Asn Leu Gln Thr Asn Glu Thr Arg Thr Val Thr
    250 255 260 265
    CAG TTC CAC TTC CTG AGT TGG TAT GAC CGA GGA GTC CCT TCC TCC TCA 869
    Gln Phe His Phe Leu Ser Trp Tyr Asp Arg Gly Val Pro Ser Ser Ser
    270 275 280
    AGG TCC CTC CTG GAC TTC CGC AGA AAA GTA AAC AAG TGC TAC AGG GGC 917
    Arg Ser Leu Leu Asp Phe Arg Arg Lys Val Asn Lys Cys Tyr Arg Gly
    285 290 295
    CGT TCT TGT CCA ATA ATT GTT CAT TGC AGT GAC GGT GCA GGC CGG AGC 965
    Arg Ser Cys Pro Ile Ile Val His Cys Ser Asp Gly Ala Gly Arg Ser
    300 305 310
    GGC ACC TAC GTC CTG ATC GAC ATG GTT CTC AAC AAG ATG GCC AAA GGT 1013
    Gly Thr Tyr Val Leu Ile Asp Met Val Leu Asn Lys Met Ala Lys Gly
    315 320 325
    GCT AAA GAG ATT GAT ATC GCA GCG ACC CTG GAG CAC TTG AGG GAC CAG 1061
    Ala Lys Glu Ile Asp Ile Ala Ala Thr Leu Glu His Leu Arg Asp Gln
    330 335 340 345
    AGA CCC GGC ATG GTC CAG ACG AAG GAG CAG TTT GAG TTC GCG CTG ACA 1109
    Arg Pro Gly Met Val Gln Thr Lys Glu Gln Phe Glu Phe Ala Leu Thr
    350 355 360
    GCC GTG GCT GAG GAG GTG AAC GCC ATC CTC AAG GCC CTG CCC CAG TGAGA 1159
    Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys Ala Leu Pro Gln
    365 370 375
    ATTC 1163
    (2) INFORMATION FOR SEQ ID NO: 7:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 376 amino acids
    (B) TYPE: amino acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (v) FRAGMENT TYPE: internal
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
    Arg His Ser Ser Gln His Arg Leu Lys Glu Lys Leu Ser Gly Leu Gly
    1 5 10 15
    Gly Asp Pro Gly Ala Asp Ala Thr Ala Ala Tyr Gln Glu Leu Cys Arg
    20 25 30
    Gln Arg Met Ala Thr Arg Pro Pro Asp Arg Pro Glu Gly Pro His Thr
    35 40 45
    Ser Arg Ile Ser Ser Val Ser Ser Gln Phe Ser Asp Gly Pro Ile Pro
    50 55 60
    Ser Pro Ser Ala Arg Ser Ser Ala Ser Ser Trp Ser Glu Glu Pro Val
    65 70 75 80
    Gln Ser Asn Met Asp Ile Ser Thr Gly His Met Ile Leu Ser Tyr Met
    85 90 95
    Glu Asp His Leu Lys Asn Lys Asn Arg Leu Glu Lys Glu Trp Glu Ala
    100 105 110
    Leu Cys Ala Tyr Gln Ala Glu Pro Asn Ser Ser Phe Val Ala Gln Arg
    115 120 125
    Glu Glu Asn Val Pro Lys Asn Arg Ser Leu Ala Val Leu Thr Tyr Asp
    130 135 140
    His Ser Arg Val Leu Leu Lys Ala Glu Asn Ser His Ser His Ser Asp
    145 150 155 160
    Tyr Ile Asn Ala Ser Pro Ile Met Asp His Asp Pro Arg Asn Pro Ala
    165 170 175
    Tyr Ile Ala Thr Gln Gly Pro Leu Pro Ala Thr Val Ala Asp Phe Trp
    180 185 190
    Gln Met Val Trp Glu Ser Gly Cys Val Val Ile Val Met Leu Thr Pro
    195 200 205
    Leu Ala Glu Asn Gly Val Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu
    210 215 220
    Gly Ser Asn Leu Tyr His Ile Tyr Glu Val Asn Leu Val Ser Glu His
    225 230 235 240
    Ile Trp Cys Glu Asp Phe Leu Val Arg Ser Phe Tyr Leu Lys Asn Leu
    245 250 255
    Gln Thr Asn Glu Thr Arg Thr Val Thr Gln Phe His Phe Leu Ser Trp
    260 265 270
    Tyr Asp Arg Gly Val Pro Ser Ser Ser Arg Ser Leu Leu Asp Phe Arg
    275 280 285
    Arg Lys Val Asn Lys Cys Tyr Arg Gly Arg Ser Cys Pro Ile Ile Val
    290 295 300
    His Cys Ser Asp Gly Ala Gly Arg Ser Gly Thr Tyr Val Leu Ile Asp
    305 310 315 320
    Met Val Leu Asn Lys Met Ala Lys Gly Ala Lys Glu Ile Asp Ile Ala
    325 330 335
    Ala Thr Leu Glu His Leu Arg Asp Gln Arg Pro Gly Met Val Gln Thr
    340 345 350
    Lys Glu Gln Phe Glu Phe Ala Leu Thr Ala Val Ala Glu Glu Val Asn
    355 360 365
    Ala Ile Leu Lys Ala Leu Pro Gln
    370 375
    (2) INFORMATION FOR SEQ ID NO: 8:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 49 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: Other
    (vii) IMMEDIATE SOURCE:
    (B) CLONE: ZC3747
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
    GGGAATAACA TGTGAATGAC AAAATAAAAT GATAGCTTGC GCTTTTGCG 49
    (2) INFORMATION FOR SEQ ID NO: 9:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 39 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: Other
    (vii) IMMEDIATE SOURCE:
    (B) CLONE: ZC8802
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
    GCGCCTCGAG CCACCATGCA GCATGCGCGG CAGCAAGAC 39
    (2) INFORMATION FOR SEQ ID NO: 10:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: Other
    (vii) IMMEDIATE SOURCE:
    (B) CLONE: ZC8803
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
    GCGCGAATTC TCACTGGGGC AGGGCCTTGA G 31
    (2) INFORMATION FOR SEQ ID NO: 11:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: Other
    (vii) IMMEDIATE SOURCE:
    (B) CLONE: ZC10011
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
    TGTACGCGGG GTTCCTC 17
    (2) INFORMATION FOR SEQ ID NO: 12:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 25 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: Other
    (vii) IMMEDIATE SOURCE:
    (B) CLONE: ZC10177
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
    GAGGAACCCC GCGTACATCG CCACC 25
    (2) INFORMATION FOR SEQ ID NO: 13:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
    Val His Cys Xaa Ala Gly Xaa Xaa Arg Xaa Gly
    1 5 10
    (2) INFORMATION FOR SEQ ID NO: 14:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 18 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
    AARGCNACNG TNGAYAAY 18
    (2) INFORMATION FOR SEQ ID NO: 15:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 3287 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (ix) FEATURE:
    (A) NAME/KEY: Coding Sequence
    (B) LOCATION: 4...3039
    (D) OTHER INFORMATION:
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
    AGG GCG CTC CCG CTG CTG TTG CTG CTA CTG CTG CTG CTG CCG CCA CGC 48
    Ala Leu Pro Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro Pro Arg
    1 5 10 15
    GTC CTG CCT GCC GCC CCC TCG TCC GTC CCC CAC GGC CGG CAG CTC CCG 96
    Val Leu Pro Ala Ala Pro Ser Ser Val Pro His Gly Arg Gln Leu Pro
    20 25 30
    GGG CGC CTG GGC TGC CTA CTC GAG GAG GGC CTC TGC GGA GCG TCC GAG 144
    Gly Arg Leu Gly Cys Leu Leu Glu Glu Gly Leu Cys Gly Ala Ser Glu
    35 40 45
    GCC TGT GTG AAC GAT GGA GTG TTT GGA AGG TGC CAG AAG GTT CCG GCA 192
    Ala Cys Val Asn Asp Gly Val Phe Gly Arg Cys Gln Lys Val Pro Ala
    50 55 60
    ATG GAC TTT TAC CGC TAC GAG GTG TCG CCC GTG GCC CTG CAG CGC CTG 240
    Met Asp Phe Tyr Arg Tyr Glu Val Ser Pro Val Ala Leu Gln Arg Leu
    65 70 75
    CGC GTG GCT TTG CAG AAA CTC TCC GGC ACA GGT TTC ACG TGG CAG GAT 288
    Arg Val Ala Leu Gln Lys Leu Ser Gly Thr Gly Phe Thr Trp Gln Asp
    80 85 90 95
    GAC TAT ACT CAG TAT GTG ATG GAC CAG GAA CTT GCA GAC CTC CCC AAA 336
    Asp Tyr Thr Gln Tyr Val Met Asp Gln Glu Leu Ala Asp Leu Pro Lys
    100 105 110
    ACC TAC CTG AGG CAT CCT GAA GCG TCC GGC CCA GCC AGG CCC TCA AAA 384
    Thr Tyr Leu Arg His Pro Glu Ala Ser Gly Pro Ala Arg Pro Ser Lys
    115 120 125
    CAC AGC ATT GGC AGT GAG AGG AGG TAC AGT CGG GAG GGC GGC GCT GCC 432
    His Ser Ile Gly Ser Glu Arg Arg Tyr Ser Arg Glu Gly Gly Ala Ala
    130 135 140
    CTG GCC AAG GCC TTC CGA CGC CAC CTG CCC TTC CTG GAG GCC CTG TCC 480
    Leu Ala Lys Ala Phe Arg Arg His Leu Pro Phe Leu Glu Ala Leu Ser
    145 150 155
    CAG GCC CCA GCT TCA GAC GCG CTC GCC AGG ACC CGG ATG GCG CAG GAC 528
    Gln Ala Pro Ala Ser Asp Ala Leu Ala Arg Thr Arg Met Ala Gln Asp
    160 165 170 175
    AGA CCC CGT GCT GAG GGT GAC GAC CGC TTC TCC AAG AGC ATC CTG ACC 576
    Arg Pro Arg Ala Glu Gly Asp Asp Arg Phe Ser Lys Ser Ile Leu Thr
    180 185 190
    TAT GTG GCC CAC ACG TCT GTG CTG ACC TAC CCT CCC GGG CCC CAG GCC 624
    Tyr Val Ala His Thr Ser Val Leu Thr Tyr Pro Pro Gly Pro Gln Ala
    195 200 205
    CAG CTC CCC GAG GAC CTC CTG CCA CGG ACC CTC AGC CAG CTC CAG CCA 672
    Gln Leu Pro Glu Asp Leu Leu Pro Arg Thr Leu Ser Gln Leu Gln Pro
    210 215 220
    GAC GAG CTC AGC CCT AAG GTG GAC AGC AGT GTG GAG AGA CAC CAT CTG 720
    Asp Glu Leu Ser Pro Lys Val Asp Ser Ser Val Glu Arg His His Leu
    225 230 235
    ATG GCA GCC CTC AGT GCC TAT GCT GCC CAG AGG CCC CCA GCT CCC CCT 768
    Met Ala Ala Leu Ser Ala Tyr Ala Ala Gln Arg Pro Pro Ala Pro Pro
    240 245 250 255
    GGG AAG GGC AGC CTG GAG CCG CAG TAC CTT CTG CGC GCC CCG TCC AGA 816
    Gly Lys Gly Ser Leu Glu Pro Gln Tyr Leu Leu Arg Ala Pro Ser Arg
    260 265 270
    ATG CCC AGG CCC TTG TTG TCG CCA GCC GTC CCC CAG AAG TGG CCT TCA 864
    Met Pro Arg Pro Leu Leu Ser Pro Ala Val Pro Gln Lys Trp Pro Ser
    275 280 285
    CCT CTG GGA GAT CCT GAA GAC CCC CCC AGC ACA GGG GAA GGA GCA CGG 912
    Pro Leu Gly Asp Pro Glu Asp Pro Pro Ser Thr Gly Glu Gly Ala Arg
    290 295 300
    ATT CAC ACT CTC CTG AAG GAC CTG CAG AGG CAG CCG GCT GAG GCG AGG 960
    Ile His Thr Leu Leu Lys Asp Leu Gln Arg Gln Pro Ala Glu Ala Arg
    305 310 315
    GGC CTG AGT GAC CTG GAG CTG GAC AGC ATG GCC GAG CTG ATG GCT GGC 1008
    Gly Leu Ser Asp Leu Glu Leu Asp Ser Met Ala Glu Leu Met Ala Gly
    320 325 330 335
    CTG ATG CAA GGC ATG GAC CAC AGA GGA GCT CTA GGC GGC CCT GGG AAA 1056
    Leu Met Gln Gly Met Asp His Arg Gly Ala Leu Gly Gly Pro Gly Lys
    340 345 350
    GCG GCC CTG GGA GAG TCT GGA GAA CAG GCG GAT GGC CCC AAG GCC GCC 1104
    Ala Ala Leu Gly Glu Ser Gly Glu Gln Ala Asp Gly Pro Lys Ala Ala
    355 360 365
    CTC CGT GGG GAA AGC TTT CCA GAT GAC GGA GTT CAG GAC GAC GAT GAC 1152
    Leu Arg Gly Glu Ser Phe Pro Asp Asp Gly Val Gln Asp Asp Asp Asp
    370 375 380
    AGA CTT TAC CAA GAG GTC CAT CGT CTG AGT GCC ACA CTC GGG GGC CTC 1200
    Arg Leu Tyr Gln Glu Val His Arg Leu Ser Ala Thr Leu Gly Gly Leu
    385 390 395
    CTG CAG GAC CAC GGG TCT CGA CTC TCG CCT GGA GCC CTC CCC TTT GCA 1248
    Leu Gln Asp His Gly Ser Arg Leu Ser Pro Gly Ala Leu Pro Phe Ala
    400 405 410 415
    AAG CCC CTC AAA ATG GAG AGG AAG AAA TCC GAG CGC CCT GAG GCT TCC 1296
    Lys Pro Leu Lys Met Glu Arg Lys Lys Ser Glu Arg Pro Glu Ala Ser
    420 425 430
    CTG TCT TCA GAA GAG GAG ACT GCC GGA GTG GAG AAC GTC AAG AGC CAG 1344
    Leu Ser Ser Glu Glu Glu Thr Ala Gly Val Glu Asn Val Lys Ser Gln
    435 440 445
    ACG TAT TCC AAA GAC CTG CTG GGG CAG CAG CCG CAT TCG GAG CCC GGG 1392
    Thr Tyr Ser Lys Asp Leu Leu Gly Gln Gln Pro His Ser Glu Pro Gly
    450 455 460
    GCA GGC GCG TTT GGG GAG CTC CAA AAC CAG ATG CCT GGG CCC TCG GAG 1440
    Ala Gly Ala Phe Gly Glu Leu Gln Asn Gln Met Pro Gly Pro Ser Glu
    465 470 475
    GAG GAG CAG AGC CTT CCA GCG GGT GCT CAG GAG GCC CTC GGC GAC GGC 1488
    Glu Glu Gln Ser Leu Pro Ala Gly Ala Gln Glu Ala Leu Gly Asp Gly
    480 485 490 495
    CTG CAA TTG GAA GTC AAG CCT TCC GAG GAA GAG GCA CGG TGC TAC ATC 1536
    Leu Gln Leu Glu Val Lys Pro Ser Glu Glu Glu Ala Arg Cys Tyr Ile
    500 505 510
    GTG ACA GAC AGA GAC CCC CTG CGC CCC GAG GAA GGA AGG CAG CTG GTG 1584
    Val Thr Asp Arg Asp Pro Leu Arg Pro Glu Glu Gly Arg Gln Leu Val
    515 520 525
    GAG GAC GTC GCC CGC CTC CTG CAG ATG CCC AGC AGC ACA TTC GCC GAC 1632
    Glu Asp Val Ala Arg Leu Leu Gln Met Pro Ser Ser Thr Phe Ala Asp
    530 535 540
    GTG GAG GTT CTC GGA CCA GCA GTG ACC TTC AAA GTG GGC GCC AAT GTC 1680
    Val Glu Val Leu Gly Pro Ala Val Thr Phe Lys Val Gly Ala Asn Val
    545 550 555
    CAG AAC GTG ACC ACT GCG GAT GTG GAG AAG GCC ACA GTT GAC AAC AAA 1728
    Gln Asn Val Thr Thr Ala Asp Val Glu Lys Ala Thr Val Asp Asn Lys
    560 565 570 575
    GAC AAA CTG GAG GAA ACC TCT GGA CTG AAA ATT CTT CAA ACC GGA GTC 1776
    Asp Lys Leu Glu Glu Thr Ser Gly Leu Lys Ile Leu Gln Thr Gly Val
    580 585 590
    GGG TCG AAA AGC AAA CTC AAG TTC CTG CCT CCT CAG GCG GAG CAA GAA 1824
    Gly Ser Lys Ser Lys Leu Lys Phe Leu Pro Pro Gln Ala Glu Gln Glu
    595 600 605
    GAC TCA ACC AAG TTC ATC GCG CTC ACC CTG GTC TCC CTC GCC TGC ATC 1872
    Asp Ser Thr Lys Phe Ile Ala Leu Thr Leu Val Ser Leu Ala Cys Ile
    610 615 620
    CTG GGC GTC CTC CTG GCC TCT GGC CTC ATC TAC TGC CTA CGC CAT AGC 1920
    Leu Gly Val Leu Leu Ala Ser Gly Leu Ile Tyr Cys Leu Arg His Ser
    625 630 635
    TCT CAG CAC AGG CTG AAG GAG AAG CTC TCG GGA CTA GGG CGC GAC CCA 1968
    Ser Gln His Arg Leu Lys Glu Lys Leu Ser Gly Leu Gly Arg Asp Pro
    640 645 650 655
    GGT GCA GAT GCC ACC GCC GCC TAC CAG GAG CTG TGC CGC CAG CGT ATG 2016
    Gly Ala Asp Ala Thr Ala Ala Tyr Gln Glu Leu Cys Arg Gln Arg Met
    660 665 670
    GCC ACG CGG CCA CCA GAC CGG CCC GAG GGC CCG CAC ACA TCC CGC ATC 2064
    Ala Thr Arg Pro Pro Asp Arg Pro Glu Gly Pro His Thr Ser Arg Ile
    675 680 685
    AGC AGC GTC TCG TCC CAG TTC AGC GAC GGG CCG ATG CCC AGC CCC TCC 2112
    Ser Ser Val Ser Ser Gln Phe Ser Asp Gly Pro Met Pro Ser Pro Ser
    690 695 700
    GCA CGC AGC AGC GCC TCG TCC TGG TCC GAG GAG CCC GTG CAG TCC AAC 2160
    Ala Arg Ser Ser Ala Ser Ser Trp Ser Glu Glu Pro Val Gln Ser Asn
    705 710 715
    ATG GAC ATC TCC ACC GGC CAC ATG ATC CTG TCC TAC ATG GAG GAC CAC 2208
    Met Asp Ile Ser Thr Gly His Met Ile Leu Ser Tyr Met Glu Asp His
    720 725 730 735
    CTG AAG AAC AAG AAC CGG CTG GAG AAG GAG TGG GAG GCG CTG TGT GCC 2256
    Leu Lys Asn Lys Asn Arg Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala
    740 745 750
    TAC CAG GCG GAG CCC AAC AGC TCA CTT GTG GCC CAG AAG GAG GAG AAT 2304
    Tyr Gln Ala Glu Pro Asn Ser Ser Leu Val Ala Gln Lys Glu Glu Asn
    755 760 765
    GTG CCC AAG AAC CGC TCC CTG GCC GTG CTG ACC TAT GAC CAC TCC CGG 2352
    Val Pro Lys Asn Arg Ser Leu Ala Val Leu Thr Tyr Asp His Ser Arg
    770 775 780
    GTC CTA CTG AAG GCG GAG AAC AGC CAC AGC CAC TCG GAC TAC ATC AAC 2400
    Val Leu Leu Lys Ala Glu Asn Ser His Ser His Ser Asp Tyr Ile Asn
    785 790 795
    GCC AGC CCC ATC ATG GAT CAC GAC CCG AGG AAC CCC GCG TAC ATC GCC 2448
    Ala Ser Pro Ile Met Asp His Asp Pro Arg Asn Pro Ala Tyr Ile Ala
    800 805 810 815
    ACC CAG GGA CCG CTG CCC GCC ACC GTG GCC GAC TTT TGG CAG ATG GTG 2496
    Thr Gln Gly Pro Leu Pro Ala Thr Val Ala Asp Phe Trp Gln Met Val
    820 825 830
    TGG GAG AGC GGC TGC GTG GTG ATC GTC ATG CTG ACA CCC CTC ACA GAG 2544
    Trp Glu Ser Gly Cys Val Val Ile Val Met Leu Thr Pro Leu Thr Glu
    835 840 845
    AAC GGC GTC CGG CAG TGC TAC CAC TAC TGG CCA GAT GAA GGC TCC AAC 2592
    Asn Gly Val Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu Gly Ser Asn
    850 855 860
    CTC TAC CAC ATC TAT GAG GTG AAC CTG GTC TCC GAG CAC ATC TGG TGC 2640
    Leu Tyr His Ile Tyr Glu Val Asn Leu Val Ser Glu His Ile Trp Cys
    865 870 875
    GAG GAC TTT CTG GTG AGG AGC TTC TAT CTG AAG AAC CTG CAG ACC AAC 2688
    Glu Asp Phe Leu Val Arg Ser Phe Tyr Leu Lys Asn Leu Gln Thr Asn
    880 885 890 895
    GAG ACG CGC ACC GTG ACC CAG TTC CAC TTC CTG AGT TGG TAT GAC CGA 2736
    Glu Thr Arg Thr Val Thr Gln Phe His Phe Leu Ser Trp Tyr Asp Arg
    900 905 910
    GGA GTC CCC TCC TCC TCA AGA TCC CTC CTG GAC TTC CGC AGA AAA GTA 2784
    Gly Val Pro Ser Ser Ser Arg Ser Leu Leu Asp Phe Arg Arg Lys Val
    915 920 925
    AAC AAG TGC TAC AGG GGC CGT TCT TGT CCA ATA ATT GTT CAT TGC AGT 2832
    Asn Lys Cys Tyr Arg Gly Arg Ser Cys Pro Ile Ile Val His Cys Ser
    930 935 940
    GAC GGT GCA GGC CGG AGC GGC ACC TAC GTC CTG ATC GAC ATG GTT CTC 2880
    Asp Gly Ala Gly Arg Ser Gly Thr Tyr Val Leu Ile Asp Met Val Leu
    945 950 955
    AAC AAG ATG GCC AAA GGT GCT AAA GAG ATT GAT ATC GCA GCA ACC CTG 2928
    Asn Lys Met Ala Lys Gly Ala Lys Glu Ile Asp Ile Ala Ala Thr Leu
    960 965 970 975
    GAG CAC TTG AGG GAC CAG AGA CCC GGC ATG GTC CAG ACG AAG GAG CAG 2976
    Glu His Leu Arg Asp Gln Arg Pro Gly Met Val Gln Thr Lys Glu Gln
    980 985 990
    TTT GAG TTC GCG CTG ACA GCC GTG GCT GAA GAG GTG AAT GCC ATC CTC 3024
    Phe Glu Phe Ala Leu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu
    995 1000 1005
    AAG GCC CTT CCC CAG TGAGCAGCGG CCTCGGGGCC TCGGGGGAGC CCCCACCCCC C 3080
    Lys Ala Leu Pro Gln
    1010
    GGATGTCGTC AGGAATCGTG ATCTGACTTT AATTGTGTGT CTTCTATTAT AACTGCATAG 3140
    TAATAGGGCC CTTAGCTCTC CCGTAGTCAG CGCAGTTTAG CAGTTAAGCA GTTAAAATGT 3200
    GTATTTTTGT TTAATCCAAC AATAATAAAG AGAGATTTGT GGAAAAATCC CAAAAAAAAA 3260
    AAAAAAAAAA AAAAAAAAAA ACTCGAG 3287
    (2) INFORMATION FOR SEQ ID NO: 16:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 1012 amino acids
    (B) TYPE: amino acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (v) FRAGMENT TYPE: internal
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
    Ala Leu Pro Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro Pro Arg Val
    1 5 10 15
    Leu Pro Ala Ala Pro Ser Ser Val Pro His Gly Arg Gln Leu Pro Gly
    20 25 30
    Arg Leu Gly Cys Leu Leu Glu Glu Gly Leu Cys Gly Ala Ser Glu Ala
    35 40 45
    Cys Val Asn Asp Gly Val Phe Gly Arg Cys Gln Lys Val Pro Ala Met
    50 55 60
    Asp Phe Tyr Arg Tyr Glu Val Ser Pro Val Ala Leu Gln Arg Leu Arg
    65 70 75 80
    Val Ala Leu Gln Lys Leu Ser Gly Thr Gly Phe Thr Trp Gln Asp Asp
    85 90 95
    Tyr Thr Gln Tyr Val Met Asp Gln Glu Leu Ala Asp Leu Pro Lys Thr
    100 105 110
    Tyr Leu Arg His Pro Glu Ala Ser Gly Pro Ala Arg Pro Ser Lys His
    115 120 125
    Ser Ile Gly Ser Glu Arg Arg Tyr Ser Arg Glu Gly Gly Ala Ala Leu
    130 135 140
    Ala Lys Ala Phe Arg Arg His Leu Pro Phe Leu Glu Ala Leu Ser Gln
    145 150 155 160
    Ala Pro Ala Ser Asp Ala Leu Ala Arg Thr Arg Met Ala Gln Asp Arg
    165 170 175
    Pro Arg Ala Glu Gly Asp Asp Arg Phe Ser Lys Ser Ile Leu Thr Tyr
    180 185 190
    Val Ala His Thr Ser Val Leu Thr Tyr Pro Pro Gly Pro Gln Ala Gln
    195 200 205
    Leu Pro Glu Asp Leu Leu Pro Arg Thr Leu Ser Gln Leu Gln Pro Asp
    210 215 220
    Glu Leu Ser Pro Lys Val Asp Ser Ser Val Glu Arg His His Leu Met
    225 230 235 240
    Ala Ala Leu Ser Ala Tyr Ala Ala Gln Arg Pro Pro Ala Pro Pro Gly
    245 250 255
    Lys Gly Ser Leu Glu Pro Gln Tyr Leu Leu Arg Ala Pro Ser Arg Met
    260 265 270
    Pro Arg Pro Leu Leu Ser Pro Ala Val Pro Gln Lys Trp Pro Ser Pro
    275 280 285
    Leu Gly Asp Pro Glu Asp Pro Pro Ser Thr Gly Glu Gly Ala Arg Ile
    290 295 300
    His Thr Leu Leu Lys Asp Leu Gln Arg Gln Pro Ala Glu Ala Arg Gly
    305 310 315 320
    Leu Ser Asp Leu Glu Leu Asp Ser Met Ala Glu Leu Met Ala Gly Leu
    325 330 335
    Met Gln Gly Met Asp His Arg Gly Ala Leu Gly Gly Pro Gly Lys Ala
    340 345 350
    Ala Leu Gly Glu Ser Gly Glu Gln Ala Asp Gly Pro Lys Ala Ala Leu
    355 360 365
    Arg Gly Glu Ser Phe Pro Asp Asp Gly Val Gln Asp Asp Asp Asp Arg
    370 375 380
    Leu Tyr Gln Glu Val His Arg Leu Ser Ala Thr Leu Gly Gly Leu Leu
    385 390 395 400
    Gln Asp His Gly Ser Arg Leu Ser Pro Gly Ala Leu Pro Phe Ala Lys
    405 410 415
    Pro Leu Lys Met Glu Arg Lys Lys Ser Glu Arg Pro Glu Ala Ser Leu
    420 425 430
    Ser Ser Glu Glu Glu Thr Ala Gly Val Glu Asn Val Lys Ser Gln Thr
    435 440 445
    Tyr Ser Lys Asp Leu Leu Gly Gln Gln Pro His Ser Glu Pro Gly Ala
    450 455 460
    Gly Ala Phe Gly Glu Leu Gln Asn Gln Met Pro Gly Pro Ser Glu Glu
    465 470 475 480
    Glu Gln Ser Leu Pro Ala Gly Ala Gln Glu Ala Leu Gly Asp Gly Leu
    485 490 495
    Gln Leu Glu Val Lys Pro Ser Glu Glu Glu Ala Arg Cys Tyr Ile Val
    500 505 510
    Thr Asp Arg Asp Pro Leu Arg Pro Glu Glu Gly Arg Gln Leu Val Glu
    515 520 525
    Asp Val Ala Arg Leu Leu Gln Met Pro Ser Ser Thr Phe Ala Asp Val
    530 535 540
    Glu Val Leu Gly Pro Ala Val Thr Phe Lys Val Gly Ala Asn Val Gln
    545 550 555 560
    Asn Val Thr Thr Ala Asp Val Glu Lys Ala Thr Val Asp Asn Lys Asp
    565 570 575
    Lys Leu Glu Glu Thr Ser Gly Leu Lys Ile Leu Gln Thr Gly Val Gly
    580 585 590
    Ser Lys Ser Lys Leu Lys Phe Leu Pro Pro Gln Ala Glu Gln Glu Asp
    595 600 605
    Ser Thr Lys Phe Ile Ala Leu Thr Leu Val Ser Leu Ala Cys Ile Leu
    610 615 620
    Gly Val Leu Leu Ala Ser Gly Leu Ile Tyr Cys Leu Arg His Ser Ser
    625 630 635 640
    Gln His Arg Leu Lys Glu Lys Leu Ser Gly Leu Gly Arg Asp Pro Gly
    645 650 655
    Ala Asp Ala Thr Ala Ala Tyr Gln Glu Leu Cys Arg Gln Arg Met Ala
    660 665 670
    Thr Arg Pro Pro Asp Arg Pro Glu Gly Pro His Thr Ser Arg Ile Ser
    675 680 685
    Ser Val Ser Ser Gln Phe Ser Asp Gly Pro Met Pro Ser Pro Ser Ala
    690 695 700
    Arg Ser Ser Ala Ser Ser Trp Ser Glu Glu Pro Val Gln Ser Asn Met
    705 710 715 720
    Asp Ile Ser Thr Gly His Met Ile Leu Ser Tyr Met Glu Asp His Leu
    725 730 735
    Lys Asn Lys Asn Arg Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala Tyr
    740 745 750
    Gln Ala Glu Pro Asn Ser Ser Leu Val Ala Gln Lys Glu Glu Asn Val
    755 760 765
    Pro Lys Asn Arg Ser Leu Ala Val Leu Thr Tyr Asp His Ser Arg Val
    770 775 780
    Leu Leu Lys Ala Glu Asn Ser His Ser His Ser Asp Tyr Ile Asn Ala
    785 790 795 800
    Ser Pro Ile Met Asp His Asp Pro Arg Asn Pro Ala Tyr Ile Ala Thr
    805 810 815
    Gln Gly Pro Leu Pro Ala Thr Val Ala Asp Phe Trp Gln Met Val Trp
    820 825 830
    Glu Ser Gly Cys Val Val Ile Val Met Leu Thr Pro Leu Thr Glu Asn
    835 840 845
    Gly Val Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu Gly Ser Asn Leu
    850 855 860
    Tyr His Ile Tyr Glu Val Asn Leu Val Ser Glu His Ile Trp Cys Glu
    865 870 875 880
    Asp Phe Leu Val Arg Ser Phe Tyr Leu Lys Asn Leu Gln Thr Asn Glu
    885 890 895
    Thr Arg Thr Val Thr Gln Phe His Phe Leu Ser Trp Tyr Asp Arg Gly
    900 905 910
    Val Pro Ser Ser Ser Arg Ser Leu Leu Asp Phe Arg Arg Lys Val Asn
    915 920 925
    Lys Cys Tyr Arg Gly Arg Ser Cys Pro Ile Ile Val His Cys Ser Asp
    930 935 940
    Gly Ala Gly Arg Ser Gly Thr Tyr Val Leu Ile Asp Met Val Leu Asn
    945 950 955 960
    Lys Met Ala Lys Gly Ala Lys Glu Ile Asp Ile Ala Ala Thr Leu Glu
    965 970 975
    His Leu Arg Asp Gln Arg Pro Gly Met Val Gln Thr Lys Glu Gln Phe
    980 985 990
    Glu Phe Ala Leu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys
    995 1000 1005
    Ala Leu Pro Gln
    1010
    (2) INFORMATION FOR SEQ ID NO: 17:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 28 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: Other
    (vii) IMMEDIATE SOURCE:
    (B) CLONE: ZC11653
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
    CGGAATTCCT CTGTGGTCCA TGCCTTGC 28
    (2) INFORMATION FOR SEQ ID NO: 18:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 38 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
    GCGCCATGAA CTTGGTGGAG TCTTCTTGCT CCGCCTGA 38
    (2) INFORMATION FOR SEQ ID NO: 19:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 38 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
    AGCTGCCTCC TCCCTCTGTC CCACTCCTGT CTGCAAGA 38
    (2) INFORMATION FOR SEQ ID NO: 20:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 30 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
    CTTGGGTGTG TAGAAGAAGC CACGTTCCCC 30
    (2) INFORMATION FOR SEQ ID NO: 21:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 2464 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (ix) FEATURE:
    (A) NAME/KEY: Coding Sequence
    (B) LOCATION: 2...2455
    (D) OTHER INFORMATION:
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
    T CAC ACG TCT GTG CTG ACC TAC CCT CCC GGG CCC CGG ACC CAG CTC CAC 49
    His Thr Ser Val Leu Thr Tyr Pro Pro Gly Pro Arg Thr Gln Leu His
    1 5 10 15
    GAG GAC CTC CTG CCA CGG ACC CTC GGC CAG CTC CAG CCA GAT GAG CTC 97
    Glu Asp Leu Leu Pro Arg Thr Leu Gly Gln Leu Gln Pro Asp Glu Leu
    20 25 30
    AGC CCT AAG GTG GAC AGT GGT GTG GAC AGA CAC CAT CTG ATG GCG GCC 145
    Ser Pro Lys Val Asp Ser Gly Val Asp Arg His His Leu Met Ala Ala
    35 40 45
    CTC AGT GCC TAT GCT GCC CAG AGG CCC CCA GCT CCC CCC GGG GAG GGC 193
    Leu Ser Ala Tyr Ala Ala Gln Arg Pro Pro Ala Pro Pro Gly Glu Gly
    50 55 60
    AGC CTG GAG CCA CAG TAC CTT CTG CGT GCA CCC TCA AGA ATG CCC AGG 241
    Ser Leu Glu Pro Gln Tyr Leu Leu Arg Ala Pro Ser Arg Met Pro Arg
    65 70 75 80
    CCT TTG CTG GCA CCA GCC GCC CCC CAG AAG TGG CCT TCA CCT CTG GGA 289
    Pro Leu Leu Ala Pro Ala Ala Pro Gln Lys Trp Pro Ser Pro Leu Gly
    85 90 95
    GAT TCC GAA GAC CCC TCT AGC ACA GGC GAT GGA GCA CGG ATT CAT ACC 337
    Asp Ser Glu Asp Pro Ser Ser Thr Gly Asp Gly Ala Arg Ile His Thr
    100 105 110
    CTC CTG AAG GAC CTG CAG AGG CAG CCG GCT GAG GTG AGG GGC CTG AGT 385
    Leu Leu Lys Asp Leu Gln Arg Gln Pro Ala Glu Val Arg Gly Leu Ser
    115 120 125
    GGC CTG GAG CTG GAC GGC ATG GCT GAG CTG ATG GCT GGC CTG ATG CAA 433
    Gly Leu Glu Leu Asp Gly Met Ala Glu Leu Met Ala Gly Leu Met Gln
    130 135 140
    GGC GTG GAC CAT GGA GTA GCT CGA GGC AGC CCT GGG AGA GCG GCC CTG 481
    Gly Val Asp His Gly Val Ala Arg Gly Ser Pro Gly Arg Ala Ala Leu
    145 150 155 160
    GGA GAG TCT GGA GAA CAG GCG GAT GGC CCC AAG GCC ACC CTC CGT GGA 529
    Gly Glu Ser Gly Glu Gln Ala Asp Gly Pro Lys Ala Thr Leu Arg Gly
    165 170 175
    GAC AGC TTT CCA GAT GAC GGA GTG CAG GAC GAC GAT GAT AGA CTT TAC 577
    Asp Ser Phe Pro Asp Asp Gly Val Gln Asp Asp Asp Asp Arg Leu Tyr
    180 185 190
    CAA GAG GTC CAT CGT CTG AGT GCC ACA CTC GGG GGC CTC CTG CAG GAC 625
    Gln Glu Val His Arg Leu Ser Ala Thr Leu Gly Gly Leu Leu Gln Asp
    195 200 205
    CAC GGG TCT CGA CTC TTA CCT GGA GCC CTC CCC TTT GCA AGG CCC CTC 673
    His Gly Ser Arg Leu Leu Pro Gly Ala Leu Pro Phe Ala Arg Pro Leu
    210 215 220
    GAC ATG GAG AGG AAG AAG TCC GAG CAC CCT GAG TCT TCC CTG TCT TCA 721
    Asp Met Glu Arg Lys Lys Ser Glu His Pro Glu Ser Ser Leu Ser Ser
    225 230 235 240
    GAA GAG GAG ACT GCC GGA GTG GAG AAC GTC AAG AGC CAG ACG TAT TCC 769
    Glu Glu Glu Thr Ala Gly Val Glu Asn Val Lys Ser Gln Thr Tyr Ser
    245 250 255
    AAA GAT CTG CTG GGG CGG CAG CCG CAT TCG GAG CCC GGG GCC GCT GCG 817
    Lys Asp Leu Leu Gly Arg Gln Pro His Ser Glu Pro Gly Ala Ala Ala
    260 265 270
    TTT GGG GAG CTC CAA AAC CAG ATG CCT GGG CCC TCG AAG GAG GAG CAG 865
    Phe Gly Glu Leu Gln Asn Gln Met Pro Gly Pro Ser Lys Glu Glu Gln
    275 280 285
    AGC CTT CCA GCG GGT GCT CAG GAG GCC CTC AGC GAC GGC CTG CAA TTG 913
    Ser Leu Pro Ala Gly Ala Gln Glu Ala Leu Ser Asp Gly Leu Gln Leu
    290 295 300
    GAG GTC CAG CCT TCC GAG GAA GAG GCG CGG GGC TAC ATC GTG ACA GAC 961
    Glu Val Gln Pro Ser Glu Glu Glu Ala Arg Gly Tyr Ile Val Thr Asp
    305 310 315 320
    GGA GAC CCC CTG CGC CCC GAG GAA GGA AGG CGG CTG GTG GAG GAC GTC 1009
    Gly Asp Pro Leu Arg Pro Glu Glu Gly Arg Arg Leu Val Glu Asp Val
    325 330 335
    GCC CGC CTC CTG CAG GTG CCC AGC AGC GCG TTC GCT GAC GTG GAG GTT 1057
    Ala Arg Leu Leu Gln Val Pro Ser Ser Ala Phe Ala Asp Val Glu Val
    340 345 350
    CTC GGA CCA GCA GTG ACC TTC AAA GTG AGC GCC AAT GTC CAA AAC GTG 1105
    Leu Gly Pro Ala Val Thr Phe Lys Val Ser Ala Asn Val Gln Asn Val
    355 360 365
    ACC ACT GAG GAT GTG GAG AAG GCC ACA GTT GAC AAC AAA GAC AAA CTG 1153
    Thr Thr Glu Asp Val Glu Lys Ala Thr Val Asp Asn Lys Asp Lys Leu
    370 375 380
    GAG GAA ACC TCT GGA CTG AAA ATT CTT CAA ACC GGA GTC GGG TCG AAA 1201
    Glu Glu Thr Ser Gly Leu Lys Ile Leu Gln Thr Gly Val Gly Ser Lys
    385 390 395 400
    AGC AAA CTC AAG TTC CTG CCT CCT CAG GCG GAG CAA GAA GAC TCC ACC 1249
    Ser Lys Leu Lys Phe Leu Pro Pro Gln Ala Glu Gln Glu Asp Ser Thr
    405 410 415
    AAG TTC ATC GCG CTC ACC CTG GTC TCC CTC GCC TGC ATC CTG GGC GTC 1297
    Lys Phe Ile Ala Leu Thr Leu Val Ser Leu Ala Cys Ile Leu Gly Val
    420 425 430
    CTC CTG GCC TCT GGC CTC ATC TAC TGC CTC CGC CAT AGC TCT CAG CAC 1345
    Leu Leu Ala Ser Gly Leu Ile Tyr Cys Leu Arg His Ser Ser Gln His
    435 440 445
    AGG CTG AAG GAG AAG CTC TCG GGA CTA GGG GGC GAC CCA GGT GCA GAT 1393
    Arg Leu Lys Glu Lys Leu Ser Gly Leu Gly Gly Asp Pro Gly Ala Asp
    450 455 460
    GCC ACT GCC GCC TAC CAG GAG CTG TGC CGC CAG CGT ATG GCC ACG CGG 1441
    Ala Thr Ala Ala Tyr Gln Glu Leu Cys Arg Gln Arg Met Ala Thr Arg
    465 470 475 480
    CCA CCA GAC CGA CCT GAG GGC CCG CAC ACG TCA CGC ATC AGC AGC GTC 1489
    Pro Pro Asp Arg Pro Glu Gly Pro His Thr Ser Arg Ile Ser Ser Val
    485 490 495
    TCA TCC CAG TTC AGC GAC GGG CCG ATC CCC AGC CCC TCC GCA CGC AGC 1537
    Ser Ser Gln Phe Ser Asp Gly Pro Ile Pro Ser Pro Ser Ala Arg Ser
    500 505 510
    AGC GCC TCA TCC TGG TCC GAG GAG CCT GTG CAG TCC AAC ATG GAC ATC 1585
    Ser Ala Ser Ser Trp Ser Glu Glu Pro Val Gln Ser Asn Met Asp Ile
    515 520 525
    TCC ACC GGC CAC ATG ATC CTG TCC TAC ATG GAG GAC CAC CTG AAG AAC 1633
    Ser Thr Gly His Met Ile Leu Ser Tyr Met Glu Asp His Leu Lys Asn
    530 535 540
    AAG AAC CGG CTG GAG AAG GAG TGG GAA GCG CTG TGC GCC TAC CAG GCG 1681
    Lys Asn Arg Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala Tyr Gln Ala
    545 550 555 560
    GAG CCC AAC AGC TCG TTC GTG GCC CAG AGG GAG GAG AAC GTG CCC AAG 1729
    Glu Pro Asn Ser Ser Phe Val Ala Gln Arg Glu Glu Asn Val Pro Lys
    565 570 575
    AAC CGC TCC CTG GCC GTG CTG ACC TAT GAC CAC TCC CGG GTC CTG CTG 1777
    Asn Arg Ser Leu Ala Val Leu Thr Tyr Asp His Ser Arg Val Leu Leu
    580 585 590
    AAG GCG GAG AAC AGC CAC AGC CAC TCA GAC TAC ATC AAC GCT AGC CCC 1825
    Lys Ala Glu Asn Ser His Ser His Ser Asp Tyr Ile Asn Ala Ser Pro
    595 600 605
    ATC ATG GAT CAC GAC CCG AGG AAC CCC GCG TAC ATC GCC ACC CAG GGA 1873
    Ile Met Asp His Asp Pro Arg Asn Pro Ala Tyr Ile Ala Thr Gln Gly
    610 615 620
    CCG CTG CCC GCC ACC GTG GCT GAC TTT TGG CAG ATG GTG TGG GAG AGC 1921
    Pro Leu Pro Ala Thr Val Ala Asp Phe Trp Gln Met Val Trp Glu Ser
    625 630 635 640
    GGC TGC GTG GTG ATC GTC ATG CTG ACA CCC CTC GCG GAG AAC GGC GTC 1969
    Gly Cys Val Val Ile Val Met Leu Thr Pro Leu Ala Glu Asn Gly Val
    645 650 655
    CGG CAG TGC TAC CAC TAC TGG CCG GAT GAA GGC TCC AAT CTC TAC CAC 2017
    Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu Gly Ser Asn Leu Tyr His
    660 665 670
    ATC TAT GAG GTG AAC CTG GTC TCC GAG CAC ATC TGG TGT GAG GAC TTC 2065
    Ile Tyr Glu Val Asn Leu Val Ser Glu His Ile Trp Cys Glu Asp Phe
    675 680 685
    CTG GTG AGG AGC TTC TAT CTG AAG AAC CTG CAG ACC AAC GAG ACG CGC 2113
    Leu Val Arg Ser Phe Tyr Leu Lys Asn Leu Gln Thr Asn Glu Thr Arg
    690 695 700
    ACC GTG ACG CAG TTC CAC TTC CTG AGT TGG TAT GAC CGA GGA GTC CCT 2161
    Thr Val Thr Gln Phe His Phe Leu Ser Trp Tyr Asp Arg Gly Val Pro
    705 710 715 720
    TCC TCC TCA AGG TCC CTC CTG GAC TTC CGC AGA AAA GTA AAC AAG TGC 2209
    Ser Ser Ser Arg Ser Leu Leu Asp Phe Arg Arg Lys Val Asn Lys Cys
    725 730 735
    TAC AGG GGC CGT TCT TGT CCA ATA ATT GTT CAT TGC AGT GAC GGT GCA 2257
    Tyr Arg Gly Arg Ser Cys Pro Ile Ile Val His Cys Ser Asp Gly Ala
    740 745 750
    GGC CGG AGC GGC ACC TAC GTC CTG ATC GAC ATG GTT CTC AAC AAG ATG 2305
    Gly Arg Ser Gly Thr Tyr Val Leu Ile Asp Met Val Leu Asn Lys Met
    755 760 765
    GCC AAA GGT GCT AAA GAG ATT GAT ATC GCA GCG ACC CTG GAG CAC TTG 2353
    Ala Lys Gly Ala Lys Glu Ile Asp Ile Ala Ala Thr Leu Glu His Leu
    770 775 780
    AGG GAC CAG AGA CCC GGC ATG GTC CAG ACG AAG GAG CAG TTT GAG TTC 2401
    Arg Asp Gln Arg Pro Gly Met Val Gln Thr Lys Glu Gln Phe Glu Phe
    785 790 795 800
    GCG CTG ACA GCC GTG GCT GAG GAG GTG AAC GCC ATC CTC AAG GCC CTG 2449
    Ala Leu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys Ala Leu
    805 810 815
    CCC CAG TGAGAATTC 2464
    Pro Gln
    (2) INFORMATION FOR SEQ ID NO: 22:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 818 amino acids
    (B) TYPE: amino acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (v) FRAGMENT TYPE: internal
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
    His Thr Ser Val Leu Thr Tyr Pro Pro Gly Pro Arg Thr Gln Leu His
    1 5 10 15
    Glu Asp Leu Leu Pro Arg Thr Leu Gly Gln Leu Gln Pro Asp Glu Leu
    20 25 30
    Ser Pro Lys Val Asp Ser Gly Val Asp Arg His His Leu Met Ala Ala
    35 40 45
    Leu Ser Ala Tyr Ala Ala Gln Arg Pro Pro Ala Pro Pro Gly Glu Gly
    50 55 60
    Ser Leu Glu Pro Gln Tyr Leu Leu Arg Ala Pro Ser Arg Met Pro Arg
    65 70 75 80
    Pro Leu Leu Ala Pro Ala Ala Pro Gln Lys Trp Pro Ser Pro Leu Gly
    85 90 95
    Asp Ser Glu Asp Pro Ser Ser Thr Gly Asp Gly Ala Arg Ile His Thr
    100 105 110
    Leu Leu Lys Asp Leu Gln Arg Gln Pro Ala Glu Val Arg Gly Leu Ser
    115 120 125
    Gly Leu Glu Leu Asp Gly Met Ala Glu Leu Met Ala Gly Leu Met Gln
    130 135 140
    Gly Val Asp His Gly Val Ala Arg Gly Ser Pro Gly Arg Ala Ala Leu
    145 150 155 160
    Gly Glu Ser Gly Glu Gln Ala Asp Gly Pro Lys Ala Thr Leu Arg Gly
    165 170 175
    Asp Ser Phe Pro Asp Asp Gly Val Gln Asp Asp Asp Asp Arg Leu Tyr
    180 185 190
    Gln Glu Val His Arg Leu Ser Ala Thr Leu Gly Gly Leu Leu Gln Asp
    195 200 205
    His Gly Ser Arg Leu Leu Pro Gly Ala Leu Pro Phe Ala Arg Pro Leu
    210 215 220
    Asp Met Glu Arg Lys Lys Ser Glu His Pro Glu Ser Ser Leu Ser Ser
    225 230 235 240
    Glu Glu Glu Thr Ala Gly Val Glu Asn Val Lys Ser Gln Thr Tyr Ser
    245 250 255
    Lys Asp Leu Leu Gly Arg Gln Pro His Ser Glu Pro Gly Ala Ala Ala
    260 265 270
    Phe Gly Glu Leu Gln Asn Gln Met Pro Gly Pro Ser Lys Glu Glu Gln
    275 280 285
    Ser Leu Pro Ala Gly Ala Gln Glu Ala Leu Ser Asp Gly Leu Gln Leu
    290 295 300
    Glu Val Gln Pro Ser Glu Glu Glu Ala Arg Gly Tyr Ile Val Thr Asp
    305 310 315 320
    Gly Asp Pro Leu Arg Pro Glu Glu Gly Arg Arg Leu Val Glu Asp Val
    325 330 335
    Ala Arg Leu Leu Gln Val Pro Ser Ser Ala Phe Ala Asp Val Glu Val
    340 345 350
    Leu Gly Pro Ala Val Thr Phe Lys Val Ser Ala Asn Val Gln Asn Val
    355 360 365
    Thr Thr Glu Asp Val Glu Lys Ala Thr Val Asp Asn Lys Asp Lys Leu
    370 375 380
    Glu Glu Thr Ser Gly Leu Lys Ile Leu Gln Thr Gly Val Gly Ser Lys
    385 390 395 400
    Ser Lys Leu Lys Phe Leu Pro Pro Gln Ala Glu Gln Glu Asp Ser Thr
    405 410 415
    Lys Phe Ile Ala Leu Thr Leu Val Ser Leu Ala Cys Ile Leu Gly Val
    420 425 430
    Leu Leu Ala Ser Gly Leu Ile Tyr Cys Leu Arg His Ser Ser Gln His
    435 440 445
    Arg Leu Lys Glu Lys Leu Ser Gly Leu Gly Gly Asp Pro Gly Ala Asp
    450 455 460
    Ala Thr Ala Ala Tyr Gln Glu Leu Cys Arg Gln Arg Met Ala Thr Arg
    465 470 475 480
    Pro Pro Asp Arg Pro Glu Gly Pro His Thr Ser Arg Ile Ser Ser Val
    485 490 495
    Ser Ser Gln Phe Ser Asp Gly Pro Ile Pro Ser Pro Ser Ala Arg Ser
    500 505 510
    Ser Ala Ser Ser Trp Ser Glu Glu Pro Val Gln Ser Asn Met Asp Ile
    515 520 525
    Ser Thr Gly His Met Ile Leu Ser Tyr Met Glu Asp His Leu Lys Asn
    530 535 540
    Lys Asn Arg Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala Tyr Gln Ala
    545 550 555 560
    Glu Pro Asn Ser Ser Phe Val Ala Gln Arg Glu Glu Asn Val Pro Lys
    565 570 575
    Asn Arg Ser Leu Ala Val Leu Thr Tyr Asp His Ser Arg Val Leu Leu
    580 585 590
    Lys Ala Glu Asn Ser His Ser His Ser Asp Tyr Ile Asn Ala Ser Pro
    595 600 605
    Ile Met Asp His Asp Pro Arg Asn Pro Ala Tyr Ile Ala Thr Gln Gly
    610 615 620
    Pro Leu Pro Ala Thr Val Ala Asp Phe Trp Gln Met Val Trp Glu Ser
    625 630 635 640
    Gly Cys Val Val Ile Val Met Leu Thr Pro Leu Ala Glu Asn Gly Val
    645 650 655
    Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu Gly Ser Asn Leu Tyr His
    660 665 670
    Ile Tyr Glu Val Asn Leu Val Ser Glu His Ile Trp Cys Glu Asp Phe
    675 680 685
    Leu Val Arg Ser Phe Tyr Leu Lys Asn Leu Gln Thr Asn Glu Thr Arg
    690 695 700
    Thr Val Thr Gln Phe His Phe Leu Ser Trp Tyr Asp Arg Gly Val Pro
    705 710 715 720
    Ser Ser Ser Arg Ser Leu Leu Asp Phe Arg Arg Lys Val Asn Lys Cys
    725 730 735
    Tyr Arg Gly Arg Ser Cys Pro Ile Ile Val His Cys Ser Asp Gly Ala
    740 745 750
    Gly Arg Ser Gly Thr Tyr Val Leu Ile Asp Met Val Leu Asn Lys Met
    755 760 765
    Ala Lys Gly Ala Lys Glu Ile Asp Ile Ala Ala Thr Leu Glu His Leu
    770 775 780
    Arg Asp Gln Arg Pro Gly Met Val Gln Thr Lys Glu Gln Phe Glu Phe
    785 790 795 800
    Ala Leu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys Ala Leu
    805 810 815
    Pro Gln
    (2) INFORMATION FOR SEQ ID NO: 23:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 2736 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
    CAGGACAGAC CCCGTGCTGA GGGTGACGAC CGCTTCTCCA AGAGCATCCT GACCTATGTG 60
    GCCCACACGT CTGTGCTGAC CTACCCTCCC GGGCCCCAGG CCCAGCTCCC CGAGGACCTC 120
    CTGCCACGGA CCCTCAGCCA GCTCCAGCCA GACGAGCTCA GCCCTAAGGT GGACAGCAGT 180
    GTGGAGAGAC ACCATCTGAT GGCAGCCCTC AGTGCCTATG CTGCCCAGAG GCCCCCAGCT 240
    CCCCCTGGGA AGGGCAGCCT GGAGCCGCAG TACCTTCTGC GCGCCCCGTC CAGAATGCCC 300
    AGGCCCTTGT TGTCGCCAGC CGTCCCCCAG AAGTGGCCTT CACCTCTGGG AGATCCTGAA 360
    GACCCCCCCA GCACAGGGGA AGGAGCACGG ATTCACACTC TCCTGAAGGA CCTGCAGAGG 420
    CAGCCGGCTG AGGCGAGGGG CCTGAGTGAC CTGGAGCTGG ACAGCATGGC CGAGCTGATG 480
    GCTGGCCTGA TGCAAGGCAT GGACCACAGA GGAGCTCTAG GCGGCCCTGG GAAAGCGGCC 540
    CTGGGAGAGT CTGGAGAACA GGCGGATGGC CCCAAGGCCG CCCTCCGTGG GGAAAGCTTT 600
    CCAGATGACG GAGTTCAGGA CGACGATGAC AGACTTTACC AAGAGGTCCA TCGTCTGAGT 660
    GCCACACTCG GGGGCCTCCT GCAGGACCAC GGGTCTCGAC TCTCGCCTGG AGCCCTCCCC 720
    TTTGCAAAGC CCCTCAAAAT GGAGAGGAAG AAATCCGAGC GCCCTGAGGC TTCCCTGTCT 780
    TCAGAAGAGG AGACTGCCGG AGTGGAGAAC GTCAAGAGCC AGACGTATTC CAAAACCTGC 840
    TGGGGCAGCA GCCGCATTCG GAGCCCGGGG CAGGCGCGTT TGGGGAGCTC CAAACCAGAT 900
    GCCTGGGCCC TCGGAGGAGG AGCAGAGCCT TCCAGCGGGT GCTCAGGAGG CCCTCGGCGA 960
    CGGCTGCAAT TGGAAGTCAA GCCTTCCGAG GAAGAGGCAC GGTGCTACAT CGTGACAGAC 1020
    AGAGACCCCC TGCGCCCCGA GGAAGGAAGG CAGCTGGTGG AGGACGTCGC CCGCCTCCTG 1080
    CAGATGCCCA GCAGCACATT CGCCGACGTG GAGGTTCTCG GACCAGCAGT GACCTTCAAA 1140
    GTGGGCGCCA ATGTCCAGAA CGTGACCACT GCGGATGTGG AGAAGGCCAC AGTTGACAAC 1200
    AAAGACAAAC TGGAGGAAAC CTCTGGACTG AAAATTCTTC AAACCGGAGT CGGGTCGAAA 1260
    AGCAAACTCA AGTTCCTGCC TCCTCAGGCG GAGCAAGAAG ACTCAACCAA GTTCATCGCG 1320
    CTCACCCTGG TCTCCCTCGC CTGCATCCTG GGCGTCCTCC TGGCCTCTGG CCTCATCTAC 1380
    TGCCTACGCC ATAGCTCTCA GCACAGGCTG AAGGAGAAGC TCTCGGGACT AGGGCGCGAC 1440
    CCAGGTGCAG ATGCCACCGC CGCCTACCAG GAGCTGTGCC GCCAGCGTAT GGCCACGCGG 1500
    CCACCAGACC GGCCCGAGGG CCCGCACACA TCCCGCATCA GCAGCGTCTC GTCCCAGTTC 1560
    AGCGACGGGC CGATGCCCAG CCCCTCCGCA CGCAGCAGCG CCTCGTCCTG GTCCGAGGAG 1620
    CCCGTGCAGT CCAACATGGA CATCTCCACC GGCCACATGA TCCTGTCCTA CATGGAGGAC 1680
    CACCTGAAGA ACAAGAACCG GCTGGAGAAG GAGTGGGAGG CGCTGTGTGC CTACCAGGCG 1740
    GAGCCCAACA GCTCACTTGT GGCCCAGAAG GAGGAGAATG TGCCCAAGAA CCGCTCCCTG 1800
    GCCGTGCTGA CCTATGACCA CTCCCGGGTC CTACTGAAGG CGGAGAACAG CCACAGCCAC 1860
    TCGGACTACA TCAACGCCAG CCCCATCATG GATCACGACC CGAGGAACCC CGCGTACATC 1920
    GCCACCCAGG GACCGCTGCC CGCCACCGTG GCCGACTTTT GGCAGATGGT GTGGGAGAGC 1980
    GGCTGCGTGG TGATCGTCAT GCTGACACCC CTCACAGAGA ACGGCGTCCG GCAGTGCTAC 2040
    CACTACTGGC CAGATGAAGG CTCCAACCTC TACCACATCT ATGAGGTGAA CCTGGTCTCC 2100
    GAGCACATCT GGTGCGAGGA CTTTCTGGTG AGGAGCTTCT ATCTGAAGAA CCTGCAGACC 2160
    AACGAGACGC GCACCGTGAC CCAGTTCCAC TTCCTGAGTT GGTATGACCG AGGAGTCCCC 2220
    TCCTCCTCAA GATCCCTCCT GGACTTCCGC AGAAAAGTAA ACAAGTGCTA CAGGGGCCGT 2280
    TCTTGTCCAA TAATTGTTCA TTGCAGTGAC GGTGCAGGCC GGAGCGGCAC CTACGTCCTG 2340
    ATCGACATGG TTCTCAACAA GATGGCCAAA GGTGCTAAAG AGATTGATAT CGCAGCAACC 2400
    CTGGAGCACT TGAGGGACCA GAGACCCGGC ATGGTCCAGA CGAAGGAGCA GTTTGAGTTC 2460
    GCGCTGACAG CCGTGGCTGA AGAGGTGAAT GCCATCCTCA AGGCCCTTCC CCAGTGAGCA 2520
    GCGGCCTCGG GGCCTCGGGG GAGCCCCCAC CCCCCGGATG TCGTCAGGAA TCGTGATCTG 2580
    ACTTTAATTG TGTGTCTTCT ATTATAACTG CATAGTAATA GGGCCCTTAG CTCTCCCGTA 2640
    GTCAGCGCAG TTTAGCAGTT AAGCAGTTAA AATGTGTATT TTTGTTTAAT CCAACAATAA 2700
    TAAAGAGAGA TTTGTGGAAA AATCCCAAAA AAAAAA 2736
    (2) INFORMATION FOR SEQ ID NO: 24:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 738 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
    ACGTGGCAGG ATGACTATAC TCAGTATGTG ATGGACCAGG AACTTGCAGA CCTCCCCAAA 60
    ACCTACCTGA GGCATCCTGA AGCGTCCGGC CCAGCCAGGC CCTCAAAACA CAGCATTGGC 120
    AGTGAGAGGA GGTACAGTCG GGAGGGCGGC GCTGCCCTGG CCAAGGCCTT CCGACGCCAC 180
    CTGCCCTTCC TGGAGGCCCT GTCCCAGGCC CCAGCTTCAG ACGCGCTCGC CAGGACCCGG 240
    ATGGCGCAGG ACAGACCCCG TGCTGAGGGT GACGACCGCT TCTCCAAGAG CATCCTGACC 300
    TATGTGGCCC ACACGTCTGT GCTGACCTAC CCTCCCGGGC CCCAGGCCCA GCTCCCCGAG 360
    GACCTCCTGC CACGGACCCT CAGCCAGCTC CAGCCAGACG AGCTCAGCCC TAAGGTGGAC 420
    AGCAGTGTGG AGAGACACCA TCTGATGGCA GCCCTCAGTG CCTATGCTGC CCAGAGGCCC 480
    CCAGCTCCCC CTGGGAAGGG CAGCCTGGAG CCGCAGTACC TTCTGCGCGC CCCGTCCAGA 540
    ATGCCCAGGC CCTTGTTGTC GCCAGCCGTC CCCCAGAAGT GGCCTTCACC TCTGGGAGAT 600
    CCTGAAGACC CCCCCAGCAC AGGGGAAGGA GCACGGATTC ACACTCTCCT GAAGGACCTG 660
    CAGAGGCAGC CGGCTGAGGC GAGGGGCCTG AGTGACCTGG AGCTGGACAG CATGGCCGAG 720
    CTGATGGCTG GCCTGATG 738
    (2) INFORMATION FOR SEQ ID NO: 25:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 932 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
    GCCGGCAGCT CCCGGGGCGC CTGGGCTGCC TACTCGAGGA GGGCCTCTGC GGAGCGTCCG 60
    AGGCCTGTGT GAACGATGGA GTGTTTGGAA GGTGCCAGAA GGTTCCGGCA ATGGACTTTT 120
    ACCGCTACGA GGTGTCGCCC GTGGCCCTGC AGCGCCTGCG CGTGGCTTTG CAGAAACTCT 180
    CCGGCACAGG TTTCACGTGG CAGGATGACT ATACTCAGTA TGTGATGGAC CAGGAACTTG 240
    CAGACCTCCC CAAAACCTAC CTGAGGCATC CTGAAGCGTC CGGCCCAGCC AGGCCCTCAA 300
    AACACAGCAT TGGCAGTGAG AGGAGGTACA GTCGGGAGGG CGGCGCTGCC CTGGCCAAGG 360
    CCTTCCGACG CCACCTGCCC TTCCTGGAGG CCCTGTCCCA GGCCCCAGCT TCAGACGCGC 420
    TCGCCAGGAC CCGGATGGCG CAGGACAGAC CCCGTGCTGA GGGTGACGAC CGCTTCTCCA 480
    AGAGCATCCT GACCTATGTG GCCCACACGT CTGTGCTGAC CTACCCTCCC GGGCCCCAGG 540
    CCCAGCTCCC CGAGGACCTC CTGCCACGGA CCCTCAGCCA GCTCCAGCCA GACGAGCTCA 600
    GCCCTAAGGT GGACAGCAGT GTGGAGAGAC ACCATCTGAT GGCAGCCCTC AGTGCCTATG 660
    CTGCCCAGAG GCCCCCAGCT CCCCCTGGGA AGGGCAGCCT GGAGCCGCAG TACCTTCTGC 720
    GCGCCCCGTC CAGAATGCCC AGGCCCTTGT TGTCGCCAGC CGTCCCCCAG AAGTGGCCTT 780
    CACCTCTGGG AGATCCTGAA GACCCCCCCA GCACAGGGGA AGGAGCACGG ATTCACACTC 840
    TCCTGAAGGA CCTGCAGAGG CAGCCGGCTG AGGCGAGGGG CCTGAGTGAC CTGGAGCTGG 900
    ACAGCATGGC CGAGCTGATG GCTGGCCTGA TG 932
    (2) INFORMATION FOR SEQ ID NO: 26:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 999 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
    CTGTTGCTGC TACTGCTGCT GCTGCCGCCA CGCGTCCTGC CTGCCGCCCC CTCGTCCGTC 60
    CCCCACGGCC GGCAGCTCCC GGGGCGCCTG GGCTGCCTAC TCGAGGAGGG CCTCTGCGGA 120
    GCGTCCGAGG CCTGTGTGAA CGATGGAGTG TTTGGAAGGT GCCAGAAGGT TCCGGCAATG 180
    GACTTTTACC GCTACGAGGT GTCGCCCGTG GCCCTGCAGC GCCTGCGCGT GGCTTTGCAG 240
    AAACTCTCCG GCACAGGTTT CACGTGGCAG GATGACTATA CTCAGTATGT GATGGACCAG 300
    GAACTTGCAG ACCTCCCCAA AACCTACCTG AGGCATCCTG AAGCGTCCGG CCCAGCCAGG 360
    CCCTCAAAAC ACAGCATTGG CAGTGAGAGG AGGTACAGTC GGGAGGGCGG CGCTGCCCTG 420
    GCCAAGGCCT TCCGACGCCA CCTGCCCTTC CTGGAGGCCC TGTCCCAGGC CCCAGCTTCA 480
    GACGCGCTCG CCAGGACCCG GATGGCGCAG GACAGACCCC GTGCTGAGGG TGACGACCGC 540
    TTCTCCAAGA GCATCCTGAC CTATGTGGCC CACACGTCTG TGCTGACCTA CCCTCCCGGG 600
    CCCCAGGCCC AGCTCCCCGA GGACCTCCTG CCACGGACCC TCAGCCAGCT CCAGCCAGAC 660
    GAGCTCAGCC CTAAGGTGGA CAGCAGTGTG GAGAGACACC ATCTGATGGC AGCCCTCAGT 720
    GCCTATGCTG CCCAGAGGCC CCCAGCTCCC CCTGGGAAGG GCAGCCTGGA GCCGCAGTAC 780
    CTTCTGCGCG CCCCGTCCAG AATGCCCAGG CCCTTGTTGT CGCCAGCCGT CCCCCAGAAG 840
    TGGCCTTCAC CTCTGGGAGA TCCTGAAGAC CCCCCCAGCA CAGGGGAAGG AGCACGGATT 900
    CACACTCTCC TGAAGGACCT GCAGAGGCAG CCGGCTGAGG CGAGGGGCCT GAGTGACCTG 960
    GAGCTGGACA GCATGGCCGA GCTGATGGCT GGCCTGATG 999
    (2) INFORMATION FOR SEQ ID NO: 27:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 1011 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
    GCGCTCCCGC TGCTGTTGCT GCTACTGCTG CTGCTGCCGC CACGCGTCCT GCCTGCCGCC 60
    CCCTCGTCCG TCCCCCACGG CCGGCAGCTC CCGGGGCGCC TGGGCTGCCT ACTCGAGGAG 120
    GGCCTCTGCG GAGCGTCCGA GGCCTGTGTG AACGATGGAG TGTTTGGAAG GTGCCAGAAG 180
    GTTCCGGCAA TGGACTTTTA CCGCTACGAG GTGTCGCCCG TGGCCCTGCA GCGCCTGCGC 240
    GTGGCTTTGC AGAAACTCTC CGGCACAGGT TTCACGTGGC AGGATGACTA TACTCAGTAT 300
    GTGATGGACC AGGAACTTGC AGACCTCCCC AAAACCTACC TGAGGCATCC TGAAGCGTCC 360
    GGCCCAGCCA GGCCCTCAAA ACACAGCATT GGCAGTGAGA GGAGGTACAG TCGGGAGGGC 420
    GGCGCTGCCC TGGCCAAGGC CTTCCGACGC CACCTGCCCT TCCTGGAGGC CCTGTCCCAG 480
    GCCCCAGCTT CAGACGCGCT CGCCAGGACC CGGATGGCGC AGGACAGACC CCGTGCTGAG 540
    GGTGACGACC GCTTCTCCAA GAGCATCCTG ACCTATGTGG CCCACACGTC TGTGCTGACC 600
    TACCCTCCCG GGCCCCAGGC CCAGCTCCCC GAGGACCTCC TGCCACGGAC CCTCAGCCAG 660
    CTCCAGCCAG ACGAGCTCAG CCCTAAGGTG GACAGCAGTG TGGAGAGACA CCATCTGATG 720
    GCAGCCCTCA GTGCCTATGC TGCCCAGAGG CCCCCAGCTC CCCCTGGGAA GGGCAGCCTG 780
    GAGCCGCAGT ACCTTCTGCG CGCCCCGTCC AGAATGCCCA GGCCCTTGTT GTCGCCAGCC 840
    GTCCCCCAGA AGTGGCCTTC ACCTCTGGGA GATCCTGAAG ACCCCCCCAG CACAGGGGAA 900
    GGAGCACGGA TTCACACTCT CCTGAAGGAC CTGCAGAGGC AGCCGGCTGA GGCGAGGGGC 960
    CTGAGTGACC TGGAGCTGGA CAGCATGGCC GAGCTGATGG CTGGCCTGAT G 1011
    (2) INFORMATION FOR SEQ ID NO: 28:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 28 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (vii) IMMEDIATE SOURCE:
    (B) CLONE: ZC11654
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
    CGGAATTCCT CTGTGGTCCA TGCCTTGC 28
    (2) INFORMATION FOR SEQ ID NO: 29:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 30 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (vii) IMMEDIATE SOURCE:
    (B) CLONE: ZC11197
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
    AAATTAATAC GACTCACTAT AGGGAGACCG 30
    (2) INFORMATION FOR SEQ ID NO: 30:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 1210 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
    AGTGCTGCTC ACTCTGGTGG CCCTGGCAGG TGTGGCTGGG CTGCTGGTGG CTCTGGCTGT 60
    GGCTCTGTGT GTGCGGCAGC ATGCGCGGCA GCAAGACAAG GAGCGCCTGG CAGCCCTGGG 120
    GCCTGAGGGG GCCCATGGTG ACACTACCTT TGAGTACCAG GACCTGTGCC GCCAGCACAT 180
    GGCCACGAAG TCCTTGTTCA ACCGGGCAGA GGGTCCACCG GAGCCTTCAC GGGTGAGCAG 240
    TGTGTCCTCC CAGTTCAGCG ACGCAGCCCA GGCCAGCCCC AGCTCCCACA GCAGCACCCC 300
    GTCCTGGTGC GAGGAGCCGG CCCAAGCCAA CATGGACATC TCCACGGGAC ACATGATTCT 360
    GGCATACATG GAGGATCACC TGCGGAACCG GGACCGCCTT GCCAAGGAGT GGCAGGCCCT 420
    CTGTGCCTAC CAAGCAGAGC CAAACACCTG TGCCACCGCG CAGGGGGAGG GCAACATCAA 480
    AAAGAACCGG CATCCTGACT TCCTGCCCTA TGACCATGCC CGCATAAAAC TGAAGGTGGA 540
    GAGCAGCCCT TCTCGGAGCG ATTACATCAA CGCCAGCCCC ATTATTGAGC ATGACCCTCG 600
    GATGCCAGCC TACATAGCCA CGCAGGGCCC GCTGTCCCAT ACCATCGCAG ACTTCTGGCA 660
    GATGGTGTGG GAGAGCGGCT GCACCGTCAT CGTCATGCTG ACCCCGCTGG TGGAGGATGG 720
    TGTCAAGCAG TGTGACCGCT ACTGGCCAGA TGAGGGTGCC TCCCTCTACC ACGTATATGA 780
    GGTGAACCTG GTGTCGGAGC ACATCTGGTG CGAGGACTTT CTGGTGCGGA GCTTCTACCT 840
    GAAGAACGTG CAGACCCAGG AGACGCGCAC GCTCACGCAG TTCCACTTCC TCAGCTGGCC 900
    GGCAGAGGGC ACACCGGCCT CCACGCGGCC CCTGCTGGAC TTCCGCAGGA AGGTGAACAA 960
    GTGCTACCGG GGCCGCTCCT GCCCCATCAT CGTGCACTGC AGTGATGGTG CGGGGAGGAC 1020
    CGGCACCTAC ATCCTCATCG ACATGGTCCT GAACCGCATG GCAAAAGGAG TGAAGGAGAT 1080
    TGACATCGCT GCCACCCTGG AGCATGTCCG TGACCAGCGG CCTGGCCTTG TCCGCTCTAA 1140
    GGACCAGTTT GAATTTGCCC TGACAGCCGT GGCGGAGGAA GTGAATGCCA TCCTCAAGGC 1200
    CCTGCCCCAG 1210
    (2) INFORMATION FOR SEQ ID NO: 31:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 1263 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
    TCACACGTCT GTGCTGACCT ACCCTCCCGG GCCCCGGACC CAGCTCCACG AGGACCTCCT 60
    GCCACGGACC CTCGGCCAGC TCCAGCCAGA TGAGCTCAGC CCTAAGGTGG ACAGTGGTGT 120
    GGACAGACAC CATCTGATGG CGGCCCTCAG TGCCTATGCT GCCCAGAGGC CCCCAGCTCC 180
    CCCCGGGGAG GGCAGCCTGG AGCCACAGTA CCTTCTGCGT GCACCCTCAA GAATGCCCAG 240
    GCCTTTGCTG GCACCAGCCG CCCCCCAGAA GTGGCCTTCA CCTCTGGGAG ATTCCGAAGA 300
    CCCCTCTAGC ACAGGCGATG GAGCACGGAT TCATACCCTC CTGAAGGACC TGCAGAGGCA 360
    GCCGGCTGAG GTGAGGGGCC TGAGTGGCCT GGAGCTGGAC GGCATGGCTG AGCTGATGGC 420
    TGGCCTGATG CAAGGCGTGG ACCATGGAGT AGCTCGAGGC AGCCCTGGGA GAGCGGCCCT 480
    GGGAGAGTCT GGAGAACAGG CGGATGGCCC CAAGGCCACC CTCCGTGGAG ACAGCTTTCC 540
    AGATGACGGA GTGCAGGACG ACGATGATAG ACTTTACCAA GAGGTCCATC GTCTGAGTGC 600
    CACACTCGGG GGCCTCCTGC AGGACCACGG GTCTCGACTC TTACCTGGAG CCCTCCCCTT 660
    TGCAAGGCCC CTCGACATGG AGAGGAAGAA GTCCGAGCAC CCTGAGTCTT CCCTGTCTTC 720
    AGAAGAGGAG ACTGCCGGAG TGGAGAACGT CAAGAGCCAG ACGTATTCCA AAGATCTGCT 780
    GGGGCGGCAG CCGCATTCGG AGCCCGGGGC CGCTGCGTTT GGGGAGCTCC AAAACCAGAT 840
    GCCTGGGCCC TCGAAGGAGG AGCAGAGCCT TCCAGCGGGT GCTCAGGAGG CCCTCAGCGA 900
    CGGCCTGCAA TTGGAGGTCC AGCCTTCCGA GGAAGAGGCG CGGGGCTACA TCGTGACAGA 960
    CGGAGACCCC CTGCGCCCCG AGGAAGGAAG GCGGCTGGTG GAGGACGTCG CCCGCCTCCT 1020
    GCAGGTGCCC AGCAGCGCGT TCGCTGACGT GGAGGTTCTC GGACCAGCAG TGACCTTCAA 1080
    AGTGAGCGCC AATGTCCAAA ACGTGACCAC TGAGGATGTG GAGAAGGCCA CAGTTGACAA 1140
    CAAAGACAAA CTGGAGGAAA CCTCTGGACT GAAAATTCTT CAAACCGGAG TCGGGTCGAA 1200
    AAGCAAACTC AAGTTCCTGC CTCCTCAGGC GGAGCAAGAA GACTCCACCA AGTTCATCGC 1260
    GCA 1263
    (2) INFORMATION FOR SEQ ID NO: 32:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 758 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
    GAATTCGGCT TAAGGCGACG GTGGACAACA AAGACAAACT GGAGGAAACC TCTGGACTGA 60
    AAATTCTTCA AACCGGAGTC GGGTCGAAAA GCAAACTCAA GTTCCTGCCT CCTCAGGCGG 120
    AGCAAGAAGA CTCCACCAAG TTCATCGCGC TCACCCTGGT CTCCCTCGCC TGCATCCTGG 180
    GCGTCCTCCT GGCCTCTGGC CTCATCTACT GCCTCCGCCA TAGCTCTCAG CACAGGCTGA 240
    AGGAGAAGCT CTCGGGACTA GGGGGCGACC CAGGTGCAGA TGCCACTGCC GCCTACCAGG 300
    AGCTGTGCCG CCAGCGTATG GCCACGCGGC CACCAGACCG ACCTGAGGGC CCGCACACGT 360
    CACGCATCAG CAGCGTCTCA TCCCAGTTCA GCGACGGGCC GATCCCCAGC CCCTCCGCAC 420
    GCAGCAGCGC CTCATCCTGG TCCGAGGAGC CTGTGCAGTC CAACATGGAC ATCTCCACCG 480
    GCCACATGAT CCTGTCCTAC ATGGAGGACC ACCTGAAGAA CAAGAACCGG CTGGAGAAGG 540
    AGTGGGAAGC GCTGTGCGCC TACCAGGCGG AGCCCAACAG CTCGTTCGTG GCCCAGAGGG 600
    AGGAGAACGT GCCCAAGAAC CGCTCCCTGG CCGTGCTGAC CTATGACCAC TCCCGGGTCC 660
    TGCTGAAGGC GGAGAACAGC CACAGCCACT CAGACTACAT CAACGCTAGC CCCATCATGG 720
    ATCACGACCC GAGGAACCCC GCGTACAAAG CCGAATTC 758
    (2) INFORMATION FOR SEQ ID NO: 33:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 1150 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
    AAGCTTCCAC CATGCGCCAT AGCTCTCAGC ACAGGCTGAA AGAGAAGCTC TCGGGACTAG 60
    GGGGCGACCC AGGTGCAGAT GCCACTGCCG CCTACCAGGA GCTGCGCCGC CAGCGTATGG 120
    CCACGCGGCC ACCAGACCGA CCTGAGGGCC CGCACACGTC ACGCATCAGC AGCGTCTCAT 180
    CCCAGTTCAG CGACGGGCCG ATCCCCAGCC CCTCCGCACG CAGCAGCGCC TCATCCTGGT 240
    CCGAGGAGCC TGTGCAGTCC AACATGGACA TCTCCACCGG CCACATGATC CTGTCCTACA 300
    TGGAGGACCA CCTGAAGAAC AAGAACCGGC TGGAGAAGGA GTGGGAAGCG CTGTGCGCCT 360
    ACCAGGCGGA GCCCAACAGC TCGTTCGTGG CCCAGAGGGA GGAGAACGTG CCCAAGAACC 420
    GCTCCCTGGC CGTGCTGACC TATGACCACT CCCGGGTCCT GCTGAAGGCG GAGAACAGCC 480
    ACAGCCACTC AGACTACATC AACGCTAGCC CCATCATGGA TCACGACCCG AGGAACCCCG 540
    CGTACATCGC CACCCAGGGA CCGCTGCCCG CCACCGTGGC TGACCTTTGG CAGATGGTGT 600
    GGGAGAGCGG CTGCGTGGTG ATCGTCATGC TGACACCCCT CGCGGAGAAC GGCGTCCGGC 660
    AGTGCTACCA CTACTGGCCG GATGAAGGCT CCAATCTCTA CCACATCTAT GAGGTGAACC 720
    TGGTCTCCGA GCACATCTGG TGTGAGGACT TCCTGGTGAG GAGCTTCTAT CTGAAGAACC 780
    TGCAGACCAA CGAGACGCGC ACCGTGACGC AGTTCCACTT CCTGAGTTGG TATGACCGAG 840
    GAGTCCCTTC CTCCTCAAGG TCCCTCCTGG ACTTCCGCAG AAAAGTAAAC AAGTGCTACA 900
    GGGGCCGTTC TTGTCCAATA ATTGTTCATT GCAGTGACGG TGCAGGCCGG AGCGGCACCT 960
    ACGTCCTGAT CGACATGGTT CTCAACAAGA CGGCCAAAGG TGCTAAAGAG ATTGATATCG 1020
    CAGCGACCCT GGAGCACTTG AGGGACCAGA GACCCGGCAT GTCCAGACGA AGGAGCAGTT 1080
    TGAGTTCGCG CTGACAGCCG TGGCTGAGGA GGTGAACGCC ATCCTCAAGG CCCTGCCCCA 1140
    GTGAGAATTC 1150
    (2) INFORMATION FOR SEQ ID NO: 34:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 2328 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
    GAATTCGGCT TGAGGAACCC CGCGTACATC GCCACCCAGG GACCGCTGCC CGCCACCGTG 60
    GCTGACTTTT GGCAGATGGT GTGGGAGAGC GGCTGCGTGG TGATCGTCAT GCAGACACCC 120
    CTCGCGGAGA ACGGCGTCCG GCAGTGCTAC CACTACTGGC CGGATGAAGG CTCCAATCTC 180
    TACCACATCT ATGAGGTGAA CCTGGTCTCC GAGCACATCT GGTGTGAGGA CTTCCTGGTG 240
    AGGAGCTTCT ATCTGAAGAA CCTGCAGACC AACGAGACGC GCACCGTGAC GCAGTTCCAC 300
    TTCCTGAGTT GGTTTGACCG AGGAGTCCCT TCCTCCTCAA GGTCCCTCCT GGACTTCCGC 360
    AGAAAAGTAA ACAAGTGCTA CAGGGGCCGT TCTTGTCCAA TAATTGTTCA TTGCAGTGAC 420
    GGTGCAGGCC GGAGCGGCAC CTACGTCCTG ATCGACATGG TTCTCAACAA GATGGCCAAA 480
    GGTGCTAAAG AGATTGATAT CGCAGCGACC CTGGAGCACT TGAGGGACCA GAGACCCGGC 540
    ATGGTCCAGA CGAAGGAGTA GTTTGAGTTC GCGCTGACAG CCGTGGCTGA GGAGGTGAAC 600
    GCCATCCTCA AGGCCCTTCC CCAGTGAGCG GCAGCCTCAG GGGCCTCAGG GGAGCCCCCA 660
    CCCCACGGAT GTTGTCAGGA ATCATGATCT GACTTTAATT GTGTGTCTTC TATTATAACT 720
    GCATAGTAAT AGGGCCCTTA GCTCTCCCGT AGTCAGCGCA GTTTAGCAGT TAAAAGTGTA 780
    TTTTTGTTTA ATCAAACAAT AATAAAGAGA GATTTGTGGA AAAATCCAGT TACGGGTGGA 840
    GGGGAATCGG TTCATCAATT TTCACTTGCT TAAAAAAAAT ACTTTTTCTT AAAGCACCCG 900
    TTCACCTTCT TGGTTGAAGT TGTGTTAACA ATGCAGTAGC CAGCACGTTC GAGGCGGTTT 960
    CCAGGAAGAG TGTGCTTGTC ATCTGCCACT TTCGGGAGGG TGGATCCACT GTGCAGGAGT 1020
    GGCCGGGGAA GCTGGCAGCA CTCAGTGAGG CCGCCCGGCA CACAAGGCAC GTTTGGCATT 1080
    TCTCTTTGAG AGAGTTTATC ATTGGGAGAA GCCGCGGGGA CAGAACTGAA CGTCCTGCAG 1140
    CTTCGGGGCA AGTGAGACAA TCACAGCTCC TCGCTGCGTC TCCATCAACA CTGCGCCGGG 1200
    TACCATGGAC GGCCCCGTCA GCCACACCTG TCAGCCCAAG CAGAGTGATT CAGGGGCTCC 1260
    CCGGGGGCAG GCACCTGTGC ACCCCATGAG TAGTGCCCAC TTGAGGCTGG CACTCCCCTG 1320
    ACCTCACCTT TGCAAAGTTA CAGATGCACC CCAACATTGA GATGTGTTTT TAATGTTAAA 1380
    ATATTGATTT CTACGTTATG AAAACAGATG CCCCCGTGAA TGCTTACCTG TGAGATAACC 1440
    ACAACCAGGA AGAACAAATC TGGGCATTGA GCAAGCTATG AGGGTCCCCG GGAGCACACG 1500
    AACCCTGCCA GGCCCCCGCT GGCTCCTCCA GGCACGTCCC GGACCTGTGG GGCCCCAGAG 1560
    AGGGGACATT TCCCTCCTGG GAGAGAAGGA GATCAGGGCA ACTCGGAGAG GGCTGCGAGC 1620
    ATTTCCCTCC CGGGAGAGGA GATCAGGGCG ACCTGCACGC ACTGCGTAGA GCCTGGAAGG 1680
    GAAGTGAGAA ACCAGCCGAC CGGCCCTGCC CCTCTTCCCG GGATCACTTA ATGAACCACG 1740
    TGTTTTGACA TCATGTAAAC CTAAGCACGT AGAGATGATT CGGATTTGAC AAAATAACAT 1800
    TTGAGTATCC GATTCGCCAT CACCCCCTAC CCCAGAAATA GGACAATTCA CTTCATTGAC 1860
    CAGGATGATC ACATGGAAGG CGGCGCAGAG GCAGCTGCGT GGGCTGCAGA TTTCCTGTGT 1920
    GGGGTTCAGC GTAGAAAACG CACCTCCATC CCGCCCTTCC CACAGCATTC CTCCATCTTA 1980
    GATAGATGGT ACTCTCCAAA GGCCCTACCA GAGGGAACAC GGCCTACTGA GCGGACAGAA 2040
    TGATGCCAAA ATATTGCTTA TGTCTCTACA TGGTATTGTA ATGAATATCT GCTTTAATAT 2100
    AGCTATCATT TCTTTTCCAA AATTACTTCT CTCTATCTGG AATTTAATTA ATCGAAATGA 2160
    ATTTATCTGA ATATAGGAAG CATATGCCTA CTTGTAATTT CTAACTCCTT ATGTTTGAAG 2220
    AGAAACCTCC GGTGTGAGAT ATACAAATAT ATTTAATTGT GTCATATTAA ACTTCTGATT 2280
    TCACCAAAAA AAAAAAAAAA AAAAAAAAAA AAAGCGGCCG CTGAATTC 2328

Claims (48)

We claim:
1. An isolated polynucleotide comprising a DNA segment encoding a mammalian islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of:
a) a polypeptide of SEQ ID NO:16 from Leu, amino acid residue 636 to Gln, amino acid residue 1012:
b) a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818; and
c) allelic variants of (a) or (b)
wherein the polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM.
2. An isolated polynucleotide according to claim 1, wherein said isolated polynucleotide encodes a mammalian islet cell antigen polypeptide selected from the group consisting of:
a) a polypeptide of SEQ ID NO:16 from Phe, amino acid residue 612 to Gln, amino acid residue 1012;
b) a polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue 818; and
c) allelic variants of (a) or (b).
3. An isolated polynucleotide according to claim 1, wherein said isolated polynucleotide encodes a mammalian islet cell antigen polypeptide selected from the group consisting of:
a) a polypeptide of SEQ ID NO:16 from Ala, amino acid residue 1 to Gln, amino acid residue 1012;
b) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acid residue 818; and
c) allelic variants of (a) or (b).
4. An isolated polynucleotide according to claim 1, wherein said isolated polynucleotide is a DNA molecule selected from the group consisting of:
a) a DNA molecule comprising the coding sequence of SEQ ID NO:15 from nucleotide 1909 to nucleotide 3039;
b) a DNA molecule comprising the coding sequence of SEQ ID NO:21 from nucleotide 1325 to nucleotide 2455;
c) allelic variants of (a) or (b); and
d) complements of polynucleotide molecules that specifically hybridize to (a), (b) or (c).
5. An isolated polynucleotide according to claim 1, wherein said isolated polynucleotide is a DNA molecule selected from the group consisting of:
a) a DNA molecule comprising the coding sequence of SEQ ID NO:15 from nucleotide 1837 to nucleotide 3039;
b) a DNA molecule comprising the coding sequence of SEQ ID NO:21 from nucleotide 2 to nucleotide 2455;
c) allelic variants of (a) or (b); and
d) complements of polynucleotide molecules that specifically hybridize to (a), (b) or (c).
6. An isolated polynucleotide according to claim 1, wherein said isolated polynucleotide is a DNA molecule selected from the group consisting of:
a) a DNA molecule comprising the coding sequence of SEQ ID NO:15 from nucleotide 4 to nucleotide 3039;
b) a DNA molecule comprising the coding sequence of SEQ ID NO:21 from nucleotide 1254 to nucleotide 2455;
c) allelic variants of (a) or (b); and
d) complements of polynucleotide molecules that specifically hybridize to (a), (b) or (c).
7. An isolated polynucleotide according to claim 1 which encodes a full length mammalian islet cell antigen polypeptide comprising the sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
8. An isolated polynucleotide comprising a DNA segment encoding a mammalian islet cell antigen polypeptide according to claim 1, wherein said mammalian islet cell antigen polypeptide is a primate islet cell antigen polypeptide.
9. A DNA construct comprising a first DNA segment encoding a human islet cell antigen polypeptide operably linked to additional DNA segments required for the expression of said first DNA segment, wherein said first DNA segment encodes a human islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of:
a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;
b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818;
c) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818; and
d) allelic variants of (a), (b) or (c)
wherein said mammalian islet cell antigen polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM.
10. A DNA construct according to claim 9 herein said first DNA segment comprises a nucleotide sequence selected from the group consisting of:
a) a DNA molecule comprising the coding sequence of SEQ. ID NO:21 from nucleotide 1325 to nucleotide 2455;
b) a DNA molecule comprising the coding sequence of SEQ ID NO:21 from nucleotide 1253 to nucleotide 2455;
c) a DNA molecule comprising the coding sequence of SEQ ID NO:21 from nucleotide 2 to nucleotide 2455;
d) naturally occurring allelic variants of (a), (b) or (c); and
e) complements of polynucleotide molecules that specifically hybridize to (a), (b), (c) or (d).
11. A DNA construct according to claim 9, wherein said first segment encodes a full length mammalian islet cell antigen comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
12. A DNA construct comprising a first DNA segment encoding a mammalian islet cell antigen according to claim 9, wherein said mammalian islet cell antigen polypeptide is a primate islet cell antigen polypeptide.
13. A host cell containing a DNA construct comprising a first DNA segment encoding a mammalian islet cell antigen polypeptide operably linked to additional DNA segments required for the expression of said first DNA segment, wherein said first DNA segment encodes a human islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of:
a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;
b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818;
c) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818; and
d) allelic variants of (a), (b) or (c)
wherein said mammalian islet cell antigen polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM.
14. A host cell according to claim 13, wherein said first DNA segment comprises a nucleotide sequence selected from the group consisting of:
a) a DNA molecule comprising the coding sequence of SEQ ID NO:21 from nucleotide 1325 to nucleotide 2455;
b) a DNA molecule comprising the coding sequence of SEQ ID NO:21 from nucleotide 1253 to nucleotide 2455;
c) a DNA molecule comprising the coding sequence of SEQ ID NO:21 from nucleotide 2 to nucleotide 2455;
d) naturally occurring allelic variants of (a), (b) or (c); and
e) complements of polynucleotide molecules that specifically hybridize to (a), (b), (c) or (d).
15. A host cell according to claim 13, wherein said first DNA segment encodes a full length mammalian islet cell antigen polypeptide comprising the sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
16. A host cell containing a DNA construct comprising a first DNA segment encoding a mammalian islet cell antigen polypeptide according to claim 13, wherein said mammalian islet cell antigen polypeptide is a primate islet cell antigen polypeptide.
17. An isolated mammalian islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of:
a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;
b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818;
c) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818; and
d) allelic variants of (a), (b) or (c)
wherein said mammalian islet cell antigen polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM.
18. An isolated mammalian islet cell antigen polypeptide according to claim 17, wherein said isolated mammalian islet cell antigen polypeptide is a full length mammalian islet cell antigen polypeptide comprising the sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
19. An isolated mammalian islet cell antigen polypeptide according to claim 17, wherein said mammalian islet cell antigen polypeptide is a primate islet cell antigen polypeptide.
20. A method for producing a mammalian islet cell antigen polypeptide comprising the steps of:
culturing a host cell containing a DNA construct comprising a first DNA segment operably linked to additional DNA segments required for the expression of said first DNA segment, wherein said first DNA segment encodes a human islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of:
a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;
b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818;
c) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818; and
d) allelic variants of (a), (b) or (c)
wherein said mammalian islet cell antigen polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM; and
isolating said mammalian islet cell antigen polypeptide.
21. A method for producing a mammalian islet cell antigen polypeptide according to claim 20, wherein said first DNA segment comprises a nucleotide sequence selected from the group consisting of:
a) a DNA molecule comprising the coding sequence of SEQ ID NO:21 from nucleotide 1325 to nucleotide 2455;
b) a DNA molecule comprising the coding sequence of SEQ ID NO:21 from nucleotide 1253 to nucleotide 2455;
c) a DNA molecule comprising the coding sequence of SEQ ID NO:21 from nucleotide 2 to nucleotide 2455;
d) naturally occurring allelic variants of (a), (b) or (c); and
e) complements of polynucleotide molecules that specifically hybridize to (a), (b), (c) or (d).
22. A method for producing a mammalian islet cell antigen polypeptide according to claim 20, wherein said first DNA segment encodes a full length mammalian islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
23. A method for producing a human islet cell antigen polypeptide according to claim 20, wherein said host cell is a bacterial cell or a cultured human cell.
24. A method for determining the presence of an autoantibody to a human islet cell antigen polypeptide in a biological sample comprising the steps of:
contacting a biological sample with a human islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of:
a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;
b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818;
c) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818; and
d) allelic variants of (a), (b) or (c),
under conditions conducive to immune complex formation, and
detecting the presence of immune complex formation between said human islet cell antigen polypeptide and said autoantibody to a human islet cell antigen, thereby determining the presence of an autoantibody to said human islet cell antigen in said biological sample.
25. The method of determining the presence of an autoantibody to a human islet cell antigen polypeptide according to claim 24, wherein said human islet cell antigen polypeptide is detectably labeled.
26. A method of determining the presence of an autoantibody to a human islet cell antigen polypeptide according to claim 24, wherein said human islet cell antigen polypeptide is a full length human islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
27. A method for predicting the clinical course of IDDM in a patient comprising:
testing a biological sample from a patient for the presence of human islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of:
a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;
b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818;
c) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818; and
d) allelic variants of (a), (b) or (c), wherein said polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM and
classifying said patient for clinical course of diabetes based on the presence or absence of mammalian islet cell antigen polypeptides in said sample.
28. A method for predicting the clinical course of IDDM according to claim 27, wherein said patient is further tested for one or more additional predictive markers associated with risk of or protection from IDDM.
29. A method for predicting the clinical course of IDDM according to claim 27, wherein said predictive marker is an autoantibody to an antigen selected from the group consisting of GAD65, IA-2/ICA512, or insulin.
30. A method for predicting the clinical course of IDDM according to claim 27, wherein said predictive marker is a genotype selected from the group consisting of HLA DR and HLA DQ.
31. A method for predicting the clinical course of IDDM according to claim 27, wherein said predictive marker is a polymorphic region in the 5′ flanking region of a human insulin gene.
32. A method of predicting the clinical course of IDDM according to claim 27, wherein said human islet cell antigen polypeptide is a full length human islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
33. A method of treating a patient to prevent an autoimmune response to a human islet cell antigen polypeptide, the method comprising inducing immunological tolerance in said patient by administering a human islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of:
a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;
b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818;
c) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818; and
d) allelic variants of (a), (b) or (c),
that specifically binds a human islet cell antigen receptor on immature or mature T or B lymphocytes.
34. A method of treating a patient to prevent an autoimmune response to a human islet cell antigen polypeptide according to claim 33, wherein said human islet cell antigen polypeptide is a full length human islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
35. A probe which comprises an oligonucleotide of at least about 16 nucleotides, wherein said oligonucleotide is at least 85% identical to a sequence of the human islet cell antigen DNA sequence of SEQ ID NOs: 15 or 21.
36. An isolated antibody which specifically binds to a human islet cell antigen polypeptide, wherein said human islet cell antigen polypeptide comprising an amino acid sequence selected from the group consisting of:
a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;
b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818;
c) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818; and
d) allelic variants of (a), (b) or (c).
37. An isolated antibody according to claim 36, wherein said isolated antibody is a monoclonal antibody.
38. An isolated antibody according to claim 36, wherein said human islet cell antigen polypeptide is a full length human islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
39. A hybridoma which produces a monoclonal antibody which specifically binds to a human islet cell antigen polypeptide, wherein said human islet cell antigen polypeptide comprises an amino acid sequence selected from the group consisting of:
a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;
b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818;
c) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818; and
d) allelic variants of (a), (b) or (c).
40. A hybridoma according to claim 39, wherein said human islet cell antigen polypeptide is a full length human islet cell antigen polypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.
41. A diagnostic kit for use in detecting an autoantibody to pancreatic β-islet cells, comprising a container containing a human islet cell antigen polypeptide wherein said human islet cell antigen polypeptide comprises an amino acid sequence selected from the group consisting of:
a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;
b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818;
c) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818; and
d) allelic variants of (a), (b) or (c),
wherein said polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM, and one or more containers containing additional reagents.
42. A pharmaceutical composition which comprises a human islet cell antigen polypeptide, wherein said human islet cell antigen polypeptide comprises an amino acid sequence selected from the group consisting of:
a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;
b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue 818;
c) a polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid residue 818; and
d) allelic variants of (a), (b) or (c), wherein said polypeptide forms an immune complex with an autoantibody from a patient at risk of or predisposed to develop IDDM in combination with a pharmaceutically acceptable carrier or vehicle.
43. A method for monitoring the disease state in a patient comprising:
testing a biological sample from a patient for the presence of mammalian islet cell antigen post-translationally modified polypeptides;
determining the concentration of said polypeptides; and
correlating levels of said polypeptides in said sample with the disease state in a patient.
44. A method for monitoring the disease state in a patient according to claim 43 wherein said human islet cell antigen post-translationally modified polypeptide comprise the sequence of SEQ ID NO:22 from His, amino acid residue 1 to Glu, amino acid residue 227.
45. A method for monitoring the disease state in a patient according to claim 43 wherein said biological sample is plasma or serum.
46. A method for monitoring the disease state in a patient comprising:
exposing T cells to islet cell antigen 1851 peptides;
detecting T cell reactivity; and
correlating T cell reactivity with disease state.
47. A method for monitoring the disease state according to claim 46 wherein said T cells are from peripheral blood mononuclear cells from a prediabetic patient.
48. A method for monitoring the disease state according to claim 46 wherein said disease state is conversion from prediabetes to diabetes.
US10/124,089 1996-03-06 2002-04-16 Islet cell antigen 1851 Abandoned US20030166067A1 (en)

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