MXPA01004006A - Secreted protein zsig49 - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zsig49, a novel secreted protein. The polypeptides and polynucleotides encoding them are highly expressed in pancreas tissue and have been mapped to human chromosome 1q24.1. The present invention provides methods for identifying abnormalities in human chromosome 1q and polymorphisms in an zsig49 gene that resides on chromosome 1q at a locus linked with a heritable form of Type II diabetes.

Description

PROTEIN ZSIG49 SECRETED BACKGROUND OF THE INVENTION Proteins secreted from cells can act as intercellular signaling molecules, which control ontogeny and maintenance of tissue form and function. These secreted proteins control, among other things, the proliferation, differentiation, migration and expression of cells of multicellular organisms and act in concert to form cells, tissues and organs, and repair and regenerate damaged tissue. Examples of secreted proteins include hormones and idiopathic polypeptide growth factors including steroid hormones (eg estrogen, terone), parathyroid hormone, follicle-stimulating hormone, interleukins, platelet-derived growth factor (PDGF), growth factor. epidermal (EGF), macr of ago-granulocyte (GM-CSF), erit ropoyet ina (EPO) and calcitonin colony stimulating factor, among others. Hormones and growth factors influence cellular metabolism by binding to receptors. The receptors could be integral membrane proteins that bind to the signaling pathways REF: 128098 within the cell, such as systems of the second messenger. Other classes of receptors are soluble molecules, such as transcription factors. There is a continuing need to discover new proteins, such as the hormones and growth factors described above. The present invention provides novel secreted proteins, agonists, antagonists and receptors of such proteins, as well as related compositions and methods, as well as other uses that should be apparent to those skilled in the art of these teachings.

BRIEF DESCRIPTION OF THE INVENTION In one aspect, the invention provides an isolated polypeptide comprising a contiguous sequence of 50 amino acid residues of SEQ ID NO: 10. In one embodiment, the contiguous sequence is 100 amino acid residues of SEQ ID NO: 10. NO: 10 In another embodiment, the contiguous sequence is 200 amino acid residues of SEQ ID NO: 10. In another embodiment, the polypeptide further comprises an affinity tag or binding domain. In another aspect, the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, from amino acid residue 34 to amino acid residue 467, wherein the polypeptide is specifically bound to an antibody to which a specific binding is polypeptide of SEQ ID NO: 10. In one embodiment, the polypeptide comprises a sequence of the amino acid residues that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10, from the residue of amino acid 34 to the residue of amino acid 467, wherein the polypeptide is specifically bound to an antibody to which a polypeptide of SEQ ID NO: 10. specifically binds. In another embodiment, the amino acid identity percent is determined using a FASTA program with ktup = 1 , maximum opening of the spacing = 10, maximum extension of the spacing = 1, and the substitution matrix = blosum62, with other parameters established as absent. In yet another embodiment, any difference between the sequence of the amino acid encoded by the polynucleotide molecule and the corresponding amino acid sequence of SEQ ID NO: 10 is due to a substitution of the conservative amino acid. The invention also provides an isolated polypeptide selected from the group consisting of: a) a polypeptide comprising amino acid residues 34-63 of SEQ ID NO: 2; b) a polypeptide comprising amino acid residues 64-467 of SEQ ID NO: 10; c) a polypeptide comprising amino acid residues 58-461 of SEQ ID NO: 12; d) a polypeptide of SEQ ID NO: 2, from the residue of amino acid 34 to the residue of amino acid 77; e) a polypeptide of SEQ ID NO: 10, from the residue of amino acid 34 to the residue of amino acid 467; f) a polypeptide of SEQ ID NO: 12, from the residue of amino acid 28 to the residue of amino acid 461; g) a polypeptide of SEQ ID NO: 2; h) a polypeptide of SEQ ID NO: 10; ei) a polypeptide of SEQ ID NO: 12. The invention further provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, from the residue of amino acid 1 to the residue of amino acid 33. In another aspect, the invention provides an isolated polynucleotide encoding a polypeptide comprising a contiguous sequence of 50 amino acid residues of SEQ ID NO: 10. In one embodiment, the contiguous sequence is 100 amino acid residues of SEQ ID NO: 10. In another embodiment , the contiguous sequence is 200 amino acid residues of SEQ ID NO: 10. In yet another embodiment, the polypeptide it further comprises an affinity tag or binding domain. In another aspect, the invention provides an isolated polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, from amino acid residue 34 to the residue of amino acid 467, wherein the polypeptide specifically binds to an antibody to which a polypeptide of SEQ ID NO: 10. specifically binds. In one embodiment, the polypeptide comprises an amino acid residue sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10, from amino acid residue 34 to amino acid residue 467, wherein the polypeptide specifically binds to an antibody to which a polypeptide of SEQ ID NO: 10 binds specifically. mode, the amino acid identity percent is determined using a FASTA program with ktup = 1, maximum spacing aperture = 10, maximum spacing extension = 1, and the substitution matrix = blosum62, with other parameters established as absent. In yet another embodiment, any difference between the amino acid sequence encoded by the polynucleotide molecule and the sequence of the corresponding amino acid of SEQ ID NO: 10 is due to a substitution of the conservative amino acid. In another aspect, the invention provides an isolated polynucleotide selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising amino acid residues 34-63 of SEQ ID NO: 2; b) a polynucleotide encoding a polypeptide comprising amino acid residues 64-467 of SEQ ID NO: 10; c) a polynucleotide encoding a polypeptide comprising amino acid residues 58-461 of SEQ ID NO: 12; d) a polynucleotide encoding a polypeptide of SEQ ID NO: 2, from the residue of amino acid 34 to the residue of amino acid 77; e) a polynucleotide encoding a polypeptide of SEQ ID NO: 10, from the residue of amino acid 34 to the residue of amino acid 467; f) a polynucleotide encoding a polypeptide of SEQ ID NO: 12, from the residue of amino acid 28 to the residue of amino acid 461; g) a polynucleotide encoding a polypeptide of SEQ ID NO: 2; h) a polynucleotide encoding a polypeptide of SEQ ID NO: 10; i) a polynucleotide encoding polypeptide of SEQ ID NO: 12; j) a polynucleotide comprising nucleotide 167 to nucleotide 1567 of SEQ ID NO: 9; k) a polynucleotide comprising nucleotide 1 to nucleotide 1383 of SEQ ID NO: 12; 1) an id polynucleot sequence complementary to a), b), c), d), e), f), g), h), i), j) or k); and m) a degenerate polynucleotide sequence of a), b), c), d), e), f), g), h) or i). The invention also provides an isolated polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, from amino acid residue 1 to amino acid residue 33. In a further aspect, the invention provides a variant zsig49 polypeptide, wherein the amino acid sequence of the variant polypeptide shares an identity with the amino acid sequence of SEQ ID NO: 10 selected from the group consisting of at least 80% identity, at least 90% identity, at least 95% identity, or more than 95% identity, and wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NO: 10 is due to one or more substitutions of the conservative amino acid. In another aspect, the invention provides a polynucleotide molecule that encodes a fusion protein consisting essentially of a first portion and a second portion linked by a peptide bond, the first portion comprises a polypeptide as described above; and the second portion comprises another polypeptide. The invention also provides a polynucleotide encoding a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1-33 of SEQ ID NO: 10, wherein the secretory signal sequence is linked operably to an additional polypeptide. In a further aspect, the patent provides an expression vector comprising the following operably linked elements: a transcription promoter; a segment of DNA encoding a polypeptide as described above; and a transcription terminator. In one embodiment, the polypeptide further comprises a secretory signal sequence operably linked to the polypeptide. In a related embodiment, the secretory signal sequence comprises amino acid residues 1-33 of SEQ ID NO: 2. In another embodiment, the DNA segment encodes an amino terminal or carboxy terminal polypeptide covalently linked to an affinity tag. The patent also provides a cell cultured in which an expression vector has been introduced as described above, wherein the cultured cell expresses the polypeptide encoded by the polynucleotide segment. Also provided is a method for producing a polypeptide comprising: culturing a cell into which an expression vector has been introduced as described above; whereby the cell expresses the polypeptide encoded by the segment of the polynucleotide; and recovering the expressed polypeptide. In one embodiment, the expression vector further comprises a secretory signal sequence operably linked to the polypeptide; the cultured cell secretes the polypeptide in a culture medium, and the polypeptide is recovered from the culture medium. In another aspect, the invention provides an antibody or antibody fragment that specifically binds to a polypeptide as described above. In one embodiment, the antibody is selected from the group consisting of: a) monoclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b) and d) human monoclonal antibody. In a related embodiment, the antibody fragment is selected from the group consisting of F (ab '), F (ab), Fab' Fab, Fv, scFv and the minimum recognition unit. In another embodiment, an anti-i-idiotypic antibody that specifically binds to the antibody described above is provided. A polypeptide is also provided as described above in combination with a pharmaceutically acceptable carrier. In another aspect, the invention provides a method for detecting an abnormality on chromosome 1 in a subject comprising: (a) amplifying nucleic acid molecules encoding a polypeptide as described in RNA isolated from a biological sample of the subject and ( b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates an abnormality on chromosome 1. In one embodiment, the detection step is carried out by comparing the nucleotide sequence of the nucleic acid molecules amplified with respect to the nucleotide sequence of SEQ ID NO: 9, wherein a difference between the nucleotide sequence of the amplified nucleic acid molecules and the corresponding nucleotide sequence of SEQ ID NO: 9 is indicative of abnormality on chromosome 1 In another embodiment, the amplification is carried out by the polymerase chain reaction or reaction in reverse transcriptase-polymerase chain. In another aspect, there is provided a method for detecting an abnormality on chromosome 1 in a subject comprising: (a) amplifying the nucleic acid molecules encoding a polypeptide as described above of the RNA isolated from a biological sample of the subject, ( b) transcribing the amplified nucleic acid molecules to express the mRNA, (c) translating the mRNA to produce polypeptides, and (d) detecting a mutation in the polypeptides, wherein the presence of a mutation indicates an abnormality on chromosome 1. invention also provides a method for diagnosing a metabolic disease or susceptibility to a metabolic disease in an individual, wherein the disease is related to the expression or activity of a polypeptide as described above in the individual, comprising the step of determining the presence of an alteration in the nucleotide sequence that encodes the polypeptide in the genome of the individual, in where the presence of an alteration in the nucleotide sequence indicates metabolic disease or susceptibility to a metabolic disease. In another aspect, the invention provides a method for diagnosing a metabolic disease or susceptibility to a metabolic disease in an individual, comprising: (a) amplifying the nucleic acid molecules encoding a polypeptide as described above of the RNA isolated from a biological sample of the individual, and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates metabolic disease or susceptibility to a metabolic disease. A method is also provided for diagnosing a metabolic disease or susceptibility to a metabolic disease in an individual, comprising (a) amplifying the nucleic acid molecules encoding a polypeptide as described above of the RNA isolated from a biological sample of the subject, ( b) transcribing the amplified nucleic acid molecules to produce mRNA, (c) translating the mRNA to produce the polypeptides, and (d) detecting a mutation in the polypeptides, wherein the presence of a mutation indicates metabolic disease or susceptibility to a disease metabolic In one modality, the metabolic disease is diabetes. In a related embodiment, the metabolic disease is type II diabetes and the individual is an Indian Pima.

The invention further provides a method for detecting the presence of zsig49 polypeptide RNA in a biological sample, comprising the steps of: (a) contacting a nucleic acid probe under hybridization conditions with either (i) RNA molecules test samples isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the nucleic acid probe has a nucleotide sequence comprising a portion of the nucleotide sequence of nucleotides 167-1567 of SEQ ID NO: 9 or its complement, or the nucleotide sequence of nucleotides 1-1383 of SEQ ID NO: 12 or its complement, and (b) detect the formation of hybrids of the nucleic acid probe and either the test RNA molecules or nucleic acid molecules synthesized, wherein the presence of the hybrids indicates the presence of the zsig49 polypeptide RNA in the biological sample. The invention also provides a method for detecting the presence of a polypeptide as described above in a biological sample, comprising the steps of: (a) contacting the biological sample with an antibody or antibody fragment, which binds specifically with a polypeptide, which consists of the amino acid sequence of SEQ ID NO: 10, wherein the contact is made under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any bound antibody or bound antibody fragment. In one embodiment, the antibody or antibody fragment further comprises a detectable label selected from the group consisting of radioisotope, fluorescent label, chemiluminescent label, enzyme label, bioluminescent label, and colloidal gold. In another aspect, the invention provides a kit for the detection of a gene encoding a polypeptide, comprising: a first container comprising a polynucleotide molecule as described above; and a second container comprising one or more reagents capable of indicating the presence of the polynucleotide molecule. The invention also provides a kit for the detection of a gene encoding a polypeptide, comprising: a first container comprising an antibody as described above; and a second container comprising one or more reagents capable of indicating the presence of the antibody.

DETAILED DESCRIPTION OF THE INVENTION Before establishing the invention in detail, it could be helpful for the understanding thereof to define the following terms: The term "affinity tag" is used herein to represent a peptide segment that can bind to a polypeptide, to provide for the purification or detection of the polypeptide or to provide sites for binding of the polypeptide to a substrate. Primarily, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO, J. 4: 1075, 1985, Nilsson et al., Methods Enz vmol. 198: 3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Nati.

Acad. Sci. USA 82: 7952-4, 1985), substance P, Flag ™ peptide (Hopp et al., Biot echnolosv 6: 1204-10, 1988, available from Eastman Kodak Co., New Haven, CT), streptavidin binding peptide. or another antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107. 1991. Affinity marks which encode the DNAs are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ). The term "allelic variant" represents any of two or more alternative forms of a gene that occupies the same chromosomal locus. Allelic variation originates naturally through mutation, and could result in phenotypic polymorphism within populations. Mutations of genes may be moderate (no change in the encoded polypeptide) or they could encode the polypeptides having the altered amino acid sequence. The term allelic variant is also used herein to represent a protein encoded by an allelic variant of a gene. The terms "amino-terminal" and "carboxyl-terminal" are used herein to represent positions within polypeptides and proteins. Where the context permits, these terms are used with reference to a particular sequence or portion of a polypeptide or protein to represent proximity or relative position. For example, a certain carboxyl-terminal sequence with respect to a reference sequence within a protein is located close to the carboxyl terminus of the sequence of reference, but not necessarily in the carboxyl term of the complete protein. The term "complements of polynucleotide molecules" represents polynucleotide molecules having a complementary base sequence and reverse orientation, as compared to a reference sequence. For example, sequence 51 ATGCACGGG 3 'is complementary to 5' CCCGTGCAT 3 '. The term "contig" represents a polynucleotide having a contiguous path of sequence identical or complementary to another polynucleotide. The contiguous sequences are said to "overlap" a given path of the polynucleotide sequence either in its entirety or along a partial path of the polynucleotide. For example, representative contigs with respect to the polynucleotide sequence 5'-ATGGCTTAGCTT-3 'are 5' -TAGCTTgagtct-3 'and 3' -gtcgacTACCGA-5 '. The term "corresponding to", when applied to the positions of the amino acid residues in the sequences, means the corresponding positions in a plurality of sequences when the sequences are optimally aligned. The term "degenerate nucleotide sequence" represents a nucleotide sequence that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). The degenerate codons contain different triplets of nucleotides, but they encode the same amino acid residue (i.e., the triplets GAU and GAC each encode Asp). The term "expression vector" represents a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest operably linked to additional segments that provide its transcription. Such additional segments could include promoter and terminator sequences, and could optionally include one or more origins of replication, one or more selected markers, an enhancer, a polyadenylation signal, and the like. The expression vectors are derived, in general, from the plasmid or viral DNA or could contain elements of both. The term "isolated", when applied to a polynucleotide molecule, represents that the polynucleotide has been removed from its natural genetic environment and thus is free of other foreign or undesired coding sequences, and is in the proper form for in use in genetically engineered protein production systems.

Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but could include the naturally occurring 5 'and 3' untranslated regions, such as promoters and terminators. The identification of the associated regions will be apparent to one skilled in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985). When applied to a protein, the term "isolated" indicates that the protein is in a condition other than its native environment, such as part of the 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 protein in a highly purified form, i.e., greater than 95% pure, more preferably greater than 99% pure. The term "operably linked", when referring to the DNA segments, represents that the segments are arranged so that they function in accordance with their intended purposes, e.g. the transcription starts at the promoter and proceeds through the coding segment towards the terminator.

The term "ortholog" (or "homologous species") represents a polypeptide or protein obtained from a species having homology to a polypeptide or analogous protein of a different species. The ortholog is the functional counterpart of a polypeptide or protein of a different species. The differences of the sequences between the orthologs are the result of the evolution of the species. The term "polynucleotide" represents a single or double stranded polymer of deoxyribonucleotide or rnucleotide bases that are read from the 5 'to the 31 end. Polynucleotides include RNA and DNA, and could be isolated from natural sources, synthesized in vi, or prepared from a combination of natural or synthetic molecules. The sizes of the polynucleotides are expressed as the base pairs (abbreviated "pb"), nucleotides ("nt") or kilobases ("kb"). When the context permits, the last two terms could describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules, it is used to represent the global length and it will be understood that it is equivalent to the term "base pairs". It will be recognized by the skilled artisan that the two strands of a double-stranded polynucleotide could differ slightly in length, and that the ends thereof could be staggered as a result of the enzymatic cut; in this way, all nucleotides in a double-stranded polynucleotide molecule could not be paired. Such unpaired ends, in general, shall not exceed 20 nt in length. A "polypeptide" is a polymer of amino acid residues linked by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides". "Probes and / or primers" as used herein, may be RNA or DNA. The DNA can be cDNA or genomic DNA. The polynucleotide probes and primers are single or double stranded DNA or RNA, generally synthetic oligonucleotides, but could be generated from the cloned cDNA or genomic sequences or their complements. The analytical probes, in general, will be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) may be used. PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt. Short polynucleotides can be used when a small region of the gene is blank for analysis. For the analysis total of genes, a polynucleotide probe could comprise a complete exon or more. The probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, radionuclide, fluorophore, chemoluminizer, paramagnetic particle, and the like, which are commercially available from many sources (such as Molecular Probes, Inc., Eugene , OR, and Amersham Corp., Arlington Heights, IL), using techniques that are well known in the art. The term "promoter" represents a portion of a gene that contains the DNA sequences that provide for the binding of the RNA polymerase and the initiation of transcription. Promoter sequences, commonly, but not always, are found in the 5 'non-coding regions of the genes. A "protein is a macromolecule comprising one or more polypeptide chains A protein could also comprise non-peptidic components, such as carbohydrate groups Carbohydrates and other non-peptidic substituents could be added to a protein by the cell in which the protein it is produced, and will vary with the cell type, proteins are defined here in terms of their conformational amino acid structures, substituents such as carbohydrate groups, Generally, they are not specified, but nevertheless they could be present. The term "receptor" represents a protein associated by cells that binds to a bioactive molecule (i.e., a ligand) and regulates the effect of the ligand on the cell. Membrane-linked receptors are characterized by a multi-domain structure comprising an extracellular ligand binding domain and an intracellular effector domain that is typically involved in signal transduction. The binding of the ligand to the receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecules in the cell. This interaction instead leads to an alteration in the cell's metabolism. Metabolic cases that bind to r ecept or-1 igand interactions include gene transcription, phosphorylation, dephosphorylation, increased cyclic AMP production, cellular calcium mobilization, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and phospholipid hydrolysis. In general, the receptors can be membrane, cytosolic or nuclear bonds; monomeric (e.g., hormone receptor that stimulates the thyroid, beta receptor- adrenergic) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, er it ropoyet receptor and IL-6 receptor). The term "secretory signal sequence" represents a DNA sequence encoding a polypeptide (a "secretory peptide") that, as a component of a higher polypeptide, directs the higher polypeptide through a secretory pathway of a cell in which it is synthesized. The upper peptide is commonly cut to remove the secretory peptide during transit through the secretory pathway. The term "binding variant" is used herein to represent alternative forms of the RNA transcribed from a gene. Binding variation arises naturally through the use of alternative binding sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and could result in several mRNAs transcribed from the same gene. Binding variants could encode polypeptides having the altered amino acid sequence. The term binding variant is also used herein to represent a protein encoded by a binding variant of a MRNA transcribed from a gene. The molecular weights and the lengths of the polymers determined by the imprecise analytical methods (e.g., elect rofores is in gel), will be understood as approximate values. When such value is expressed as "around" X or "approximately" X, the set value of X will be understood to be accurate to ± 10%. All references cited herein are incorporated by reference in their entirety. The present invention is based in part on the discovery of a new DNA sequence (SEQ ID NO: 9) and the corresponding deduced polypeptide sequence (SEQ ID NO: 10), for a secreted protein that maps to human chromosome 1. The polypeptide of the present invention has been designated z s ig49. The novel polynucleotides encoding the zsig49 polypeptide of the present invention were initially identified by consulting an EST database for the secretory signal sequences characterized by a 5 'side methionine start site, a hydrophobic region of approximately 13 amino acids and a cut (SEQ ID NO: 3, where the cut appears between the amino acid residues of alanine and glycine) in an effort to select for secreted proteins. The polypeptides corresponding to ESTs that satisfy the search criteria were compared with the known sequences to identify the secreted proteins that had homology with the known ligands. An EST sequence was discovered and determined to be new. The EST sequence of an islet cell library. A clone considered likely containing the entire coding region was used for sequencing, and revealed the 3 'end of a poly-A + message. A putative signal sequence is intact with a 5 'side of stoppage of the predicted start methionine (residue 1 of SEQ ID NOs: 2 and 10). The alignment of the murine (SEQ ID NO: 12) and human (SEQ ID NO: 1) DNA sequences indicated that the human sequence could also be extended in the 3 'direction. A series of 3 'RACE PCRs were carried out and the human cDNA sequence was extended to 1704 bp (SEQ ID NO: 9). The deduced amino acid sequence is shown in SEQ ID NO: 10. Analysis of the DNA encoding a zsig49 polypeptide (SEQ ID NO: 10) revealed an open reading frame encoding 467 amino acid residues comprising a putative signal sequence (residues 1-33 of SEQ ID NO: 10) and the predicted mature sequence of 434 amino acid residues (residues 34 to 467 of SEQ ID NO: 10). A dibasic site (lys-lys) is found in residues 62-63 of SEQ ID NO: 10. Cysteine residues are found in amino acid residues 42, 44, 81, 90, 95, 100, 130, 165 , 207, 240, 262, 390, 393 and 396 of SEQ ID NO: 10. The patent also provides a murine ortholog of human zsig49. Analysis of the polynucleotide sequence (SEQ ID NO: 12) encoding the murine ortholog (SEQ ID NO: 13) revealed a putative signal sequence (amino acid residues 1-27 of SEQ ID NO: 13), and a mature sequence of amino acid residues 434 (amino acid residues 28-461 of SEQ ID NO: 13). As in humans, there is a dibasic site (lys-lys) found in residues 56-57 of SEQ ID NO: 13 and the cysteine residues corresponding to amino acid residues 35, 75, 84, 89, 94, 124, 159, 201, 234, 256, 384, 387 and 390 of SEQ ID NO: 13. Multimeric complexes can be formed through the intermolecular disulfide bonds between zsig49 and a second polypeptide. Dimeric proteins within the present invention are formed by intermolecular disulfide bonds formed between the cysteine residues. These proteins include homodimers and heterodimer. In the latter case, the second polypeptide can be an ortholog or homolog of zsig49 or another similar protein. The protein could also be one that has a cysteine residue available for disulfide bond formation. The precursor proteins are cut or processed in the active form through the action of sprohormonal convertases (endoproteases). The most relevant cutting or processing site is a prohormonal converting amino acid dibasic site. There are only a few combinations of the dibasic amino acid, including lys-lys, arg-arg, arg-lys and lys-arg. Non-dibasic cutting and processing sites have also been observed, for example, Asn-Arg is a non-dibasic site found in gastrin. The zsig49 polypeptides could be processed at the lys-lys dibasic site (amino acid residues 62-62 of SEQ ID NO: 2 and 10 or amino acid residues 56-57 of SEQ ID NO: 13) by the prohormonal convertases in a active form. Known prohormonal convertases include, but are not limited to, prohormone convertase 3 (PC3), converta sa prohormonal 2 (PC2), furine or similar family convertases. of furin, such as prohormone convertase 4 (PC4) and PACE4. Therefore, the present invention provides polypeptides or fragments of polypeptides modified in post-processed form having the amino acid sequence of amino acid residue 34 to amino acid residue 63 of SEQ ID NOs: 2 or 110; the amino acid sequence of amino acid residue 64 to amino acid residue 77 of SEQ ID NO: 2; the amino acid sequence of amino acid residues 64 to 467 of SEQ ID NO: 10; amino acid sequence 28 to amino acid residue 57 of SEQ ID NO: 13; or the amino acid sequence of amino acid residue 58 to amino acid residue 461 of SEQ ID NO: 13. Examples of post-translational modifications include protolytic cleavage, glycosylation, disulfide bond, and hydroxylation. Analysis of the tissue distribution of the corresponding mRNA with respect to the zsig49 DNA sequence of SEQ ID NO: 1, by Northern blot analysis and Dot blot showed strong expression in pancreas, slightly decreased expression in testes, obvious expression in stomach, liver, pituitary gland, thyroid and salivary. Weaker transcript was detected in the prostate, spinal cord, gland adrenal, small intestine, trachea, spleen, thymus, peripheral / lymphocyte / lymphocytes and lymph node. There are two main transcripts at approximately 2 kb and 5 kb. While the 2 kb transcript is the main transcript in the testes, the 5 kb transcript is the main transcript in the other tissues. These results suggest that SEQ ID NO: 1 could be the result of an incompletely bound mRNA. The present invention further provides polynucleotide molecules, which include DNA and RNA molecules, which encode the zsig49 proteins. The polynucleotides of the present invention include the sense strand; the anti-sense strand; and DNA as double-stranded, which have both sense and antisense strands that are hybridized together by their respective hydrogen bonds. A representative DNA sequence encoding a zsig49 protein is set forth in SEQ ID NO: 10. DNA sequences encoding other zsig49 proteins can be readily generated by those skilled in the art based on the genetic code. The DNA sequences of the counterpart can be generated by substitution of U for T. Those skilled in the art will easily recognize that, from the point of view of the degeneracy of the genetic code, considerable variation of the sequence between these polynucleotide molecules is possible. SEQ ID NO: 4 is a degenerate DNA sequence encompassing all of the DNAs encoding the partial zsig49 polypeptide of SEQ ID NO: 2, SEQ ID NO: 11 is a degenerate DNA sequence encompassing all of the DNAs encoding the zsig49 polypeptide of SEQ ID NO: 10, and SEQ ID NO: 14 is a degenerate DNA sequence encompassing all of the DNAs that encode the zsig49 sequence of the murine ortholog of SEQ ID NO: 13. Those skilled in the art will recognize that the degenerate sequences of SEQ ID NOs: 4, 11 and 14 also provide all of the RNA sequences encoding SEQ ID NOs : 2, 10 and 13 substituting U for T. In this manner, the polynucleotides encoding the zsig49 polypeptide comprising nucleotide 1 to nucleotide 231 of SEQ ID NO: 4, nucleotide 1 to nucleotide 1401 are contemplated by the present invention. of SEQ ID NO: 11 and nucleotide 1 to nucleotide 1383 of SEQ ID NO: 14 and their RNA equivalents. Table 1 establishes the one-letter codes used within SEQ ID NOs: 4, 11 and 14 which represent the positions of the degenerate nucleotides. The "resolutions" are the nucleotides represented by a letter of the code. The "complement" indicates the code for the complementary nucleotide (s). For example, the code Y represents C or T, and its complement R represents A or G, being A complementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Nucleotide Complement AATCCGGGGCCTTAARA / GYC / TYC / TRA / GMA / CKG / TKG / TMA / C s C / GSC / G w A / TWA / THA / C / TDA / G / TBC / G / TVA / C / GVA / C / GBC / G / TDA / G / THA / C / TNA / C / G / TNA / C / G / T The degenerate codons used in SEQ ID NOs:, 11 and 14, which encompass all possible codons for a given amino acid, are set forth in Table 2.

TABLE 2 Amino Acid Code Codons Codon of a degenerate letter Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT SN Thr T ACÁ ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN Wing A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAÁ CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG He I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Amino acid c or d i go Codons of a degenerate letter Phe F TTC TTT TTY Tyr and TAC TAT TAY Trp W TGG TGG Ter • TAA TAG TGA TRR Asn / As P B RAY Glu / Gln Z SAR Any X NNN One skilled in the art will appreciate that some ambiguity is introduced in the determination of a degenerate codon, representative of all possible codons that encode each amino acid. For example, the degenerate codon for serine (WSN), in some circumstances, can encode arginine (AGR), and the degenerate codon for arginine (MGN), in some circumstances, can encode serine (AGY). There is a similar relationship between the codons that encode phenylalanine and leucine. In this way, some polynucleotides encompassed by the degenerate sequence could encode the variant amino acid sequences, but one skilled in the art can easily identify such sequences variants with reference to the amino acid sequences of SEQ ID NOs: 2, 10 and 14. Variant sequences can easily be tested for functionality as described herein. An expert in the art will also appreciate that different species can exhibit "preferential codon usage". In general, see, Grantham, et al., Nuc. Acids Res. 8_: 1893-912, 1980; Haas, et al. Curr. Biol. 6 .: 315-24, 1996; Wain-Hobson, et al., Gene 13: 355-64, 1981; Grosjean and Fiers, Gene 18: 199-209, 1982; Holm, Nuc. Acids Res. 14: 3075-87, 1986; Ikemura, J. Mol. Biol. 158: 573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codons" is a term of the art that is referred to as translation codons of proteins that are most frequently used in adult cells. a certain species, favoring in this way one or a few representatives of the possible codons that code for each amino acid (See Table 2). For example, the amino acid threonine (Thr) could be encoded by ACA, ACC, ACG or ACT, but in mammalian cells, ACC is the most commonly used codon; In other species, for example, insect cells, yeast, viruses or bacteria, the different Thr codons could be preferential. The preferential codons for a Particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of preferential codon sequences in the recombinant DNA, for example, can improve the production of the protein, making the translation of the protein more efficient within a particular cell type or species. Therefore, the sequence of the degenerate codon described in SEQ ID NOs: 4, 11 and 14 serve as a template to optimize the expression of polynucleotides in various cell types and species commonly used in the art and are described herein. Sequences containing preferential codons can be tested and optimized for expression in several species, and be tested for functionality as described here. The formation of the hybridization map by radiation is a somatic cell technique developed to construct contiguous high-resolution maps of mammalian chromosomes (Cox et al., Science 250: 245-50, 1990). Partial or complete knowledge of a gene sequence allows for the designation of PCR primers suitable for use with hybrid mapping panels by chromosomal radiation. Hybrid radiation mapping panels are commercially available, which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH panel (Research Genetics, Inc., Huntsville, AL). These panels allow rapid PCR-based chromosomal localizations and gene sorting, marked sequence sites (STSs) and other non-polymorphic and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between the newly discovered genes of interest and the previously mapped markers. Accurate knowledge of a gene position can be useful in a number of ways including: 1) determining whether a sequence is part of an existing contig and obtaining the additional surrounding genetic sequences in various forms, such as YAC-, BAC- clones or cDNA, 2) provide a possible candidate gene for a hereditary disease that shows binding to the same chromosomal region and 3) for organisms of the cross-referenced model, such as the mouse, it could be beneficial to help determine what function it might have a particular gene. The formation of the hybridization map by radiation showed that the maps of zsig49 9.76 cR 3000 are far from of the D1S2635 marker in the GeneBridge map panel 4 RH and 62 cR_10,000 are distant from the SHGC-6236 marker in the Stanford G3 RH panel. The use of the surrounding markers places zsig49 in the chromosomal region lq24. A susceptibility of the locus for prostate cancer (HPC1) located in chromosome lq24 has been localized, and a locus susceptibility for type II diabetes mellitus has also been localized with respect to the q arm of chromosome 1. Diabetes mellitus of Type II has a substantial genetic component (Barnett et al., Diabetoloqia 2_0: 87, 1981, Knowler et al., Am. J. Epidemiol 113: 144-56, 1981, Hanson et al., Am. J. Hum. Genet 5_7_: 160-70, 1995). Genes have been identified that predispose certain forms of diabetes, including several loci for Type I diabetes and for early-onset diabetes in young people (Froguel et al., Nature 356: 162, 1992; Davies et al., Nature 371: 130, 1994, Yamagata et al., Nature 384: 455, 1996, Stoffers et al., Nat. Genet 17: 138, 1997 and Elbein et al., Diabetes 48: 1775-82, 1999). Although genetic defects have been identified in rare syndromes of type II diabetes mellitus, specific defects have not yet been defined as pathogenic in common forms of this disease. The model The mathematician has suggested that type II diabetes mellitus is a polygenic disease (DeFronzo, Diabetes Reviews 5: 177, 1997; Lowe, "Diabetes Mellitus," Principies of Molecular Medicine, (Jameson, ed.), pages 433-442 (Human Press Inc. 1998)). A binding analysis indicates that a locus susceptible to diabetes resides on chromosome Iq (Hanson et al., Am., J. Hum. Genet, 63: 1130-8, 1998). In the Stanford G3 RH panel, the zsig49 gene was found to form the map 5 cR_10,000 (1 cR_10,000 = -25 kb) distal from a marker of the potential loci susceptible to diabetes, D1S1677, identified by Hanson et al., ibid. Hanson's study was an investigation of the genome width for the loci linked to diabetes and the body mass index in Pima Indians, a Native American population with a high prevalence of Type II diabetes and obesity (Bennett et al., Lancet 2: 125 1971); Knowler et al., Am. J. Clin. Nutr. 53 (Suppl): 1543S 1991). Therefore, the nucleotide sequences encoding the zsig49 gene can be used in the diagnosis or prognosis of the metabolic disease, such as diabetes. These methods are also appropriate for the diagnosis or prognosis of diabetes in the Pima Indians. The present invention provides reagents for the use in diagnostic applications. For example, the zsig49 gene, a probe comprising zsig49 DNA or RNA, or a subsequence thereof, can be used to determine whether the zsig49 gene is present on chromosome 1 or if a mutation has occurred. Chromosomal aberrations detectable at the zsig49 gene locus include, but are not limited to, aneuploidy, changes in the number of the copy of the gene, insertions, changes of the restriction site and rearrangements. These aberrations can be presented within the coding sequence, within the introns or within the flanking sequences that include the 5 'side promoter and regulatory regions, and could manifest as physical alterations within a coding sequence or changes in the level of gene expression. In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as described above, under the conditions wherein the polynucleotide will hybridize to the complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product with a reaction product of control. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples for use in the present invention include genomic DNA, cDNA and RNA. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NOs: 1, 9 or 12, the complements of SEQ ID NOs: 1, 9 or 12 or an RNA equivalent thereof. Appropriate test methods in this regard include molecular genetic techniques known to those skilled in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, chain reaction by ligation (Barany, PCR Methods and Applications 1: 5-16 1991), ribonuclease protection tests, use of single nucleotide polymorphisms (SNPs) (Zhao et al., Am. J. Hum. Genet. 63.:225-40 , 1998) and other genetic binding analysis techniques known in the art (Sambrook et al., Ibid., Ausubel et al., Ibid., Marian, Chest 108: 225-65, 1995). The ribonuclease protection assays (see, e.g., Ausubel et al., Ibid., Chapter 4) comprise the hybridization of an RNA probe to a sample of RNA from the patient, after which the reaction product (hybrid RNA-RNA) is exposed to RNase. The hybridized regions of RNA are protected from digestion. In PCR tests, a genetic sample from the patient is inoculated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or quantity of the product recovered are indicative of mutations in the patient. Another technique based on PCR that can be used in the analysis of single-strand conformational polymorphism (SSCP) (Hayashi, PCR Methods and Applications 1: 3-8, 1991).

Zsig49 is expressed in organs of the endocrine system, pancreas, testes, thymus gland, adrenal gland, thyroid gland and pituitary gland, suggesting an associated metabolic activity. The hormones released by the endocrine tissues regulate production, growth and development, provide defense against stress and maintain and regulate a metabolic balance in the body. Zsig49 is also expressed in other tissues, such as the stomach and small intestine, that secrete hormones in response to the entry and digestion of food. The expression of zsig49 is the strongest in the pancreas. The acinar cells of the pancreas are involved in the production of secretory fluids led to the small intestine for use during digestion. The islets of Langerhans (islets) are the site of synthesis of hormones that affect metabolism and neurological functions. For example, in islets, mature cells produce glucagon, mature cells produce glucagon, mature β cells produce insulin, and mature d cells produce somatostatin. Glucagon and insulin coordinate the flow of endogenous glucose, free fatty acids, amino acids and other substrate molecules to ensure that the energy needed is satisfied at baseline and during exercise. In addition, they coordinate the efficient disposition of nutrient input from food. Other products similar to the islet cell hormones (which include amylin, pancrea s t a t ina, somatostatin, and pancreatic polypeptide) could play subsidiary roles in the regulation of metabolism. The ability of zsig49 to modulate the energy balance of mammals could be evaluated by monitoring one or more of the following metabolic functions: adinopogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, thermogenesis, oxygen utilization or the like.

These metabolic functions are monitored by techniques (tests or animal models) known to those skilled in the art. Such methods of the present invention comprise incubating cells to study the polypeptide ± zsig49, monoclonal antibody, agonist or antagonist thereof and observe changes in adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake or the like. For example, the glycoregulatory effects of insulin are predominantly exerted in liver tissue, skeletal muscle and adipose tissue. Insulin binds to its cellular receptor in these three tissues and initiates the specific actions of the tissue that result, for example, in the inhibition of glucose production and the stimulation of glucose utilization. In the liver, insulin stimulates glucose uptake and inhibits gluconeogenesis and glycogenolysis. In the skeletal and adipose tissue, insulin acts to stimulate the taking, storage and utilization of glucose. One could also make use of zsig49 polypeptides, agonists and / or antagonists in the prevention or treatment of pancreatic conditions, characterized by dysfunction associated with pathological regulation of glucose levels in the blood, insulin resistance or digestive function. As used herein, the terms "treat" or "treatment" will be understood to include the reduction of symptoms as well as the effects on the fundamental disease process. In particular, diabetes mellitus is a metabolic disorder caused by a complete or partial lack of insulin. The most predominant forms are type I or insulin-dependent diabetes, and non-insulin-dependent diabetes, type II. Diabetes can also result from secondary causes that interrupt or limit insulin production, such as pancreatectomy or pancreatic insufficiency due to pancreatic disease, hypersecretion of hormones antagonistic to insulin or the administration of drugs that interfere with carbohydrate metabolism. The onset could also be due to impaired glucose tolerance. The use of the zsig49 polypeptides, agonists and / or antagonists could be done to treat diabetes or alleviate or eliminate associated symptoms related to elevated glucose levels. The zsig49 polypeptides could find application, for example, in the maintenance and / or regulation of blood sugar levels. Animal models, such as mice NOD, a spontaneous model system for insulin-dependent diabetes mellitus (IDDM) and a transgenic mouse model of viral induction (Herrath et al., J. Clin Invest. 98: 1324, 1996), is available for study the induction of non-sensitivity. The administration of zsig49 polypeptides before or after the onset of the disease can be monitored by urine glucose test. The stimulation of the proliferation or differentiation of the pancreatic cells can be measured either by administration of the zsig49 polypeptides to the cultured pancreatic cells, or by administering molecules of the present invention to the appropriate animal model. Such reagents would be useful for the culture of the islets, and for this reason their component cells include the cells, the β cells and the d cells. Cultivated islets would provide a source for islet cells for transplantation, an alternative for complete transplantation of the pancreas. Tests that measure cell proliferation or differentiation are well known in the art. For example, tests that measure proliferation include such tests as chemosensory ibid to the neutral red dye (Cavanaugh et al., Investiaational New Druss 8_: 347-54, 1990), the incorporation of radiolabeled nucleotides (Cook et al., Analytical Biochem. 179: 1-7, 1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol.Methods 82: 169-79, 1985), and the use of tetrazolium salts (Mosmann, J. Immunol., Methods 65: 55-63, 1983; Alley et al. ., Cancer Res. 4.-589-601, 1988; Marshall et al., Growth Rea. 5: 69-84, 1995; and Scudiero et al., Cancer Res. 48: 4827-33, 1988). Tests that measure differentiation include, for example, measuring cell surface markers associated with the specific expression of tissue stages, enzymatic activity, functional activity, or morphological changes (Watt, FASEB, 5: 281-4, 1991; , Di f ferent ion 5_7_: 63-75, 1994; Raes, Adv. Cell Biol. Technol. Bioprocesses, 161-71, 1989). In the testes, gametes undergo spermatogenesis and mature into terminally differentiated cells (sperm or sperm). Additionally, Leydig cells in the testes secrete androgens that are involved in the development of male sexual characteristics and activity. The factors involved in the regulation of the maturation of sperm cells and the interaction of the sperm of the egg are of therapeutic value for the treatment of conditions associated with fertility and for the contraceptive use of the male. The factors that influence the maturation process of the gamete could enter directly from the Sertoli cells that are in contact with the spermatogenic cells. Other paracrine and endocrine factors occur outside the seminiferous tubes, as in the interstitial Leydig cells, and are transported in the microenvironment of the sperm cells by the transport and binding of the proteins that are expressed by the Sertoli cells in the seminiferous tubes. It is believed that factors that play an important role in the process of spermatogenic cell maturation include testo terona, Leydig factor, IGF-1, inhibin homologs, insulin and activin. The proliferation or differentiation of the testicular cells can be measured in vi tro, by administering zsig49 polypeptides to the cultured testicular cells or by administering molecules of the present invention to the appropriate animal model Cultured testicular cells include dolphin DB1; Tes cells (CRL-6258); mouse spg GC-1 cells (CRL-2053); TM3 cells (CRL-1714); TM4 cells (CRL- 1715) and pig ST cells (CRL-1746), available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD. Tests that measure cell proliferation or differentiation are well known in the art and are discussed here. Artificial tests are well known in the art to evaluate the effect of zsig49 polypeptides on the testes. For example, the compounds can be injected intraperitoneally for a specific duration of time. After the treatment period, the animals are sacrificed and the testicles are removed and weighed. The testicles are homogenized and the counts of the sperm heads are made (Meistrich et al., Exp. Cell Res. 99: 72-8, 1976). Other activities may be analyzed, for example, chemotaxic activity, which could be associated with the proteins of the present invention. For example, factors of the late stage in spermatogenesis could be involved in the interactions of the egg sperm and sperm motility. Activities, such as improving the viability of cryopreserved sperm, stimulate the acrosome reaction, improve sperm motility and improve sperm egg interactions, could be associated with the proteins of the sperm. present invention. The tests evaluating such activities are known (Rosenberger, J. Androl, 11: 89-96, 1990, Fuchs, Zentralbl Gvnakol 11: 117-20, 1993, Neurwinger et al., Androloaia 22: 335-9, 1990; et al., Human Repro 3: 856-60, 1988; and Jockenhovel, Androloaia 22: 171-178, 1990; Lessing et al., Fertile Steril 4: 406-9 (1985); Zaneveld, InMale Infertility Chapter 11, Comhaire Ed., Champan &Hall, London 1996). These activities are expected to result in improved fertility and successful reproduction. The polypeptides of the present invention could exert regulatory effects on male gametes, reproductive development and testicular functions through the inhibition of retalimentation of the hypothalamus and the anterior putuitaria. The proteins of the testes, such as activins and inhibins, have been shown to regulate the secretion of active molecules that include the hormone follicles t imulant e (FSH) by the pituitary (Ying, Endod., Rev. 9 .: 267-93 , 1988, Plant et al., Hum. Reprod. 8. 41-44, 1993). Testosterone reduces the amount of gonadot ropin released from the hypothalamus. The polypeptides of the present invention could be evaluated for transcription and hormone-dependent expression, using methods known in the art. For example, zsig49 polypeptides can be tested for androgen regulated expression using transgenic mice as described in Allison et al., Mol. Cell. Biol. 9 .: 2254-7, 1989, castration and steroid therapy (Heyns et al., Ibid. And Page and Parker, Mol. Cell, Biol. 27_: 343-55, 1982) and hormonal suppression (Passapera et al. al., ibid and Castro et al., ibid.). If desired, the development of the zsig49 polypeptide in this respect can be compared with other androgen proteins, such as testosterone. Therapeutic use can be made of the zsig49 polypeptides, agonists and antagonists by inducing or releasing the suppression of the feedback mechanism in the treatment of reproductive dysfunctions. The zsig49 polypeptides, agonists and / or antagonists of the present invention, could have applications in the improvement of fertilization during aided reproduction in humans and animals. Such aided breeding methods are known in the art and include artificial insemination, iv fertilization, embryo transfer and intimalphalphage transfer of gamete. Such methods are useful to help patients who may have physiological or metabolic disorders that prevent their natural conception. Such methods are also used in animal breeding programs, such as cattle breeding, zoo or racing horses and could be used in methods for the creation of transgenic animals. The Dot blot analysis indicated the expression of zsig49 in the salivary gland. The salivary glands synthesize and secrete a number of proteins that have various biological functions. Such proteins facilitate the lubrication of the oral cavity (e.g., proline-rich mucins and proteins), re-mineralization (eg, staterin and proteins rich in ionic proline), digestion (eg, amylase, lipase and proteases), provide anti-microbial integrity maintenance capabilities (eg, proteins). rich in proline, lysozyme, histatins and lact operoxidase) and mucosa (eg, mucins). In addition, saliva is a rich source of growth factors synthesized by the salivary glands. For example, saliva is known to contain epidermal growth factor (EGF), nerve growth factor (NGF), transforming growth factor-alpha (TGF-), transforming growth factor-beta (TGF-β), insulin , growth factors similar to insulin I and II (IGF-I and IGF- II) and fibroblast growth factor (FGF). See, for example, Zelles et al., J. Dental Res. 74: 1826-32, 1995. The synthesis of growth factors by the salivary glands is believed to be androgen-dependent and is necessary for the health of the oral cavity and the gastrointestinal tract. In addition to the expression of the salivary glands, zsig49 is also expressed in the stomach and small intestine. This suggests that polypeptides of zsig49, agonists or antagonists thereof could be used therapeutically to aid digestion. To verify the presence of this ability in the zsig49, agonist or antagonist polypeptides of the present invention, such zsig49 polypeptides, agonists or antagonists are evaluated with respect to their ability to break down the starch according to the procedures known in the art. . If desired, the development of the zsig49 polypeptide in this respect can be compared to digestive enzymes, such as amylase, lipase, proteases and the like. In addition, zsig49 polypeptides or agonists or antagonists thereof could be evaluated in combination with one or more digestive enzymes to identify the synergistic effects. Also, polypeptides of zsig49, agonists or antagonists thereof could be therapeutically useful to promote wound healing. To verify the presence of this ability in polypeptides of zsig49, agonists or antagonists of the present invention, such polypeptides of zsig49, agonists or antagonists are evaluated with respect to their ability to facilitate wound healing according to procedures known in the art. . If desired, the development of zsig49 polypeptide in this respect can be compared to growth factors, such as EGF, NGF, TGF-, TGF-β, insulin, IGF-I, IGF-II, fibroblast growth factor ( FGF) and the like. In addition, zsig49 polypeptides or agonists or antagonists thereof could be evaluated in combination with one or more growth factors to identify inertial effects. In addition, zsig49 polypeptides, agonists or antagonists thereof could be therapeutically useful for antimicrobial applications. To verify the presence of this ability in the polypeptides of zsig49, agonists or antagonists thereof of the present invention, such polypeptides of zsig49, agonists or antagonists thereof are evaluated with respect to their anti-microbial properties according to procedures known in the art. See, for example, Barsum et al., Eur. Respir. J. 8 .: 709-14, 1995; Sandovsky-Losica et al., J. Med. Vet. Mvcol. (England) 28: 279-87, 1990; Mehentee et al., J. Gen. Microbiol (England) 135 (Pt. 8): 2181-8, 1989; Segal and Savage, (J. Med. Vet. Mvcol 24: 477-9, 1986 and the like.) If desired, the development of the zsig49 polypeptide in this respect can be compared to known proteins that are functional in this respect, such as proteins rich in proline, lysozyme, histatins, lactoperoxidase or the like.In addition, the polypeptides of zsig49, agonists or antagonists thereof could be evaluated in combination with one or more anti- microbial agents to identify the synergistic effects. antimicrobial agents could be directly driven or indirectly driven, such agents operate via membrane association mechanisms or by pore formation of the action of attaching directly to the offending microbe.The antimicrobial agents can also act by via an enzymatic mechanism, breaking the protective microbial substances or the cell wall / membrane thereof. anti-microbial, able to inhibit the proliferation or action of the microorganism or to interrupt the integrity of the microorganism or by the mechanism established here, are useful in methods to prevent contamination in cell culture by microbes susceptible to such antimicrobial activity. Such techniques involve culturing cells in the presence of an effective amount of the zsig49 polypeptide or an agonist or antagonist thereof. As well, the zsig49 polypeptides or agonists thereof could be used as reagents of cell cultures in the studies of the infection of exogenous microorganisms, such as bacterial, viral or fungal infection. Such radicals could also be used in animal models of infection. Also, the adhesion properties by microorganisms of zsig49 polypeptides or agonists thereof can be studied under a variety of conditions in binding tests and the like. In addition, zsig49 polypeptides, agonists or antagonists thereof could be therapeutically useful for the maintenance of mucosal integrity. To verify the presence of this capacity in the zsig49 polypeptides, agonists or antagonists of the present invention, such zsig49 polypeptides, agonists or antagonists are evaluated with respect to their maintenance of mucosal integrity according to procedures known in the art. See, for example, Zahm et al., Eur. Respir. J. 8: 381-6, 1995, which describes the methods for measuring the viscoelastic properties and surface properties of the mucosa, as well as for evaluating mucosal transport by coughing and ciliary activity. If desired, the development of the zsig49 polypeptide in this respect can be compared to mucins and the like. In addition, zsig49 polypeptides or agonists or antagonists thereof could be evaluated in combination with mucins to identify synergistic effects. In addition, the zsig49 polypeptides are expressed in the prostate. The prostate gland is regulated by androgens and shares other properties with the salivary glands. For example, the salivary glands and the prostate gland are classified with slow replicators with respect to their proliferative capacity. See, for example, Zajicek, Med. Hvpotheses 7 (10): 1241-51, 1981. Such slow replicators exhibit similar ontogeny and proceed during regeneration and neoplasia through similar stages. The prostate gland also appears to produce growth factors, such as EGF and NGF and other biologically important proteins, such as kallikreins. See, for example, Hiramatsu et al., Biochem. Int. 17 (2): 311-7, 1988, Harper et al., J. Biol. Chem. 257 (14): 8541-8, 1982 and Brady et al., Biochemistry 25 (12): 5203-10, 1988. The prostate gland also functions as an androgen dependent. The zsig49 gene was located on the human chromosome lq24 which is also the location of a locus susceptibility for prostate cancer (HPC1). Prostate dysfunction, such as prostate adenocarcinoma or the like, could also be detected using the zsig49 polypeptides. The present invention also provides methods for studying or identifying new prohormonal endoproteases or pro-transfons, enzymes that process protein precursors and prohormones. Prohormonal convertases sometimes exhibit tissue specificity. As a result, zsig49 polypeptides, which are expressed at high levels in pancreatic tissue, are probably processed by prohormonal convertases that exhibit specificity of the pancreatic, such as PC2 and PC3. In such methods of the present invention, zsig49 polypeptides or fragments (substrate) could be incubated with known or expected prohormonal convertases (enzyme), which produce a fragment of 30 amino acid residue from amino acid residue 34 to amino acid residue 63 (product ). The enzyme and substrate are incubated together or co-expressed in a test cell for a sufficient time to achieve cleavage / processing of the zsig49 polypeptide or fragment or fusion thereof. The detection and / or quantification of continuous cutting products, using the procedures that are known in the art. For example, enzyme kinetics techniques, which measure the cutting rate, can be used to study or identify prohormonal convertases capable of cleaving the zsig49 polypeptides, fragments or fusion proteins of the present invention. The agonists or antagonists of the zsig49 polypeptides described above are incubated within the scope of the present invention. Agonists could be identified using a method comprising providing cells sensitive to a zsig49 polypeptide, fragment or fusion; cultivate the cells in Presence of a test compound and compare the cell response with the cell grown in the presence of the zsig49 polypeptide, and select the test compounds for which the cell response is of the same type. As described herein, the described polypeptides can be used to construct the zsig49 variants and the functional fragments of zsig49. Such variants and fragments are considered to be zsig49 agonists. Another type of zsig49 agonist is provided by the anti-i-idiotipo antibodies., and fragments thereof, which, for example, mimic the zsig49 RNA binding domain. The zsig49 agonists can also be constructed using combinatorial libraries. Methods for constructing and screening phage display and other combinatorial libraries are provided, for example, by Kay et al., Phage Display of Peptides and Proteins (Academic Press 1996), Verdine, U.S. Pat. No. 5,783,384, Kay, et al., U.S. Pat. No. 5,747,334 and Kauffman et al., U.S. Pat. No. 5,723,323. Zsig49 can also be used to identify inhibitors (antagonists) of its activity. Such method comprises providing cells responsive to a zsig49 polypeptide, culturing a first portion of the cells in the presence of the zsig49 polypeptide, culturing a second portion of the cells in the presence of the zsig49 polypeptide and a test compound and detecting a decrease in a cellular response of the second portion of the cells as compared to the first portion of the cells. In addition to the tests described here, samples can be tested for the inhibition of zsig49 activity within a variety of tests designed to measure receptor binding or stimulation / inhibition of zsig49-dependent cellular responses. For example, the sensitive cell lines of zsig49 can be transferred with a reporter gene construct that is sensitive to the cell path stimulated by zsig49. Constructs of the reporter gene of this type are known in the art, and will generally comprise a zsig49 DNA response element operably linked to a gene encoding a test protein, such as luciferase. DNA response elements may include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE), insulin response element (IRÉ) (Nasrin et al., Proc. Nati). Acad Sci. USA 87: 5273-7, 1990) and serum response elements (SRE) (Shaw et al., Cell 56: 563-72, 1989). The cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19): 9063-6; 1988 and Habener, Molec. Endocrinol 4 (8): 1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 5_6_: 335-44; 1989. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit zsig49 activity in target cells, as evidenced by a decrease in zsig49 stimulation of receptor gene expression. Tests of this type will detect compounds that directly block the binding of zsig49 to cell surface receptors, as well as compounds that block processes in the cell pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of the binding of zsig49 to the receptor using the labeling of zsig49 with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FITC and the like). In tests of this type, the ability of a test sample to inhibit the binding of labeled zsig49 to the receptor is indicative of the inhibitory activity, which can be confirmed by secondary tests. The receptors used within the binding tests could be cellular receptors or isolated, immobilized receptors.

Useful antagonists of the zsig49 polypeptides may also include antibodies directed against an epitope of the zsig49 polypeptide. In the preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NOs: 1, 9 or 12, other probes, primers, fragments and polynucleotide sequences mentioned herein or sequences complementary thereto. Hybridization of polynucleotides is well known in the art and is widely used for many applications, see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; Ausubel et al., Eds., Current Protocols in Molecular Bioloqy, John Wiley and Sons, Inc., NY, 1987; Berger and Kimmel, eds., Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, 1987 and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26: 227-59, 1990. Hybridization of polynucleotides exploits the ability of the single-stranded complementary sequences to form a double helix hybrid. Such hybrids include DNA-DNA, RNA-RNA and DNA-RNA. Hybridization occurs between sequences that contain some degree of complementarity. Hybrids can tolerate mismatched base pairs in the double helix, but the stability of the hybrid is influenced by the degree of mismatch. The Tm of the hybrid unpaired decreases by 1 * C for every 1-1.5% of base pair mismatch. Varying the severity of the hybridization conditions allows control over the degree of mismatch that will be present in the hybrid. The degree of severity increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases. Severe hybridization conditions encompass temperatures of about 5-25'C below the thermal melting point (Tm) of the hybrid and a hybridization buffer having up to Na + 1M. Higher degrees of severity at lower temperatures can be achieved with the addition of formamide which reduces the Tm of the hybrid to approximately 1 C for each 1% formamide in the buffer solution. In general, such severe conditions encompass temperatures of 20-70'C and a hybridization buffer containing up to 6X SSC and 0-50% formamide. A higher degree of severity can be achieved at temperatures of 40-70 ° C with a hybridization buffer that has up to 4X SSC and 0-50% formamide. Highly severe conditions typically encompass temperatures of 42-70'C with a Hybridization buffer that has up to IX SSC and 0-50% formamide. The different degrees of severity can be used during hybridization and washing to achieve the maximum specific binding for the target sequence. Typically, washings after hybridization are performed at increased degrees of severity to remove the unhybridized polynucleotide probes from the hybridized complexes. The above conditions are understood to serve as a guide, and are well within the capabilities of one skilled in the art to adapt these conditions for use with a particular polypeptide hybrid. The Tm for a specific white sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a perfectly aligned probe sequence. These conditions that influence the Tm include the size and content of base pairs of the polynucleotide probe, the ionic strength of the hybridization solution and the presence of destabilizing agents in the hybridization solution. The numerous equations for calculating Tra are known in the art, see for example (Sambrook et al., Ibid., Ausubel et al., Ibid., Berger and Kimmel, ibid. And Wetmur, ibid.) And are specific for DNA, RNA and hybrids DNA-RNA and the sequences of polynucleotide probes of variant length. Sequence analysis programming elements such as Oligo 4.0 (publicly available) and Primer Premier (PREMIER Biosoft International, Palo Alto, CA) as well as Internet sites are available tools to analyze a given sequence and calculate Tm based on the criteria defined by the user. Such programs can also analyze a given sequence under the defined conditions and suggest the appropriate probe sequences. Typically, hybridization of the longer polynucleotide sequences, > 50 bp is made at temperatures of approximately 20-25'C below the calculated Tra. For smaller probes, < 50 bp, hybridization is typically carried out at Tm or below 5-10'C. This allows the maximum hybridization rate for the DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the speed and stability of hybrid formation. The sequences of smaller probes, < 50 bp, come into balance quickly with complementary sequences, but could form less stable hybrids. Incubation times from minutes to hours can be used to achieve hybrid formation. The sequence probes more long equilibrium more slowly, but form more stable complexes even at lower temperatures. Incubations are allowed to proceed overnight or longer. In general, the incubations are carried out for a period equal to three times the calculated Cot time. The time Cot, the time it takes for the re-associated polynucleotide sequences, can be calculated for a particular sequence by methods known in the art. The composition of the base pair of the polynucleotide sequence will affect the thermal stability of the hybrid complex, thereby influencing the choice of the hybridization temperature and the ionic strength of the hybridization buffer. The A-T pairs are less stable than the G-C pairs in aqueous solutions containing NaCl. Therefore, the higher the G-C content, the more stable the hybrid. Although the distribution of residues G and C within the sequence also contribute positively to the stability of the hybrid. The composition of the base pair can be manipulated to alter the Tm of a given sequence, for example, 5-methylodesoxytine can be replaced by thymidine to increase the Tm. The 7-desazo-2'-deoxyguanosine can be replaced by guanosine to reduce the dependence on Tm.

The ionic concentration of the hybridization buffer also affects the stability of the hybrid. Hybridization dampers, in general, contain blocking agents, such as Denhardt's solution (Sigma Chemical Co., St. Louis, MO.), denatured salmon sperm DNA, tRNA, powdered milk (BLOTTO), heparin or SDS, and a source of Na +, such as SSC (IX SSC: 0.15 M NaCl, 15 mM sodium citrate) or SSPE (IX SSPE: 1.8 M NaCl, 10 mM NaH2P04, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration of the buffer, the stability of the hybrid increases. Typically, hybridization buffers contain between 10 mM-1 M Na +. Premixed hybridization solutions are also available from commercial sources, such as Clontech Laboratories (Palo Alto, CA) and Promega Corporation (Madison, Wl) for use according to the manufacturer's instruction. In addition to the destabilizing or denaturing agents, such as formamide, tyalkyl ammonium salts, guanidinium cations or thiocyanate cations for the hybridization solution, the solution will alter the Tm of a hybrid. Typically, formamide is used at a concentration of up to 50% to allow incubations to be carried out at temperatures inferior and more convenient. Formamide also acts to reduce non-specific background when using RNA probes. As previously observed, the isolated zsig49 polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. In general, it is preferred to isolate RNA from the lymph node, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA. Total RNA can be prepared using guanidine HCl extraction followed by isolation by a CaCl gradient centrifugation (Chirgwin et al., Biochemistry 18: 52-94, 1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Nati. Acad.

Sci. USA 6_9: 1408-12, 1972). The complementary DNA (CDNA) is prepared from poly (A) + RNA using the known methods. The polynucleotides encoding the zsig49 polypeptides are then identified and isolated by, for example, hybridization or PCR. The polynucleotides of the present invention can also be synthesized using the automated equipment. The current method of choice is the phosphoramidite method. If double-stranded DNA synthesized chemically is required for an application, such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 bp) is technically simple and can be done by synthesizing the complementary strands and then hybridizing. For the production of higher genes (> 300 bp), however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical synthesis of DNA is seldom 100%. To overcome this problem, synthetic (double-stranded) genes are assembled in the modular form from single-stranded fragments that are 20 to 100 nucleotides in length. The methods of gene synthesis are well known in the art. See, for example, Glick and Pasternak, Molecular Biotechnology, Principies & Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994; Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984; and Cumie et al., Proc. Nati Acad. Sci. USA 87: 633-7, 1990. The present invention also provides the counterpart polypeptides and polynucleotides of other species (orthologs). These orthologous polynucleotides can be used, inter alia, to prepare the respective orthologous proteins. These species include, but they are not limited to mammals, birds, amphibians, reptiles, fish, insects and other vertebrate and invertebrate species. Of particular interest are orthologs of zsig49 from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate proteins. Orthologs of human proteins can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a type of tissue or cell that expresses the protein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences described herein. A mRNA library of a positive cell or tissue line is then prepared. A cDNA encoding the zsig49 polypeptide can then be isolated by a variety of methods, such as by probing with a full or partial cDNA of human or with one or more groups of degenerate probes based on the described sequences. A cDNA can also be cloned using the polymerase chain reaction or PCR (Mullis, U.S. Patent 4,683,202), using the designed primers of the sequences described herein. In an additional method, the library cDNA can be used to transform or transfer host cells, and expression of the cDNA of interest can be detected with an antibody to zsig49. Similar techniques can also be applied for the isolation of genomic clones. Those skilled in the art will recognize that the sequences described in SEQ ID NOs: 1, 9 and 12 and SEQ ID NOs: 2, 10 and 13 represent a single allele of the human zsig49 gene and polypeptide and a single allele of the gene and murine zsig49 polypeptide, and such allelic variation and alternative binding is expected to occur. In addition, allelic variants can be cloned by probing cDNA or the genomic libraries of different individuals according to standard procedures. Allelic variants of the DNA sequences shown in SEQ ID NOs: 1, 9 and 12, including those containing silent mutations and those in which the mutations result in amino acid sequence changes, are within the scope of the invention. present invention, as are proteins that are allelic variants of SEQ ID NOs: 2, 10 and 13. The generated cDNAs of the alternately linked mRNAs which maintain the properties of the zsig49 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. The allelic variants and binding variants of these sequences can be cloned by probing the cDNA or genomic libraries of different individuals or tissues according to the standard procedures known in art. The present invention also provides isolated zsig49 polypeptides that are substantially homologous to the polypeptides of SEQ ID NOS: 2, 10 and 13, and their homologous / orthologous species. The term "substantially homologous" is used herein to mean polypeptides having 50%, preferably 60%, more preferably at least 80%, of sequence identity with respect to the sequences shown in SEQ ID NOS: 2, 10 and 13 or your orthologs Such polypeptides will more preferably be at least 90% identical, and more preferably 95% or more identical to SEQ ID NOS: 2, 10 and 13 or their orthologs. The percent identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Biol. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Nati Acad. Sci. USA 89: 10915-9, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment records using a maximum aperture spacing of 10, a maximum extension spacing of 1, and the registration matrix "blosum 62" of Henikoff and Henikoff (ibid) as shown in Table 3 (amino acids are indicated by the codes of a standard letter). Then the percent of identity is calculated as follows: Total number of identical alignments x 100 [length of the upper sequence plus the number of spaces entered in the upper sequence to align the two sequences] Table 3 ARNDCQEGHILKMFPSTWYA 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 10 E -1 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 - 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 Ol 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 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 20 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -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 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 - 1 -2 -2 0 -3 -1 The identity of the sequence of the polynucleotide molecules is determined by similar methods using a ratio as described above. Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lipman is a method of protein alignment appropriate for the examination of the level of identity shared by an amino acid sequence described here, and the amino acid sequence of a putative variant of zsig49. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat. Acad. Sci. USA 85: 2444, 1988, and by Pearson, Meth. Enzvmol 183: 63, 1990. Briefly, FASTA first characterizes the similarity of the sequence by identifying regions shared by the sequence of doubt (eg, SEQ ID NOS: 2, 10, and 13) and a test sequence that has either the highest density high of the identities (if the variable ktup is 1) or "pairs of identities (if ktup = 2), s consider the substitutions, insertions or deletions of conservative amino acids.The ten regions with the highest density of identities are then re-register by comparing the similarity of all amino acids using a matrix of amino acid substitution, and the ends of the regions are "arranged" to include only the residues that contribute to the highest record. If there are several regions with records larger than the "cut" value (calculated by a predetermined formula based on the length of the sequence and the ktup value), then the initial arranged regions are examined to determine if the regions can be joined to form a approximate alignment with the spaces. Finally, the highest registration regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 444, 1970); Sellers, SIAM J. Appl. Math. 2j6: 787, 1974), which allows the insertions and deletions of amino acids. The preferred parameters for the FASTA analysis are: ktup = 1, maximum opening of the spacing = 10, maximum extension of the spacing = 1, and substitution matrix = BLOSUM62. These parameters can be entered into a FASTA program by modifying the record matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzvmol 183: 63, 1990. FASTA can also be used to determine the identity of the sequence of acid molecules nucleic acids using a ratio as described above. For comparisons of the id nucleotide sequences, the ktup value may be in the range between one to six, preferably three to six, more preferably three, with other parameters set as absent. The present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared to the amino acid sequences of SEQ ID NOS: 2, 10 or 13. The BLOSUM62 table is an amino acid substitution matrix. derived from approximately 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 related protein groups (Hanikoff and Hanikoff, Proc. Nat. Acad. Sci. USA 89: 10915, 1992). Therefore, substitution frequencies of BLOSUM62 can be used to define conservative amino acid substitutions that could be introduced into the amino acid sequences of the present invention. As used herein, the language "conservative amino acid substitutions" refers to a substitution represented by a BLOSUM62 value greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2 or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (eg, 1, 2 or 3), whereas substitutions of Conservative amino acids are characterized by a BLOSUM62 value of at least 2 (eg, 2 or 3). The substantially homologous proteins and polypeptides are characterized in that they have one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is, substitutions of conservative amino acids (see Table 4) and other substitutions that do not significantly affect the folding and activity of the protein or polypeptide; small eliminations, typically from one to about 30 amino acids; 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 an affinity tag. Polypeptides comprising affinity tags may further comprise a proteolytic cleavage site between the zsig49 polypeptide and the affinity tag. Such preferred sites include thrombin cutting sites and cutting sites of factor Xa.

Table 4 Conservative amino acid substitutions Basic: arginine lysine histidine Acid acid glutamic acid aspartic acid Polar glutamine asparagine Hydrophobic leucine isoleucine valine Aromatic pheni lalanine t ript orthodon tyrosine Small: glycine alanine serine threonine methionine The proteins of the present invention can also comprise amino acid residues that do not occur naturally. Amino acids that do not occur naturally include, without limitation, t rans-3-methyproline, 2,4-methaneproline, cis-4-hydroxyproline, t rans-4-hydroxyproline, N-methyl-glycine, alo-t reonine, methyl threonine, hydroxyethyl-cysteine, hydroxyethyhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidin carboxylic acid, dehydroproline, 3- and 4-methyprimproline, 3,3-dimethylproline, ter-leucine, norvaline, 2-azaphenyl- alanine, 3-azaphenylalanine, 4 -a zafeni lalanine and 4-fluorophenylalanine. Various methods are known in the art for the incorporation of amino acid residues that do not occur naturally in proteins. For example, an IV system can be employed, wherein nonsense mutations are suppressed using chemically aminoacylated rRNA suppressors. Methods for synthesizing the amino acids and aminoacylating the ANRt are known in the art. The transcription and translation of the plasmids containing the nonsense mutations is carried out in a cell-free system, comprising an extract of E. col i S30 and commercially available enzymes and other reagents. The Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113: 2722, 1991; Ellman et al., Methods Enzvmol. 202: 301, 1991; Chung et al., Science 259: 806-9, 1993; and Chung et al., Proc. Nati Acad. Sci. USA 90: 10145-9, 1993). In a second method, translation is carried out in Xen opu s oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor RNAs (Turcatti et al., J. Biol. Chem. 271: 1999-1-8, 1996). In a third method, the cells of E. co li are cultured in the absence of a natural amino acid to be replaced (eg, phenylalanine) and in the presence of naturally occurring non-naturally occurring amino acids (eg, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine) . The amino acid that does not occur naturally is incorporated into the protein instead of its natural counterpart. See, Koide et al., Biochem. 3_3: 7470-6, 1994. Naturally occurring amino acid residues can be converted to species that do not occur naturally by chemical modification in vi t ro. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, amino acids that do not occur naturally and unnatural amino acids could be replaced by amino acid residues zsig49. The essential amino acids in the zsig49 polypeptides of the present invention can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). . In the latter technique, simple mutations of alanine are introduced into each residue in the molecule, and the resulting mutant molecules are tested for biological activity (eg, adhesion modulation, modulation by differentiation or the like) to identify amino acid residues that they are critical for the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699-708, 1996. The ligand-receptor sites or other biological interaction can also be determined by physical analysis of the structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity-labeled, in conjunction with the mutation of the site amino acids of putative contact. See, for example, de Vos et al., Science 255: 306-12, 1992; Smith et al., J. Mol. Biol. 22: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. Multiple amino acid substitutions can be made and tested using the known methods of mutagenesis and screening, such as those described by Reidhaar-Olson and Sauer (Science 241: 53-7, 1988) or Bowie and Sauer (Proc. Nati, Acad. Sci. USA 86: 2152-6, 1989). Briefly, these authors describe methods for simultaneously randomizing two or more positions in a polypeptide, selecting the functional polypeptide and then sequencing the muti-ligated peptides to determine the spectrum of substitutions available at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 3_0_: 10832 -7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and site-directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988). The zsig49 DNA variants described and the polypeptide sequences can be generated by mixing DNA as described by Stemmer, Nature 370: 389-91. 1994, Stemmer, Proc. Nati Acad.

Sci. USA 91: 10747-51, 1994 and WIPO publication WO 97/20078. Briefly, variant DNAs are generated by homologous recombination by the random fragmentation of a parent DNA followed by re-assembly using PCR, resulting in point mutations introduced randomly. This technique can be modified using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and testing provides rapid "evolution" of the sequences by selecting the desirable mutations, while simultaneously selecting against the deleterious changes. Mutagenesis methods as described above can be combined with automated, highly computerized screening methods to detect the activity of cloned, mutagenized polypeptides in host cells. The mutagenized DNA molecules encoding the active polypeptides (e.g., receptor binding) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of waste of individual amino acids in a polypeptide of interest, and can be applied to polypeptides of unknown structure. The polypeptides of the present invention comprise at least 15 contiguous amino acid residues of SEQ ID NOS: 2, 10 or 13. In certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50 or more contiguous residues of the SEC ID NOS: 2, 10 or 13, up to the complete predicted mature polypeptides (residues 34-467 of SEQ ID NO: 10 or residues 28-461 of SEQ ID NO: 13) or residues 34-77 of the SEC ID NO: 2, or the primary translation products (residues 1 to 461 of SEQ ID NO: 10 or residues 1 to 461 of SEQ ID NO: 13) or residues 1-77 of SEQ ID NO: 2 As described in more detail below, these polypeptides may further comprise additional idi polypeptide sequences, not zsig49. Such fragments or peptides could comprise an "immunogenic epitope", which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen. Peptides carrying immunogenic epitopes can be identified using standard methods (see, for example, Geysen et al., Proc. Nati, Acad. Sci. USA 81: 3998, 1983).

In contrast, the polypeptide or peptide fragments could comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind. Certain epitopes consist of a linear or contiguous pathway of amino acids, and the antigensity of such an epitope is not interrupted by denatured agents. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et al., Science 219: 660, 1983). Therefore, the peptides and polypeptides carrying antigenic epitopes of the present invention are useful for raising antibodies that bind to the polypeptides described herein. Such peptides and polypeptides that carry epitopes can be produced by fragmentation of a zsig49 polypeptide, or by chemical synthesis of peptides, as described herein. In addition, epitopes can be selected by phage display from random peptide libraries (see, for example, Lane and Stephen, Curr Opin, Immunol 5: 268, 1993), and Cortese et al., Curr. Opin. Biotechnol. 1: 616, 1996). The standard methods to identify epitopes and producing antibodies from small peptides comprising an epitope are described, for example, by Mole, "Epitope Mapping," In Methods in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc. 1992), Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies", in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter and Ladyman (eds.) Pages 60-84 (Cambridge University Press 1995) and Coligan et al. (eds.), Current Protocols in Immunology, pages 9.3.1 - 9.3.5 and pages 9.4.1 -9.4.11 (John Wiley & amp; amp;; Sons 1997). Antibodies that recognize short, linear epitopes are particularly useful in analytical and diagnostic applications that employ denatured proteins, such as Western blotting (Tobin, Proc. Nat. Acad. Sci. USA 76: 4350-6, 1979), or in the analysis of fixed cells or tissue samples. Antibodies to linear epitopes are also useful for detecting fragments of zsig49, such as could be present in body fluids or cell culture media. For any zsig49 polypeptide, including variants of fusion proteins, one skilled in the art can easily generate a sequence of fully degenerate polynucleotide encoding such a variant, using the information set forth in Tables 1 and 2 above. In addition, those skilled in the art can use the standard programming elements to make the zsig49 variants based on the nucleotide and amino acid sequences described herein. Therefore, the present invention includes a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO : 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14. Appropriate forms of computer readable media include magnetic media and optically readable media . Examples of the magnetic means include a fixed or hard disk drive, a random access memory (RAM) microcircuit, a flexible disk, digital linear tape (DLT), a cache disk or a ZIP disk. Optically readable media is exemplified by compact discs (eg, CD read-only memory (ROM), rewritable CD (RW) and recordable CD), and versatile digital video discs (DVD) (eg, DVD-ROM, DVD-RAM and DVD + RW). Using the methods discussed above, a The person skilled in the art can identify and / or prepare a variety of polypeptides that are substantially homologous to residues 34 to 77 of SEQ ID NO: 2, residues 34 to 467 of SEQ ID NO: 10, residues 28 to 461 of SEQ ID NO: 13 or the allelic variants thereof and maintain the properties of the wild-type protein. Such polypeptides could include additional amino acids, such as affinity tags and the like. Such polypeptides could also include additional polypeptide segments as generally described herein. The polypeptides of the present invention, which include full-length proteins, fragments thereof and fusion proteins, can be produced in host cells genetically engineered according to conventional techniques. Suitable host cells are the types of cells that can be transformed or transferred with exogenous DNA and grown in culture and include cultured higher eukaryotic bacteria, fungi and cells. Eukaryotic cells are preferred, particularly cultured cells of multicellular organisms. Techniques for manipulating the cloned DNA molecules and introducing the exogenous DNA into a variety of host cells are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987. In general, a DNA sequence encoding a zsig49 polypeptide of the present invention is operably linked to other genetic elements required for its expression. , which include, in general, a promoter and a transcription terminator in an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that in certain systems the selectable markers could be provided in separate vectors, and the replication of the exogenous DNA could be provided by the integration into the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design at the level of the skilled artisan. Many elements are described in the literature and are available through commercial providers. To direct a zsig49 polypeptide in the secretory pathway of a host cell, a secretory signal sequence (also known as a sequence) leader, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence could be that of the zsig49 polypeptide, or it could be derived from another secreted protein (e.g., t-PA) or synthesized from n ovo. The secretory signal sequence binds to the zsig49 DNA sequence in the correct reading frame and is positioned to redirect the synthesized polypeptide into the secretory pathways for the host cell. Sequences of the secretory signal are commonly placed 5 'to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences could be placed anywhere in the DNA sequence of interest (see, eg, Welch. et al., US Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides in the path of the secretory signal. The present invention provides such fusion polypeptides. A signal fusion polypeptide can be made, wherein a secretory signal sequence derived from amino acid residues 1-33 of SEQ ID NO: 2 or residues 1-33 of SEQ ID NO: 10, is linked operably to another polypeptide using methods known in the art described herein. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably amino-terminally fused to an additional peptide to direct the additional peptide in the secretory pathway. Such constructions have numerous applications known in the art. For example, these novel fusion constructs of the secretory signal sequence can direct the secretion of an active component of a normally non-secreted protein. Such fusions could be used i n vi ve or i n vi t ro to direct the peptides through the secretory pathway. Cultured mammalian cells are suitable hosts in the present invention. 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 5_2_: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), transfection mediated by DEAE-dext rano (Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987), transfection mediated by liposomes (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993) and viral vectors (Miller and Rosman, BioTechniques 1: 980-90, 1989; Finer, Nature Med. 2: 714-16, 1996). The production of recombinant polypeptides in cultured mammalian cells is described, 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. Patent No. 4,579,821 and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells include COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573, Graham et al. al., J. Gen. Virol. 36: 59-72, 1977) and Chinese hamster ovary cell lines (eg CHO-K1; ATCC No. CCL 61). Additional appropriate cell lines are known in the art and are available from public depositaries, such as American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters, such as SV-40 or cytomegalovirus promoters, are preferred. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those of the metallotein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the major adenovirus late promoter.

If the zsig49 polypeptide is expressed in a non-endocrine or non-neuroendocrine cell, expression of the host cell will not express the prohormonal convertases PC2 and PC3, which are thought to be involved in the regulated secretory pathway. Another number of this family of endoproteases, furin, is present in most cells and is thought to be involved in the constitutive secretory pathway. Vollenweider et al. (Diabetes 44: 1075-80, 1995) have described, in general, the role of these prohormonal conversion endoproteases and specifically describe studies involving the co-transfection of COS cells with proinsulin and one of the endoproteases. Their results showed that PC3 and furin were able to cut proinsulin in both junctions; PC2 did not exhibit prohormonal cleavage to any significant degree. Without co-transduction of an endoprotia, the prohormone was not converted to any greater degree by COS cells. However, the co-t ransfection system described is not yet an exact model of the natural environment of the ß cells, since ß cells make PC2 and PC3. Also, a non-endocrine cell does not represent a native environment for the expression of PC2 and PC3. In addition, co-transfection could result in overexpression general or local of PC2 and / or PC3, in relation to the native environment of the ß cells. In a preferred embodiment, the host cells will be co-transfected with a second DNA expression construct comprising the following operably linked elements: a transcription promoter; a segment of DNA that encodes an endoprotease; and a transcription terminator, wherein the host cell expresses the DNA segment encoding the endoprotease. The drug section is used, in general, for cultured mammalian cells, in which the DNA has been inserted. Such cells are commonly referred to as "trans fect before". Cells that have been cultured in the presence of the selective agent and are capable of passing the gene of interest to their progeny are referred to as "stable transfectants". A preferred selectable marker is a gene that encodes antibiotic neomycin resistance. The selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems could also be used to increase the level of expression of the gene of interest, a process referred to as "amplification". The amplification is carried out by the culture of the trans fect before presence of a low level of the selective agent and then increasing the amount of the selective agent, to select the 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. Other drug resistance genes (e.g., hygromycin resistance, mu-t-drug resistance, puromycin acetyl transferase) can also be used. Alternate markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins, such as CD4, CD8, MHC class I, placental alkaline phosphatase could be used to classify transfected cells from non-transfected cells by such means as FACS classification or magnetic score separation technology. Other higher eukaryotic cells may also be used as hosts, which include plant cells, insect cells and bird cells. The use of Agroba c t eri um rh i z ogen is as a vector for expressing genes in plant cells have been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11: 47-58, 1987. The transformation of insect cells and the production of foreign polypeptides therein are described by Guarino et al., U.S. Pat. No. 5,162,222 and WIPO Publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from the nuclear polyhedral virus of Au t ograph a ca l i forn i ca (AcNPV). However, pFastBacl ™ can be modified to a considerable degree. The polyhedrin promoter can be removed and replaced with the basic baculovirus protein promoter (also known as the Peo r, p6.9 or MP promoter) that is expressed before baculovirus infection, and has been shown to be advantageous for expressing secreted proteins . See, Hill-Perkins and Possee, J. Gen. Virol. 71: 971-6, 1990; Bonning et al., J. Gen. Virol. 75: 1551-6, 1994; and, Cha zenbal k and Rapoport, J. Biol. Chem. 270: 1543-9, 1995. In such constructions of the transfer vector, a short or long version of the basic protein promoter can be used. In addition, the transfer vectors can be constructed, which replace the secretory signal sequences of native zsig49 with the sequences of secretory signals derived from insect proteins. For example, a secretory signal sequence of glycos i 1 trans ferase from eedies t eroid (EGT), bee mellitin (Invitrogen, Carlsbad, CA) or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructions to replace the secretory signal sequence of native zsig49. The DNA encoding the zsig49 polypeptide is inserted into the baculoviral genome in place of the AcNPV polyhedrin gene encoding the sequence by one of two methods. The first is the traditional method of homologous DNA recombination between wild-type AcNPV and a transfer vector containing zsig49 flanked by the AcNPV sequences. Appropriate insect cells, e.g., SF9 cells, are infected with wild-type AcNPV and transfected with a transfer vector comprising a zsig49 polynucleotide operably linked to a promoter, terminator and flanking sequence of the AcNPV polyhedrin gene. See, King and Possee, The Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall; O'Reilly et al. , Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. Natural recombination in an insect cell will result in a recombinant baculovirus containing zsig49 driven by the polyhedrin promoter. The recombinant viral reserves are they do it by the methods commonly used in art. The second method for making the recombinant baculovirus uses a transposon-based system described by Luckow (Luckow et al., J. Virol. 67: 4566-79, 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl ™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zsig49 polypeptide into a baculovirus genome maintained in E. col i as an upper plasmid called a "bacmido". The pFastBacl ™ transfer vector uses the AcNPV polyhedrin promoter to direct expression of the gene of interest, in this case zsig49. However, pFastBacl ™ can be modified to a considerable degree. The polyhedrin promoter can be removed and replaced with the basic baculovirus protein promoter (also known as Peor, p6.9 or MP promoter) which is expressed early in baculovirus infection and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins and Possee, J. Gen. Virol. 71: 971-6, 1990; Bonning et al., J. Gen. Virol. 7_5: 1551-6, 1994; and, Chazenbalk, G.D., and Rapoport, J. Biol. Chem. 270: 1543-9, 1995. In such transfer vector constructs, a short or long version of the basic protein promoter can be used. In addition, the transfer vectors can be constructed, which replace the secretory signal sequences of native zsig49 with the sequences of secretory signals derived from insect proteins. For example, an ecdysteroid (trans EGT), honey melitin (Invitrogen, Carlsbad, CA) or gp67 baculovirus (PharMingen, San Diego, CA) secretory signal sequence can be used in constructions to replace the signal sequence secretor of native zsig49. In addition, the transfer vectors can include a frame fusion with a DNA encoding an epitope tag in the C or N term of the expressed zsig49 polypeptide, eg, a Glu-Glu epitope tag (Grussenmeyer et al., Ibid. ). Using a technique known in the art, a transfer vector containing zsig49 is transformed into E. col i and screened for bacmides containing an interrupted lacZ gene indicative of the recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodop cells t was frugiperda, e. g. Sf9 cells. The virus Recombinant expressing zsig49 occurs subsequently. Recombinant viral stocks are made by methods commonly known in the art. The recombinant virus is used to infect host cells, typically a cell line derived from autumn worm, Spodop t era fr u gipe rda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, DC, 1994. Another suitable cell line is the High FiveO ™ cell line (Invitrogen) derived from Tri ch opl usiani (Patent US # 5,300,435). The commercially available serum free media is used for the growth and maintenance of the cells. Suitable media are Sf900 II ™ (Life Technologies) or ESF 921 ™ (Expression Systems) for Sf9 cells; and Ex-cell0405 ™ (JRH Biosciences, Lanexa, KS) or Express FiveO ™ (Life Technologies) for T cells. neither . The cells are grown from an inoculation density of about 2-5 x 10 5 cells at a density of 1-2 x 10 6 cells, at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10. , more typically close to 3. Cells infected with the recombinant virus typically produce the polypeptide Recombinant zsig49 at 12-72 hours post-infection and secrete it with varying efficiency in the medium. The culture is usually harvested 48 hours post-infection. Centrifugation is used to separate the cells from the medium (supernatant). The supernatant containing the zsig49 polypeptide is filtered through micropore filters, usually 0.45 μm pore size. The procedures used are described, in general, in the available laboratory manuals (King and Possee, ibid., O'Reilly et al., Ibid., Richardson, C. D., ibid.). Subsequent purification of the zsig49 polypeptide from the supernatant can be achieved using the methods described herein. Fungal cells, which include yeast cells, can also be used in the present invention. Yeast species of particular interest in this regard include Saccharomyces cerivisiae, Pichia pastoris and Pichia methanol ica. Methods for transforming S. cerivisiae cells with exogenous DNA and producing recombinant polypeptides thereof are described by, for example, Kawasaki, U.S. Pat. No. 4,599,311; et al., U.S. Pat. No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welchet al., U.S. Patent No. 5,037, 743 and Murray et al., U.S. Pat. No. 4,845,075. The transformed cells they are selected for the phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerivisiae is the POT1 vector system described by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected for growth in glucose-containing media. Promoters and terminators suitable for use in yeast include those of the glycolytic enzyme genes (see, eg, Kawasaki, US Patent No. 5,599,311, Kigsman et al., US Patent No. 4,615,974 and Bitter, US Patent No. 4,977,092 ) and the alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schi zosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago mayis, Pichia pastor is, Pichia methanolica, Pichia guillermondii and Candida maltose are known in the art. See, for example, Gleeson et al., J. Gen Microbiol. 132: 3459-65, and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells could be used according to the methods of McKnight et al. al., U.S. Pat. No. 4,935,349. Methods for transforming Acremonium chrysogenum are described by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are described by Lambowitz, U.S. Pat. No. 4,486,533. For example, the use of Pichia Methanolica as a host for the production of recombinant proteins is described by Raymond, U.S. Pat. No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14: 11-23, 1998 and in the international publications Nos. WO 97/17450, WO 97/17451, WO 98/02536 and WO 98/02565. DNA molecules for use in the transformation of P. methanolica will commonly be prepared as double-stranded circular plasmids, which are preferably linearized before transformation. For the production of polypeptides in P. methanolica, it is preferred that the promoter and terminator in the plasmid be from the P. methanolica gene, such as an alcohol utilization gene from P. methanolica (AUG1 or AUG2). Other useful promoters include the dihydroxyacetone synthase (DHAS), dehydrogenase (FMD) and catalase (CAT) genes. To facilitate the integration of DNA into the host chromosome, it is preferred to have the complete expression segment of the plasmid flanked at both ends by the host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimide zol carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which the utilization of the methanol genes (AUG1 and AUG2) is eliminated. For the production of the secreted proteins, the host cells deficient in the vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanol ica cells by electroporation using an exponentially decayed pulsed electric field having a field strength of 2.5 to 4.5 kV / cm, preferably about 3.75 kV / cm and a constant time (t) of 1 to 40. milliseconds, more preferably approximately 20 milliseconds. Prokaryotic host cells, which include strains of the bacterium Es ch eri ch i a co l i, Ba ci l l s and other genera are also useful for the host cells in the present invention. The techniques to transform these hosts and express the cloned foreign DNA sequences, are known in the art (see, e.g., Sambrook et al., ibid.). When a zsig49 polypeptide is expressed in bacteria, such as E. In this case, the polypeptide could be maintained in the cytoplasm, typically as insoluble granules or it could be directed into the periplasmic space by a bacterial secretion sequence. In the last last, the cells are used, and the granules are recovered and denatured, using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by interrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recover the protein, for which the need is obvious. for the Denaturing and re-folding. The adenovirus system can also be used for the production of proteins in vitro. By culturing the non-293 cells infected with adenovirus under conditions where the cells are not dividing rapidly, the cells can produce proteins for extended periods of time. For example, BHK cells are grown to confluent in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum free conditions, which allows the infected cells to survive for several weeks without significant cell division. Alternatively, 293 cells infected with the adenoviral vector can grow as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of the protein (see Garnier et al., Cvtotechnol U5_: 145-55, 1994). With any protocol, a expressed, secreted heterologous protein can be repeatedly isolated from the supernatant of the cell culture. In the production protocol of infected 293 cells, proteins also could be effectively obtained secreted The transformed and transfected host cells are cultured according to conventional procedures in a culture medium containing the nutrients and other components required for the growth of the chosen host cells. A variety of appropriate media are known in the art, including defined media and complex media and, in general, include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The media may also contain such components, such as growth factors or sera, as required. The growth medium, in general, will be selected for cells containing the exogenously added DNA, by, for example, selection of the drug or deficiency in an essential nutrient that is complemented by a selectable marker carried in the expression vector, or transfected in the host cell. The cells of P. m e th a n o l i ca are cultured in a medium comprising adequate sources of carbon, nitrogen and traces of nutrients at a temperature of about 25'C to 35'C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking small flasks or dispersing of fermenters. A preferred culture medium for P. me tha n ol i ca is YEPD (2% D-glucose, 2% Bacto ™ Peptone (Difco Laboratories, Detroit, MI), 1% Bacto ™ yeast extract (Difco) Laboratories), 0.004% adenine and 0.006% L-leucine). A novel method for testing proteins of the present invention involves viral delivery systems. Examples of viruses for this purpose include adenovirus, herpes virus, vaccine virus and adeno-associated virus (AAV). Adenoviruses, a double-stranded DNA virus, is currently the best-studied gene transfer vector for the delivery of heterologous nucleic acids (for review, see Becker et al., Meth. Cell. Biol. 4_3: 161-89 , 1994, and Douglas and Curiel, Science &Medicine 4_: 44-53, 1997). The adenovirus system offers several advantages: the adenovirus can (i) accommodate relatively large DNA inserts; (ii) grow at higher dosage; (iii) infect a wide range of mammalian cell types; and (iv) used with a large number of available vectors containing different promoters. Also, because the adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. Some disadvantages (especially for gene therapy) associated with the release of the adenovirus gene include: (i) very low efficiency integration in the host genome; (ii) existence in the mainly episomal form and (iii) the immune response of the host to the virus administered, preventing readministration of the adenoviral vector. By removing portions of the adenovirus genome, larger insertions (up to 7 kb) of the heterologous DNA can be accommodated. These insertions can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-t transfected plasmid. In one example of a system, the essential gene El has been removed from the viral vector and the virus will not replicate unless the El gene is provided by the host cell (eg, the 293 human cell line). When administered intravenously to intact animals, the adenovirus mainly targets the liver. If the adenoviral delivery system has a deletion of the El gene, the virus can not replicate in the host cells. However, host tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, the heterologous protein will be secreted). The secreted proteins will introduce the circulation in the highly vascularized liver, and can be determined the effects on the infected animal. The adenovirus system can also be used for the production of proteins in vitro. By culturing the non-293 cells infected with the adenovirus under conditions where the cells are not dividing rapidly, the cells can produce proteins for extended periods of time. For example, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum free conditions, which allows the infected cells to survive for several weeks without significant cell division. Alternatively, 293S cells infected with the adenoviral vector can grow in a suspension culture at relatively high cell density to produce significant amounts of proteins (see Garnier et al., Cvtotechnol.15: 145.-55, 1994). With any protocol, a expressed, secreted heterologous protein can be repeatedly isolated from the supernatant of the cell culture. In the production protocol of the infected 293S cells, the non-secreted proteins could also be obtained effectively.

The zsig49 polypeptides or fragments thereof could also be prepared by chemical synthesis. The zsig49 polypeptides could be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated and may or may not include a residue of the initial amino acid methionine.

The present invention also provides a variety of other mergers of polypeptides and related multimeric proteins, comprising one or more id polypept fusions. For example, a zsig49 polypeptide can be prepared as a fusion for a dimerized protein as described in U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred dimerized proteins in this regard include immunoglobulin constant region domains. The polypeptide fusions of immunoglobulin zsig49 can be expressed in cells genetically engineered to produce a variety of analogs of zyggars. Auxiliary domains can be fused to the zsig49 polypeptides to direct them to specific cells, tissues or macromolecules. For example, a zsig49 polypeptide or protein could be targeted to a predetermined cell type by fusing a zsig49 polypeptide to a ligand that specifically binds a receptor on the surface of the white cell. In this way, the polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A zsig49 polypeptide can be fused to two or more radicals, such as an affinity tag for purification and a targeting domain. The id polypept fusions may also comprise one or more cleavage sites, particularly between the domains. See, Tuan et al., Connective Tissue Research 34: 1-9, 1996. The expressed recombinant zsig49 polypeptides (or chimeric zsig49 polypeptides) can be purified using conventional methods and means of fractionation and / or purification. Precipitation by ammonium sulfate and acid or chaotic extraction could be used for the fractionation of the samples. Examples of the purification steps could include hydroxyapatite, size exclusion, FPLC and high reverse phase liquid chromatography. Suitable anion exchange media include dextrans derivatives, agarose, cellulose, polyacrylamide, special silicas and the like. DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, NJ), PEI, DEAE, QAE and Q derivatives are preferred.

Examples of chromatographic media include media derived with phenyl, butyl or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Appropriate solid supports include glass beads, silica based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like, which are insoluble under the conditions in which they are used. These supports could be modified with reactive groups that allow the binding of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate radicals. Examples of coupling chemicals include activation of cyanogen bromide, activation of N-hydroxysuccinimide, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemicals. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. The methods for joining the receptor polypeptides to the Support means are well known in the art. The selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen medium. See, for example, Affinity Chromat oaraphy: Principies & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The zsig49 polypeptides of the present invention can be isolated by exploitation of their structural characteristics. In one embodiment of the invention there is included a fusion of the polypeptide of interest and an affinity tag (eg, poly id strein, Glu-Glu, FLAG, maltose binding protein, an immunoglobulin domain) that could be constructed to facilitate purification . An example of a purification method of protein constructs having an N-terminal or C-terminal affinity tag produced from mammalian cells, such as BHK cells, involves using an antibody to the epitope of the affinity tag to purify the protein. The SDS-PAGE, Western analysis, amino acid analysis and N-terminal sequencing can be done for the purified protein to confirm its identity. Protein re-folding procedures (and optionally reoxidation) could be advantageously used. It is preferred to purify the protein until > 80% purity, more preferably up to > 90% purity, even more preferably > 95% and a pharmaceutically pure state is particularly preferred, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin. The proteins / polypeptides that bind zsig49 (such as a zsig49 binding receptor) can also be used for the purification of zsig49. The proton / polypeptide of zsig49 binding is immobilized on a solid support, such as agarose beads, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or similar materials that are stable under the conditions of use. Methods for attaching polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting medium, in general, will be configured in the form of a column, and Fluids containing the zsig49 polypeptide are passed through the column one or more times to allow the zsig49 polypeptide to bind to the receptor or ligand binding polypeptide. The bound zsig49 polypeptide is then eluted using changes in saline concentration, chaotropic agents (guanidine HCl) or pH to interrupt binding of the ligand receptor. A test system using a ligand binding receptor (or an antibody, a member of a complement / ant i-complement pair) or a binding fragment thereof, and a commercially available biodetector instrument (BIAcore ™, Pharmacia Biosensor, Piscataway, NJ). Such receptor member, antibody of a complement / ant i-complement pair or fragment is immobilized on the surface of a receptor microcircuit. The use of this instrument is described by Karlsson, J. Immunol. Methods 145: 229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234: 554-63, 1993. A receptor member, antibody or fragment is covalently linked, using the amine or sulfhydryl chemistry, to the dextran fibers that bind to the gold film in the flow cell. A test sample is passed through the cell. If a ligand, Epitope or opposite member of the complement / anticomplement pair is present in the sample, it will bind to the receptor, antibody or immobilized member, respectively, causing a change in the Refractive Index of the medium, which is detected as a change in the surface plasmon resonance of the golden movie. This system allows the determination of the speed of on and off, of which the binding affinity can be calculated, and verification of the binding stoichiometry. As used herein, the term "complement / anticomplement pair" represents non-identical radicals that form a non-covalently associated pair under the appropriate conditions. For example, biotin and avidin (or streptavidin) are prototypic members of a complement / ant i-complement pair. Other compounds of complement / ant i-complement pairs include receptor / ligand pairs, antigen / antigen (or hapten or epitope), sense / antisense polynucleotide pairs, and the like. Where the subsequent dissociation of the complement / anticomplement pair is desirable, the complement / anticomplement pair preferably has a binding affinity of <; 109 M "1. The zsig49 polypeptide and other homologues of the Ligands can also be used within the test systems known in the art. Such systems include Scatchard analysis for the determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric tests (Cunningham et al., Science 253: 545-48, 1991). Cunningham et al., Science 245: 821-25, 1991). The activity of zsig49 polypeptides can be measured by a silicon-based biodetector microphysiometer, which measures the rate of extracellular acidification or proton excretion associated with receptor binding and subsequent physiological cellular responses. An example of a device is the Cytosensor ™ microphysiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell et al., Science 257: 1906-12, 1992; Pitchford et al., Meth. Enzvmol 228: 84-108, 1997; Arimilli et al., J. Immunol. Meth. 212: 49-59. 1998; Van Liefde et al., Eur. J. Pharmacol. 346: 87-95, 1998. The microphysiometer can be used to test the adherence or non-adherence of eukaryotic cells or prokaryotes. By measuring the changes of extracellular acidification in a cellular medium over time, the microphysiometer directly measures the cellular responses to various stimuli, including zsig49 polypeptide, its agonists or antagonists. Preferably, the microphysiometer is used to measure the responses of a eukaryotic cell responsive to zsig49, compared to a control eukaryotic cell that does not respond to the zsig49 polypeptide. Eukaryotic cells sensitive to zsig49 comprise cells in which a receptor for zsig49 has been transfected; or cells naturally responsive to zsig49, such as cells derived from pancreatic tissue. The differences, measured by a change, for example, an increase or decrease in extracellular acidification, in the response of cells exposed to the zsig49 polypeptide, relative to a control, are a direct measurement of the cellular responses modulated by zsig49. In addition, such responses modulated by zsig49 can be tested under a variety of stimuli. Using the microphysiometer, there is provided a method for identifying agonists of the zsig49 polypeptide, which comprises providing cells responsive to a zsig49 polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound and detecting a change, e.g., an increase or decrease, in a cellular response of the second portion of the cells compared to the first portion of the cells . The change in cellular response is shown as a measurable change in the rate of extracellular acidification. In addition, culturing a third portion of the cells in the presence of the zsig49 polypeptide and in the absence of a test compound, can be used as a positive control for the zsig49 sensitive cells, and as a control to compare the agonist activity of a test compound. with such zsig49 polypeptide. In addition, using the microphysiometer, there is provided a method for identifying zsig49 polypeptide antagonists, which comprises providing cells responsive to a zsig49 polypeptide, culturing a first portion of the cells in the presence of zsig49 and in the absence of a test compound, culturing a second portion of the cells in the presence of zsig49 and in the presence of a test compound and detect a change, for example, an increase or decrease in a cellular response of the second portion of the cells compared to the first portion of the cells. The change in Cell response is shown as a measurable change in the rate of extracellular acidification. Antagonists and agonists, for the zsig49 polypeptide, can be quickly identified using this method. In addition, zsig49 can be used to identify cells, tissues or cell lines that respond to a path stimulated by zsig49. The microphysiometer, described above, can be used to rapidly identify ligand-sensitive cells, such as the zsig49-sensitive cells of the present invention. The cells can be cultured in the presence or absence of the zsig49 polypeptide. These cells that cause a measurable change in extracellular acidification in the presence of zsig49 are sensitive to zsig49. Such cell lines can be used to identify antagonists and agonists of the zsig49 polypeptide as described above. The nucleic acid molecules described herein can be used to detect the expression of a zsig49 gene in a biological sample. Such probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequences of SEQ ID NOS: 1, 4, 9, 11, 12, 14 or fragments thereof, as well as single-stranded nucleic acid molecules that they have the complement of the sequences nucleotides of SEQ ID NOS: 1, 4, 9, 11, 12, 14 or a fragment thereof. The probe molecules could be DNA, RNA, oligonucleotides and the like. In a basic test, a single-stranded probe molecule is incubated with RNA, isolated from a biological sample, under the conditions of temperature and ionic strength that promote base pairing between the probe and the white zsig49 RNA species. After separating the unbound probe from the hybridized molecules, the amount of hybrids is detected. Well-established hybridization methods of RNA detection include northern analysis and dot / slot blot hybridization (see, for example, Ausubel ibid. And Wu et al. (Eds.), "Analysis of Gene Expression at the RNA Level," in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can be labeled in detectable form with radioisotopes, such as 32P or 35S. Alternatively, zsig49 RNA can be detected with a non-radioactive hybridization method (see, eg, Isaac (ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Humana Press, Inc., 1993). Typically, non-radioactive detection is achieved by the enzymatic conversion of chromogenic or chemiluminescent substrates. Radicals do not Illustrative radioactive agents include biotin, fluorescein and digoxigenin. Oligonucleotide probes zsig49 are also useful for diagnosis in vivo. As an illustration, labeled 18F oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al., Nature Medicine 4: 467, 1998). Numerous diagnostic procedures take advantage of the polymerase chain reaction (PCR) to increase the sensitivity of detection methods. Standard techniques for carrying out PCR are well known (see, in general, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and alaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), (ed.), Clinical Applications of PCR (Humana Press, Inc. 1998) and Meltzer (ed.), PCR in Bioanalvsis (Humana Press, Inc. 1998)). PCR primers can be designed to amplify a sequence encoding a particular zsig49 domain or region. A PCR variation for the tests of Diagnosis is reverse transcriptase-PCR (RT-PCR). In the RT-PCR technique, the RNA is isolated from a biological sample, reverse transcribed to cDNA and the cDNA is incubated with zsig49 primers (see, for example, Wu et al. (Eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR ", in Methods in Gene Biotechnology, CRC Press, Inc., pages 15-28, 1997). PCR is then carried out and the products are analyzed using standard techniques. As an illustration, the RNA is isolated from the biological sample using, for example, the guanidinium thiocyanate cell lysis procedure described above. Alternatively, a solid phase technique can be used to isolate the mRNA from a cell-used. A reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short dT homopolymers or zsig49 anti-sense oligomers. The oligo-dT primers offer the advantage that several idlic mRNA nucleotide sequences that can provide the control target sequences are amplified.

The zrnpl sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically at least 5 bases in length.

PCR amplification products can be detected using a variety of methods. For example, the PCR products can be fractionated by gel electrophoresis and visualized by staining ethidium bromide. Alternatively, fractionated PCR products can be transferred to a membrane, hybridized with a detectably labeled zsig49 probe and examined by autoradiography. Additional alternative methods include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide detection by chemiluminescence, and the C-TRAK colorimetric test. Another method is real-time quantitative PCR (Perkin-Elmer Cetus, Norwalk, Ct.). A fluorogenic probe, consisting of an oligonucleotide with a reporter and a quenching dye attached, hybridize between the forward and reverse primers. Using the 5 'endonuclease activity of the labeled DNA polymerase, the reporter dye is separated from the quencher dye and a sequence specific signal is generated and increases as the amplification increases. The intensity of the fluorescence can be continuously monitored and quantified during the PCR reaction. Another method for detecting the expression of zsig49 is the cyclization probe (CPT) technology, in which a single-stranded DNA target is linked to an excess of the chimeric DNA-RNA-DNA probe to form a complex, the RNA portion is cut with Rnasa H, and the presence of the cut chimeric probe is detected (see, for example Beggs et al., J Clin Clinical Microbiol 34: 2985, 1996 and Bekkaoui et al., Biotechniques 20: 240, 1996). Alternative methods for the detection of zsig49 sequences can use methods, such as amplification based on nucleic acid sequence (NASBA), cooperative amplification of the templates by cross-hybridization (CATCH), and the ligase chain reaction (LCR ) (see, for example, Marshall et al., US Patent No. 5,686,272 (1997), Dyer et al., J. Virol. Methods 6.0: 161, 1996; Ehricht et al., Eur. J. Biochem. 243: 358, 1997 and Chadwick et al., J. Virol. Methods 7 __: 59, 1998). Other standard methods are known to those skilled in the art. The probes and primers of zsig49 can also be used to detect and localize the gene expression of zsig49 in tissue samples. Methods for such hybridization i n s i t u are well known to those skilled in the art (see, for example, Choo (ed.), I_n Situ Hybridizat ion Protocols, Humana Press, Inc., 1994, Wu et al., (Eds.), "Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Si tu Hybridi za t ion (RISH)", in Methods in Gene Biot echnology, CRC Press, Inc., pages 259 -278, 1997 and Wu et al. (eds.), "Localization of DNA or Abundance of mRNA by Fluorescence In Si t u Hybridization (RISH)", in Methods in Gene Biotechnology, CRC Press, Inc., pages 279-289, 1997). In another embodiment, the present invention provides methods for detecting in a sample from an individual, an abnormality on chromosome 1 associated with a disease, comprising the steps of: (a) contacting nucleic acid molecules of the sample with a nucleic acid probe that hybridizes with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, 9 or 12, its complements or fragments, under severe conditions, and (b) detecting the presence or absence of the hybridization of the probe with the nucleic acid molecules in the sample, wherein the absence of hybridization is indicative of an abnormality on chromosome 1, such as an abnormality that causes a defective glucose metabolism. The present invention also provides methods for detecting in a sample of an individual, a abnormality in the zsig49 gene associated with a disease, comprising: (a) isolating the nucleic acid molecules encoding zsig49 from the sample, and (b) comparing the nucleotide sequence of the isolated zsig49-encoding sequence with the nucleotide sequence of the SEQ ID NOS: 1, 9 or 12, wherein the difference between the sequence of the isolated zsig49 coding sequence or a polynucleotide encoding the zsig49 polypeptide generated from the isolated zsig49 coding sequence and the nucleotide sequences of SEQ ID NOS: 1, 9 or 12, is indicative of an abnormality in the zsig49 gene associated with disease or susceptibility to a disease in an individual, such as a defective glucose metabolism or diabetes. The present invention also provides methods for detecting in a sample from an individual, an abnormality in the expression of the zsig49 gene associated with disease or disease susceptibility, comprising: (a) obtaining zsig49 RNA from the sample, (b) generating zsig49 cDNA by polymerase chain reaction of zsig49 RNA and (c) comparing the nucleotide sequence of zsig49 cDNA to the nucleotide sequence of SEQ ID NOS: 1, 9 or 12, wherein a difference between the cDNA sequence of zsig49 and the nucleotide sequence of SEQ ID NO: 1, 9 or 12 is indicative of an abnormality in the expression of the zsig49 gene associated with disease or susceptibility to disease. In the additional modalities, the disease is defective glucose metabolism or diabetes. In other aspects, the present invention provides methods for detecting in a sample from an individual, an abnormality in the zsig49 gene associated with a disease, comprising: (a) contacting the nucleic acid molecules of the sample with a DNA probe. nucleic acid, wherein the probe hybridizes to a nucleic acid molecule having the nucleotide sequence of SEQ ID NOS: 1, 9 or 12, its complements or fragments, under severe conditions, and (b) detecting the presence or absence of Hybridization is indicative of an abnormality of zsig49. The absence of hybridization of the probe is associated with defective glucose metabolism. Hybridization i n s i t u provides another method to identify abnormalities of the zsig49 gene. According to this method, a zsig49 probe is labeled with a detectable label by a method known in the art. For example, the probe can be directly marked by random priming, end marking, PCR or translation of cut. Appropriate direct labels include radioactive labels, such as 32P, 3H and 35S and non-radioactive labels, such as fluorescent labels (eg, fluorescein, Texas Red, AMCA blue (7-amino-4-met-il-coumanin-3-acetate) , phosphorus yellow, rhodamine, etc.), cyanine dyes that are detectable with visible light, enzymes and the like. Probes labeled with an enzyme can be detected by means of a colorimetric reaction providing a substrate for the enzyme. In the presence of several substrates, different colors are produced by the reaction, and these colors can be visualized to separately detect multiple probes if desired. Suitable substrates for alkaline phosphatase include 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. A preferred substrate for horseradish peroxidase is diaminobenzoate. An illustrative method for detecting chromosomal abnormalities with hybridization i n s i t u is described by Wang et al., U.S. Pat. No. 5,856,089. Following this method, for example, a method for performing i n s i t u hybridization with a zsig49 probe to detect an abnormality of the chromosomal structure in a cell of a sample of Fixed tissue obtained from a patient, suspected of having a metabolic disease may comprise the steps of: (1) obtaining a fixed tissue sample from the patient, (2) pre-treating the fixed tissue sample obtained in step (1) ) with a bisulfite ionic composition, (3) digest the fixed tissue sample with proteinase, (4) perform the hybridization insitu in the cells obtained from the digested fixed tissue sample from step (3) with a probe that hybridizes specifically to the zsig49 gene, where a signal pattern of the hybridized probes is obtained, (5) comparing the signal pattern of the probe hybridized in step (4) with a predetermined signal pattern of the hybridized probe obtained when carrying performed the insitu hybridization on the cells that have a region of interest of the normal critical chromosome and (6) detecting a structural abnormality of the chromosome in the patient's cells, detecting a difference between the signal pattern l obtained in step (4) and the predetermined signal pattern. Examples of zsig49 gene abnormalities include deletions, amplifications, translocations, reversals, and imimers. The present invention also contemplates kits for performing a diagnostic test for the expression of the zsig49 gene or to detect mutations in the zsig49 gene. Such kits comprise nucleic acid probes, such as double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NOS: 1, 9 or 12 or a portion thereof, as well as single-stranded nucleic acid molecules that they have the complement of the nucleotide sequence of SEQ ID NOS: 1, 9 or 12 or a portion thereof. The probe molecules could be DNA, RNA, oligonucleotides and the like. The kits may comprise nucleic acid primers for performing the PCR or oligonucleotides to perform the ligase chain reaction. Preferably, such a kit contains all the elements necessary to perform a diagnostic test of the nucleic acid described above. A kit will comprise at least one container comprising a probe or primer of zsig49. The kit could also comprise a second container comprising one or more reagents capable of indicating the presence of the zsig49 sequences. Examples of such indicator reagents include detectable labels such as radioactive labels, fluoroerases, chemiluminescent agents and the like. A case could also comprise a means for carrying to the user such probes and primers of zsig49 that are used to detect the gene expression of zsig49. For example, written instructions could establish that surrounded nucleic acid molecules can be used to detect, either a nucleic acid molecule encoding zsig49 or a nucleic acid molecule having a nucleotide sequence that is complementary to a nucleotide sequence encoding zsig49. The written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert. Several additional diagnostic methods are well known to those skilled in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics Humana Press, Inc., 1991).; Coleman and Tsongalis, Molecular Diaqnostics, Humana Press, Inc., 1996 and Elles, Molecular Diagnosis of Genetic Diseases, Humana Press, Inc., 1996). The invention also provides anti-i-zsig49 antibodies. Antibodies to zsig49 can be obtained, for example, by using as an antigen the product of a zsig49 expression vector, or zsig49 isolate from a natural source. Anti-zsig49 antibodies particularly useful "bind specifically" with zsig49. The antibodies are considered to bind specifically if the antibodies bind to a zsig49 polypeptide, peptide or epitope with a binding affinity (Ka) of 106 M "1 or greater, preferably 107 or greater, more preferably 108 M" 1 or more. greater and more preferably 109 or greater. The binding affinity of an antibody can be readily determined by one skilled in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 5: 660, 1949). Suitable antibodies include antibodies that bind to zsig49 in particular domains. Anti-zsig49 antibodies can be produced using peptides and polypeptides that carry the antigenic zsig49 epitope. Peptides and polypeptides carrying the antigenic zsig49 epitope of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained in SEQ ID NOS: 2, 10 or 13. However, the peptides and polypeptides that comprise a higher portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids or any longer length and including the complete amino acid sequence of a polypeptide of the invention, are also useful for inducing antibodies that bind with zsig49. It is desirable that the amino acid sequence of the epitope-bearing peptide be selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophobic residues, whereas hydrophobic residues are preferably avoided). In addition, amino acid sequences containing proline residues may also be desirable for the production of the antibody. Polyclonal antibodies to the recombinant zsig49 protein or to zsig49 isolated from natural sources can be prepared using methods well known to those skilled in the art. See, for example, Green et al., "Production of Polyclonal Ant i sera," in Immunochemical Protocols (Manson, ed.), Pages 1-5 (Humana Press 1992) and Williams et al. , "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies", in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995). The immunogenicity of a zsig49 polypeptide can be increased by the use of an adjuvant, such as alum (aluminum hydroxide) or complete or incomplete Freund's adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as zsig49 fusions or a portion thereof with an immunoglobulin polypeptide or with the maltose binding protein. The idiogenic polypeptide immunogen could be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such a portion could advantageously bind or bind to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization. Although polyclonal antibodies typically originate in animals, such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, hamsters, goats or sheep, an anti-zsig49 antibody of the present invention could also be derived from a sub-human primate antibody. General techniques for diagnostically and therapeutically originating antibodies useful in baboons could be found, for example, in Goldenberg et al., International Patent Publication No. WO 91/11465 and Losman et al., Int. J. Cancer 46: 310, 1990. Antibodies may also originate in transgenic animals, such as sheep, cows, goats or transgenic pigs. The antibodies can also be expressed in yeast and fungi in the modified forms, as well as in mammalian and insect cells. Alternatively, monoclonal zsig49 antibodies can be generated. Rodent monoclonal antibodies to specific antigens could be obtained by methods known to those skilled in the art (see, for example, Kohler et al., Nature 256: 495, 1975, Colligan et al. (Eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & amp; amp; amp; amp.; Sons 1991), Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli", in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.) page 93 (Oxford University Press 1995)). Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising a zsig49 gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing B lymphocytes with cells of myeloma to produce hybridomas, cloning of hybridomas, selected positive clones that produce antibodies for the antigen, growing the clones that produce antibodies for the antigen and isolating the antibodies from the cultures of the hybridoma. In addition, an anti-zsig49 antibody of the present invention could be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice that have been designed to produce human-specific antibodies in response to antigenic challenge. In this technique, the human heavy and light chain locus elements are introduced into strains of mice derived from the embryonic root cell lines containing white breaks of the endogenous heavy chain and the light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, and mice can be used to produce hybridomas that secrete human antibodies. Methods for obtaining human antibodies from the transgenic mice are described, for example, by Green et al., Nature Genet. 7:13, 1994, Lonberg et al., Nature 368: 856, 1994 and Taylor et al., Int. Immun. 6.579, 1994. Monoclonal antibodies can be isolated and purify from hybridoma cultures using a variety of well-established techniques. Such isolation techniques include affinity chromatography with protein A sepharose, size exclusion chromatography, and ion exchange chromatography (see, for example, Coligan on pages 2.7.1-2.7.12 and pages 2.9.1-2.9. 3; Baines et al., "Purification of Immunoglobulin G (IgG)", in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)). For particular uses, it may be desirable to prepare fragments of anti-zsig49 antibodies. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be obtained by digestion of pepsin or papain whole antibodies by conventional methods. As an illustration, antibody fragments can be produced by enzymatic cleavage of the antibodies with pepsin, to provide a 5S fragment represented by F (ab ') 2. This fragment can be further cut using a thiol reducing agent to produce the 3.5S Fab 'monovalent fragments. Optionally, the cutting reaction can be carried out using a blocking group for the sulfhydryl groups which result from the cutting of disulfide bonds. As an alternative, an enzymatic cut using pepsin produces two monovalent Fab fragments and one Fe fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophvs. 8_9: 230, 1960, Porter, Biochem. J. 73: 119, 1959, Edelman et al., In Methods in Enzymology Vol. 1, page 422 (Academic Press 1967) and by Coligan, ibid. Other methods for cutting antibodies, such as heavy chain separation, to form fragments of heavy to light monovalent chains, additional fragment cutting or other enzymatic, chemical or genetic techniques, could also be used, provided the fragments are attached to a antigen that is recognized by the intact antibody. For example, the Fv fragments comprise an association of VH and VL chains. This association may be non-covalent, as described by Inbar et al., Proc. Nat. Acad. Sci. USA 6: 959, 1972. Alternatively, the variable chains can be linked by an intercellular disulfide bond or chemical crosslink, such as gluteraldehyde (see, eg, Sandhu, Crit. Rev. Biotech 12: 437, 1992 ).

The Fv fragments could comprise VH and VL chains which are connected by a peptide linker. These single chain antigen (scFv) binding proteins are prepared by constructing a structural gene comprising the DNA sequences encoding the VH and VL domains that are connected by an oligonucleotide. The structural gene is inserted into an expression vector that is subsequently introduced into a host cell, such as E. col i. The recombinant host cells synthesize a simple polypeptide chain with a linker peptide that binds the two V domains. Methods for producing scFv are described, for example, by Whitlow et al., Methods: A Companion to Methods in Enzymology 2: 97, 1991, see also, Bird et al., Science 242: 423, 1988, Ladner et al., US Patent. No. 4,946,778, Pack et al., Bio / Technolosv 11: 1271, 1993 and Sandhu, s upra.

As an illustration, an svFv can be obtained by exposing lymphocytes to the zsig49 polypeptide in vi t ro, and selecting libraries of antibody exposure in the phage or similar vectors (e.g., through the use of immobilized or labeled protein or zsig49 peptide). ). The genes that encode the polypeptides that have domains of Potential zsig49 polypeptide binding can be obtained by screening random peptide libraries exposed in the phage (phage display) or in bacteria, such as E. co l i. The nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as by means of random mutagenesis synthesis and random polynucleotide. These random peptide exposure libraries can be used to screen peptides that interact with a known blank which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide exposure libraries are known to those skilled in the art (Ladner et al., US Patent No. 5,223,409, Ladner et al., US Patent No. 4,946,778, Ladner et al., Patent. US No. 5,403,484, Ladner et al., US Patent No. 5,571,698 and Kay et al., Phage Display of Peptides and Proteins (Academic Press, Inc. 1996)) and randomized peptide exposure libraries and kits for screening such libraries. are commercially available, for example from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide exposure libraries can be screened using the zsig49 sequences described herein, to identify proteins that bind to zsig49. Another form of an antibody fragment is a peptide encoding a simple complementary determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, using the polymerase chain reaction to synthesize the variable region of the RNA of the cells that produce antibodies (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2 .: 106, 1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies", in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.) page 166 (Cambridge University Press 1995), and Ward et al., "Genetic Manipulation and Expression of Antibodies", in Monoclonal Antibodies: Principies and Applications, Birch et al., (eds.), page 137 (Wiley -Liss, Inc. 1995)). Alternatively, an anti-zsig'49 antibody it could be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determinant regions of heavy and light variable chains of mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counterparts. The use of the antibody components derived from the humanized monoclonal antibodies obviates the potential problems associated with the immunogenicity of the murine constant regions. General techniques for the cloning of murine immunoglobulin variable domains are described, for example, by Orlandi et al., Proc. Nat. Acad. Sci. USA 8_6: 3833, 1989. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321: 522, 1986, Carter et al., Proc. Nat. Acad. Sci. USA 89: 4285, 1992, Sandhu, Crit. Rev. Biotech. 12: 437, 1992, Singer et al., J. Immun. 150: 2844, 1993, Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc. 1995), Kelley, "Engineering Therapeutic Antibodies", in Protein Engineering: Principies and Practice, Cleland et al. (eds.), pages 399-434 (John Wiley & Sons, Inc. 1996) and by Queen et al., U.S. Pat. No. 5, 693, 762 (1997). Polyclonal anti-idiotypic antibodies can be prepared by immunizing animals with anti-zsig49 antibodies or antibody fragments, using standard techniques. See, for example, Green et al., "Production of Polyclonal Antisera," in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), Pages 1-12 (Humana Press 1992). Also, see Coligan, ibid. on pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotypic antibodies can be prepared using anti-zsig49 antibodies or antibody fragments as immunogens with the techniques described above. As another alternative, humanized anti-idiotypic antibodies or sub-human primate anti- idiotypic antibodies can be prepared using the techniques described above. Methods for producing anti-i-idiotypic antibodies are described, for example, by Irie, U.S. Pat. No. 5,208,146, Greene et al., U.S. Pat. No. 5,637,677 and Varthakavi and Minocha, J. Gen. Virol. 77: 1875. 1996. A variety of tests known to those skilled in the art can be used to detect antibodies and binding proteins that are they bind specifically to zsig49 proteins or peptides. Examples of tests are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such tests include: immunoelect rofores is common, radioimmunoassay, radioimmunoprecipitation, immunoabsorbent test Enzyme linked (ELISA), Dot blot or Western blot, inhibition or competition test and the overlap test. In addition, the antibodies can be screened for binding to the wild-type mutant anti-zsig49 protein or peptide. Antibodies to zsig49 could be used to label cells expressing zsig49 polypeptide; for isolating the zsig49 polypeptide by affinity purification; for diagnostic tests to determine the circulation levels of zsig49 polypeptides; to detect or quantify the soluble zsig49 polypeptide as a marker of the pathology or occult disease; in the analytical methods that use FACS; to screen libraries of expression; to generate anti-idiotypic antibodies and as neutralizing antibodies or as antagonists for the activity associated with zsig49 in vitro and in vi. The direct marks or labels Suitable include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; Indirect labels or tags could characterize the use of ina / avidin biot pairs or other complement / ant i-complement pairs as intermediaries. The antibodies could also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, and these conjugates are used for diagnostic or therapeutic applications. The present invention contemplates the use of anti-zsig49 antibodies to screen biological samples i n vi t ro for the presence of zsig49. In one type of viral test, anti-zsig49 antibodies are used in the liquid phase. For example, the presence of zsig49 in a biological sample can be tested by mixing the biological sample with a trace amount of labeled zsig49 and an anti-zsig49 antibody under conditions that promote binding between zsig49 and its antibody. The zsig49 and anti-zsig49 complexes in the sample can be separated from the reaction mixture by contacting the complex with an immobilized protein that binds to the antibody, such as an antibody to Fe or protein A from S t aphyl or co cc us The The concentration of zsig49 in the biological sample will be inversely proportional to the amount of labeled zsig49 bound to the antibody and directly related to the amount of free labeled zsig49. Although anti-rat zsig49 or human antibodies can be used to detect zsig49, human anti-zsig49 antibodies are preferred for human diagnostic tests. In vitro tests may also be performed, in which the anti-zsig49 antibody binds to a solid phase vehicle. For example, the antibody can be attached to a polymer, such as aminodexthan, to bind the antibody to an insoluble support, such as a polymer-coated bead, dish or tube. Other appropriate in vi t ro tests will be readily apparent to those skilled in the art. In another method, anti-zsig49 antibodies can be used to detect zsig49 in tissue sections prepared from a biopsy specimen. Such immunochemical detection can be used to determine the relative abundance of zsig49 and determine the distribution of zsig49 in the tissue examined. The general immunochemical techniques are well established (see, for example, Ponder, "Cell Marking Techniques and Their Application", in Mammalian Development: A Practical Approach, Monk (ed.), Pages 115-38 (IRL Press 1987), Coligan on pages 5.8.1- 5.8.8, Ausubel (1995) on pages 14.6.1 to 14.6.13 (Wiley Interscience 1990) and Manson (ed.), Methods In Molecular Biology, Vol. 10: Immunochemical Protocols (The Humana Press, Inc. 1992)). Immunochemical detection can be performed by contacting a biological sample with an anti-zsig49 antibody, and then contacting the biological sample with a det ectably labeled molecule that binds to the antibody. For example, the detectably labeled molecule may comprise an antibody moiety that binds to the anti-zsig49 antibody. Alternatively, the anti-zsig49 antibody can be conjugated with avidin / is treptavidin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin / streptavidin). Numerous variations of this basic technique are well known to those skilled in the art. Alternatively, an anti-zsig49 antibody can be conjugated with a detectable label to form an anti-zsig49 immunoconjugate. Appropriate detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme marker, a bioluminiscent marker or colloidal gold. Methods for making and detecting such detectably labeled immunoconjugates are well known to those skilled in the art, and are described in more detail below. The detectable marker can be a radioisotope that is detected by autoradiography. Isotopes that are particularly useful for the purpose of the present invention are 3 H, 125 I, 131 I, 35 S, 1 C and the like. Anti-zsig49 immunoconjugates can also be labeled with a fluorescent compound. The presence of a fluorescently labeled antibody is determined by exposure of the immunoconjugate to light of the appropriate wavelength and the resulting fluorescence is detected. Fluorescent marker compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and luorescamine. Alternatively, anti-zsig49 immunoconjugates can be detectably labeled by coupling a component of the antibody to a chemiluminescent compound. The presence of the immunoconjugate marked by chemiluminescence is determined by detecting the presence of the luminescence that originates during the course of a chemical reaction. Examples of the compounds marked by chemiluminescence include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester. Similarly, a bioluminescent compound can be used to label the anti-zsig49 immunoconjugates of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems, in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds that are useful for labeling include luciferin, luciferase and ecuorin. Alternatively, anti-zsig49 immunoconjugates can be detectably labeled by binding an anti-zsig49 antibody component to an enzyme. When the antisense-enzyme conjugate is incubated in the presence of the appropriate substrate, the enzyme radical reacts with the substrate to produce a chemical radical that can be detected, for example by means of spectrophotometry, fluoromethyl or visual media. Examples of enzymes that can be used to detectably label immunoconjugates Polyspecific lobes include β-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase. Those skilled in the art will know other suitable markers that may be employed in accordance with the present invention. The binding of the marker radicals to the anti-ZSIG49 antibodies can be carried out using standard techniques known in the art. The typical methodology in this aspect is described by Kennedy et al., Clin. Chim. Acta 70: 1, 1976), Schurs et al., Clin. Chim. Acta 1: 1, 1977, Shih et al., Int. J. Cancer 46: 1101, 1990, Stein et al., Cancer Res. 50: 1330, 1990 and Coligan, supra. In addition, the convenience and versatility of immunochemical detection can be enhanced by using anti-zsig49 antibodies that have been conjugated to avidin, streptavidin and biotin (see, for example, Wilchek et al., (Eds.), "Avidin-Biot in Technology. ", Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al.," Immunochemical Applications of Avidin-Biot in Technology ", in Methods in Molecular Biology, Vol. 10 Manson (ed.), Pages 149- 162 (The Humana Press, Inc. 1992) The methods for performing immunoprotes are well established, see, for example, Cook and Self, "Monoclonal Ant ibodies in Diagnostic Immunoassays", in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter and Ladyman (eds.), Pages 180-208, (Cambridge University Press, 1995), Perry, "The Role of Monoclonal Antibodies in the Advancement of Immunoassay Technology", in Monoclonal Antibodies : Principies and Applications, Birch and Lennox (eds.), Pages 107-120 (Wiley-Liss, Inc. 1995) and Diamandis, Immunoassay (Academic Press, Inc. 1996). Biological samples suitable for the detection of the zsig49 protein include cells, tissues or body fluids, such as urine, saliva or blood.

In a related method, anti-zsig49 antibodies labeled with biotin or FITC can be used to identify cells that link zsig49. Such binding can be detected, for example, using flow cytometry. The present invention also contemplates kits for carrying out an immunological diagnostic test for the zsig49 expression gene. Such kits comprise at least one container comprising an antibody or anti-zsig49 antibody fragment. A kit could also comprise a second container comprising one or more reagents capable of indicating the presence of the antibody or zsig49 antibody fragments. Examples of such Indicator reagents include detectable labels, such as a radioactive label, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, colloidal gold, and the like. A kit could also comprise a means for bringing to the user such antibodies or fragments of zsig49 antibodies that are used to detect the zsig49 protein. For example, written instructions that could establish such an antibody or enclosed antibody fragment can be used to detect zsig49. The written material can be applied directly to a container or the written material can be provided in the form of a packaging insert. The molecules of the present invention can be used to identify the isolated receptors that bind zsig49. For example, the proteins and peptides of the present invention can be immobilized on a column and membrane preparations run on the column (Immobili zed - Af f ini and Ligand Techniques, Hermanson et al., Eds., Academic Press, San Diego, CA , 1992, pp. 195-202). Proteins and peptides can also be radiolabelled (Mehods in Enzymol., Vol.182, "Guide to Protein Purification", Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or marked by photoaffinity (Brunner et al., Ann.Rev. Biochem. 62: 483-514, 1993 and Fedan et al., Biochem Pharmacol. 3_3: 1167-80, 1984) and the specific cell surface proteins can be identified. . For pharmaceutical use, pharmaceutically effective amounts of zsig49 therapeutic antibodies, zsig49 polypeptide small molecule antagonists or agonists or zsig49 polypeptide fragments can be formulated with pharmaceutically acceptable carriers for parenteral, oral, nasal, rectal administration, Topical, transdermal or similar, according to conventional methods. The formulations could further include one or more diluents, fillers, emulsions, preservatives, buffers, excipients and the like, and could be provided in such forms as for example liquids, powders, emulsions, suppositories, liposomes, ransdermic patches and tablets. . Slow or extended release systems, which include any number of biopolymers (biological based systems), systems employing liposomes and polymeric delivery systems, can also be used with the compositions described herein to provide a continuous or long-term source of the polypeptide, agonist or antagonist of zsig49. Such slow release systems are applicable to formulations, for example, for oral, topical and parenteral use. The term "pharmaceutically acceptable carrier or vehicle" refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredients and that is not toxic to the host or patient. One skilled in the art could formulate the compounds of the present invention in the proper manner, and in accordance with accepted practices, such as those described in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed. , 1995. As used herein, a pharmaceutically effective amount of a zsig49 polypeptide, agonist or antagonist is an amount sufficient to induce a desired biological result. The result can be the relief of the signs, symptoms or causes of a disease or any other desired alteration of a biological system. For example, an effective amount of a polypeptide of the present invention is that which provides either subjective relief of symptoms or an objectively identified improvement as observed by the physician or other qualified observer. In in particular, such an effective amount, if administered to a patient suffering from diabetes, results in a decrease in glucose levels, prevention or significant delay in onset of the disease or loss of islet infiltration in NOD mice or other beneficial effect. The dosages of the zsig49 polypeptide, in general, will be determined by the physician according to the accepted standards, taking into account the nature and severity of the condition to be treated, the patient's features, etc. The determination of the dosage is within the level of the expert in the art. The proteins could be administered for acute treatment, for a week or less, frequently over a period of one to three days or could be used in chronic treatment, for several months or years. The zsig49 polypeptides that encode the polynucleotides are useful within gene therapy applications where it is desired to increase or inhibit the activity of zsig49. If a mammal has a mutated or absent zsig49 gene, the zsig49 gene can be introduced into mammalian cells. In one embodiment, a gene encoding a zsig49 polypeptide is introduced into a viral vector. Such vectors include an attenuated DNA virus or defective, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV) and the like. Defective viruses, which completely or almost completely lack viral genes, are preferred. A defective virus is not infectious after introduction into a cell. The use of defective viral vectors allows administration to cells in a specific, localized area, without implying that such a vector can infect other cells. Examples of particular vectors include, but are not limited to, a vector of defective herpes simplex virus 1 (HSV1) (Kaplitt et al., Molec.Cell.Neurosci.2_: 320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90: 626-30, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol., 61: 3096-101, 1987; Samulski et al., J. Virol., 63: 3822-8, 1989). In another embodiment, a zsig49 gene can be introduced into a retroviral vector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346; Mann et al., Cell 33: 153, 1983; Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol. 2: 1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al., And Kuo et al., Blood 82: 845, 1993. Alternatively, the vector can be introduced by lipofection in vi vo using liposomes . Synthetic cationic lipids can be used to prepare liposomes for the transfection of a gene encoding a marker (Felgner et al., Proc. Nat 1. Acad. Sci. USA 84: 7413-7, 1987; Mackey et al. , Proc. Nati, Acad. Sci. USA 85: 8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs has certain practical advantages. The molecular target of liposomes to specific cells represents an area of benefit. More particularly, transfection directed towards particular cells represents an area of benefit. For example, transfection directed toward particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney and brain. The lipids could be chemically coupled to other molecules for the purpose of target. White peptides (e.g., hormones or neurotransmitters), proteins, such as antibodies or non-peptidic molecules can be coupled chemically to liposomes. It is possible to remove white cells from the body; to introduce the vector as a naked DNA plasmid; and then re-implant the transformed cells in the body. DNA vectors discovered for gene therapy can be introduced into the desired host cells by methods known in the art, eg, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gun of genes or use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem. 267: 963-7, 1992; Wu et al., J. Biol. Chem. 263: 14621-4, 1988. The antisense methodology can be used to inhibit the translation of the zsig49 gene, such as inhibiting cell proliferation. Polynucleotides that are complementary to a segment of a polynucleotide encoding zsig49 (eg, a polynucleotide as set forth in SEQ ID NOS: 1, 9 or 12), are designed to bind to the mRNA encoding zsig49 and to inhibit the translation of such mRNA. Such antisense polynucleotides are used to inhibit the expression of the genes encoding the zsig49 polypeptide in the cell culture or in a subject.

Transgenic mice, designed to express the zsig49 gene and mice that exhibit a complete absence of zsig49 gene function, referred to as "missing mice" (Snouwaert et al., Science 257: 1083, 1992), could also be generated (Lowell et al., Nature 366: 740-42, 1993). These mice could be used to study the zsig49 gene and the encoded protein used by a viral system. The invention is further illustrated by the following non-limiting examples.

EXAMPLES Example 1 Identification of the zsig49 cDNA sequence The polynucleotides encoding the zsig49 polypeptide of the present invention were initially identified by consulting an EST database for secretory signal sequences characterized by a 5 'side methionine start site, a hydrophobic region of about 13 amino acids and a cleavage site (SEQ ID NO: 3, wherein the cut occurs between the alanine and the glycine amino acid residues), in an effort to select the secreted proteins. The polypeptides corresponding to ESTs that meet the criteria of search, were compared with the known sequences to identify the secreted proteins that have homology with the known ligands. An EST sequence was discovered and determined to be new. The EST sequence was from an islet cell library. To identify the corresponding cDNA, a considered clone that probably contained the entire coding sequence was used for sequencing. Using a S.N.A.P. ™ Miniprep kit from Invitrogen (Invitrogen, Corp., San Diego, CA) according to the manufacturer's instructions, a 5 ml culture was prepared overnight in LB + 50 μg / ml ampicillin. The template was sequenced in an ABIPRISM ™ DNA model 377 sequencer (Perkin-Elmer Cetus, Norwalk, Ct.) Using the ABI PRISM ™ (Perkin-Elmer Corp.) dye-terminator cycle reading reaction kit. according to the manufacturer's instructions. The sequencing reactions were carried out in a Hybaid Omnigene Temperature Cycling system (National Labnet Co., Woodbridge, NY). The programming element of SEQUENCHER ™ 3.1 sequence analysis (Gene Codes Corporation, Ann Arbor, MI) was used for the analysis of the results. The resulting 952 bp sequence is described in SEQ ID NO: 1.

Example 2 Tissue distribution Northerns analyzes were performed using Human Multiple Tissue Blots from Clontech (Palo Alto, CA). A probe of approximately 120 bp (SEQ ID NO: 5) of the clone described above in Example 1 was amplified. The oligonucleotide primers ZC14887 (SEQ ID NO: 5) and ZC16136 (SEQ ID NO: 6) were used to amplify the sequence of the probe in a polymerase chain reaction as follows: 1 cycle at 95 * C for 1 minute; 35 cycles of 95'C for 30 seconds, 50'C for 30 seconds and 72 * C for 30 seconds, followed by a 2-minute extension at 72'C. The resulting DNA fragment was subjected to electrophoresis on 1% agarose gel (SEA PLAQUE GTG low melt agarose, FMC Corp., Rockland, ME), the fragment was purified using the QIAquick ™ method (Qiagen, Chatsworth, CA). The DNA probes were radioactively labeled with 32 P using the REDIPRIME® DNA marking system (Amersham, Arlington Heights, Illinois) according to the manufacturer's specifications. The probe was purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA). The EXPRESSHYB solution (Clontech, Palo Alto, CA) was used for the pre-hybridization and as a hybridization solution for the Northern blots. Hybridization was carried out overnight at 65 ° C and the spots were then washed in 2X SSC and 0.1% SDS at room temperature, followed by two washes in 0. IX SSC and 0.1% SDS at 55'C. and they were exposed to film for 48 hours.There are two main transcripts at approximately 2 kb and 5 kb, while the 2 kb transcript is the main transcript in the testicle., transcription of 5 kb in the main transcript in other tissues including pancreas, liver, stomach and thyroid. The signal intensity was the highest for the testis, with relatively less intense signals in the liver, thyroid and stomach and weak signals in the small intestine, spleen, prostate, thymus, spinal cord, trachea and lymph node. The 120 bp probe (SEQ ID NO: 5) described above was also tested with a Master Dot Blot RNA (Clontech) which contained the RNAs from various tissues that were normalized to 8 internal genes. The stain was pre-hybridized, hybridized and washed as described above. After 48 hours of exposure, the highest expression was observed in the pancreas, with stronger expression in the testicle and stomach. It was observed in the liver, pituitary gland, gland thyroid and salivary glands a lower level of expression. A weaker level was detected in the adrenal gland, small intestine, trachea, spleen, thymus, peripheral leukocytes, lymph node, and fetal tissues. 3 Chromosome assignment and placement of Zsig49 Zsig49 was mapped to chromosome 1 using the commercially available GeneBridge 4 Radiation Hybrid panel and the Stanford G3 Radiation Hybrid (RH) panel (Research Genetics, Inc., Huntsville, AL). The GeneBridge 4 Radiation Hybrid panel contains the PCRable DNAs from each of the 93 hybrid clones by radiation, plus two control DNAs (the HFL donor and the A23 receptor), while the Stanford G3 RH panel contained PCRable DNAs from each of the 83 hybrid clones by radiation, plus two control DNAs (the RM donor and the A3 receptor). The servers of the WWW p u b l i c a m e n t e d i s p o n i b l e s (ht tp: / / coal, wi.mit.edu: 8000 / cgi -bin / contig / rhmapper.pl) and http: // s gc-www. Stanford edu / RH / rhserverformnew .html) allowed chromosomal localization in relation to the respective chromosomal working markers.

For the mapping of zsig49 with the GeneBridge 4 RH panel and the Stanford G3 RH panel, reactions of 20 μl were adjusted in a 96-well microtiter plate (Stratagene, La Jolla, CA) and used in a thermal cycler "RoboCycler Gradient 96"(Stratagene). Each of the 95 reactions by PCR consisted of 2 μl of the reaction buffer by 10X KlenTaq PCR (Clontech), 1.6 μl of dNTPs mixture (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 μl of the primer antisense, ZC 16,080 (SEQ ID NO: 7), 1 μl of the antisense primer, ZC 16,079 (SEQ ID NO: 8), 2 μl of PediLoad (Research Genetics, Inc.), 0.4 μl of 50X Advantage KlenTaq Polymerase Mix (Clontech ), 25 ng of DNA from an individual or control hybrid clone and ddH20 for a total volume of 20 μl. The reactions were overloaded with an equal amount of mineral oil and sealed. The conditions of the PCR cycler were as follows: a denaturation of the initial cycle 1 of 5 minutes at 95 ° C, 35 cycles of a denaturation of 1 minute at 95 ° C, 1 minute hybridization at 66 ° C and extension of 1.5 minutes at 72 ° C, followed by a final extension of 1 cycle of 7 minutes at 72 ° C. The reactions were separated by electrophoresis to a 2% agarose gel (Life Technologies, Gaithersburg, MD).

The results of the hybrid radiation mapping showed that the zsig49 9.76 cR_3000 maps are distant from the D1S2635 marker in the GeneBridge 4 RH mapping panel and 62 cR_10,000 are distant from the SHGC-6232 marker in the Stanford G3 RH panel. The next and distant frame structures were D1S2635 and CHLC. GATA70D01, respectively. The use of the positions of the surrounding markers zsig49 in the Iq42-q43 region on the integrated LDB chromosome 1 map (The Genetic Location Data , University of Southhampton, WWW server: http: // cedar.genetics.soton.ac uk / public__html /). In an autosomal scan for the loci linked to type II diabetes mellitus and the body mass index in the Pima Indians (Hanson et al., Am. J. Hum. Genet, 63: 1130-8, 1998), a potential locus susceptible to diabetes on chromosome lq near marker D1S1677. D1S1677 was mapped on the Stanford G3 RH panel using similar conditions as described above for zsig49 and found to map only 5 cR_10,000 (1 cR_10,000 = -25 kb) close to zsig49, making zsig49 a positional gene candidate for the locus of type II diabetes mellitus.

Example 4 Murine zsig49 ortholog The human zsig49 DNA sequence (SEQ ID NO: 1) described above, was used to search for murine orthologs. A clone considered to contain probably a murine orthologous was sequenced and an alignment with human zsig49 (SEQ ID NO: 1) indicated that the murine sequence was absent at approximately 42 bp at the 5 'end. Two primers ZC24781 of 5 'RACE (SEQ ID NO: 23) and ZC24785 (SE ID NO: 24) were designed according to the murine sequence. To a final volume of 25 μl, 3 μl of small intestine or stomach cDNA diluted 1/100 as a template, 20 pmol of each of the oligonucleotide primers ZC9739 and ZC24785 and 1 U of the antibody Ex Ta q / Ta q (1: 1) was added. The 5 'RACE reactions were run as follows: 94'C for 2 minutes, followed by 5 cycles (94'C for 20 seconds, 65'C for 30 seconds, 72'C for 30 seconds) followed by 30 cycles (94'). C for 20 seconds, 64'C for 30 seconds; 72'C for 30 seconds) followed by an extension of 2 minutes at 72'C. Then a second spliced PCR randa was made. To a final volume of 25 μl was added 1 μl of the first diluted 1/50 of the PCR product as a template, 20 pmol each of the oligonucleotide primers ZC9719 (SEQ ID NO: 18) and ZC24781 (SEQ ID NO: 23) and 1 U of the antibody Ex Ta q / Ta q (1: 1). The reactions were run as follows: 94'C for 2 minutes, followed by 5 cycles (94'C for 20 seconds, 65'C for 30 seconds, 72'C for 30 seconds) followed by 35 cycles (94'C for 20 minutes). seconds, 64'C for 30 seconds, 72'C and 30 seconds) followed by an extension of 2 minutes at 72'C. The PCR products spliced from the second round were purified and sequenced as described above. Comparison of the murine DNA sequence (SEQ ID NO: 12) with the human zsig49 DNA sequence (SEQ ID NO: 1) indicated that the human sequence differed from the murine sequence by approximately 17 bp from the end 5 'which encodes the Met start.

Example 5 Extension of the cDNA Sequence of human zsig49 The alignment of the human and murine DNA sequences indicated that the human sequence could be further extended in the 3 'direction. A series of 3 'RACE PCRs and the extension of the human cDNA sequence were carried out at 1704 bp (SEQ ID NO: 9).

The 3 * RACE primers ZC24645 (SEQ ID NO: 15) and ZC24646 (SEQ ID NO: 16) were designed according to the human zsig49 sequence described by SEQ ID NO: 1. To a final volume of 25 μl was added 3 μl of 1/100 dilution of one of the following marathon cDNAs (adrenal gland of human, fetal liver, islet, pancreas, stomach, small intestine and testicle) as a template, 20 pmoles of each of the oligonucleotide primers ZC9739 (SEQ ID NO: 17) and ZC24645 (SEQ ID NO: 15), and 1 U of the antibody Ex Ta q / Ta q (1: 1). The reactions were run as follows: 94 ° C for 2 minutes, followed by 5 cycles (94 ° C for 20 seconds, 67 ° C for 1 minute) followed by 35 cycles (94 ° C for 20 seconds, 64 ° C for 30 minutes). seconds; 72'C for 1 minute) followed by a 5 minute extension at 72'C. To 25 μl of a second round of spliced PCR was added 1 μl of each of a first round PCR product diluted to 1/50 as the template, 20 pmoles of each of the oligonucleotide primers ZC9719 (SEQ ID NO: 18) and ZC24646 (SEQ ID NO: 16) and 1 U of the antibody Ex Ta q / Ta q (1: 1). The reactions were run as follows: 94 ° C for 2 minutes, followed by 5 cycles (94 ° C for 20 seconds, 69 ° C for 1 minute); followed by 35 cycles (94'C for 20 seconds, 64'C for 30 seconds; 69'C for 1 minute) followed by a 5 minute extension at 69'C. The spliced PCR products from the second round were separated on an agarose gel and purified with a Qiaquick gel purification kit (Qiagen). The purified PCR products generated from the small intestine and stomach molds were sequenced as described above. The sequence indicating the PCR product was extended and diverged from the original zsig49 clone at nucleotide 389 of SEQ ID NO: 1 and continued for approximately 400 bp before an intron was obtained. Three additional rounds of 3 'RACE were carried out as described above, using the primers ZC24780 (SEQ ID NO: 19), ZC24779 (SEQ ID NO: 20), ZC 24965 (SEQ ID NO: 21) and ZC25142 (SEC ID NO: 22) designed from the newly extended sequence. The marathon cDNA of the small intestine and stomach was used as a template. The sequencing of the resulting PCR products was done as described above. The resulting 1,704 bp sequence is described in SEQ ID NO: 9, which contains a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2. From the foregoing, it will be appreciated that, although Described specific embodiments of the invention for purposes of illustration, various modifications could be made without deviating from the spirit and scope of the invention. Therefore, the invention is not limited, except by the appended claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

LIST OF SEQUENCES < 110 > ZymoGenetics. Inc. < 120 > PROTEINA ZSIG49 SECRETADA < 130 > 98-30PC < 150 > 09 / 176,545 < 151 > 1998-10-21 < 160 > 24 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 952 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (158) ... (388) < 400 > 1 ttggggaaag agtcgcctgc ctccggaccg gagtgcagac ctctgaccct ggagtcgctc 60 ggccgctggg aaccgtcccc ttgggtcgtc gcctgggccg cccgtcgttc cccggccccg 120 aggggtccgg ctggccgcgg tgtgggtaga ggtcagc atg age ca ggg gtc cgc 175 Met Ser Gln Gly Val Arg 1 5 cgg gca ggc gct ggg cag ggg gta gcg gcc gcg gtg cag ctg ctg gtc 223 Arg Wing Gly Wing Gly Gln Gly Val Wing Wing Wing Val Gln Leu Leu Val 10 15 20 acc ctg age ttc ctg cgg age gtc gtc gag gcg cag gtc act gga gtt 271 Thr Leu Ser Phe Leu Arg Ser Val Val Glu Ala Gln Val Thr Gly Val 25 30 35 ctg gat gat tgc ttg tgt gat att gac age ate gat aac ttc aat acc 319 Leu Asp Asp Cys Leu Cys Asp lie Asp Ser lie Asp Asn Phe Asn Thr 40 45 50 tac aaa ate ttc ecc aaa ata aaa aaa ttg caa gag aga gac tat ttt 367 Tyr Lys lie Phe Pro Lys lie Lys Leu Gln Glu Arg Asp Tyr Phe 55 60 65 70 cgt tat tac aag gta agg ttg taatttttta ttctgttgat atcaaaggtt 418 Arg Tyr Tyr Lys Val Arg Leu 75 tatatgtgac ctttatgatc cttttgaaag cccatttcag ttcctctcag caccttgtgt 47Í atatetttea tcactgaatt tattatgtat tgcagtggaa acctattgat ctttttaaac 53! agtacaaatc ttagccccct tcctttgtat ggggagttcc tcatttttca gttttggttt 59Í ttaggcagag actactgtct etatagaage tgaaaatgcc acagaettac tttgtcagcc 65Í tetettataa catagttctg ccatctggac acacctactc agcctttgag ttgtgctgat 71! gtcagtgtgc tageattgtt agtggaaagg accacagcag catctttgtt ggacctcttt 778 ctgagagggc tggcaaaaca ggctgaggct ccaagtagac cactaccgac agtgatgctc 838 cagaattggt tcttaaatct agtaatagtc tactetagac ctttacaaaa taaccggtga 898 tactttaaag gcagcgagtc cctgcaacag caataaactt ccttctcctc GGGA 952 < 210 > 2 < 211 > 77 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Ser Gln Gly Val Arg Arg Wing Gly Wing Gly Gln Gly Val Wing Wing 1 5 10 15 Ala Val Gln Leu Leu Val Thr Leu Ser Phe Leu Arg Ser Val Val Glu 20 25 30 Wing Gln Val Thr Gly Val Leu Asp Asp Cys Leu Cys Asp lie Asp Ser 35 40 45 lie Asp Asn Phe Asn Thr Tyr Lys lie Phe Pro Lys lie Lys Lys Leu 50 55 60 Gln Glu Arg Asp Tyr Phe Arg Tyr Tyr Lys Val Arg Leu 65 70 75 < 210 > 3 < 211 > 15 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > cutting site < 400 > 3 Leu Leu Thr Leu Wing Leu Leu Gly Gly Pro Thr Trp Wing Gly Lys 1 5 10 15 < 210 > 4 < 211 > 231 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Degenerate nucleotide sequence encoding the zsig49 polypeptide of SEQ ID NO: 2. < 221 > variation < 222 > (1) ... (231) < 223 > Each N is independently any nucleotide. < 400 > 4 atgwsnearg gngtnmgnmg ngcnggngcn ggnearggng tngcngcngc ngtncarytn 60 ytngtnaeny tnwsnttyyt nmgnwsngtn gtngargcnc argtnacngg ngtnytngay 120 gaytgyytnt gygayathga ywsnathgay aayttyaaya cntayaarat httyccnaat 180 athaaraary tncargarmg ngaytaytty mgntaytaya argtnmgnyt n 231 < 210 > 5 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC14887 < 400 > 5 tcgatgctgt caatatcaca ca 22 < 210 > 6 < 211 > 48 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC16136 < 400 > 6 tgtgggtata agtcagcatg agccaagggg tccgccgggc aggcgctg 48 < 210 > 7 < 211 > 1 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC16080 < 400 > 7 aggggtgcag gtggtaga < 210 > 8 < 211 > 1! < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC16079 < 400 > 8 tcccgaacag ccatcatt < 210 > 9 < 211 > 1704 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (167 (1567) < 400 > 9 ggeaegaggt tggggaaaga gtcgc-ctgcc tccggaccgg agtgcagacc tctgaccctg 60 gagtcgctcg gccgctggga accgtcccct tgggtcgtcg cctgggccgc ccgtcgttcc 120 ccggccccga ggggtccggc tggccgcggt gtgggtagag gtcage atg age caa 175 Met Ser Gln 1 ggg gtc cgc cgg gca ggc gct ggg cag ggg gta gcg gcc gcg gtg cag 223 Gly Val Arg Arg Ala Gly Ala Gly Gln Gly Val Ala Ala Ala Val Gln 5 10 15 ctg ctg gtc acc ctg age ttc ctg cgg age gtc gtc gag gcg cag gtc 271 Leu Leu Val Thr Leu Ser Phe Leu Arg Ser Val Val Glu Ala Gln Val 25 30 35 act gga gtt ctg gat gat tgc ttg tgt gat att gac age ate gat aac 319 Thr Gly Val Leu Asp Asp Cys Leu Cys Asp lie Asp Ser lie Asp Asn 40 45 50 ttc aat acc tac aaa ate ttc ecc aaa ata aaa aaa ttg caa gag aga 367 Phe Asn Thr Tyr Lys lie Phe Pro Lys lie Lys Lys Leu Gln Glu Arg 55 60 65 gac tat ttt cgt tat tac aag gtt aat ctg aag cga cct tgt cct ttc 415 Asp Tyr Phe Arg Tyr Tyr Lys Val Asn Leu Lys Arg Pro Cys Pro Phe 70 75 80 tgg gca gaa gat ggc cac tgt tea ata aaa gac tgt cat gtg gag ecc 463 Trp Wing Glu Asp Gly His Cys Ser He Lys Asp Cys His Val Glu Pro 85 90 95 tgt cea gag agt aaa att ceg gtt gga ata aaa gct ggg cat tet aat 511 Cys Pro Glu Ser Lys lie Pro Val Gly He Lys Wing Gly His Ser Asn 100 105 110 115 aag tac ttg aaa atg gca aac aat acc aaa gaa tta gaa gat tgt gag 559 Lys Tyr Leu Lys Met Wing Asn Asn Thr Lys Glu Leu Glu Asp Cys Glu 120 125 130 ca gct aat aaa ctg gga gca att aac age here tta agt aat caa age 607 Gln Ala Asn Lys Leu Gly Ala He Asn Ser Thr Leu Ser Asn Gln Ser 135 140 145 aaa gaa gct ttc att gac tgg gca aga tat gat gat tea tea cgg gat cac 655 Lys Glu Wing Phe He Asp Trp Wing Arg Tyr Asp Asp Ser Arg Asp His 150 155 160 ttt tgt gaa ctt gat gat gag aga tet cea gct gct cag tat gta gac 703 Phe Cys Glu Leu Asp Asp Glu Arg Ser Pro Wing Wing Gln Tyr Val Asp 165 170 175 cta ttg ctg aac cea gag cgt tac act ggc tat aaa ggg acc tet gca 751 Leu Leu Leu Asn Pro Glu Arg Tyr Thr Gly Tyr Lys Gly Thr Ser Ala 180 185 190 195 tgg aga gtg tgg aac age ate tat gaa gag aac tgt ttc aag cct cga 799 Trp Arg Val Trp Asn Ser He Tyr Glu Glu Asn Cys Phe Lys Pro Arg 200 205 210 tet gtt tat cgt cct tta aat cct cg gcg cct age cga ggc gaa gat 847 Ser Val Tyr Arg Pro Leu Asn Pro Leu Pro Wing Pro Arg Gly Glu Asp 215 220 225 gat gga gaa tea ttc tac here tgg cta gaa ggt ttg tgt ctg gag aaa 895 Asp Gly Glu Ser Phe Tyr Thr Trp Leu Glu Gly Leu Cys Leu Glu Lys 230 235 240 aga gtc ttc tat aag ctt ata teg gga ctt cat gct age ate aat tta 943 Arg Val Phe Tyr Lys Leu He Ser Gly Leu His Wing Ser He Asn Leu 245 250 255 cat cta tgc gca aat tat ctt ttg gaa gaa acc tgg ggt aag ecc cgt 991 His Leu Cys Wing Asn Tyr Leu Leu Glu Glu Thr Trp Gly Lys Pro Ser 260 265 270 275 tgg gga cct aat att aaa gaa ttc aaa cac cgc ttt gac cct gtg gaa 1039 Trp Gly Pro Asn He Lys Glu Phe Lys His Arg Phe Asp Pro Val Glu 280 285 290 acc aag gga gag ggt cea aga agg ctc aag aat ctt tac ttt tta tac 1087 Thr Lys Gly Glu Gly Pro Arg Arg Leu Lys Asn Leu Tyr Phe Leu Tyr 295 300 305 ttg att gag ctt cga gct ttg tea aag gtg gct cea tat ttt gag cgc 1135 Leu He Glu Leu Arg Ala Leu Ser Lys Val Ala Pro Tyr Phe Glu Arg 310 315 320 tea att gtc gat ctt tac act gga aat gca gaa gat gat gct gac here 1183 Ser He Val Asp Leu Tyr Thr Gly Asn Wing Glu Glu Asp Wing Asp Thr 325 330 335 aaa act ctt cta ctg aat ate ttt ca a gat aa tcc ttt ecc atg 1231 Lys Thr Leu Leu Leu Asn He Phe Gln Asp Thr Lys Ser Phe Pro Met 340 345 350 355 falls ttt gat gag aaa tcc atg ttt gca ggt gac aaa aaa ggg gcc aag 1279 His Phe Asp Glu Lys Ser Met Phe Wing Gly Asp Lys Lys Gly Wing Lys 360 365 370 tea cta aag gag gaa ttc cga tta cat ttc aag aat ate tcc cgt ata 1327 Ser Leu Lys Glu Glu Phe Arg Leu His Phe Lys Asn He Ser Arg He 375 380 385 atg gac tgt gtt gga tgt gac aaa tgc aga tta tgg gga aaa tta cag 1375 Met Asp Cys Val Gly Cys Asp Lys Cys Arg Leu Trp Gly Lys Leu Gln 390 395 400 act cag ggt tta gga act gcc ctg aag ata tta ttc tet gaa aaa gaa 1423 Thr Gln Gly Leu Gly Thr Ala Leu Lys He Leu Phe Ser Glu Lys Glu 405 410 415 ate caa aag ctt cea gag aat agt cea tet aaa ggc ttc ca ctc acc 1471 He Gln Lys Leu Pro Glu Asn Ser Pro Ser Lys Gly Phe Gln Leu Thr 420 425 430 435 'cga cag gaa ata gtt gct ctt tta aat gct ttt gga agg ctt tet here 1519 Arg Gln Glu He Val Ala Leu Leu Asn Ala Phe Gly Arg Leu Ser Thr 440 445 450 agt ata aga gac tta cag aat ttt aaa gtc tta tta ca ca ag agg 1567 Ser He Arg Asp Leu Gln Asn Phe Lys Val Leu Leu Gln His Ser Arg 455 460 465 taataaaggc ttttatgtgt ctaactagag acataaagtg actgtggaaa gccttttaat 1627 tatggacatt catcagaaag acactaatct gacttcaaga attctgaact attaaataga 1687 aaatttaaat getcaac 1704 < 210 > 10 < 211 > 467 < 212 > PRT < 213 > Homo sapiens < 400 > 10 Met Ser Gln Gly Val Arg Arg Wing Gly Wing Gly Gln Gly Val Wing Wing 1 5 10 15 Ala Val Gln Leu Leu Val Thr Leu Ser Phe Leu Arg Ser Val Val Glu 20 25 30 Wing Gln Val Thr Gly Val Leu Asp Asp Cys Leu Cys Asp lie Asp Ser 35 40 45 lie Asp Asn Phe Asn Thr Tyr Lys lie Phe Pro Lys lie Lys Lys Leu 50 55 60 Gln Glu Arg Asp Tyr Phe Arg Tyr Tyr Lys Val Asn Leu Lys Arg Pro 65 70 75 80 Cys Pro Phe Trp Wing Glu Asp Gly His Cys Ser lie Lys Asp Cys His 85 90 95 Val Glu Pro Cys Pro Glu Ser Lys lie Pro Val Gly lie Lys Ala Gly 100 105 110 His Ser Asn Lys Tyr Leu Lys Met Wing Asn Asn Thr Lys Glu Leu Glu 115 120 125 Asp Cys Glu Gln Wing Asn Lys Leu Gly Ala lie Asn Ser Thr Leu Ser 130 135 140 Asn Gln Ser Lys Glu Wing Phe lie Asp Trp Wing Arg Tyr Asp Asp Ser 145 150 155 160 Arg Asp His Phe Cys Glu Leu Asp Asp Glu Arg Ser Pro Ala Wing Gln 165 170 175 Tyr Val Asp Leu Leu Leu Asn Pro Glu Arg Tyr Thr Gly Tyr Lys Gly 180 185 190 Thr Ser Wing Trp Arg Val Trp Asn Ser lie Tyr Glu Glu Asn Cys Phe 195 200 205 Lys Pro Arg Ser Val Tyr Arg Pro Leu Asn Pro Leu Pro Pro Arg 210 215 220 Gly Glu Asp Asp Gly Glu Ser Phe Tyr Thr Trp Leu Glu Gly Leu Cys 225 230 235 240 Leu Glu Lys Arg Val Phe Tyr Lys Leu lie Ser Gly Leu His Ala Ser 245 250 255 lie Asn Leu His Leu Cys Wing Asn Tyr Leu Leu Glu Glu Thr Trp Gly 260 265 270 Lys Pro Ser Trp Gly Pro Asn lie Lys Glu Phe Lys His Arg Phe Asp 275 280 285 Pro Val Glu Thr Lys Gly Glu Gly Pro Arg Arg Leu Lys Asn Leu Tyr 290 295 300 Phe Leu Tyr Leu lie Glu Leu Arg Ala Leu Ser Lys Val Ala Pro Tyr 305 310 315 320 Phe Glu Arg Ser lie Val Asp Leu Tyr Thr Gly Asn Wing Glu Glu Asp 325 330 335 Wing Asp Thr Lys Thr Leu Leu Leu Asn lie Phe Gln Asp Thr Lys Ser 340 345 350 Phe Pro Met His Phe Asp Glu Lys Ser Met Phe Ala Gly Asp Lys Lys 355 360 365 Gly Ala Lys Ser Leu Lys Glu Glu Phe Arg Leu His Phe Lys Asn lie 370 375 380 Ser Arg lie Met Asp Cys Val Gly Cys Asp Lys Cys Arg Leu Trp Gly 385 390 395 400 Lys Leu Gln Thr Gln Gly Leu Gly Thr Ala Leu Lys lie Leu Phe Ser 405 410 415 Glu Lys Glu Lie Gln Lys Leu Pro Glu Asn Ser Pro Ser Lys Gly Phe 420 425 430 Gln Leu Thr Arg Gln Glu lie Val Wing Leu Leu Asn Wing Phe Gly Arg 435 440 445 Leu Ser Thr Ser lie Arg Asp Leu Gln Asn Phe Lys VaL Leu Leu Gln 450 455 460 His Ser Arg 465 < 210 > 11 < 211 > 1401 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Degenerate polynucleotide encoding the polypeptide of SEQ ID NO: 10 < 221 > variation < 222 > (1) ... (1401) < 223 > Each N is independently T, A, G or C. < 400 > 11 atgwsnearg gngtnmgnmg ngcnggngcn ggnearggng tngcngcngc ngtncarytn 60 ytngtnaeny tnwsnttyyt nmgnwsngtn gtngargcnc argtnacngg ngtnytngay 120 gaytgyytnt gygayathga ywsnathgay aayttyaaya cntayaarat httyccnaar 180 athaaraary tncargarmg ngaytaytty mgntaytaya argtnaayyt naarmgnccn 240 tgyccnttyt gggcngarga yggncaytgy wsnathaarg aytgycaygt ngarccntgy 300 ccngarwsna aratheengt nggnathaar gcnggncayw snaayaarta yytnaaratg 360 gcnaayaaya cnaargaryt ngargaytgy garcargena ayaarytngg ngcnathaay 420 wsnacnytnw snaaycarws naargargen ttyathgayt gggcnmgnta ygaygaywsn 480 mgngaycayt tytgygaryt ngaygaygar mgnwsnccng engencarta ygtngayytn 540 ytnytnaayc cngarmgnta yacnggntay aarggnacnw sngcntggmg ngtntggaay 600 wsnathtayg argaraaytg yttyaarccn mgnwsngtnt aymgnccnyt naayccnytn 660 gnggngarga gcnccnwsnm ygayggngar wsnttytaya rggnytntgy cntggytnga 720 ytngaraarm gngtnttyta yaarytnath wsnggnytnc aygcnwsnat haayytncay 780 ytntgygnca aytayytnyt ngargaracn tggggnaarc cnwsntgggg nccnaayath 840 aargarttya arcaymgntt ygayccngtn garacnaarg gngarggncc nmgnmgnytn 900 aaraayytnt ayttyytnta yytnathgar ytnmgngvny tnwsnaargt ngenecntay 960 ttygarmgnw snathgtnga yytntayacn ggnaaygcng argargaygc ngayacnaar 1020 acnytnytny tnaayathtt yeargayaen aarwsnttyc cnatgcaytt ygaygaraar 1080 wsnatgttyg cnggngayaa raarggngcn aarwsnytna argargartt ymgnytncay 1140 ttyaaraaya thwsnmgnat hatggaytgy gtnggntgyg ayaartgymg nytntggggn 1200 aarytncara cncarggnyt nggnacngcn ytnaarathy tnttywsnga raargarath 1260 caranertnc cngaraayws nccnwsnaar ggnttycary tnacnmgnca rgararhgtn 1320 gcnytnytna aygcnttygg nmgnytnwsn acnwsnathm gngayytnca raayttyaar 1380 gtnytnytnc arcaywsnmg n 1401 < 210 > 12 < 211 > 1584 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > CDS < 222 > (1) ... (1383) < 400 > 12 cgg gcc gtt act ggg cag ggg gcg gcg gcc gcg gtg caa ctg ctt gtc 48 Arg Ala Val Thr Gly Gln Gly Ala Ala Ala Ala Gl Gln Leu Leu Val 1 5 10 15 acc ctg age ttc ctc tea agt ctg gtc aag act cag gtg act gga gtt 96 Thr Leu Ser Phe Leu Ser Ser Leu Val Lys Thr Gln Val Thr Gly Val 20 25 30 ctg gat gat tgc tta tgt gac att gac age att gat aaa ttc aac acc 144 Leu Asp Asp Cys Leu Cys Asp He Asp Ser He Asp Lys Phe Asn Thr 35 40 45 tac aaa ate ttt ecc aaa ata aag aag tta caa gaa cga gac tat ttt 192 Tyr Lys He Phe Pro Lys He Lys Lys Leu Gln Glu Arg Asp Tyr Phe 50 55 60 cgt tat tac aag gtt aat ctg aaa cga cea tgt cct ttc tgg gca gaa 240 Arg Tyr Tyr Lys Val Asn Leu Lys Arg Pro Cys Pro Phe Trp Wing Glu 65 70 75 80 gat ggc cac tgc tea ata aaa gac tgt cat gtg gag ecc tgt cea gaa 288 Asp Gly His Cys Ser He Lys Asp Cys His Val Glu Pro Cys Pro Glu 85 90 95 agt aaa att cea gtt gga att aaa gcc ggg cgt tea aat aag tac teg 336 Ser Lys He Pro Val Gly He Lys Wing Gly Arg Ser Asn Lys Tyr Ser 100 105 110 caca gca gca aac age acc aaa gaa ctg gat gac tgt gag cag gct aac 384 Gln Wing Wing Asn Ser Thr Lys Glu Leu Asp Asp Cys Glu Gln Wing Asn 115 120 125 aaa ctg ggc gcc ate aac age acg cta agt aac gaa age aaa gaa gcg 432 Lys Leu Gly Ala He Asn Ser Thr Leu Ser Asn Glu Ser Lys Glu Ala 130 135 140 ttc att gac tgg gcg aga tat gat gat teg cag gac cac ttt tgt gaa 480 Phe He Asp Trp Wing Arg Tyr Asp Asp Ser Gln Asp His Phe Cys Glu 145 150 155 160 ctt gat gat gag cgg tet cct gct gca cag tat gtg gac ctg ctg ctg 528 Leu Asp Asp Glu Arg Ser Pro Ala Ala Gln Tyr Val Asp Leu Leu Leu 165 170 175 aac ceg gaa cgg tac act ggc tac aag ggc tcc tea gca tgg agg gtg 576 Asn Pro Glu Arg Tyr Thr Gly Tyr Lys Gly Ser Ser Wing Trp Arg Val 180 185 190 tgg aac age ate tat gaa gaa aac tgc ttc aag cct cga tet gtt tat 624 Trp Asn Ser He Tyr Glu Glu Asn Cys Phe Lys Pro Arg Ser Val Tyr 195 200 205 cgt cct tta aat cct ttg gcg ecc age aga ggg gaa gat gat gga gaa 672 Arg Pro Leu Asn Pro Leu Pro Wing Pro Arg Gly Glu Asp Asp Gly Glu 210 215 220 tea ttc tat acg tgg cta gaa ggt ttg tgt ctt gag aaa aga gtc ttc 720 Ser Phe Tyr Thr Trp Leu Glu Gly Leu Cys Leu Glu Lys Arg Val Phe 225 230 235 240 tat aag ctt ata tea gga ctc cat gcc age ate aat tta cat ctg tgt 768 Tyr Lys Leu He Ser Gly Leu His Wing Ser He Asn Leu His Leu Cys 245 250 255 gca aac tac ctt ctg gaa gaa acc tgg ggg aaa cct agt tgg gga cea 816 Ala Asn Tyr Leu Leu Glu Glu Thr Trp Gly Lys. Pro Ser Trp Gly Pro 260 265 270 aac ate aag gag ttt aga cgc cgc ttt gac cct gtg gaa here aag ggg 864 Asn He Lys Glu Phe Ar.g Arg Arg Phe Asp Pro Val Glu Thr Lys Gly 275 280 285 gaa ggt cea agg agg cta aag aac ctg tac ttt tta tac ttg ata gag 912 Glu Gly Pro Arg Arg Leu Lys Asn Leu Tyr Phe Leu Tyr Leu He Glu 290 295 300 ctc cgt gct ttg tea aag gtg gcc cct tac ttt gag cgc teg att gtt 960 Leu Arg Ala Leu Ser Lys Val Wing Pro Tyr Phe Glu Arg Ser He Val 305 310 315 320 gat ctc tat act ggc aat gtg gaa gat gat gcc gac acc aag acc ctt 1008 Asp Leu Tyr Thr Gly Asn Val Glu Asp Asp Wing Asp Thr Lys Thr Leu 325 330 335 ctc ctc age ate ttt cag gat aa aa tcc ttt cct atg cac ttc gat 1056 Leu Leu Ser He Phe Gln Asp Thr Lys Ser Phe Pro Met His Phe Asp 340 345 350 gag aaa tcc atg ttt gca ggt gac aaa aag ggg gcc aag tea tta aag 1104 Glu Lys Ser Met Phe Wing Gly Asp Lys Lys Gly Wing Lys Ser Leu Lys 355 360 365 gaa gaa ttc cgg tta cat ttc aag aac ate tcc cgg ate atg gac tgt 1152 Glu Glu Phe Arg Leu His Phe Lys Asn He Ser Arg He Met Asp Cys 370 375 380 gtt ggg tgc gat aaa tgc aga ctg tgg ggg aaa ctg cag act cag ggt 1200 Val Gly Cys Asp Lys Cys Arg Leu Trp Gly Lys Leu Gln Thr Gln Gly 385 390 395 400 tta gga act gcc ttg aag ate ctc ttc tet gaa aag gaa ate caa aac 1248 Leu Gly Thr Ala Leu Lys He Leu Phe Ser Glu Lys Glu He Gln Asn 405 410 415 ctt ceg gag aac age cea tcc aaa ggc ttc cag ctc act cgg cag gaa 1296 Leu Pro Glu Asn Ser Pro Ser Lys Gly Phe Gln Leu Thr Arg Gln Glu 420 425 430 ate gtt gtt ctt tta aat gct ttt gga aga ctt tet here age ata aga 1344 He Val Ala Leu Leu Asn Ala Phe Gly Arg Leu Ser Thr Ser He Arg 435 440 445 gaa tta cag aac ttt aaa gcg ttg ttg cag cag agg agg taatgaagac 1393 Glu Leu Gln Asn Phe Lys Ala Leu Leu Gln His Arg Arg 450 455 460 ttttctatgt cttcatagac atagcagact gtatgaagcc ttttagcctt ggacactggg 1453 caaagagact acatgtctaa gacttcaaga attctgaact ctttaagaga aaattcaaat 1513 gtccacttga atatttatga tctttaatag aataccaatt agagatattt ataaatcctc 1573 gtgccgaatt c 1584 < 210 > 13 < 211 > 461 < 212 > PRT < 213 > Mus musculus < 400 > 13 Arg Ala Val Thr Gly Gln Gly Ala Ala Ala Ala Val Gln Leu Leu Val 1 5 10 15 Thr Leu Be Phe Leu Be Ser Leu Val Lys Thr Gln Val Thr Gly Val 20 25 30 Leu Asp Asp Cys Leu Cys Asp lie Asp Ser lie Asp Lys Phe Asn Thr 35 40 45 Tyr Lys lie Phe Pro Lys lie Lys Lys Leu Gln Glu Arg Asp Tyr Phe 50 55 60 Arg Tyr Tyr Lys Val Asn Leu Lys Arg Pro Cys Pro Phe Trp Wing Glu 65 70 75 80 Asp Gly His Cys Ser lie Lys Asp Cys His Val Glu Pro Cys Pro Glu 85 90 95 Ser Lys lie Pro Val Gly lie Lys Wing Gly Arg Ser Asn Lys Tyr Ser 100 105 110 Gln Ala Ala Asn Ser Thr Lys Glu Leu Asp Asp Cys Glu Gln Ala Asn 115 120 125 Lys Leu Gly Ala lie Asn Ser Thr Leu Ser Asn Glu Ser Lys Glu Ala 130 135 140 Phe Lie Asp Trp Wing Arg Tyr Asp Asp Ser Gln Asp His Phe Cys Glu 145 150 155 160 Leu Asp Asp Glu Arg Ser Pro Wing Wing Gln Tyr Val Asp Leu Leu Leu 165 170 175 Asn Pro Glu Arg Tyr Thr Gly Tyr Lys Gly Ser Ser Wing Trp Arg Val 180 185 190 Trp Asn Ser lie Tyr Glu Glu Asn Cys Phe Lys Pro Arg Ser Val Tyr 195 200 205 Arg Pro Leu Asn Pro Leu Pro Pro Arg Gly Glu Asp Asp Gly Glu 210 215 220 Ser Phe Tyr Thr Trp Leu Glu Gly Leu Cys Leu Glu Lys Arg Val Phe 225 230 235 240 Tyr Lys Leu lie Ser Gly Leu His Wing Ser lie Asn Leu His Leu Cys 245 250 255 Wing Asn Tyr Leu Leu Glu Glu Thr Trp Gly Lys Pro Ser Trp Gly Pro 260 265 270 Asn lie Lys Glu Phe Arg Arg Arg Phe Asp Pro Val Glu Thr Lys Gly 275 280 285 Glu Gly Pro Arg Arg Leu Lys Asn Leu Tyr Phe Leu Tyr Leu lie Glu 290 295 300 Leu Arg Ala Leu Ser Lys Val Ala Pro Tyr Phe Glu Arg Ser lie Val 305 310 315 320 Asp Leu Tyr Thr Gly Asn Val Glu Asp Asp Wing Asp Thr Lys Thr Leu 325 330 335 Leu Leu Ser lie Phe Gln Asp Thr Lys Ser Phe Pro Met His Phe, Asp 340 345 350 Glu Lys Ser Met Phe Wing Gly Asp Lys Lys Gly Wing Lys Ser Leu Lys 355 360 365 Glu Glu Phe Arg Leu His Phe Lys Asn lie Ser Arg lie Met Asp Cys 370 375 380 Val Gly Cys Asp Lys Cys Arg Leu Trp Gly Lys Leu Gln Thr Gln Gly 385 390 395 400 Leu Gly Thr Ala Leu Lys lie Leu Phe Ser Glu Lys Glu Lie Gln Asn 405 410 415 Leu Pro Glu Asn Ser Pro Ser Lys Gly Phe Gln Leu Thr Arg Gln Glu 420 425 430 lie Val Ala Leu Leu Asn Ala Phe Gly Arg Leu Ser Thr Ser lie Arg 435 440 445 Glu Leu Gln Asn Phe Lys Ala Leu Leu Gln His Arg Arg 450 455 460 < 210 > 14 < 211 > 1383 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Degenerate polynucleotide encoding the polypeptide of SEQ ID NO: 13 < 221 > variation < 222 > (1) ... (1383) < 223 > Each N is independently A, T, G or C. < 400 > 14 mgngengtna cnggncargg ngengengen gcngtncary tnytngtnac nytnwsntty 60 ytnwsnwsny tngtnaarac ncargtnacn ggngtnytng aygaytgyyt ntgygayath 120 ayaarttyaa gaywsnathg yacntayaar athttyccna arathaaraa rytncargar 180 mgngaytayt tymgntayta yaargtnaay ytnaarmgne cntgyccntt ytgggcngar 240 gayggncayt gywsnathaa rgaytgycay gtngarccnt gyccngarws naaratheen 300 gtnggnatha argcnggnmg nwsnaayaar taywsncarg cngcnaayws nacnaargar 360 ytngaygayt gygarcarge naayaarytn ggngcnatha aywsnacnyt nwsnaaygar 420 wsnaargarg cnttyathga ytgggcnmgn taygaygayw sneargayea yttytgygar 480 ytngaygayg armgnwsncc ngengencar taygtngayy tnytnytnaa yccngarmgn 540 tayacnggnt ayaarggnws nwsngcntgg mgngtntgga aywsnathta ygargaraay 600 tgyttyaarc cnmgnwsngt ntaymgnccn ytnaayccny tngcnccnws nmgnggngar 660 gaygayggng arwsnttyta yacntggytn garggnytnt gyytngaraa rmgngtntty 720 tayaarytna thwsnggnyt ncaygcnwsn athaayytnc ayytntgygc naaytayytn 780 cntggggnaa ytngargara rccnwsntgg ggnccnaaya thaargartt ymgnmgnmgn 840 ttygayccng tngaracnaa rggngarggn ccnmgnmgny tnaaraayyt ntayttyytn 900 tayytnathg arytnmgngc nytnwsnaar gtngcnccnt ayttygar g nwsnathgtn 960 gayytntaya cnggnaaygt ngargaygay gengayacna aracnytnyt nytnwsnath 1020 ttyeargaya cnaarwsntt yecnatgeay ttygaygara arwsnatgtt ygcnggngay 1080 aaraarggng cnaarwsnyt naargargar ttymgnytnc ayttyaaraa yathwsnmgn 1140 athatggayt gygtnggntg ygayaartgy mgnytntggg gnaarytnca racncarggn 1200 ytnggnacng cnytnaarat hytnttywsn garaargara thcaraayyt neengaraay 1260 arggnttyca wsnccnwsna rytnacnmgn cargarathg tngc nytnyt naaygcntty 1320 ggnmgnytnw snacnwsnat hmgngarytn caraayttya argcnytnyt ncarcay gn 1380 mgn 1383 < 210 > 15 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC24645 < 400 > 15 tgctggtcac cctgagcttc ctg 23 < 210 > 16 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC24646 < 400 > 16 tcgaggcgca ggtcactgga gtt 23 < 210 > 17 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC9739 < 400 > 17 ccatcctaat acgactcact atagggc 27 < 210 > 18 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC9719 < 400 > 18 actcactata gggctcgagc ggc 23 < 210 > 19 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC24780 < 400 > 19 tagacctatt gctgaaccca gagcg 25 < 210 > 20 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC24779 < 400 > 20 cactggctat aaagggacct ctgca 25 < 210 > 21 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC24965 < 400 > 21 gccgaggcga agatgatgga gaatc 25 < 210 > 22 < 211 > 31 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC25142 < 400 > 22 agaatatctc ccgtataatg gactgtgttg g 31 < 210 > 23 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC24781 < 400 > 2. 3 gaggaagctc agggtgacaa gcagt 25 < 210 > 24 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC24785 < 400 > 24 gcaatcatcc agaactccag tcacc 25

Claims (37)

175 CLAIMS Having described the invention as above, the content of the following claims is claimed as property.

1. An isolated polypeptide, characterized in that it comprises a contiguous sequence of 50 amino acid residues of SEQ ID NO: 10.

2. An isolated polypeptide according to claim 1, characterized in that the contiguous sequence is 100 amino acid residues of SEQ ID NO: 10.

3. An isolated polypeptide according to claim 1, characterized in that the contiguous sequence is 200 amino acid residues of SEQ ID NO: 10.

4. An isolated polypeptide comprising a sequence of amino acid residues that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, from the residue of amino acid 34 to amino acid residue 467, characterized in that the polypeptide binds specifically to an antibody to which it specifically binds a polypeptide of SEQ ID NO: 10.

5. An isolated polypeptide according to claim 4, characterized in that the polypeptide comprises a sequence of amino acid residues that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10, from the amino acid residue 34 to amino acid residue 467, wherein the polypeptide that specifically binds to an antibody to which a polypeptide of SEQ ID NO: 10 specifically binds.

6. An isolated polypeptide of claim 4, characterized in that the percent identity of the amino acid is determined using a FASTA program with ktup = 1, maximum spacing aperture = 10, maximum extension spacing = 1, and substitution matrix = blosum62 , with other parameters established as absent.

7. An isolated polypeptide according to claim 4, characterized in that any difference between the amino acid sequence encoded by the polynucleotide molecule and the corresponding amino acid sequence of SEQ ID NO: 10 is due to a conservative amino acid substitution.

8. An isolated polypeptide according to claim 1, characterized in that it further comprises an affinity tag or domain of 177 link.

9. An isolated polypeptide, characterized in that it is selected from the group consisting of: a) a polypeptide comprising amino acid residues 34-63 of SEQ ID NO: 2; b) a polypeptide comprising amino acid residues 64-467 of SEQ ID NO: 10; c) a polypeptide comprising amino acid residues 58-461 of SEQ ID NO: 12; d) a polypeptide of SEQ ID NO: 2, from amino acid residue 34 to amino acid residue 77; e) a polypeptide of SEQ ID NO: 10, from amino acid residue 34 to amino acid residue 467; f) a polypeptide of SEQ ID NO: 12, from amino acid residue 28 to amino acid residue 461; g) a polypeptide of SEQ ID NO: 2; h) a polypeptide of SEQ ID NO: 10; and i) a polypeptide of SEQ ID NO: 12.

10. An isolated polypeptide, characterized in that it comprises the amino acid sequence of SEQ ID NO: 2, from amino acid residue 1 to amino acid residue 33.

11. An isolated polynucleotide, characterized in that it encodes a polypeptide comprising a 178 contiguous sequence of 50 amino acid residues of the SEQ ID NO: 10.

12. An isolated polynucleotide according to claim 11, characterized in that the contiguous sequence is 100 amino acid residues of SEQ ID NO: 10.

13. An isolated polynucleotide according to claim 11, characterized in that the contiguous sequence is 200 amino acid residues of SEQ ID NO: 10.

14. An isolated polynucleotide, characterized in that it encodes a polypeptide comprising a sequence of amino acid residues that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, from amino acid residue 34 to amino acid residue 467, wherein the polypeptide that specifically binds to an antibody to which a polypeptide of SEQ ID NO: 10.

15. An isolated polynucleotide according to claim 14, characterized in that the polypeptide comprises a sequence of residues of amino acid that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10, from amino acid residue 34 to the amino acid residue 179 467, wherein the polypeptide that specifically binds to an antibody to which a polypeptide of SEQ ID NO: 10 specifically binds.

16. An isolated polynucleotide of claim 14, characterized in that the amino acid identity percent is determined using a FASTA program with ktup = 1, maximum spacing aperture = 10, maximum extension spacing = 1, and substitution matrix = blosum62 , with other parameters established as absent.

17. An isolated polynucleotide according to claim 14, characterized in that any difference between the amino acid sequence encoded by the polynucleotide molecule and the corresponding amino acid sequence of SEQ ID NO: 10 is due to a conservative amino acid substitution.

18. An isolated polynucleotide according to claim 11, characterized in that the polypeptide further comprises an affinity tag or binding domain.

19. An isolated polynucleotide, characterized in that it is selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising amino acid residues 34-63 of the SEC 180 ID NO: 2; b) a polynucleotide encoding a polypeptide comprising amino acid residues 64-467 of SEQ ID NO: 10; c) a polynucleotide encoding a polypeptide comprising amino acid residues 58-461 of SEQ ID NO: 12; d) a polynucleotide encoding a polypeptide of SEQ ID NO: 2, from amino acid residue 34 to amino acid residue 77; e) a polynucleotide encoding a polypeptide of SEQ ID NO: 10, from amino acid residue 34 to amino acid residue 467; f) a polynucleotide encoding a polypeptide of SEQ ID NO: 12, from the amino acid residue 28 to amino acid residue 461; g) a polynucleotide encoding a polypeptide of SEQ ID NO: 2; h) a polynucleotide encoding a polypeptide of SEQ ID NO: 10; i) a polynucleotide encoding a polypeptide of SEQ ID NO: 12; j) a polynucleotide comprising nucleotide 167 to nucleotide 1567 of SEQ ID NO: 9; k) a polynucleotide comprising the nucleotide 181 1 to nucleotide 1383 of SEQ ID NO: 12; 1) a polynucleotide sequence complementary to a), b), c), d), e), f), g), h), i), j) o); and m) a degenerate polynucleotide sequence of a), b), c), d), e), f), g), h) or i).

An isolated polynucleotide, characterized in that it encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, from amino acid residue 1 to amino acid residue 33.

21. A variant zsig49 polypeptide, characterized in that it comprises the sequence Amino Acid of the variant polypeptide shares an identity with the amino acid sequence of SEQ ID NO: 10 selected from the group consisting of at least 80% identity, at least 90% identity, at least 95% identity or greater than 95 % identity, wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NO: 10 is due to one or more conservative amino acid substitutions.

22. A polynucleotide molecule, characterized in that it encodes a fusion protein consisting essentially of a first portion and a second portion bound by a peptide bond the first portion comprises a polypeptide in accordance with 182 claim 1, and the second portion comprises another polypeptide.

23. A polynucleotide that encodes a fusion protein, characterized in that it comprises a secretory signal sequence having the amino acid sequence of amino acid residues 1-33 of SEQ ID NO: 10, wherein the secretory signal sequence is linked operably to an additional polypeptide.

24. An expression vector, characterized in that it comprises the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide according to claim 1, and a transcription terminator.

25. An expression vector according to claim 24, characterized in that it further comprises a secretory signal sequence operably linked to the polypeptide.

26. An expression vector according to claim 25, characterized in that the secretory signal sequence comprises amino acid residues 1-33 of SEQ ID NO: 2.

27. An expression vector according to 183 claim 24, characterized in that the DNA segment encodes a polypeptide covalently linked to the amino terminal or carboxy terminal to an affinity tag.

28. A cultured cell into which an expression vector according to claim 24 has been introduced, characterized in that the cultured cell expresses the polypeptide encoded by the idlic polynucleotide segment.

29. A method for producing a polypeptide, characterized in that it comprises: culturing a cell into which an expression vector has been introduced according to claim 24, whereby the cell expresses the polypeptide encoded by the polynucleotide segment; and recovering the expressed polypeptide.

30. A method for producing a polypeptide according to claim 29, characterized in that the expression vector further comprises a secretory signal sequence operably linked to the polypeptide; the cultured cell secretes the polypeptide in a culture medium and the polypeptide is recovered from the culture medium.

31. An antibody or antibody fragment, 184 characterized in that it specifically binds to a polypeptide according to claim 1.

32. An antibody according to claim 31, characterized in that the antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b); and d) human monoclonal antibody.

33. An antibody fragment according to claim 32, characterized in that the antibody fragment is selected from the group consisting of F (ab ') / F (ab), Fab', Fv, scFv and minimum recognition unit.

34. An anti-idiototype antibody, characterized in that it binds specifically to the antibody of claim 31.

35. A polypeptide according to claim 1, characterized in that it is in combination with a pharmaceutically acceptable carrier.

36. A kit for the detection of a gene encoding a polypeptide, characterized in that it comprises: 185 a first container comprising a polynucleotide molecule according to claim 11, and a second container comprising one or more reagents capable of indicating the presence of the polynucleotide molecule.

37. A kit for the detection of a gene encoding a polypeptide, characterized in that it comprises: a first container comprising an antibody according to claim 31, and a second container comprising one or more reagents capable of indicating the presence of the antigen icuerpo.