WO2001038505A2 - Secreted protein, zalpha37 - Google Patents

Abstract

Novel soluble, membrane bound, and cytosolic UDP-glycosyltransferase enzyme polypeptides, polynucleotides encoding the polypeptides, antibodies and related compositions and methods are disclosed. The polypeptides may be used for detecting substrates, agonists and antagonists. The polypeptides, polynucleotides and antibodies may also be used in methods that modulate cell-cell interactions, and glycoprotein biosynthesis and glycolipid metabolism, and growth factor and hormone regulation, as well as methods of detection and diagnosis of disease.

Description

Description SECRETED PROTEIN, ZALPHA37

BACKGROUND OF THE INVENTION

UDP-Glycosyltransferase molecules transfer carbohydrate molecules to glycoproteins during biosynthesis. This family includes carbohydrate transferring enzymes, such as glucuronosyltransferases, sialyltransferases, fucosyltransferases, and galactosyltransferases.

Galactosyltransferases promote the transfer of an activated galactose residue in UDP-galactose to the monosaccharide N-acetylglucosamine. This transfer is a step in the biosynthesis of the carbohydrate portion of galactose-containing glycoproteins, such as oligosaccharides and glycolipids, in animal tissues. One subgroup of the galactosyltransferases is the beta-l,3-galactosyltransferases, which are characterized by the elongation of type I oligosaccharide chains. Additionally, the beta- 1,3-galactosyltransferases are found on glycoproteins and glycolipids, are important precursors of blood group antigens, and are present in soluble oligosaccharides of human milk. Similar to other members of galactosyltransferases, the beta- 1,3- galactosyltransferases require a divalent cation (Mn ) to function.

A member of the galactosyltransferase superfamily has recently been identified in Drosophila. This molecule, Brainiac (brn), also known as a Neurogenic Secreted Signaling Peptide (NSSP), is involved in contact and adhesion between germ- line and follicle cells (Amado, M. et al., J. Biol. Chem. 273. 21: 12770-12778, 1998). Germline Brainiac activity has been shown to be essential for development of follicular epithelium (Goode, S. et al., Dev. Biol. 178:35-50, 1996). Additionally, brn is required continuously throughout oogenesis, beginning in the germarium at the time that follicle cells envelop the oocyte-nurse cell complex and continuing stages when the eggshell is produced. The expression of brn in the germline continuously throughout oogenesis is consistent with bra's role in developing the follicular epithelium around each germline cyst, as well as for dorsal-ventral patterning of the follicular epithelium during later phases of oogenesis. See Goode, S. et al., Development. 116: 177-192, 1992.

UDP galactose ceramide galactosyltransferases transfer galactose to ceramide in the formation of galactocerebrosides. This is the key enzymatic step in the biosynthesis of the galactcerebrosides. Cerebrosides are the most abundant of the myelin membrane sphingolipids which, along with phospholipids and cholesterol, are the main lipid constituents of the lipid bilayer in the myelin membrane. One member of this subgroup, CGT, is involved in complex biosynthesis in the myelination of oϋgodendrocytes (Bosio, A. et al., Genomics 34:69-75, 1996).

Related to CGT are UDP glucuronosyltransferase enzymes. These microsomal proteins catalyze the transfer of glucuronic acid to a wide variety of lipid- soluble endogenous and exogenous compounds, including bilirubin, steroids, drugs, and environmental pollutants, rendering them more polar, generally water soluble, less toxic, and more easily excreted from the body (Beaulieu, M. et al., DNA and Cell Biology 16(T0 : 1143-1154). UGT2B17 belongs to this subgroup and is the predominate protein for glucuronidating androgens in the human prostate.

Expression of glycosyltransferases varies within the cell. Some galactosyltransferases are found in the Golgi apparatus. Additionally, galactosyltransferases have been shown to be expressed on the cell surface, where their function is theorized to participate in cellular interactions, perhaps as enzymes, or enzyme-like complementary molecules as well as secreted substrates. As a cell surface carbohydrate, galactosyltransferases have been implicated in varied biology such as cell migration, contact inhibition, tissue interactions, neuronal specificity, fertilization, embryonic cell adhesions, limb bud morphogenesis, mesenchyme development, immune recognition, growth control, and tumor metastasis. See, for example, Shur, B.D., Mol Cell Bioc. 61:143-158, 1984. CGT (supra) is localized to the endoplasmic reticulum.

A deficiency of beta-l,3-galactosyltransferase enzymes has been noticed in the Tn-syndrome. This syndrome is a rarely acquired disorder affecting all hemopoietic lineages, and is characterized by the expression of the Tn and the sialosyl- Tn antigens on the cell surface. The Tn is aN-acetylgalactosamine linked O- glycosidically to threonine or serine residues of membrane proteins. These antigens bind naturally occurring serum antibodies thereby leading to mild hemolytic anemia and pronounced thrombopenia. Thus, the blood cells in the Tn-syndrome are expected to carry less sialic acid if galactose can not be transferred to N-Acetylgalactosamine. The expression of Tn and sialosyl-Tn antigens as a consequence of incomplete or disordered gylcan biosynthesis has been recognized as a cancer-associated phenomenon. Tn and sialosyl-Tn antigens are among the most investigated cancer-associated carbohydrate antigens. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein. DESCRIPTION OF THE INVENTION

These and other aspects of the invention will become evident upon reference to the following detailed description of the invention. Within one aspect the invention provides an isolated polypeptide comprising residues 20 to 483 of SEQ ID NO:2. Within an embodiment, the isolated polypeptide the polypeptide comprises residues 1 to 483 of SEQ ID NO:2. Within another embodiment, the isolated polypeptide comprises residues 1 to 523 of SEQ ID NO:2. Within another embodiment, the invention provides an antibody which specifically binds to the polypeptide comprising residues 1 to 523 of SEQ ID NO:2.

Within another aspect, the invention provides an isolated polypeptide selected from the group consisting of: a polypeptide comprising residues 1 to 19 of SEQ ID NO:2; a polypeptide comprising residues 35 to 49 of SEQ ID NO:2; a polypeptide comprising residues 50 to 483 of SEQ ID NO:2; a polypeptide comprising residues 484 to 511 of SEQ ID NO:2; a polypeptide comprising residues 518 to 521 of SEQ ID NO:2; a polypeptide comprising residues 20 to 483 of SEQ ID NO:2; and a polypeptide comprising residues 20 to 523 of SEQ ID NO:2.

Within another aspect, the invention provides an isolated polynucleotide encoding a polypeptide wherein the polypeptide comprises residues 20 to 483 of SEQ ID NO:2. Within an embodiment, the isolated polynucleotide comprises residues 1 to comprises residues 1 to 523 of SEQ ID NO:2.

Within another aspect, the invention provides an isolated polynucleotide encoding a polypeptide molecule wherein the polypeptide is selected from the group consisting of: a polypeptide comprising residues 1 to 19 of SEQ ID NO:2; a polypeptide comprising residues 35 to 49 of SEQ ID NO:2; a polypeptide comprising residues 50 to 483 of SEQ ID NO:2; a polypeptide comprising residues 484 to 511 of SEQ ID NO:2; a polypeptide comprising residues 518 to 521 of SEQ ID NO:2; a polypeptide comprising residues 20 to 483 of SEQ ID NO:2; and a polypeptide comprising residues 20 to 523 of SEQ ID NO:2.

Within another aspect, the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment wherein the DNA segment is a polynucleotide encoding the polypeptide of claim 1; and a transcription terminator. Within an embodiment, the DNA segment contains an affinity tag. Within another embodiment, the invention provides a cultured cell into which has been introduced the expression vector, wherein said cell expresses the polypeptide encoded by the DNA segment. Within a further embodiment, the invention provides a method of producing a polypeptide comprising culturing the cell, whereby said cell expresses the polypeptide encoded by the DNA segment; and recovering the polypeptide.

Within another aspect, the invention provides a method of producing an antibody comprising the following steps in order: inoculating an animal with a polypeptide selected from the group consisting of: a polypeptide comprising residues 1 to 19 of SEQ ID NO:2; a polypeptide comprising residues 35 to 49 of SEQ ID NO:2; a polypeptide comprising residues 50 to 483 of SEQ ID NO:2; a polypeptide comprising residues 484 to 511 of SEQ ED NO:2; a polypeptide comprising residues 518 to 521 of SEQ ID NO:2; a polypeptide comprising residues 20 to 483 of SEQ ID NO:2; and a polypeptide comprising residues 20 to 523 of SEQ ID NO:2, wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal. Within an embodiment, the antibody produced binds to residues 1 to 523 of SEQ ID NO:2. Within an embodiment, the antibody is a monoclonal antibody.

Within another aspect, the invention provides a method of producing an antibody comprising the following steps in order: inoculating an animal with an epitope bearing portion of a polypeptide wherein the epitope bearing portion is selected from the group consisting of: a polypeptide consisting of residues 66 to 75 of SEQ ID NO:2; a polypeptide consisting of residues 80 to 93 of SEQ ID NO:2; a polypeptide consisting of residues 101 to 109 of SEQ ID NO:2; a polypeptide consisting of residues 272 to 284 of SEQ ID NO:2; a polypeptide consisting of residues 307 to 316 of SEQ ID NO:2; a polypeptide consisting of residues 427 to 434 of SEQ ID NO:2; a polypeptide consisting of residues 515 to 523 of SEQ ID NO:2; a polypeptide consisting of residues 44 to 55 of SEQ ID NO:2; a polypeptide consisting of residues 67 to 75 of SEQ ID NO:2; a polypeptide consisting of residues 81 to 96 of SEQ ID NO:2; a polypeptide consisting of residues 100 to 108 of SEQ ID NO:2; a polypeptide consisting of residues 129 to 139 of SEQ ID NO:2; a polypeptide consisting of residues 211 to 235 of SEQ ID NO:2; a polypeptide consisting of residues 273 to 289 of SEQ ID NO:2; a polypeptide consisting of residues 304 to 318 of SEQ ID NO:2; a polypeptide consisting of residues 332 to 342 of SEQ ID NO:2; a polypeptide consisting of residues 391 to 397 of SEQ ID NO:2; a polypeptide consisting of residues 427 to 435 of SEQ ID NO:2; a polypeptide consisting of residues 468 to 484 of SEQ ID NO: 2; a polypeptide consisting of residues

514 to 523 of SEQ ID NO:2; a polypeptide consisting of residues 66 to 73 of SEQ ID NO:2; a polypeptide consisting of residues 125 to 139 of SEQ ID NO:2; a polypeptide consisting of residues 211 to 233 of SEQ ID NO:2; a polypeptide consisting of residues 274 to 283 of SEQ ID NO:2; a polypeptide consisting of residues 305 to 316 of SEQ ID NO:2; a polypeptide consisting of residues 447 to 452 of SEQ ID NO:2; a polypeptide consisting of residues 470 to 484 of SEQ ID NO:2; and a polypeptide consisting of residues 516 to 523 of SEQ ID NO:2, wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal. Within an embodiment, the antibody produced binds to a residues 1 to 523 of SEQ ID NO:2. Within another embodiment, the antibody is a monoclonal antibody. Within another aspect, the invention provides a method of forming a reversible enzyme-substrate complex comprising; providing an enzyme wherein the receptor comprises residues 20 to 483 of SEQ ID NO:2; and contacting the enzyme with a substrate; whereby the enzyme binds the substrate. Within an embodiment, the the substrate is selected from the group consisting of: glycoproteins; glycolipids; and antibodies.

Within another aspect the invention provides an isolated polypeptide comprising residues 23 to 483 of SEQ ID NO:5. Within an embodiment, the polypeptide comprises residues 1 to 483 of SEQ ID NO:5. Within another embodiment the polypeptide comprises residues 1 to 523 of SEQ ID NO:5.

Within another aspect the invention provides an isolated polypeptide selected from the group consisting of: a polypeptide comprising residues 1 to 22 of SEQ ID NO:5; a polypeptide comprising residues 28 to 42 of SEQ ID NO:5; a polypeptide comprising residues 43 to 483 of SEQ ID NO:5; a polypeptide comprising residues 484 to 511 of SEQ ID NO:5; a polypeptide comprising residues 518 to 521 of SEQ ID NO:5; a polypeptide comprising residues 23 to 483 of SEQ ID NO:5; and a polypeptide comprising residues 23 to 523 of SEQ ID NO:5.

Within another aspect is provided an isolated polynucleotide encoding a polypeptide wherein the polypeptide comprises residues 23 to 483 of SEQ ID NO:5. Within an embodiment, the polypeptide molecule comprises residues 1 to 483 of SEQ

ID NO:5. Within another embodiment, the polypeptide molecule comprises residues 1 to 523 of SEQ ID NO:5.

Within another aspect the invention provides an isolated polynucleotide encoding a polypeptide molecule wherein the polypeptide is selected from the group consisting of: a polypeptide comprising residues 1 to 19 of SEQ ID NO:5; a polypeptide comprising residues 28 to 42 of SEQ ID NO: 5; a polypeptide comprising residues 43 to 483 of SEQ ID NO:5; a polypeptide comprising residues 484 to 511 of SEQ ID NO:5; a polypeptide comprising residues 518 to 521 of SEQ ID NO:5; a polypeptide comprising residues 23 to 48 3 of SEQ ID NO:5; and a polypeptide comprising residues 23 to 523 of SEQ ID NO:5. Within another aspect the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment wherein the DNA segment is a polynucleotide encoding the polypeptide of claim 1; and a transcription terminator. Within an embodiment, the DNA segment contains an affinity tag. Within another embodiment, is provided a cultured cell into which has been introduced the expression vector, wherein said cell expresses the polypeptide encoded by the DNA segment. Within another embodiment, is provided a method of producing a polypeptide comprising culturing the cell, whereby said cell expresses the polypeptide encoded by the DNA segment; and recovering the polypeptide.

Within another aspect of the invention, is provided a method of producing an antibody comprising the following steps in order: inoculating an animal with a polypeptide selected from the group consisting of: a polypeptide comprising residues 1 to 22 of SEQ ID NO:5; a polypeptide comprising residues 29 to 42 of SEQ ID NO:5; a polypeptide comprising residues 43 to 483 of SEQ ID NO:5; a polypeptide comprising residues 484 to 511 of SEQ ID NO:5; a polypeptide comprising residues 518 to 521 of SEQ ID NO:5; a polypeptide comprising residues 23to 483 of SEQ ID NO:5; and a polypeptide comprising residues 23 to 523 of SEQ ID NO:5, wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal. Within another embodiment, the invention provides a method of producing an antibody comprising the following steps in order: inoculating an animal with an epitope bearing portion of a polypeptide wherein the epitope bearing portion is selected from the group consisting of: a polypeptide consisting of residues 66 to 75 of SEQ ID NO:5; a polypeptide consisting of residues 80 to 93 of SEQ ID NO:5; a polypeptide consisting of residues 101 to 109 of SEQ ED NO:5; a polypeptide consisting of residues 272 to 284 of SEQ ED NO:5; a polypeptide consisting of residues 307 to 316 of SEQ ID NO: 5; a polypeptide consisting of residues 427 to 434 of SEQ ED NO:5; a polypeptide consisting of residues 515 to 523 of SEQ ED NO:5; a polypeptide consisting of residues 44 to 55 of SEQ ID NO:5; a polypeptide consisting of residues 67 to 75 of SEQ ED NO:5; a polypeptide consisting of residues 81 to 96 of SEQ ID NO: 5; a polypeptide consisting of residues 100 to 108 of SEQ ED NO:5; a polypeptide consisting of residues 129 to 139 of SEQ ED NO:5; a polypeptide consisting of residues 211 to 235 of SEQ ID NO:5; a polypeptide consisting of residues 273 to 289 of SEQ ID NO:5; a polypeptide consisting of residues 304 to 318 of SEQ ID NO:5; a polypeptide consisting of residues 332 to 342 of SEQ ID NO:5; a polypeptide consisting of residues 391 to 397 of SEQ ED NO:5; a polypeptide consisting of residues 427 to 435 of SEQ ID NO:5; a polypeptide consisting of residues 468 to 484 of SEQ ID NO:5; a polypeptide consisting of residues 514 to 523 of SEQ ID NO:5; a polypeptide consisting of residues 66 to 73 of SEQ ED NO:5; a polypeptide consisting of residues 125 to 139 of SEQ ID NO:5; a polypeptide consisting of residues 211 to 233 of SEQ ED NO:5; a polypeptide consisting of residues 274 to 283 of SEQ ID NO:5; a polypeptide consisting of residues 305 to 316 of SEQ ED NO:5; a polypeptide consisting of residues 447 to 452 of SEQ ID NO:5; a polypeptide consisting of residues 470 to 484 of SEQ JD NO:5; and a polypeptide consisting of residues 516 to 523 of SEQ ED NO:5, wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal. Within an embodiment, the antibody produced by the binds to a residues 1 to 523 of SEQ ED NO:5. Within another embodiment, the antibody is selected from : (a) polyclonal antibody, (b) murine monoclonal antibody, (c) humanized antibody derived from (b), and (d) human monoclonal antibody, a monoclonal antibody. Within another embodiment, the antibody specifically binds to a polypeptide of residues 1 to 483 of SEQ ID NO:5.

Within another aspect the invention provides a method of forming a reversible enzyme-substrate complex comprising; providing an enzyme wherein the enzyme comprises residues 23 to 483 of SEQ ID NO: 5; and contacting the enzyme with a substrate; whereby the enzyme binds the substrate. Within an embodiment, the substrate is selected from the group consisting of: glycoproteins; glycolipids; and antibodies.

Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms: The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, 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 Enzymol. 198:3. 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu- Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985) (SEQ ID NO:7), substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-1210, 1988), streptavidin binding peptide, maltose binding protein (Guan et al., Gene 67:21- 30, 1987), cellulose binding protein, thioredoxin, ubiquitin, T7 polymerase, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags and other reagents are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA; Eastman Kodak, New Haven, CT).

The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide. The term "complements of a polynucleotide molecule" is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.

The term "corresponding to", when applied to positions of amino acid residues in sequences, means corresponding positions in a plurality of sequences when the sequences are optimally aligned.

The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and

GAC triplets each encode Asp). The term "expression vector" is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).

An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. The isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. The polypeptides are provided in a highly purified form, i.e. greater than 95% pure, or greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.

"Operably linked" means that two or more entities are joined together such that they function in concert for their intended purposes. When referring to DNA segments, the phrase indicates, for example, that coding sequences are joined in the correct reading frame, and transcription initiates in the promoter and proceeds through the coding segment(s) to the terminator. When referring to polypeptides, "operably linked" includes both covalently (e.g., by disulfide bonding) and non-covalently (e.g., by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions) linked sequences, wherein the desired function(s) of the sequences are retained.

The term "ortholog" or "species homolog", denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.

A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length. A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".

The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.

A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell.

Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule (i.e., a substrate) and mediates the effect of the ligand on the cell. Membrane-bound receptors are characterized by a multi-domain or multi-peptide structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta- adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and EL-6 receptor).

The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

A "segment" is a portion of a larger molecule (e.g., polynucleotide or polypeptide) having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5' to the 3' direction, encodes the sequence of amino acids of the specified polypeptide.

A "soluble enzyme" is a polypeptide that is not bound to a cell membrane. Soluble enzymes are most commonly substrate-binding enzyme polypeptides that lack transmembrane and cytoplasmic domains. Soluble enzymes can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate. Many cell-surface enzymes have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs. Enzyme polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring. For the purposes of this application a membrane- bound enzyme may have intracellular enzyme capabilities.

The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated 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 upon the discovery of a novel

2797 bp DNA sequence (SEQ ID NO:l) and corresponding polypeptide sequence (SEQ

ID NO:2) which have homology to members of the UDP-glycosyltransferase family.

The polypeptides and polynucleotides encoding them have been designated human zalpha37 (zalpha37h). These polynucleotides have also been identified in the kidney and liver and as such the polynucleotides and polypeptides are also called Kidney and Liver Polypeptides (KLP). Additionally, zalpha37 has been observed in fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary. Sequence analysis of a deduced amino acid sequence indicates that zalpha37h is a type I protein having a signal sequence (residues 1 to 19 of SEQ ID NO:2); an catalytic domain (residues 20 to 483 of SEQ ED NO:2); a transmembrane domain (residues 484 to 511 of SEQ ID NO:2); and an endoplasmic reticulum retention signal (residues 518 to 521 of SEQ JD NO:2). Within the catalytic domain, there is an alpha helix comprising residues 35 to 49 of SEQ ID NO:2. There is a potential N-glycosylation site at residue 52 of SEQ JD NO:2. The mouse ortholog of zalpha37 has also been identified from a mouse kidney cDNA library, and designated (zalpha37m). The polynucleotide and polypeptide sequences for zalpha37m are shown in SEQ JD NOs: 4 and 5, respectively. The degenerate sequence of zalpha37m is shown in SEQ ED NO:6. Sequence analysis of a deduced amino acid sequence indicates that zalpha37m is a type I protein having a signal sequence (residues 1 to 22 of SEQ ID NO: 5); a catalytic domain (residues 23 to 483 of SEQ ID NO:5); a transmembrane domain (residues 484 to 511 of SEQ ED NO: 5); and an endoplasmic reticulum retention signal (residues 518 to 521 of SEQ ID NO:5). Within the catalytic domain, there is an alpha helix comprising 28 to 42 of SEQ ID NO:5. There is a potential N-glycosylation site at residue 70 of SEQ ID NO:5. Analysis of the tissue distribution of human zalpha37 can be performed by the Northern blotting technique using Human Multiple Tissue and Master Dot Blots. Such blots are commercially available (Clontech, Palo Alto, CA) and can be probed by methods known to one skilled in the art. Also see, for example, Wu W. et al., Methods in Gene Biotechnology, CRC Press LLC, 1997. Portions of the polynucleotides of the present invention have been identified in a normal kidney cDNA library as well as a liver tumor and a diseased liver cDNA libraries.

The failure of tumor cell-tumor cell adhesion is believed to be a contributing factor in tumor metastases. See, for example, Zetter, Cancer Biology, 4: 219-29, 1993. Metastases, in turn, are generally associated with poor prognosis for cancer treatment. The metastatic process involves a variety of cellular events, including angiogenesis, tumor cell invasion of the vascular or lymphatic circulation, tumor cell arrest at a secondary site; tumor cell passage across the vessel wall into the parenchymal tissue, and tumor cell proliferation at the secondary site. Thus, both positive and negative regulation of adhesion are necessary for metastasis. That is, tumor cells must break away from the primary tumor mass, travel in circulation and adhere to cellular and/or extracellular matrix elements at a secondary site. Molecules capable of modulating cell-cell and cell-matrix adhesion, such as the UDP- glycosyltransferases, are therefore sought for the study, diagnosis, prevention and or treatment of metastases.

Beta-l,3-galactosyltransferases have limited homology to each other. In contrast to other glycosyltransferases, they do not appear to be localized to the same chromosomes. Additionally, a member of this family has recently been identified in Drosophila. This molecule, Brainiac (brn), also known as a Neurogenic Secreted Signaling Peptide (NSSP), is involved in contact and adhesion between germ-line and follicle cells (Amado, M. et al., J. Biol. Chem. 273, 21: 12770-12778, 1998). Germline Brainiac activity has been shown to be essential for development of follicular epithelium (Goode, S. et al., Dev. Biol. 178:35-50, 1996). Additionally, brn is required continuously throughout oogenesis, beginning in the germarium at the time that follicle cells envelop the oocyte-nurse cell complex and continuing stages when the eggshell is produced. The expression of brn in the germline continuously throughout oogenesis is consistent with bra's role in developing the follicular epithelium around each germline cyst, as well as for dorsal-ventral patterning of the follicular epithelium during later phases of oogenesis. See Goode, S. et al., Development. 116: 177-192, 1992.

The present invention further provides polynucleotide molecules, including DNA and RNA molecules, encoding zalpha37 proteins. The polynucleotides of the present invention include the sense strand; the anti-sense strand; and the DNA as double-stranded, having both the sense and anti-sense strand annealed together by their respective hydrogen bonds. Representative DNA sequences encoding zalpha37 proteins are set forth in SEQ ID NOs:l, 3, 4 and 6. DNA sequences encoding other zalpha37 proteins can be readily generated by those of ordinary skill in the art based on the genetic code.

The present invention provides polynucleotide molecules, including DNA and RNA molecules, that encode the zalpha37 polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NOs:3 and 6 are degenerate DNA sequences that encompasse all

DNAs that encode the zalpha37 polypeptide of SEQ ID NOs:2 and 5, respectively. Those skilled in the art will recognize that the degenerate sequences of SEQ ID NOs:3 and 6 also provide all RNA sequences encoding SEQ ID NOs:2 and 5, respectively by substituting U for T. Thus, zalpha37 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 2797 of SEQ ID NO:l, and nucleotide 1 to nucleotide 2212 of SEQ ID NO:4, and their RNA equivalents are contemplated by the present invention. Table 1 sets forth the one-letter codes used within SEQ ID NOs:3 and 6 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.

TABLE 1

Nucleotide Resolution Nucleotide Complement

A A T T

C C G G

G G C C

T T A A

R A|G Y C|T

Y C|T R A|G

M A|C K G|T

K G|T M A|C

S C|G S C|G

W A|T W A|T

H A|C|T D A|G|T

B C|G|T V A|C|G

V A|C|G B C|G|T

D A|G|T H A|C|T

N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ED NOs:3 and 6, encompassing all possible codons for a given amino acid, are set forth in Table 2.

TABLE 2

One

Amino Letter Codons Degenerate

Acid Code Codon

Cys C TGC TGT TGY

Ser s AGC AGT TC A TCC TCG TCT WSN

Thr T ACA ACC ACG ACT ACN

Pro P CCA CCC CCG CCT CCN

Ala 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

Gin Q CAA 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

De I ATA ATC ATT ATH

Leu L CTA CTC CTG CTT TTA TTG YTN

Val V GTA GTC GTG GTT GTN

Phe F TTC TTT TTY

Tyr Y TAC TAT TAY

Trp W TGG TGG

Ter TAA TAG TGA TRR

Asn|Asp B RAY

Glu|Gln Z SAR

Any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of SEQ ID NOs:2 and 5. Variant sequences can be readily tested for functionality as described herein.

One of ordinary skill in the art will also appreciate that different species can exhibit "preferential codon usage." 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. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequences disclosed in SEQ ID NOs:3 and 6 serve as templates for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.

Within embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NOs:l and 4, or a sequence complementary thereto under stringent conditions. Polynucleotide hybridization is well known in the art and 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 Biology. 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. Polynucleotide hybridization exploits the ability of single stranded complementary sequences to form a double helix hybrid. Such hybrids include DNA-DNA, RNA-RNA and DNA-RNA.

As an illustration, a nucleic acid molecule encoding a variant zalpha37 polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs:l or 4 (or their complements) at 65 °C overnight in

ExpressHyb™ Hybridization Solution (CLONTECH Laboratories, Inc., Palo Alto,

CA). One of skill in the art can devise variations of these hybridization conditions.

Following hybridization, the nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under highly stringent conditions. Typical stringent washing conditions include washing in a solution of 0.5x - 2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 55 - 65°C. That is, nucleic acid molecules encoding a variant zalpha37 polypeptide hybridize with a nucleic acid molecule having the nucleotide sequences of SEQ ID NOs:l or 4, (or their complements) under stringent washing conditions, in which the wash stringency is equivalent to O.lx - 2x SSC with 0.1% SDS at 55 - 65°C, including O.lx SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C. One of skill in the art can readily devise equivalent conditions, for example, by substituting SSPE for SSC in the wash solution.

The present invention also contemplates zalpha37 variant nucleic acid molecules that can be identified using two criteria: a determination of the similarity between the encoded polypeptides with the amino acid sequences of SEQ ID NOs:2 or 5 (as described below), and a hybridization assay, as described above. Such zalpha37 variants include nucleic acid molecules that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or 4 (or their complements) under stringent washing conditions, in which the wash stringency is equivalent to O.lx - 2x SSC with 0.1% SDS at 55 - 65°C, and (2) that encode a polypeptide having about 65%, 80%, 90%, 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO:2 or 5. Alternatively, zalpha37 variants can be characterized as nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or 4 (or their complements) under highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, and (2) that encode a polypeptide having about 65%, 80%, 90%, 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ED NOs:2 or 5.

The highly conserved amino acids in the catalytic domain of human and mouse zalpha37 can be used as a tool to identify new family members. For instance, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding the conserved catalytic domain from RNA obtained from a variety of tissue sources or cell lines. In particular, highly degenerate primers designed from the human and mouse zalpha37 sequences are useful for this purpose. Such regions of human are from residue 381 to residue 386 of SEQ ID NOs:2 or 5; from residue 351 to residue 356 of SEQ ID NOs:2 or 5, and from residue 458 to residue 463 of SEQ ID NOs:2 or 5.

As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zalpha37 RNA. Such tissues and cells can be identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include tissues of kidney and liver. Polymerase chain reactions on cDNA libraries from cell lines and tissues show that the human zalpha37 can also be found in fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary.

Total RNA can be prepared using guanidine isothiocyante extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al.,

Biochemistry 18:52-94, 1979). Poly (A)+ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).

Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using known methods.

In the alternative, genomic DNA can be isolated. Polynucleotides encoding zalpha37 polypeptides are then identified and isolated by, for example, hybridization or PCR.

Full-length clones zalpha37 polypeptides can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron. Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with antibodies to zalpha37 or other specific binding partners.

The invention also provides isolated and purified zalpha37 polynucleotide probes. Such polynucleotide probes can be RNA or DNA. DNA can be either cDNA or genomic DNA. Polynucleotide probes are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences and will generally comprise at least 16 nucleotides, more often from 17 nucleotides to 25 or more nucleotides, sometimes 40 to 60 nucleotides, and in some instances a substantial portion, domain or even the entire zalpha37 gene or cDNA. The synthetic oligonucleotides of the present invention have at least 75% identity to a representative zalpha37 DNA sequence (SEQ ED NOS:l, 3, 4 or 6) or their complements. The invention also provides oligonucleotide probes or primers comprising at least 14 contiguous nucleotides of a polynucleotide of SEQ ED NOs: 1, 3, 4, or 6 or a sequence complementary to SEQ ID NOs: 1, 3, 4, or 6.

Regions from which to construct probes include the 5' and/or 3' coding sequences, substrate binding regions, and signal sequences, and the like. Techniques for developing polynucleotide probes and hybridization techniques are known in the art, see for example, Ausubel et al., eds., Current Protocols in Molecular Biology. John Wiley and Sons, Inc., NY, 1991. For use as probes, the molecules can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp.,

Arlington Heights, EL, using techniques that are well known in the art. Such probes can also be used in hybridizations to detect the presence or quantify the amount of zalpha37 gene or mRNA transcript in a sample. zalpha37 polynucleotide probes could be used to hybridize to DNA or RNA targets for diagnostic purposes, using such techniques such as fluorescent in situ hybridization (FISH) or immunohistochemistry. Polynucleotide probes can be used to identify genes encoding zalpha37-like proteins. For example, zalpha37 polynucleotides can be used as primers and/or templates in PCR reactions to identify other novel members of the UDP-glycosyltransferase family. Such probes can also be used to screen libraries for related sequences encoding novel UDP- glycosyltransferases. Such screening would be carried out under conditions of low stringency which would allow identification of sequences which are substantially homologous, but not requiring complete homology to the probe sequence. Such methods and conditions are well known in the art, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989. Such low stringency conditions could include hybridization temperatures less than 42°C, formamide concentrations of less than 50% and moderate to low concentrations of salt. Libraries may be made of genomic DNA or cDNA. Polynucleotide probes are also useful for Southern, Northern, or dot blots, colony and plaque hybridization and in situ hybridization. Mixtures of different zalpha37 polynucleotide probes can be prepared which would increase sensitivity or the detection of low copy number targets, in screening systems.

In addition, such polynucleotide probes could be used to hybridize to counterpart sequences on individual chromosomes. Chromosomal identification and/or mapping of the zalpha37 gene could provide useful information about gene function and disease association. Many mapping techniques are available to one skilled in the art, for example, mapping somatic cell hybrids, and fluorescence in situ hybridization (FISH). One method is radiation hybrid mapping. Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows the designing of PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Commercially available radiation hybrid mapping panels which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL), are available. These panels enable rapid, PCR based, chromosomal localizations and ordering of genes, sequence-tagged sites (STSs), and other non- polymorphic- and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers. The precise knowledge of a gene's position can be useful in a number of ways including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms such as YAC-, BAC- or cDNA clones, 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region, and 3) for cross-referencing model organisms such as mouse which may be beneficial in helping to determine what function a particular gene might have.

Human and mouse zalpha37 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a human and mouse zalpha37 gene. In view of the tissue-specificity of the cDNA observed for human and mouse zalpha37, this gene region is expected to provide for specific expression in tissues of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary. Promoter elements from a human and mouse zalpha37 gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of zalpha37 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous zalpha37 gene in a cell is altered by introducing into the zalpha37 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The targeting sequence is a zalpha37 5' non-coding sequence that permits homologous recombination of the construct with the endogenous zalpha37 locus, whereby the sequences within the construct become operably linked with the endogenous zalpha37 coding sequence. In this way, an endogenous zalpha37 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.

The polynucleotides of the present invention can also be synthesized using DNA synthesizers. Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA 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 straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 bp), however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. See Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-356 (1984) and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7. 1990. The present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are zalpha37 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human and mouse zalpha37 can be cloned using 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 tissue or cell type that expresses human or mouse zalpha37 as disclosed herein. Such tissue would include, for example, tissues of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A zalpha37-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human and mouse zalpha37 sequences disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to human or mouse zalpha37 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequences disclosed in

SEQ JD NOs:l and 4 represent single alleles of human and mouse zalpha37 and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequences shown in SEQ ID NOs: 1, and 4 including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NOs: 2 and 5. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the human and mouse zalpha37 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.

The present invention also provides isolated zalpha37 polypeptides that are substantially similar to the polypeptides of SEQ ID NOs:2 or 5 and their orthologs.

Such polypeptides will be about 65%, 80%, 90% identical, or 95% or more identical to SEQ ID NOs:2 or 5 and their orthologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16,

1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9. 1992.

Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as:

Total number of identical matches x lOO

[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences] 27

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>H r- H 1

£- H CN to H 1

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1 1 I 1

^ CN CN o ro CN H CN H H

1 1 1 1 1 1

H -* CN ro H o ro CN H ro H CO

1 1 1 1 1 1

X oo ro ro r4 CN H CN H CN CN CN to

1 1 1 1 1 1 1

CD V£> CN CN CO t CN σ CN CN to CO 1 1 1 1 1 1 1 1 1

W IT) CM o ro ro H CN CO H o H ro CN CN

1 1 1 1 1 1 1 1 σ to C CN o ro CN H O ro H σ H CN H CN 1 1 1 1 1 1 1 1 1

P υ> to o CN H H ro <# H ro ro o H <-r- to to

1 1 1 1 1 1 1 1 1 1 1 a «3 H to o o o H ro ro O CN ro CN i-l σ CN ro

1 1 1 1 1 1 1 1 tϋ in O CN to H o CN o ro CN CN ro CN H to CN t 1 1 1 1 1 1 1 1 1 1 1 1 1 dj ^t» H CN CN o H o CN H H H CN H σ CO CN o

1 1 1 1 1 1 1 1 1 1 1

< OJ S Q U σ w CD K H ι 2 Pn CM CO E→ IS >H >

Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed 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 suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zalpha37. The FASTA algorithm is described by Pearson and Lipman, Proc. NatT Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzvmol. 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NOs:2 and/or 5) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring 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. 26:787 (1974)), which allows for amino acid insertions and deletions. Illustrative parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzvmol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from four to six. The present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared with the amino acid sequences of SEQ ED NOs:2 or 5. The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89: 10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. As used herein, the language "conservative amino acid substitution" refers to a substitution represented by a BLOSUM62 value of 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. Conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

Conservative amino acid changes in a zalpha37 gene can be introduced by substituting nucleotides for the nucleotides recited in SEQ ID NOs:l or 4. Such "conservative amino acid" variants can be obtained, for example, by oligonucleotide- directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A Practical Approach (JRL Press 1991)). The ability of such variants to promote cell-cell interactions can be determined using a standard method, such as the assay described herein. Alternatively, a variant zalpha37 polypeptide can be identified by the ability to specifically bind anti-human and anti- mouse zalpha37 antibodies.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites of enzyme-substrate interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904. 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related UDP-glycosyltransferase-like molecules. Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region- directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed human and mouse zalpha37 DNA and polypeptide sequences can be generated through DNA shuffling, as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747- 51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by 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 assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes. Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides (e.g., substrate binding activity) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

Regardless of the particular nucleotide sequence of a variant zalpha37 gene, the gene encodes a polypeptide that is characterized by its substrate binding activity, or by the ability to bind specifically to an anti- zalpha37 antibody. More specifically, variant zalpha37 genes encode polypeptides which exhibit at least 72%, and greater than 75, 80, or 90%, of the activity of polypeptide encoded by the human and mouse zalpha37 gene described herein.

Variant zalpha37 polypeptides or substantially homologous zalpha37 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are of a minor nature, that is conservative amino acid substitutions and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and 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. The present invention thus includes polypeptides of from 523 to 1650 amino acid residues that comprise a sequence that is about 65%, 85%, 90%, and 95% or more identical to the corresponding region of SEQ ED NOs:2 or 5. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zalpha37 polypeptide and the affinity tag. Such sites include thrombin cleavage sites and factor Xa cleavage sites.

For any zalpha37 polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above. Moreover, those of skill in the art can use standard software to devise zalpha37 variants based upon the nucleotide and amino acid sequences described herein. Accordingly, the present invention includes a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ JD NOs:l 2, 3, 4, 5, and 6. Suitable forms of computer-readable media include magnetic media and optically-readable media. Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZEP disk. Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain(s) conferring a biological function may be swapped between human and mouse zalpha37 of the present invention with the functionally equivalent domain(s) from another family member, such as CGT, or UGT2B17. Such domains include, but are not limited to, conserved motifs such as the catalytic, transmembrane, and endoplasmic reticulum rentention domains. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known UDP glycosyltransferase family proteins (e.g. CGT, or UGT2B17), depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.

Moreover, using methods described in the art, polypeptide fusions, or hybrid zalpha37 proteins, are constructed using regions or domains of the inventive human and mouse zalpha37 in combination with those of other UDP galactosyltransferase molecules, (e.g. CGT, or UGT2B17), or heterologous proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure. Auxiliary domains can be fused to zalpha37 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., tissues of the kidney, liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary). For example, a protease domain could be targeted to a predetermined cell type (kidney, liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary) by fusing it to the catalytic domain (residues 20 to 483 of SEQ ID NO:2, or residues 23 to 483 of SEQ ED NO:5), or a portion thereof. In this way, polypeptides, polypeptide fragments and proteins can be targeted for therapeutic or diagnostic purposes. Such N terminal, or portions thereof can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34: 1-9, 1996.

Polypeptide fusions of the present invention will generally contain not more than about 2,300 amino acid residues, not more than about 1,800 residues, or not more than about 1,600 residues, and will in many cases be considerably smaller. For example, residues of zalpha37 polypeptide can be fused to E. coli .-galactosidase (1,021 residues; see Casadaban et al., J. Bacteriol. 143:971-980. 1980), a 10-residue spacer, and a 4-residue factor Xa cleavage site. In a second example, residues of zalpha37 polypeptide can be fused to maltose binding protein (approximately 370 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.

The invention also provides soluble zalpha37 proteins, used to form fusion or chimeric proteins with human Ig, as His-tagged proteins, or FLAG™-tagged proteins. One such construct is comprises residues 20 to 483 of SEQ ED NO:2, or reisudues 23 to 483 of SEQ ID NO:5, fused to human Ig. zalpha37 or zalpha37-Ig chimeric proteins are used, for example, to identify the zalpha37 substrates, including the natural substrate, as well as agonists and antagonists of the natural substrate. Using labeled soluble zalρha37, cells expressing the substrate are identified by fluorescence immunocytometry or immunohistochemistry. The soluble fusion proteins or soluble Ig fusion protein is useful in studying the distribution of the substrate on tissues or specific cell lineages, and to provide insight into enzyme-substrate biology. In an alternative approach, a soluble zalpha37 enzyme extracellular substrate-binding region can be expressed as a chimera with immunoglobulin heavy chain constant regions, typically an F_c fragment, which contains two constant region domains and a hinge region, but lacks the variable region. Such fusions are typically secreted as multimeric molecules, wherein the Fc portions are disulfide bonded to each other and two enzyme polypeptides are arrayed in close proximity to each other. Fusions of this type can be used to affinity purify the cognate substrate from solution, as an in vitro assay tool, to block signals in vitro by specifically titrating out substrate, and as antagonists in vivo by administering them to block substrate stimulation. To purify substrate, a zalpha37-Ig fusion protein (chimera) is added to a sample containing the substrate under conditions that facilitate enzyme-substrate binding (typically near- physiological temperature, pH, and ionic strength). The chimera-substrate complex is then separated by the mixture using protein A, which is immobilized on a solid support (e.g., insoluble resin beads). The substrate is then eluted using conventional chemical techniques, such as with a salt or pH gradient. In the alternative, the chimera itself can be bound to a solid support, with binding and elution carried out as above. For use in assays, the chimeras are bound to a support via the F_c region and used in an ELISA format.

To direct the export of a zalpha37 polypeptide from the host cell, the zalpha37 DNA is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide or a zalpha37 secretory peptide (i.e., residues 1 to 19 of SEQ ED NO:2, or residues 1 to 22 of SEQ JD NO:5). To facilitate purification of the secreted polypeptide, a C-terminal extension, such as a poly-histidine tag, substance P, Flag peptide (Hopp et al., Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., New Haven, CT), maltose binding protein, or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the zalpha37 polypeptide.

The present invention also includes "functional fragments" of zalpha37 polypeptides and nucleic acid molecules encoding such functional fragments. Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a zalpha37 polypeptide. As an illustration, DNA molecules having the nucleotide sequence of SEQ D NO: 1 or SEQ ED NO:4 can be digested with Bal3l nuclease to obtain a series of nested deletions. The fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for cell-cell interactions, or for the ability to bind anti-zalpha37 antibodies. One alternative to exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment. Alternatively, particular fragments of the zalpha37 gene can be synthesized using the polymerase chain reaction.

Standard methods for identifying functional domains are well-known to those of skill in the art. For example, studies on the truncation at either or both termini of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover, standard techniques for functional analysis of proteins are described by, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993), Content et al., "Expression and preliminary deletion analysis of the 42 kDa 2-5A synthetase induced by human interferon," in Biological Interferon Systems, Proceedings of ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72 (Nijhoff 1987), Herschman, "The EGF Enzyme," in Control of Animal Cell Proliferation, Vol. I, Boynton et al., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al., Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al., Biochem. Pharmacol. 50: 1295 (1995), and Meisel et al., Plant Molec.

Biol. 30:1 (1996).

The present invention also contemplates functional fragments of an zalpha37 gene that has amino acid changes, compared with the amino acid sequences of SEQ ID NOs:2 and 5. A variant zalpha37 gene can be identified on the basis of structure by determining the level of identity with nucleotide sequences of SEQ ID NOs:l and 4, and amino acid sequences of SEQ ID NOs:2 and 5, as discussed above. An alternative approach to identifying a variant gene on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zalpha37 gene can hybridize to a nucleic acid molecule having the nucleotide sequences of SEQ ID NOs: 1 or 4, as discussed above. Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptide fragments or variants of SEQ ID NOs:2 and 5 or that retain the glycosyltransferase activity of the wild-type human and mouse zalpha37 protein. Such polypeptides may include additional amino acids from, for example, a secretory signal, an catalytic domain, a transmembrane, and endoplasmic reticulum retention domains, including amino acids responsible for intracellular signaling; fusion domains; affinity tags; and the like.

Within the polypeptides of the present invention are polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NOs:2 and 5. An "epitope" is a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002, 1984. Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least 6 amino acid residues in length. Relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al., Science 219:660-666, 1983. Antibodies that recognize short, linear epitopes are particularly useful in analytic and diagnostic applications that employ denatured protein, such as Western blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or in the analysis of fixed cells or tissue samples. Antibodies to linear epitopes are also useful for detecting fragments of zalpha37, such as might occur in body fluids or cell culture media.

Antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to a zalpha37 protein. Antigenic, epitope-bearing polypeptides contain a sequence of at least six, or at least nine, or from 15 to about 30 contiguous amino acid residues of a zalpha37 protein (e.g., SEQ JD NOs:2 and 5). Polypeptides comprising a larger portion of a zalpha37 protein, i.e. from 30 to 50 residues up to the entire sequence, are included. The amino acid sequence of the epitope-bearing polypeptide can be selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided. Such regions include the N terminal, including the secretory sequence, the catalytic domain, the transmembrane domain, or the endoplasmic reticulum retention signal domain of human and mouse zalpha37 and fragments thereof. Polypeptides in this regard include those comprising residues 1 to 19 of SEQ ED NO:2; residues 20 to 34 of SEQ ED NO:2; residues 35 to 49 of SEQ ED NO:2; residues 50 to 483 of SEQ ID NO:2; residues 484 to 511 of SEQ ID NO:2; residues 512 to 518 of SEQ ID NO:2; residues 1 to 34 of SEQ ID NO:2; residues 20 to 483 of SEQ ID NO:2; residues 484 to 523 of SEQ ID NO:2; residues 1 to 523 of SEQ ID NO:2; residues 20 to 523 of SEQ ID NO:2; residues 1 to 22 of SEQ ID NO:5; residues 23 to 42 of SEQ ID NO:5;. residues 28 to 43 of SEQ JD NO:5; residues 49 to 483 of SEQ ID NO:5; residues 23 to 483 of SEQ ID NO:5; residues 484 to 511 of SEQ ID NO:5; and residues 518 to 523 of SEQ ED NO:5.

The present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a zalpha37 polypeptide described herein.

Such fragments or peptides may 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. Immunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen et al., Proc. NatT Acad. Sci. USA 81:3998 (1983)).

In contrast, polypeptide fragments or peptides may 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 stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing 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)). Accordingly, antigenic epitope- bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein.

Antigenic epitope-bearing peptides and polypeptides contain at least four to ten amino acids, or at least ten to fifteen amino acids, or 15 to 30 amino acids of SEQ ID NOs:2 or 5. Such epitope-bearing peptides and polypeptides can be produced by fragmenting a zalpha37 polypeptide, or by chemical peptide synthesis, as described herein. Moreover, epitopes can be selected by phage display of random peptide libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616 (1996)). Standard methods for identifying epitopes and producing antibodies from small peptides that comprise 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 & Sons 1997). Zalpha37 polypeptides can also be used to prepare antibodies that specifically bind zalpha37 epitopes, peptides or polypeptides. The zalpha37 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response. One of skill in the art would recognize that antigenic, epitope-bearing polypeptides contain a sequence of at least 6, or at least 9, and at least 15 to about 30 contiguous amino acid residues of a zalpha37 polypeptide (e.g., SEQ ID NOs:2 or 5). Polypeptides comprising a larger portion of a zalpha37 polypeptide, i.e., from 30 to 10 residues up to the entire length of the amino acid sequence are included. Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein. Suitable antigens include the zalpha37 polypeptides as shown in SEQ ED NO:2 from amino acid number 1 to amino acid number 523, the zalpha37 polypeptides as shown in SEQ ID NO: 5 from amino acid number 1 to amino acid number 523, or a contiguous 9 to 523 amino acid fragment thereof.

As an illustration, potential antigenic sites in human and mouse zalpha37 were identified using the Jameson-Wolf method, Jameson and Wolf, CABIOS 4:181, (1988), as implemented by the PROTEAN program (version 3.14) of LASERGENE (DNASTAR; Madison, WI). Default parameters were used in this analysis.

Suitable antigens of the human zalpha37 as predicted by the Jameson- Wolf method include residue 66 to_. residue 75 of SEQ ID NO:2; residue 80 to residue 93 of SEQ ED NO:2; residue 101 to residue 109 of SEQ ID NO:2; residue 272 to residue 284 of SEQ ID NO:2; residue 307 to residue 316 of SEQ ID NO:2; residue 427 to residue 434 of SEQ ID NO:2; and residue 515 to residue 523 of SEQ ID NO:2. Hydrophilic peptides, such as those predicted by one of skill in the art from a hydrophobicity plot are also immonogenic. zalpha37 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: residue 44 to residue 55 of SEQ ID NO:2; residue 67 to residue 75 of SEQ ID NO:2 residue 81 to residue 96 of SEQ ID NO:2; residue 100 to residue 108 of SEQ ID NO:2 residue 129 to residue 139 of SEQ ID NO:2; residue 211 to residue 235 of SEQ D NO:2; residue 273 to residue 289 of SEQ ID NO:2; residue 304 to residue 318 of SEQ ID NO:2; residue 332 to residue 342 of SEQ ID NO:2; residue 391 to residue 397 of SEQ ID NO:2; residue 427 to residue 435 of SEQ ID NO:2; residue 468 to residue 484 of SEQ ID NO:2; and residue 514 to residue 523 of SEQ ID NO:2. Additionally, antigens can be generated to portions of the polypeptide which are likely to be on the surface of the folded protein. These antigens include: residue 66 to residue 73 of SEQ ED NO:2; residue 125 to residue 139 of SEQ ID NO:2; residue 211 to residue 233 of SEQ ID NO:2; residue 274 to residue 283 of SEQ ID NO:2; residue 305 to residue 316 of SEQ ID NO:2; residue 447 to residue 452 of SEQ ED NO:2; residue 470 to residue 484 of SEQ JD NO:2; and residue 516 to residue 523 of SEQ ID NO:2.

Suitable antigens of the mouse zalpha37 as predicted by the Jameson- Wolf method include residue 47 to residue 53 of SEQ ID NO:5; residue 58 to residue 64 of SEQ ED NO:5; residue 57 to residue 73 of SEQ JD NO:5; residue 66 to residue 72 of SEQ ED NO:5; residue 79 to residue 92 of SEQ ED NO:5; residue 94 to residue 106 of SEQ ED NO:5; residue 100 to residue 106 of SEQ ID NO:5; residue 123 to residue 130 of SEQ ID NO:5; residue 133 to residue 138 of SEQ ID NO:5; residue 156 to residue 161 of SEQ ID NO:5; residue 211 to residue 217 of SEQ ID NO:5; residue 220 to residue 225 of SEQ ED NO:5; residue 228 to residue 236 of SEQ ED NO:5; residue 271 to residue 277 of SEQ ED NO:5; residue 271 to residue 286 of SEQ ID NO:5; residue 279 to residue 286 of SEQ ID NO:5; residue 289 to residue 295 of SEQ ID NO:5; residue 308 to residue 318 of SEQ ID NO:5; residue 337 to residue 342 of SEQ ID NO:5; residue 334 to residue 341 of SEQ ID NO:5; residue 391 to residue 405 of SEQ ID NO:5; residue 392 residue 397 of SEQ ID NO:5; residue 426 to residue 435 of SEQ ED NO:5; residue 441 to residue 454 of SEQ ED NO:5; and residue 515 to residue 523 of SEQ ID NO:5. Hydrophilic peptides, such as those predicted by one of skill in the art from a hydrophobicity plot are also immonogenic. Zalpha37m hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: residue 63 to residue 71 of SEQ ED NO:5; residue 61 to residue 111 of SEQ ED NO:5; residue 80 to residue 95 of SEQ ED NO:5; residue 99 to residue 105 SEQ ED NO:5; residue 124 to residue 138 of SEQ ED NO:5; residue 189 to residue 200 of SEQ ED NO:5; residue 210 to residue 220 of SEQ ID NO:5; residue 211 to residue 219 of SEQ ID NO:5; residue 223 to residue 232 of SEQ ID NO:5; residue 276 to residue 285 of SEQ ID NO:5; residue 332 to residue 338 of SEQ ID NO:5; residue 332 to residue 341 of SEQ ED NO:5; residue 391 to residue 399 of SEQ ID NO:5; residue 427 to residue 437 of SEQ ID NO:5; residue 428 to residue 434 of SEQ ID NO:5; residue 443 to residue 451 of SEQ ID NO: 5; residue 473 to residue 485 of SEQ ED NO:5; residue 476 to residue 483 of SEQ ID NO:5; and residue 515 to residue 523 of SEQ ID NO:5. Additionally, antigens can be generated to portions of the polypeptide which are likely to be on the surface of the folded protein. These antigens include: residue 64 to residue 72 of SEQ ID NO:5; residue 79 to residue 92 of SEQ ED NO:5; residue 98 to residue 106 of SEQ ED NO:5; residue 132 to residue 138 of SEQ JD NO:5; residue 210 to residue 217 of SEQ ED NO:5; residue 220 to residue 225 of SEQ JD NO:5; residue 274 to residue 283 of SEQ ID NO:5; residue 334 to residue 339 of SEQ ID NO:5; residue 427 to residue 435 of SEQ ID NO:5; residue 442 to residue 452 of SEQ ID NO:5; residue 474 to residue 484 of SEQ ED NO:5; and residue 518 to residue 523 of SEQ ID NO:5.

Antibodies from an immune response generated by inoculation of an animal with the antigens listed above can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example. Current Protocols in Immunology. Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hvbridoma Antibodies: Techniques and Applications. CRC Press, Inc., Boca Raton, FL, 1982. As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zalpha37 polypeptide or a fragment thereof. The immunogenicity of a zalpha37 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions zalpha37 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included. Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered" antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.

Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zalpha37 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zalpha37 protein or peptide). Genes encoding polypeptides having potential zalpha37 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a substrate or enzyme, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO. 5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from CLONTECH Laboratories, Inc., (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using the zalpha37 sequences disclosed herein to identify proteins which bind to zalpha37. These "binding proteins" which interact with zalpha37 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. These binding proteins can also act as zalpha37 "antagonists" to block zalpha37 substrate binding in vitro and in vivo. These anti-zalpha37 binding proteins would be useful for modulating, for example, glycoprotein and glycolipid metabolism, growth factor and hormone regulation, and cell-cell interactions.

As used herein, the term "binding proteins" additionally includes antibodies to zalpha37 polypeptides, the cognate substrate of zalpha37 polypeptides, proteins useful for purification of zalpha37 polypeptides, and proteins associated with the N terminal region (residues 20 to 483 of SEQ ED NO:2, or residues 23 to 483 of SEQ ED NO.5). Antibodies are determined to be specifically binding if they exhibit a threshold level of binding activity (to zalpha37 polypeptide, peptide or epitope) of at least 10-fold greater than the binding affinity to a control (non-zalpha37 ) polypeptide. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660- 672, 1949).

A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to zalpha37 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zalpha37 protein or polypeptide. Antibodies to zalpha37 may be used for immunohistochemical tagging cells that express zalpha37; for isolating zalpha37 by affinity purification; for diagnostic assays for determining circulating levels of zalpha37 polypeptides; for detecting or quantitating soluble zalpha37 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zalpha37 in vitro and in vivo. Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates. Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Moreover, antibodies to zalpha37 or fragments thereof may be used in vitro to detect denatured zalpha37 or fragments thereof in assays, for example, Western Blots or other assays known in the art. The soluble zalpha37 is useful in studying the distribution of substrates in tissues or specific cell lineages, and to provide insight into enzyme/substrate biology. Using labeled zalpha37, cells expressing the substrate are identified by fluorescence immunocytometry or immunocytochemistry. Application may also be made of the specificity of UDP-glycosyltransferases for their substrates.

Antibodies can be made to soluble, zalpha37 polypeptides which are His or FLAG™ tagged. Alternatively, such polypeptides form a fusion protein with Human Ig. In particular, antiserum containing polypeptide antibodies to His-tagged, or FLAG™-tagged soluble zalpha37 can be used in analysis of tissue distribution of zalpha37 by immunohistochemistry on human or primate tissue. These soluble zalpha37 polypeptides can also be used to immunize mice in order to produce monoclonal antibodies to a soluble zalpha37 polypeptide. Monoclonal antibodies to a soluble zalpha37 polypeptide can also be used to mimic substrate/enzyme coupling, resulting in activation or inactivation of the substrate/enzyme pair. For instance, it has been demonstrated that cross-linking anti-soluble CD40 monoclonal antibodies provides a stimulatory signal to B cells that have been sub-optimally activated with anti-IgM or LPS, and results in proliferation and immunoglobulin production. These same monoclonal antibodies act as antagonists when used in solution by blocking activation of the receptor. Monoclonal antibodies to zalpha37 can be used to determine the distribution, regulation and biological interaction of the zalpha37 and its substrate pair on specific cell lineages identified by tissue distribution studies. Soluble zalpha37 or antibodies to zalpha37 can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For instance, polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (substrate or antigen, respectively, for instance). More specifically, zalpha37 polypeptides or anti- zalpha37 antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti-complementary molecule.

Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like. Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance). Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion. For these purposes, biotin/streptavidin is an exemplary complementary/ anticomplementary pair.

In another embodiment, polypeptide-toxin fusion proteins or antibody- toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues). Alternatively, a fusion protein including only the secretory, catalytic, transmembrane, or endoplasmic reticulum retention domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest. Similarly, the corresponding substrate to zalpha37 can be conjugated to a detectable or cytotoxic molecule and provide a generic targeting vehicle for cell/tissue-specific delivery of generic anti-complementary-detectable/ cytotoxic molecule conjugates.

In another embodiment, zalpha37-cytokine fusion proteins or antibody- cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary), if the zalpha37 polypeptide or anti- zalpha37 antibody targets hyperproliferative tissues from these organs. (See, generally, Hornick et al., Blood 89:4437-47, 1997). They described fusion proteins that enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine. Suitable zalpha37 polypeptides or anti- zalpha37 antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediates improved target cell lysis by effector cells. Suitable cytokines for this purpose include interleukin 2 and granulocyte- macrophage colony-stimulating factor (GM-CSF), for instance.

In yet another embodiment, if the zalpha37 polypeptide or anti- zalpha37 antibody targets vascular cells or tissues, such polypeptide or antibody may be conjugated with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis. Such therapeutic approach poses less danger to clinicians who administer the radioactive therapy. The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action. Zalpha37 polynucleotides and/or polypeptides may be useful for regulating the maturation of UDP-glycosyltransferase substrate-bearing cells, such as fibroblasts, lymphocytes and hematopoietic cells. Zalpha37 polypeptides will also find use in mediating metabolic or physiological processes in vivo. The effects of a compound on proliferation and differentiation can be measured in vitro using cultured cells. Bioassays and ELISAs are available to measure cellular response to zalpha37, in particular are those which measure changes in cytokine production as a measure of cellular response (see for example, Current Protocols in Immunology ed. John E. Coligan et al., NIH, 1996). Assays to measure other cellular responses, including glycoprotein and glycolipid biosynthesis and metabolism, growth factor and hormone regulation, cell-cell interactions are known in the art.

The zalpha37 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 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 zalpha37 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within 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 within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

To direct a zalpha37 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be derived from another secreted protein (e.g., t-PA) or a zalpha37 secretory peptide (residues 1 to 19 of SEQ ID NO:2, or reisudes 1 to 22 of SEQ ED NO:5). Alternatively, it can be synthesized de novo. The secretory signal sequence is operably linked to the zalpha37 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). The endoplasmic reticulum retention signal (residues 518 to 521 of SEQ ED NOs: 2 or 5) can also be substituted with a different endoplasmic reticulum retention signal from another UDP-glycosyltransferase (CGT, for example).

The catalytic domain of zalpha37 can be substituted by a heterologous sequence providing a different catalytic domain. In this case, the fusion product can be secreted, and the secretory peptide of zalpha37 can direct the new catalytic domain to the transmembrane and/or endoplasmic reticulum of the cell. This substituted catalytic domain can be chosen from the catalytic domains represented by the UDP- glycosyltransferase protein families. Similarly, the catalytic domain of zalpha37 protein can be used to substitute the catalytic domain of a different UDP-glycosyltransferase. In these cases, the fusion products can be soluble or membrane-bound proteins.

Cultured mammalian cells are suitable hosts within 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 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manasas, VA. In general, strong transcription promoters can be used such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." One selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G- 418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. One amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins, such as CD4, CD8, Class I MHC, or placental alkaline phosphatase, may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa calif ornica nuclear polyhedrosis virus (AcNPV). See, King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide. London, Chapman & Hall; O'Reilly, D.R. et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D.,

Ed., Baculovirus Expression Protocols. Methods in Molecular Biology. Totowa, NJ, Humana Press, 1995. A second method of making recombinant zalpha37 baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J Virol 67:4566-79, 1993). This system, which utilizes transfer vectors, 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 zalpha37 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." The pFastBacl™ transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case zalpha37 . However, pFastBacl™ can be modified to a considerable degree. The polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D., J. Gen. Virol. 71:971-6, 1990; Bonning, B.C. et al., J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk, G.D., and Rapoport, B., 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. Moreover, transfer vectors can be constructed which replace the native zalpha37 secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdysteroid Glycosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native zalpha37 secretory signal sequence. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zalpha37 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using a technique known in the art, a transfer vector containing zalpha37 is transformed into E. coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses zalpha37 is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.

The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveO™ cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent #5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 H™ (Life Technologies) or ΕSF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa, KS) or Express FiveO™ (Life Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2-5 x 10⁵ cells to a density of 1-2 x 10⁶ cells at which time a recombinant viral stock is added at a multiplicity of infection (MOT) of 0.1 to 10, more typically near 3. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R.D., ibid.; O'Reilly, D.R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification of the zalpha37 polypeptide from the supernatant can be achieved using methods described herein.

Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). One vector system for use in Saccharomyces cerevisiae is the POTl vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and 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, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in U.S. patents 5,716,808, 5,736,383, 5,854,039, and 5,888,768.

Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing a zalpha37 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, 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 disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell. P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. One culture medium for P. methanolica is YΕPD (2% D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit, MI), 1% Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, tran_-3-methylproline, 2,4-methanoproline, cw-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, α//o-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRΝAs. Methods for synthesizing amino acids and aminoacylating tRΝA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722. 1991; Εllman et al., Methods Enzvmol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Νatl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRΝA and chemically aminoacylated suppressor tRΝAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incoφorated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. 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, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zalpha37 amino acid residues.

The polypeptides of the present invention are purified to >80% purity, to 90% purity, to >95% purity, and or a pharmaceutically pure state, 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. A purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. Expressed recombinant zalpha37 proteins (including chimeric polypeptides and multimeric proteins) are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid. Maltose binding protein fusions are purified on an amylose column according to methods known in the art.

The polypeptides of the present invention can be isolated by a combination of procedures including, but not limited to, anion and cation exchange chromatography, size exclusion, and affinity chromatography. For example, immobilized metal ion adsorption (MAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzvmol., Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification.

Zalpha37 polypeptides, or fragments thereof, can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149. 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, IL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3. 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides.

Using methods known in the art, zalpha37 proteins can be prepared as monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.

The activity of zalpha37 polypeptides can be measured using a variety of assays that measure, for example, cell-cell interactions, glycolipid and glycoprotein biosynthesis, development, and other biological functions associated with UDP- glycosyltransferase family members or with UDP-glycosyltransferase interactions, such as, differentiation, and proliferation for example. Of particular are changes in the transfer of galactosyl molecules in glycoprotein and glycolipid synthesis and in cell-cell interactions in kidney, liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary tissue as well as cell lines derived from these tissues. Such assays are well known in the art. For a general reference, see Kolbinger, F. et al.; J. Biol. Chem. 273: 433-440, 1998; Amado, M. et al., J. Biol. Chem. 273:12770-12778, 1998; Hennet, T. et al., J. Biol. Chem. 273:58-65. 1998; and Ram B.P., and Munjal, D.D., CRC Crit Rev. Biochem. 17:257-311, 1985. Specific assays include, but are not limited to bioassays measuring cell migration, contact inhibition, tissue interactions, fertilization, embryonic cell adhesions, limb bud morphogenesis, mesenchyme development, immune recognition, growth control, tumor metastasis and suppression, glycoprotein and glycolipid biosynthesis, growth factor and hormone regulation, and conjugation of lipid-soluble compounds.

Additional activities likely associated with the polypeptides of the present invention include proliferation of cells of the kidney, liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary directly or indirectly through other growth factors; action as a chemotaxic factor; and as a factor for expanding pancreas and mesenchymal stem cell and precursor populations. Proteins, including alternatively spliced peptides, of the present invention are useful for tumor diagnosis and staging, tumor suppression, lipid metabolism, modulating the activity or levels of other proteins, including growth factors and hormones, as well as modulating growth and differentiation either working in isolation, or in conjunction with other molecules (growth factors, cytokines, etc.) in liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary and peripheral blood. Alternative splicing of zalpha37 may be cell-type specific and confer activity to specific substrates.

Another assay of interest measures or detects changes in proliferation, differentiation, and development. Additionally, the effects of a zalpha37 polypeptides on cell-cell interactions of epithelial cells, tumor cells and cells of the kidney, liver, esophagus, lung, and ovary in particular, would be of interest to measure. Yet other assays examines changes in glycoprotein and glycolipid synthesis.

The activity of molecules of the present invention can be measured using a variety of assays that, for example, measure neogenesis or hyperplasia (i.e., proliferation) of tissues of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary. Additional activities likely associated with the polypeptides of the present invention include proliferation of endothelial cells, fibroblasts, and lymphoid cells directly or indirectly through other growth factors; action as a chemotaxic factor for endothelial cells, fibroblasts and/or phagocytic cells; and factor for expanding mesenchymal stem cell and precursor populations.

Proliferation can be measured using cultured kidney, liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, and salivary gland or in vivo by administering molecules of the claimed invention to an appropriate animal model. Generally, proliferative effects are observed as an increase in cell number and therefore, may include inhibition of metastasis, as well as mitogenesis. Cultured cells include kidney fibroblasts, liver tumors, and diseased liver from primary cultures, as well as carcinomas of esophagus, liver, lung, and ovary. Established cell lines are easily identifiable by one skilled in the art and are available from ATCC (Manasas, VA). Assays measuring cell proliferation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radiolabelled 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 82:169-179, 1985), and use of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988). To determine if zalpha37 is a chemotractant in vivo, zalpha37 can be given by intradermal or intraperitoneal injection. Characterization of the accumulated leukocytes at the site of injection can be determined using lineage specific cell surface markers and fluorescence immunocytometry or by immunohistochemistry (Jose, J. Exp. _____• 179:881-87, 1994). Release of specific leukocyte cell populations from bone marrow into peripheral blood can also be measured after zalpha37 injection.

Differentiation is a progressive and dynamic process, beginning with pluripotent stem cells and ending with terminally differentiated cells. Pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that are lost when commitment to a cell lineage is made. Progenitor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cell products and enzymes and enzyme-like complementary molecules. The stage of a cell population's differentiation is monitored by identification of markers present in the cell population. For example, myocytes, osteoblasts, adipocytes, chrondrocytes, fibroblasts and reticular cells are believed to originate from a common mesenchymal stem cell (Owen et al., Ciba Fdn. Symp. 136:42-46, 1988). Markers for mesenchymal stem cells have not been well identified (Owen et al., J. of Cell Sci. 87:731-738, 1987), so identification is usually made at the progenitor and mature cell stages. The novel polypeptides of the present invention are useful for studies to isolate mesenchymal stem cells and kidney fibroblast progenitor cells, both in vivo and ex vivo.

There is evidence to suggest that factors that stimulate specific cell types down a pathway towards terminal differentiation or dedifferentiation affect the entire cell population originating from a common precursor or stem cell. Thus, zalpha37 polypeptides may stimulate inhibition or proliferation of endocrine and exocrine cells of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary.

Molecules of the present invention may, while stimulating proliferation or differentiation of fibroblasts, inhibit proliferation or differentiation of adipocytes, by virtue of their effect on common precursor/stem cells. The novel polypeptides of the present invention are useful to study neural and epithelial stem cells and liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, and salivary gland, progenitor cells, both in vivo and ex vivo.

Assays measuring differentiation include, for example, measuring cell- surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991;

Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol.

Bioprocesses. 161-171, 1989).0

The zalpha37 polypeptides of the present invention can be used to study proliferation or differentiation in liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary. Such methods of the present invention generally comprise incubating cells derived from these tissues in the presence and absence of zalpha37 polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in cell proliferation or differentiation. Cell lines from these tissues are commercially available from, for example, American Type Culture Collection (Manasas, VA).

Proteins, including alternatively spliced peptides, and fragments, of the present invention are useful for studying cell-cell interactions, glycolipid and glycoprotein biosynthesis, growth factor and hormone regulation, development, fertility and other biological functions associated with UDP-glycosyltransferase family members. zalpha37 molecules, variants, and fragments can be applied in isolation, or in conjunction with other molecules (growth factors, cytokines, etc.) in liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary.

Proteins of the present invention are useful for delivery of therapeutic agents such as, but not limited to, proteases, radionuclides, chemotherapy agents, and small molecules. Effects of these therapeutic agents can be measured in vitro using cultured cells, ex vivo on tissue slices, or in vivo by administering molecules of the claimed invention to the appropriate animal model. An alternative in vivo approach for assaying proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, lentivirus, vaccinia virus and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see T.C. Becker et al., Meth. Cell Biol. 43:161-89, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44-53, 1997). The adenovirus system offers several advantages: adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co- transfected plasmid. In an exemplary system, the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary). When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.

Moreover, adenoviral vectors containing various deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector. Such adenoviruses are El deleted, and in addition contain deletions of E2A or E4 (Lusky, M. et al., J. Virol. 72:2022-2032, 1998; Raper, S.E. et al., Human Gene Therapy 9:671- 679, 1998). In addition, deletion of E2b is reported to reduce immune responses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998). Moreover, by deleting the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated. Generation of so called "gutless" adenoviruses where all viral genes are deleted are particularly advantageous for insertion of large inserts of heterologous DNA. For review, see Yeh, P. and Perricaudet, M., FASEB J. 11 :615-623, 1997.

The adenovirus system can also be used for protein production in vitro. By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, 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 infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al., Cytotechnol. 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293S cell production protocol, non- secreted proteins may also be effectively obtained.

As a membrane bound protein, the activation of zalpha37 polypeptide can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with zalpha37 binding and subsequent physiologic cellular responses. An exemplary 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, H.M. et al., Science 257:1906-1912, 1992; Pitchford, S. et al.. Meth. Enzvmol. 228:84-108. 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59, 1998; Van Liefde, I. Et al., Eur. J. Pharmacol. 346:87-95, 1998. The microphysiometer can be used for assaying adherent or non- adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including agonists, substrates ligands, or antagonists of the zalpha37 polypeptide. The microphysiometer is used to measure responses of a zalpha37-expressing eukaryotic cell, compared to a control eukaryotic cell that does not express zalpha37 polypeptide. Zalpha37-expressing eukaryotic cells comprise cells into which zalpha37 has been transfected, as described herein, creating a cell that is responsive to zalpha37-modulating stimuli; or cells naturally expressing zalpha37, such as zalpha37-expressing cells derived from, for example, kidney, liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary tissues. Differences, measured by a change in extracellular acidification, relative to a control, are a direct measurement of zalpha37- modulated cellular responses. Moreover, such zalpha37-modulated responses can be assayed under a variety of stimuli. Also, using the microphysiometer, there is provided a method of identifying agonists and antagonists of zalpha37 polypeptide, comprising providing cells expressing a zalpha37 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 in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change extracellular acidification rate. Antagonists and agonists, including the natural substrate for zalpha37 polypeptide, can be rapidly identified using this method. Similarly, the soluble zalpha37 polypeptide can also be measured by the microphysiometer. A zalpha37-responsive eukaryotic cell, comprise cells into which the substrate for zlpha37 has been transfected creating a cell that is responsive to zalpha37; or cells naturally responsive to zalpha37 such as cells derived from kidney, liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary tissues.

Moreover, zalpha37 can be used to identify cells, tissues, or cell lines which respond to a zalpha37-modulated pathway. The microphysiometer, described above, can be used to rapidly identify cells responsive to zalpha37 of the present invention. Cells can be cultured in the presence or absence of zalpha37 polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of zalpha37 substrate are responsive to zalpha37. Such cell lines, can be used to identify substrates, antagonists and agonists of zalpha37 polypeptide as described above.

As a protein found in the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary, zalpha37, its substrates, agonists (including the native catalytic domain) and antagonists have enormous potential in both in vitro and in vivo applications. Compounds identified as zalpha37 agonists and antagonists are useful for studying cell-cell interactions, glycolipid and glycoprotein biosynthesis, growth factor and hormone regulation, development, and other biological functions associated with UDP-glycosyltransferase family members or with UDP-glycosyltransferase interactions in vitro and in vivo. For example, zalpha37 and agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with cytokines and hormones to replace serum that is commonly used in cell culture. Agonists are thus useful in specifically promoting the growth and/or development of cells of the myeloid and lymphoid lineages in culture. Additionally, zalpha37 polypeptides and zalpha37 agonists, including small molecules are useful as a research reagent, such as for the expansion, differentiation, and/or cell-cell interactions of liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary. zalpha37 polypeptides are added to tissue culture media for these cell types.

Compounds identified as zalpha37 agonists are useful for modifying the proliferation and development of target cells in vitro and in vivo. For example, agonist compounds are useful alone or in combination with cytokines and hormones as components of defined cell culture media. Agonists are thus useful in specifically mediating the growth and/or development of zalpha37-bearing cells in culture. Antagonists are useful as research reagents for characterizing substrate-enzyme interaction.

The invention also provides antagonists, which either bind to zalpha37 polypeptides or, alternatively, to a substrate to which zalpha37 polypeptides bind, thereby inhibiting or eliminating the function of zalpha37. Such zalpha37 antagonists would include antibodies; polypeptides which bind either to the zalpha37 polypeptide or to its substrate; natural or synthetic analogs of zalpha37 substrates which retain the ability to bind the substrate but do not result in glycoprotein or glycolipid synthesis or metabolism, or growth factor and hormone regulation. Such analogs could be peptides or peptide-like compounds. Natural or synthetic small molecules which bind to zalpha37 polypeptides and prevent glyprotein or glycolipid synthesis or metabolism, or growth factor and hormone regulation are also contemplated as antagonists. Also contemplated are soluble zalpha37 enzymes. As such, zalpha37 antagonists would be useful as therapeutics for treating certain disorders where blocking glycosylation of substrate would be beneficial.

Zalpha37 polypeptides may be used within diagnostic systems to detect the presence of substrate polypeptides. Antibodies or other agents that specifically bind to zalpha37 or its substrate may also be used to detect the presence of circulating enzyme or substrate polypeptides. Such detection methods are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay. Immunohistochemically labeled zalpha37 antibodies can be used to detect zalpha37 enzyme and/or substrates in tissue samples. zalpha37 levels can also be monitored by such methods as RT-PCR, where zalpha37 mRNA can be detected and quantified. The information derived from such detection methods would provide insight into the significance of zalpha37 polypeptides in various diseases, and as such would serve as diagnostic tools for diseases for which altered levels of zalpha37 are significant. Altered levels of zalpha37 enzyme polypeptides may be indicative of pathological conditions including, for example, cancer, cirrhosis, disorders of the liver, hormonal regulation and development, and fertility.

Antagonists are also useful as research reagents for characterizing sites of interactions between members of complement/anti-complement pairs as well as sites of cell-cell interactions. Inhibitors of zalpha37 activity (i.e., zalpha37 antagonists) include anti- zalpha37 antibodies and soluble zalpha37 polypeptides (such as residues 20 to 483 in SEQ ED NO:2, or residues 23 to 483 of SEQ ID NO:5 ), as well as other peptidic and non-peptidic agents (including ribozymes). Zalpha37 can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of zalpha37. In addition to those assays disclosed herein, samples can be tested for inhibition of zalpha37 activity within a variety of assays designed to measure enzyme/substrate binding or the stimulation inhibition of zalpha37-dependent cellular responses. For example, zalpha37-responsive cell lines can be transfected with a reporter gene construct that is responsive to a zalpha37- modulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a DNA response element operably linked to a gene encoding an assayable protein, such as luciferase, or a metabolite, such as cyclic AMP. DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE), insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563-72, 1989). 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 56:335-44; 1989. One reporter gene construct would contain a catalytic domain that, upon binding a UDP-glycosyltransferase substrate, would signal intracellularly through, for example, a SRE reporter. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zalpha37 on the target cells, as evidenced by a decrease in zalpha37 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block zalpha37 binding to a substrate (or the anti-complementary member of a complementary/anti- complementary pair) as well as compounds that block processes in the cellular pathway subsequent to complement/anti-complement binding. In the alternative, compounds or other samples can be tested for direct blocking of zalpha37 binding to a substrate using zalpha37 tagged with a detectable label (e.g., ¹²⁵I, biotin, horseradish peroxidase, FITC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled zalpha37 to its substrate is indicative of inhibitory activity, which can be confirmed through secondary assays. UDP-glycosyltransferases used within binding assays may be cellular UDP-glycosyltransferases, soluble UDP- glycosyltransferases, or isolated, immobilized UDP-glycosyltransferases.

Also, zalpha37 polypeptides, agonists or antagonists thereof may be therapeutically useful for promoting wound healing, for example, in liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, and salivary gland. To verify the presence of this capability in zalpha37 polypeptides, agonists or antagonists of the present invention, such zalpha37 polypeptides, agonists or antagonists are evaluated with respect to their ability to facilitate wound healing according to procedures known in the art. If desired, zalpha37 polypeptide performance in this regard can be compared to growth factors, such as EGF, NGF, TGF-α, TGF-β, insulin, IGF-I, IGF-π, fibroblast growth factor (FGF) and the like. In addition, zalpha37 polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more growth factors to identify synergistic effects.

A zalpha37 polypeptide can also be used for purification of substrate. The polypeptide is immobilized on a solid support, such as beads of agarose, cross- linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use. Methods for linking 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 will generally be configured in the form of a column, and fluids containing substrates are passed through the column one or more times to allow substrates to bind to the enzyme polypeptide. The substrate is then eluted using changes in salt concentration, chaotropic agents (guanidine HC1), or pH to disrupt substrate-enzyme binding.

An assay system that uses a substrate-binding enzyme (or an antibody, one member of a complementary/ anti-complementary pair or other cell-surface binding protein) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed. Such enzyme, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a enzyme chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. A enzyme, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If a substrate, epitope, or opposite member of the complementary/anti-complementary pair is present in the sample, it will bind to the immobilized substrate, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated.

Substrate binding enzyme polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

A "soluble protein" is a protein that is not bound to a cell membrane. Soluble proteins are most commonly substrate-binding enzyme polypeptides that lack transmembrane and cytoplasmic domains. Soluble proteins can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate, or immunoglobulin constant region sequences. Many cell-surface proteins have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs. Proteins are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.

Soluble forms of zalpha37 polypeptides may act as antagonsits to or agonists of zalpha37 polypeptides, and would be useful to modulate the effects of zalpha37 in liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary. The soluble of zalpha37 does not contain a transmembrane domain (i.e., the polypeptide of residues 20 to 483 of SEQ ID NO:2, or the polypeptide of residues 23 to 483 of SEQ ID NO:5) may act as an agonist or antagonist of zalpha37 activity. Since polypeptides of this nature are not anchored to the membrane, they can act at sites distant from the tissues in which they are expressed. Thus, the activity of the soluble form of zalpha37 polypeptides can be more wide spread than its membrane-anchored counterpart. Both isoforms would be useful in studying the effects of the present invention in vitro and in vivo.

Molecules of the present invention can be used to identify and isolate UDP-glycosyltransferases, or members of complement/anti-complement pairs involved in cell-cell interactions, glycoprotein and glycolipid biosynthesis and metabolism, and growth factor and hormone regulation. For example, proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Substrate Techniques, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202). Proteins and peptides can also be radiolabeled (Methods in Enzvmol., vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or photoaffinity labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33:1167-80. 1984) and substrate proteins can be identified.

The molecules of the present invention will be useful in modulating cell- cell interactions, and glycolipid and glycoprotein biosynthesis and metabolism, and growth factor and hormone regulation. The polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders associated with tumor growth, hormonal regulation, lipid metabolism and fertility. The molecules of the present invention can be used to modulate cell-cell interactions including, for example, cell adhesion, and cell fusion. Additionally they can be used to modulate glycolipid and glycoprotein biosynthesis and metabolism, or growth factor and hormone regulation or to treat or prevent development of pathological conditions in such diverse tissue as liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary kidney. In particular, certain diseases may be amenable to such diagnosis, treatment or prevention. These diseases include, but are not limited to cancer, cholesterolemia, hypertension, hepatitis, cirrhosis, polycystic kidney disease, prostate hypeφlasia, ovary carcinoma, esophageal carcinoma, liver carcinoma, lung carcinoma, testicular carcinoma and diseases associated with defects in glycosyltransferase activities. The molecules of the present invention can be used to modulate inhibition and proliferation of tissues in the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary.

Polynucleotides encoding zalpha37 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zalpha37 activity. If a mammal has a mutated or absent zalpha37 gene, the zalpha37 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zalpha37 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, heφes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective heφes simplex virus 1 (HSV1) vector (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 zalpha37 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S. Patent 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 vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the puφose of targeting. Targeted peptides (e.g., hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.

Similarly, the zalpha37 polynucleotides (SEQ ED NOs:l, 3, 4, or 6) can be used to target specific tissues such as tissues of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary. It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid; and then to re-implant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun 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. Various techniques, including antisense and ribozyme methodologies, can be used to inhibit zalpha37 gene transcription and translation, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of a zalpha37-encoding polynucleotide (e.g., a polynucleotide as set forth in SEQ ID NOs:l, 3, 4, or 6) are designed to bind to zalpha37-encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of zalpha37 polypeptide-encoding genes in cell culture or in a subject.

Mice engineered to express the zalpha37 gene, referred to as "transgenic mice," and mice that exhibit a complete absence of zalpha37 gene function, referred to as "knockout mice," may also be generated (Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature 366:740-42, 1993; Capecchi, M.R., Science 244: 1288-1292, 1989; Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 1986). For example, transgenic mice that over-express zalpha37, either ubiquitously or under a tissue- specific or tissue-restricted promoter can be used to ask whether over-expression causes a phenotype. For example, over-expression of a wild-type zalpha37 polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which zalpha37 expression is functionally relevant and may indicate a therapeutic target for the zalpha37 , its agonists or antagonists. For example, a transgenic mouse to engineer is one that over-expresses the soluble zalpha37 polypeptide (approximately amino acids 1 to 483 of SEQ ID NO:2 or

5), or the membrane-bound enzyme, (residues 1 to 511 of SEQ JD NO:2 or 5). Moreover, such over-expression may result in a phenotype that shows similarity with human diseases. Similarly, knockout zalpha37 mice can be used to determine where zalpha37 is absolutely required in vivo. The phenotype of knockout mice is predictive of the in vivo effects of that a zalpha37 antagonist, such as those described herein, may have. The human zalpha37 cDNA can be used to isolate murine zalpha37 mRNA, cDNA and genomic DNA, which are subsequently used to generate knockout mice. These mice may be employed to study the zalρha37 gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human diseases. Moreover, transgenic mice expression of zalpha37 antisense polynucleotides or ribozymes directed against zalpha37 , described herein, can be used analogously to transgenic mice described above.

Zalpha37 polypeptides, variants, and fragments thereof, may be useful as replacement therapy for disorders associated with UDP glycosyltransferases, including disorders related to, for example, tumors and disease states of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, esophagus, lung, and ovary, hypertension, elevated cholesterol, defective myelination, and clearance o lipid soluble compounds, including, but not limited to, bilirubin, steroids, drugs, and environmental pollutants. A less widely appreciated determinant of tissue moφhogenesis is the process of cell rearrangement: Both cell motility and cell-cell adhesion are likely to play central roles in moφhogenetic cell rearrangements. Cells need to be able to rapidly break and probably simultaneously remake contacts with neighboring cells. See Gumbiner, B.M., Cell 69:385-387, 1992. As a secreted protein in tissues of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary, zalpha37 can play a role in intercellular rearrangement in these and other tissues.

Zalpha37 gene may be useful to as a probe to identify humans who have a defective zalpha37 gene. The strong expression of zalpha37 in tissues of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland esophagus, lung, and ovary suggests that zalpha37 polynucleotides or polypeptides can be used as measured as an indication of aberrant growth in these tissues. Thus, polynucleotides and polypeptides of zalpha37, and mutations to them, can be used a diagnostic indicators of cancer and tumor stage in these tissues. The zalpha37 polypeptide is expressed in tissues of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary. Thus, the polypeptides of the present invention are useful in studying cell adhesion and the role thereof in metastasis and may be useful in preventing metastasis, in particular metastasis in tumors of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, esophagus, lung, and ovary. Similarly, polynucleotides and polypeptides of zalρha37 may be used to replace their defective counteφarts in tumor or diseased tissues. Thus, zalpha37 polypeptide pharmaceutical compositions of the present invention may be useful in prevention or treatment of disorders associated with pathological regulation or the expansion of these tissues. The polynucleotides of the present invention may also be used in conjunction with a regulatable promoter, thus allowing the dosage of delivered protein to be regulated.

The activity of molecules of the present invention can be measured using a variety of assays that measure cell differentiation and proliferation as well as assays that measure cell contractility. Such assays are well known in the art. Zalpha37 is expressed in contractile tissues, such as testis. The effects of Zalpha37 polypeptide, its antagonists and agonists, on tissue contractility can be measured in vitro using a tensiometer with or without electrical field stimulation. Such assays are known in the art and can be applied to tissue samples, such as aortic rings, vas deferens, ilium, uterine and other contractile tissue samples, as well as to organ systems, such as atria, and can be used to determine whether Zalpha37 polypeptide, its agonists or antagonists, enhance or depress contractility. Molecules of the present invention are hence useful for treating dysfunction associated with contractile tissues or can be used to suppress or enhance contractility in vivo. As such, molecules of the present invention have utility in treating, infertility, in vitro fertilization, birth control, and treating impotence or other male reproductive dysfunction.

The effect of the Zalpha37 polypeptides, antagonists and agonists of the present invention on contractility of tissues, including for example, testis, gastrointestinal tissues, uterus, prostate, and heart can be measured in a tensiometer that measures contractility and relaxation in tissues. See, Dainty et al., J. Pharmacol. 100:767, 1990; Rhee et al., Neurotox. 16: 179, 1995; Anderson, M.B., Endocrinol. 114:364-368, 1984; and Downing, S.J. and Sherwood, O.D, Endocrinol. 116:1206- 1214, 1985. For example, measuring vasodilatation of aortic rings is well known in the art. Briefly, aortic rings are taken from 4 month old Sprague Dawley rats and placed in a buffer solution, such as modified Krebs solution (118.5 mM NaCl, 4.6 mM KC1, 1.2 mM MgSO₄.7H₂O, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂.2H₂O, 24.8 mM NaHCO₃ and 10 mM glucose). One of skill in the art would recognize that this method can be used with other animals, such as rabbits, other rat strains, Guinea pigs, and the like. The rings are then attached to an isometric force transducer (Radnoti Inc., Monrovia, CA) and the data recorded with a Ponemah physiology platform (Gould Instrument systems, Inc., Valley View, OH) and placed in an oxygenated (95% O₂, 5% CO₂) tissue bath containing the buffer solution. The tissues are adjusted to 1 gram resting tension and allowed to stabilize for about one hour before testing. The integrity of the rings can be tested with norepinepherin (Sigma Co., St. Louis, MO) and Carbachol, a muscarinic acetylcholine agonist (Sigma Co.). After integrity is checked, the rings are washed three times with fresh buffer and allowed to rest for about one hour. To test a sample for vasodilatation, or relaxation of the aortic ring tissue, the rings are contracted to two grams tension and allowed to stabilize for fifteen minutes. A Zalpha37 polypeptide sample is then added to 1, 2 or 3 of the 4 baths, without flushing, and tension on the rings recorded and compared to the control rings containing buffer only. Enhancement or relaxation of contractility by Zalpha37 polypeptides, their agonists and antagonists is directly measured by this method, and it can be applied to other contractile tissues such as uterus, prostate, and heart.

The activity of molecules of the present invention can be measured using a variety of assays that measure stimulation of gastrointestinal cell contractility, modulation of nutrient uptake and/or secretion of digestive enzymes. Of particular interest are changes in contractility of smooth muscle cells. For example, the contractile response of segments of mammalian duodenum or other gastrointestinal smooth muscles tissue (Depoortere et al., J. Gastrointestinal Motility 1: 150-159, 1989, incoφorated herein by reference). An exemplary in vivo assay uses an ultrasonic micrometer to measure the dimensional changes radially between commissures and longiturdinally to the plane of the valve base (Hansen et al., Society of Thoracic Surgeons 60:S384-390, 1995).

Gastric motility is generally measured in the clinical setting as the time required for gastric emptying and subsequent transit time through the gastrointestinal tract. Gastric emptying scans are well known to those skilled in the art, and briefly, comprise use of an oral contrast agent, such as barium, or a radiolabeled meal. Solids and liquids can be measured independently. A test food or liquid is radiolabeled with an isotope (e.g. ^99mTc), and after ingestion or administration, transit time through the gastrointestinal tract and gastric emptying are measured by visualization using gamma cameras (Meyer et al., Am. J. Dig. Pis. 21:296, 1976; Collins et al., Gut 24: 1117, 1983; Maughan et al., Diabet. Med. 13 9 Supp. 5:S6-10. 1996 and Horowitz et al., Arch. Intern. Med. 145:1467-1472, 1985). These studies may be performed before and after the administration of a promotility agent to quantify the efficacy of the drug.

The tissue specificity of Zalpha37 expression suggests a role in spermatogenesis, a process that is similar to the development of blood cells (hematopoiesis). Briefly, spermatogonia undergo a maturation process similar to the differentiation of hematopoietic stem cells. In view of the tissue specificity observed for Zalpha37, agonists and antagonists have enormous potential in both in vitro and in vivo applications. Zalpha37 polypeptides, agonists and antagonists may also prove useful in modulating spermatogenesis and thus aid in overcoming infertility. Antagonists are useful as research reagents for characterizing sites of ligand-receptor interaction. In vivo, Zalpha37 polypeptides, agonists or antagonists may find application in the treatment of male infertility or as a male contraceptive agents.

The Zalpha37 polypeptides, antagonists of agonists, of the present invention can also modulate sperm capacitation. Before reaching the oocyte or egg and initiating an egg-sperm interaction, the sperm must be activated. The sperm undergo a gradual capacitation, lasting up to 3 or 4 hours in vitro, during which the plasma membrane of the sperm head and the outer acrosomal membrane fuse to form vesicles that facilitate the release of acrosomal enzymes. The acrosomal membrane surrounds the acrosome or acrosomal cap which is located at the anterior end of the nucleus in the sperm head. In order for the sperm to fertilize egg the sperm must penetrate the oocyte. To enable this process the sperm must undergo acrosomal exocytosis, also known as the acrosomal reaction, and release the acrosomal enzymes in the vicinity of the oocyte. These enzymes enable the sperm to penetrate the various oocyte layers, (the cumulus oophorus, the corona radiata and the zona pellucida). The released acrosomal enzymes include hyaluronidase and proacrosin, in addition to other enzymes such as proteases. During the acrosomal reaction, proacrosin is converted to acrosin, the active form of the enzyme, which is required for and must occur before binding and penetration of the zona pellucida is possible. A combination of the acrosomal lytic enzymes and sperm tail movements allow the sperm to penetrate the oocyte layers. Numerous sperm must reach the egg and release acrosomal enzymes before the egg can finally be fertilized. Only one sperm will successfully bind to, penetrate and fertilize the egg, after which the zona hardens so that no other sperm can penetrate the egg (Zaneveld, in Male Infertility Chapter 11, Comhaire (Ed.), Chapman & Hall, London, 1996). Peptide hormones, such as insulin homologs are associated with sperm activation and egg-sperm interaction. For instance, capacitated sperm incubated with relaxin show an increased percentage of progressively motile sperm, increased zona penetration rates, and increased percentage of viable acrosome-reacted sperm (Carrell et al., Endocr. Res. 21:697-707, 1995). Localization of Zalpha37 to the testis suggests that the Zalpha37 polypeptides described herein play a role in these and other reproductive processes.

Accordingly, proteins of the present invention can have applications in enhancing fertilization during assisted reproduction in humans and in animals. Such assisted reproduction methods are known in the art and include artificial insemination, in vitro fertilization, embryo transfer and gamete intrafallopian transfer. Such methods are useful for assisting men and women who have physiological or metabolic disorders preventing natural conception or can be used to enhance in vitro fertilization. Such methods are also used in animal breeding programs, such as for livestock breeding and could be used as methods for the creation of transgenic animals. Proteins of the present invention can be combined with sperm, an egg or an egg-sperm mixture prior to fertilization of the egg. In some species, sperm capacitate spontaneously during in vitro fertilization procedures, but normally sperm capacitate over an extended period of time both in vivo and in vitro. It is advantageous to increase sperm activation during such procedures to enhance the likelihood of successful fertilization. The washed sperm or sperm removed from the seminal plasma used in such assisted reproduction methods has been shown to have altered reproductive functions, in particular, reduced motility and zona interaction. To enhance fertilization during assisted reproduction methods sperm is capacitated using exogenously added compounds. Suspension of the sperm in seminal plasma from normal subjects or in a "capacitation media" containing a cocktail of compounds known to activate sperm, such as caffeine, dibutyl cyclic adenosine monophosphate (dbcAMP) or theophylline, have resulted in improved reproductive function of the sperm, in particular, sperm motility and zonae penetration (Park et al., Am. J. Obstet. Gvnecol. 158:974-9, 1988; Vandevoort et al., Mol. Repro. Develop. 37:299-304, 1993; Vandevoort and Overstreet, J. Androl. 16:327-33, 1995). The presence of immunoreactive relaxin in vivo and in association with cryopreserved semen, was shown to significantly increase sperm motility (Juang et al., Anim. Reprod. Sci. 20:21-9, 1989; Juang et al., Anim. Reprod. Sci. 22:47-53, 1990). Porcine relaxin stimulated sperm motility in cryopreserved human sperm (Colon et al., Fertil. Steril. 46:1133-39, 1986; Lessing et al., Fertil. Steril. 44:406-9, 1985) and preserved ability of washed human sperm to penetrate cervical mucus in vitro (Brenner et al., Fertil. Steril. 42:92-6, 1984). Polypeptides of the present invention can used in such methods to enhance viability of cryopreserved sperm, enhance sperm motility and enhance fertilization, particularly in association with methods of assisted reproduction.

In cases where pregnancy is not desired, Zalpha37 polypeptide or polypeptide fragments may function as germ-cell-specific antigens for use as components in "immunocontraceptive" or "anti-fertility" vaccines to induce formation of antibodies and/or cell mediated immunity to selectively inhibit a process, or processes, critical to successful reproduction in humans and animals. The uses of sperm and testis antigens in the development of immunocontraceptives have been described (O'Hern et al., Biol Reprod. 52:311-39, 1995; Diekman and Herr, Am. J. Reprod. Immunol. 37:111-17, 1997; Zhu and Naz. Proc. Natl. Acad. Sci. USA 94:4704- 9,1997). A vaccine based on human chorionic gonadotrophin (HCG) linked to a diphtheria or tetanus carrier was in clinical trials (Talwar et al., Proc. Natl. Acad. Sci. USA 91:8532-36, 1994). A single injection resulted in production of high titer antibodies that persisted for nearly a year in rabbits (Stevens, Am. J. Reprod. Immunol.

29:176-88, 1993). Such methods of immunocontraception using vaccines could include a Zalpha37 testes-specific protein or fragment thereof. The Zalpha37 protein or fragments can be conjugated to a carrier protein or peptide, such as tetanus or diphtheria toxoid. An adjuvant, as described above, can be included and the protein or fragment can be noncovalently associated with other molecules to enhance intrinsic immunoreactivity. Methods for administration and methods for determining the number of administrations are known in the art. Such a method might include a number of primary injections over several weeks followed by booster injections as needed to maintain a suitable antibody titer.

The polypeptides, antagonists, agonists, nucleic acid and/or antibodies of the present invention may be used in treatment of disorders associated with gonadal development, pubertal changes, fertility, neuralgia associated with reproductive phenomena, male sexual dysfunction, impotency, prostate cancer, testicular cancer, gastrointestinal mobility and dysfunction. The molecules of the present invention may used to modulate or to treat or prevent development of pathological conditions in such diverse tissue as fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary. In particular, certain syndromes or diseases may be amenable to such diagnosis, treatment or prevention. Moreover, natural functions, such as spermatogenesis, may be suppressed or controlled for use in birth control by molecules of the present invention. Molecules expressed in the testis, such as Zalpha37 polypeptides, may modulate hormones, hormone receptors, growth factors, or cell-cell interactions, of the reproductive cascade or be involved in spermatogenesis, or testis development, would be useful as markers for cancer of reproductive organs and as therapeutic agents for hormone-dependent cancers, by inhibiting hormone-dependent growth and/or development of tumor cells. Human reproductive system cancers such as testicular and prostate cancers are common. Moreover, receptors for steroid hormones involved in the reproductive cascade are found in human tumors and tumor cell lines (breast, prostate, endometrial, ovarian, kidney, and pancreatic tumors) (Kakar et al., Mol. Cell. Endocrinol.. 106:145-49, 1994; Kakar and Jennes, Cancer Letts.. 98:57-62, 1995). Thus, expression of Zalpha37 in reproductive tissues suggests that polypeptides of the present invention would be useful in diagnostic methods for the detection and monitoring of reproductive cancers.

Diagnostic methods of the present invention involve the detection of Zalpha37 polypeptides in the serum or tissue biopsy of a patient undergoing analysis of reproductive function or evaluation for possible reproductive cancers, e.g., testicular or prostate cancer. Such polypeptides can be detected using immunoassay techniques and antibodies, described herein, which are capable of recognizing Zalpha37 polypeptide epitopes. More specifically, the present invention contemplates methods for detecting Zalpha37 polypeptides comprising:

Exposing a test sample potentially containing Zalpha37 polypeptides to an antibody attached to a solid support, wherein said antibody binds to a first epitope of a Zalpha37 polypeptide;

Washing the immobilized antibody-polypeptide to remove unbound contaminants;

Exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a Zalpha37 polypeptide, wherein the second antibody is associated with a detectable label; and

Detecting the detectable label. Altered levels of Zalpha37 polypeptides in a test sample, such as serum, semen, urine, sweat, saliva, biopsy, and the like, can be monitored as an indication of reproductive function or of reproductive cancer or disease, when compared against a normal control.

Additional methods using probes or primers derived, for example, from the nucleotide sequences disclosed herein can also be used to detect Zalpha37 expression in a patient sample, such as a blood, urine, semen, saliva, sweat, biopsy, tissue sample, or the like. For example, probes can be hybridized to tumor tissues and the hybridized complex detected by in situ hybridization. Zalpha37 sequences can also be detected by PCR amplification using cDNA generated by reverse translation of sample mRNA as a template (PCR Primer A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Press, 1995). When compared with a normal control, both increases and decreases of Zalpha37 expression in a patient sample, relative to that of a control, can be monitored and used as an indicator or diagnostic for disease.

Moreover, the activity and effect of KLP and mouse zalpha37 on tumor progression and metastasis can be measured in vivo. Several syngeneic mouse models have been developed to study the influence of polypeptides, compounds or other treatments on tumor progression. In these models, tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor. The cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models. Tumor models include the Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No. CRL-6323), amongst others. These are both commonly used tumor lines, syngeneic to the C57BL6 mouse, that are readily cultured and manipulated in vitro. Tumors resulting from implantation of either of these cell lines are capable of metastasis to the lung in C57BL6 mice. The Lewis lung carcinoma model has recently been used in mice to identify an inhibitor of angiogenesis (O'Reilly MS, et al. C l 79: 315-328,1994). C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one time injection of recombinant adenovirus. Three days following this treatment, 10 to 10 cells are implanted under the dorsal skin. Alternatively, the cells themselves may be infected with recombinant adenovirus, such as one expressing KLP and mouse zalpha37, before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically. The mice normally develop visible tumors within 5 days. The tumors are allowed to grow for a period of up to 3

3 weeks, during which time they may reach a size of 1500 - 1800 mm in the control treated group. Tumor size and body weight are carefully monitored throughout the experiment. At the time of sacrifice, the tumor is removed and weighed along with the lungs and the liver. The lung weight has been shown to correlate ell with metastatic tumor burden. As an additional measure, lung surface metastases are counted. The resected tumor, lungs and liver are prepared for histopathological examination, immunohistochemistry, and in situ hybridization, using methods known in the art and described herein. The influence of the expressed polypeptide in question, e.g., KLP and mouse zalpha37, on the ability of the tumor to recruit vasculature and undergo metastasis can thus be assessed. In addition, aside from using adenovirus, the implanted cells can be transiently transfected with KLP and mouse zalpha37. Moreover, purified KLP and mouse zalpha37 or KLP and mouse zalpha37-conditioned media can be directly injected in to this mouse model, and hence be used in this system. Use of stable KLP and mouse zalpha37 transfectants as well as use of induceable promoters to activate KLP and mouse zalpha37 expression in vivo are known in the art and can be used in this system to assess KLP and mouse zalpha37 induction of metastasis. For general reference see, O'Reilly MS, et al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.

Prostatic hypeφlasia is an almost universal phenomenon is aging men, and is characterized by enlargement of the prostate, often resulting in obstruction of the urethra. See Fauci, et al., Harrison's Principles of Internal Medicine. Fourteenth Edition, Mc-Graw-Hill, 1998, p. 596 to 588. As a protein that is expressed in prostate tissue, polypeptides and polynucleotides can be used to treat or diagnose prostate related disorders, such as, for example, prostate hypeφlasia. Diagnostically, an increase, or decrease in zalpha37 molecules, including polypeptides, polynucleotides and fragments thereof, can signal an enlargement, or shrinkage, respectively, of said tissue. Assays to detect polynucleotides and polypeptides of the present invention are discussed elsewhere in this application and are widely known in the art. Additionally, antagonists, including antibodies would find use as a means of treating the enlargement of the prostate. Such methods are also widely known in the art. UGT2B17, a UDP-glucuronosyltransfease enzyme that is expressed in prostate tissue where it may be responsible for glucouronidation, and deactivation of androgens, thus regulating the level of androgens in this tissue. (Beaulieu, M. et al., 1 Biol. Chem. 271(37):22855-22862, 1996) As a protein that shares homology to UGT3B17, and is expressed in prostate tissue, zalpha37 can be useful as a regulator of steroid hormones in this tissue. Thus, a such treatment could include gene therapy, or surgical or systemic application of zalpha37 polypeptides and/or polynucleotides.

Zalpha37 has homology to the galactosyltransferases, which have been associated with various disease and carcinomic states. For example, galactosyltransferase has been discovered to be exclusively associated with carcinomic cells and premalignant cells in prostatic hypeφlasia (Amselgruber, W.M. et al., Nutrition 11(5 Suppl):638-421; 1995). Remarkable elevation of galactosyltransferase activity was observed in sera of patients with cancer, especially those with blood cancer. (Nishiwaki, S. et al., Cancer Res. 52(7): 1875-80. 1992). In this study a high incidence was observed in the progressive stage in esophagus, stomach, colorectal, and testis cancer, and the enzyme level in sera of patients with benign disease was elevated.

After effective therapy the enzyme activity decreased to below detectable levels. Release of galactosyltransferase from cancer cells was also observed by Strous (see Strous G.J., Crit. Rev. Biochem 21(2): 119-51; 1986) in which an increased level of galactosyltransferase enzyme activity was reported in patients suffering from breast and ovary cancer. Thus, methods of detecting galactosyltransferase enzymes have a use in detecting and monitoring disease, hypeφlasia, and cancer. Methods of detecting zalpha in tissues of the kidney, liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, esophagus, lung, and ovary, as well as in peripheral blood, serum, ascites, milk and saliva can aid in diagnosing, staging, and monitoring such diseases. Tn-syndrome, also called Permanent Mixed-Field Polyagglutinability, is a very rare acquired disorder affecting all hematopoietic lineages. This syndrome is characterized by the expression of the Tn and sialosyl-Tn antigens on the cell surface. The Tn antigen has been identified as an unsubstituted α-linked N-acetyl- galactosamine linked O-glycosidically to threonine or serine residues of membrane proteins. In healthy blood, this sugar is substituted by galactose and sialic acid to form a tetrasaccharide. This Tn antigen may be a result of a deficiency in β,l,3,galactosyltransferase. Expression of the Tn antigen along with the sialosyl-Tn antigen and a TF antigen (characterized by a deficiency in α,2,3,sialyl- transferase) have been recognized as a cancer-associated phenomenon for many years. See Berger, E.G. et al., Transfus. Clin. Biol. 2:103-108, 1994.

Thus, the study of this syndrome has been useful in elucidating the biology of carbohydrate glycosylation disorders and the appearance of cryptantigens on the cell surface, and cancer. Highly specific and complex tumor glycan antigens are likely of great interest in studying tissue specific tumors and zalpha37 can be useful for studying tumors in kidney, liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, esophagus, lung, and ovary tumors.

Itzkowitz, et al., looked at the expression of these cryptantigens in tissues from normal, chronic pancreatitic, and pancreatic cancer patients. The sialosyl- Tn antigen is expressed in 97% of malignant, but 0% of normal tissues. The authors suggest that normal pancreas tissue is preferentially galactosylated resulting in less silaosyl-Tn antigen. In malignant tissue, conditions favor the sialylation of Tn antigens thereby accounting for enhanced expression of sialosyl Tn over T anitgens.

In view of the presence of zalpha37 cDNA in kidney, liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary tissue, a defect in the zalpha37 gene may result in defective galactosylation of cell surface carbohydrates of nephrotic cells, leading to over sialylation of the Tn antigen. Thus, zalpha37 polypeptides would be useful as β,l,3, galactosyltransferase replacement therapy for pre-cancerous and cancer tissues. To verify the presence of such activity in zalpha37 containing normal cell lines and tumor cell lines, such cell lines are evaluated with respect to the presence of the Tn antigen according to procedures known in the art. See, for example, Berger et al., ibid., Itzkowitz et al., ibid, and the like.

Additionally, the lack of conditions favoring proper galactosylation may result in an increase in sialosyl Tn antigens in tissues expressing zalpha37, which may cause an auto-immune reaction resulting in an immune attack on the kidney, liver fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, esophagus, lung, and ovary. In these cases, zalpha37 molecules may be used to encourage proper galactosylation and limit the antigenic recognition in tissues over expressing the sialosyl Tn antigen. Similarly, a defective zalpha37 gene may result in improper glycoslation of the surface carbohydrates of the tissues of kidney, liver, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary thus affecting cell-cell interactions and possibly cell cycle regulation. Such cases could be treated by administering polypeptides of zalpha37 to mammals with such a defective gene.

Human zalpha37 polynucleotides of SEQ ED NO: 1 map to chromosome 5pl3. Thus, the present invention also provides reagents which will find use in diagnostic applications. For example, the zalpha37 gene, a probe comprising zalpha37 DNA or RNA or a subsequence thereof can be used to determine if the zalpha37 gene is present on chromosome 5pl3 or if a mutation has occurred. Detectable chromosomal aberrations at the zalpha37 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level.

Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymoφhism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).

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 disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. 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 within 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 or 3, the complement of SEQ ED NOs:l or 3, or an RNA equivalent thereof. Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymoφhism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1:5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid. ; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995). Ribonuclease protection assays (see, e.g., Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA are protected from digestion. Within PCR assays, a patient's genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient. Another PCR- based technique that can be employed is single strand conformational polymoφhism (SSCP) analysis (Havashi, PCR Methods and Applications 1:34-8. 1991).

For pharmaceutical use, the proteins of the present invention can be administered orally, rectally, parenterally (particularly intravenous or subcutaneous), intracisternally, intraperitoneally, topically (as douches, powders, ointments, drops or transdermal patch) bucally, or as a pulmonary or nasal inhalant. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, pharmaceutical formulations will include a zalpha37 protein, alone, or in conjunction with a dimeric partner, in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995. Therapeutic doses will generally be in the range of 0.1 to 100 μg/kg of patient weight per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. The proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years. In general, a therapeutically effective amount of zalpha37 is an amount sufficient to produce a clinically significant change in extracellular matrix remodeling, scar tissue formation, tumor suppression, platelet aggregation, apoptosis, myogenesis, metastasis in tissues of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, esophagus, lung, and ovary. Similarly, a therapeutically effective amount of zalpha37 is an amount sufficient to produce a clinically significant change in disorders associated with tissues of the liver, kidney, fetal kidney, fetal muscle, spinal cord, testis, bone marrow, prostate, salivary gland, as well as carcinomas of esophagus, liver, lung, and ovary. The invention is further illustrated by the following non-limiting examples.

EXAMPLES From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for puφoses of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Example 1

Extension of EST Sequence The novel KLP polypeptide-encoding polynucleotides of the present invention were initially identified by querying a database of partial sequences. A partial sequence was identified in a diseased liver cDNA library. The polynucleotide sequence (SEQ ID NO:l) of the insert corresponding to the cDNA clone was sequenced. The deduced amino acid sequence of the insert was determined to be full-length and is shown in SEQ ID NO:2. This polypeptide, and the polynucleotides encoding it, were identified as a novel member of the UDP-glycosyltransferase family, KLP. Partial sequences were additionally identified in normal kidney cDNA libraries. Example 2

Identification of the Mouse Ortholog The polypeptide-encoding polynucleotides of the mouse ortholog of the human zalpha37 polypeptides, zalpha37m, were identified by their homology to the human sequence. Clone INC2516747 was used to obtain a consensus sequence that was used to query the mouse EST database. Several potential orthologs were selected, one of which was used to search for a potential full-length clone. EST2342680 was identified in a mouse kidney cDNA library and ordered. The deduced amino acid sequence was found to contain the full-length sequence of zalpha37m (SEQ ID NO: 5). Example 3 Chromosomal Assignment and Placement of Human Zalpha37 Human Zalpha37 was mapped to chromosome 5 using the commercially available version of the "Stanford G3 Radiation Hybrid Mapping Panel" (Research Genetics, Inc., Huntsville, AL). The "Stanford G3 RH Panel" contains DNA from each of 83 radiation hybrid clones of the whole human genome, plus two control DNAs (the RM donor and the A3 recipient). A publicly available WWW server (http://shgc- www.stanford.edu) allows chromosomal localization of markers and genes.

For the mapping of human Zalpha37 with the "Stanford G3 RH Panel", 20 μl reactions were set up in a 96-well microtiter plate compatible for PCR (Stratagene, La Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the 85 PCR reactions consisted of 2 μl 10X KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 μl sense primer, ZC 25,875 (SEQ ID NO:7), 1 μl antisense primer, ZC 25,876 (SEQ ED NO:8), 2 μl "RediLoad" (Research Genetics, Inc., Huntsville, AL), 0.4 μl 50X Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and distilled water for a total volume of 20 μl. The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 94°C, 35 cycles of a 45 seconds denaturation at

94°C, 45 seconds annealing at 58°C and 1 minute AND 15 seconds extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C. The reactions were separated by electrophoresis on a 2% agarose gel (EM Science, Gibbstown, NJ) and visualized by staining with ethidium bromide. The results showed linkage of Zalpha37 to the chromosome 5 marker

SHGC-64147 with a LOD score of >12 and at a distance of 0 cR_10000 from the marker. The use of surrounding genes and markers positions human Zalpha37 in the 5pl3 chromosomal region. This region of the human chromosome is a known syntenic region for mouse chromosome region 15. Example 4 Chromosomal Assignment and Placement of Human Murine Zalpha37

Murine Zalpha37 was mapped in mouse to chromosome 15 using the commercially available mouse T31 whole genome radiation hybrid (WGRH) panel (Research Genetics, Inc., Huntsville, AL). A publicly available WWW server (http://www.genome.wi.mit.edu/cgi-bin/mouse_rh/rhmap-auto/rhmapper.cgi) allowed chromosomal localization in relationship to mapped markers on the Whitehead Institute/MIT Center for Genome Research's (WICGR) radiation hybrid map of the mouse genome. The T31 WGRH panel contains DNA from each of 100 radiation hybrid clones, plus two control DNAs (the 129 aa donor and the A23 recipient). For the mapping of murine Zalpha37 with the T31 WGRH panel, 20 μl reactions were set up in 96-well microtiter plates compatible for PCR (Stratagene, La Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the 102 PCR reactions consisted of 2 μl 10X PCR reaction buffer (Qiagen, Inc., Valencia, CA), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 μl sense primer, ZC 26,426 (SEQ ID NO:9), 5' CCC TGG GCT CTG TAG TGA 3', 1 μl antisense primer, ZC 26,427 (SEQ ID NO: 10), 5" TGG GGC CAA ACT GAC ATC 3', 2 μl "RediLoad" (Research Genetics, Inc., Huntsville, AL), 0.1 μl Qiagen HotStarTaq DNA Polymerase (5 units/μl), 25 ng of DNA from an individual hybrid clone or control and distilled water for a total volume of 20 μl. The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 15 minute denaturation at 95°C, 35 cycles of a 45 seconds denaturation at 95°C, 45 seconds annealing at 58°C and 1 minute and 15 seconds extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C. The reactions were separated by electrophoresis on a 2% agarose gel (EM Science, Gibbstown, NJ) and visualized by staining with ethidium bromide.

The results showed that murine Zalpha37 maps 7.90 cR_3000 distal of the framework marker D15Mitl l on the mouse chromosome 15 WICGR radiation hybrid map. Example 5 Tissue Distribution of Human Zalpha37 in Tissue Panels using PCR

A panel of cDNA samples from human tissues was screened for zalpha37 expression using PCR. The panel was made in-house and contained 94 cDNA samples from marathon cDNA and cDNA samples from various normal and cancerous human tissues and cell lines as shown in Table 4, below. The cDNA samples came from in-house libraries or marathon cDNA preparations of RNA that were prepared in- house, or from a commercial supplier such as Clontech (Palo Alto, CA) or Invitrogen (Carlsbad, CA). The marathon cDNAs were made using the Marathon-Ready™ Kit (Clontech, Palo Alto, CA) and standardized to ensure an equal amount of cDNA was placed inot each well. To assure quality of the panel samples, three tests for quality control (QC) were run: (1) To assess the RNA quality used for the libraries, the in- house cDNAs were tested for average insert size by PCR with vector oligos that were specific for the vector sequences for an individual cDNA library; (2) Standardization of the concentration of the cDNA in panel samples was achieved using standard PCR methods to amplify full length alpha tubulin or G3PDH cDNA; and (3) a sample was sent to sequencing to check for possible ribosomal or mitochondrial DNA contamination. The panel was set up in a 96-well format that included a human genomic DNA (Clontech, Palo Alto, CA) positive control sample. Each well contained approximately 0.2-100 pg/μl of cDNA. The PCR reactions were set up using oligos ZC25939 (SEQ ID NO: 11) and ZC25940 (SEQ ID NO: 12), Advantage 2 Taq Polymerase (Clontech, Palo Alto, CA), and Rediload dye (Research Genetics, Inc., Huntsville, AL). The amplification was carried out as follows: 1 cycle at 94°C for 2 minutes, 35 cycles of 94°C for 10 seconds, 65.0°C for 20 seconds and 68°C for 60 seconds, followed by 1 cycle at 68°C for 7 minutes. About 12.5 μl of the PCR reaction product was subjected to standard Agarose gel electrophoresis using a 2% agarose gel. The correct predicted DNA fragment size of -307 was observed in fetal kidney, fetal muscle, kidney, liver, spinal cord, five testis samples, bone marrow, two prostate samples, salivary gland, esophagus tumor, liver tumor, lung tumor, ovarian tumor and the genomic well . Table 4

Example 6

Zalpha37 Expression in Normal Ovary and Ovary Carcinoma using PCR To determine expression levels of zalpha37 in normal ovary an additional PCR was performed on cDNA prepared from normal ovary. Another cDNA sample from an ovary carcinoma, as well as a positive control were also included in the reaction. The PCR reactions were set up using oligos ZC25939 (SEQ ID NO: 11) and

ZC25940 (SEQ ID NO: 12), Advantage 2 Taq Polymerase (Clontech, Palo Alto, CA), and Rediload dye (Research Genetics, Inc., Huntsville, AL). The amplification was carried out as follows: 1 cycle at 94°C for 2 minutes, 35 cycles of 94°C for 10 seconds, 65.0°C for 20 seconds and 68°C for 60 seconds, followed by 1 cycle at 68°C for 4 minutes, and refrigeration until the reaction mixtures were loaded on an agarose gel. About 12.5 μl of the PCR reaction product was subjected to standard Agarose gel electrophoresis using a 2% agarose gel. The predicted DNA fragment size of -307 was not observed in the normal ovary or carcinomic ovary cDNA samples, but was observed in the positive control.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for puφoses of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. An isolated polypeptide chosen from: a) a polypeptide comprising residues 20 to 483 of SEQ ID NO:2;and b) a polypeptide comprising residues 23 to 483 of SEQ ED NO: 5.

2. An isolated polypeptide chosen of: a) a polypeptide comprising residues 1 to 19 of SEQ ID NO:2; b) a polypeptide comprising residues 35 to 49 of SEQ ED NO:2; c) a polypeptide comprising residues 50 to 483 of SEQ ED NO:2; d) a polypeptide comprising residues 484 to 511 of SEQ ID NO:2; e) a polypeptide comprising residues 518 to 521 of SEQ ID NO:2; f) a polypeptide comprising residues 20 to 483 of SEQ ID NO:2; g) a polypeptide comprising residues 20 to 523 of SEQ ID NO:2; h) a polypeptide comprising residues 1 to 22 of SEQ ID NO:5; i) a polypeptide comprising residues 28 to 42 of SEQ ED NO:5; j) a polypeptide comprising residues 43 to 483 of SEQ ID NO:5; k) a polypeptide comprising residues 484 to 511 of SEQ ED NO:5;

1) a polypeptide comprising residues 518 to 521 of SEQ ED NO:5; m) a polypeptide comprising residues 23 to 483 of SEQ ID NO: 5; and n) a polypeptide comprising residues 23 to 523 of SEQ ID NO:5.

3. A method of producing an antibody comprising the following steps in order: inoculating an animal with a polypeptide chosen from: a) a polypeptide comprising residues 1 to 19 of SEQ ID NO:2; b) a polypeptide comprising residues 35 to 49 of SEQ ID NO:2; c) a polypeptide comprising residues 50 to 483 of SEQ JD NO:2; d) a polypeptide comprising residues 484 to 511 of SEQ ED NO:2; e) a polypeptide comprising residues 518 to 521 of SEQ ED NO:2; f) a polypeptide comprising residues 20 to 483 of SEQ ED NO:2; g) a polypeptide comprising residues 20 to 523 of SEQ ID NO:2; h) a polypeptide comprising residues 1 to 22 of SEQ ID NO:5; i) a polypeptide comprising residues 28 to 42 of SEQ ID NO: 5; j) a polypeptide comprising residues 43 to 483 of SEQ ID NO:5; k) a polypeptide comprising residues 484 to 511 of SEQ ID NO:5; 1) a polypeptide comprising residues 518 to 521 of SEQ ED NO: 5; m) a polypeptide comprising residues 23 to 483 of SEQ ID NO:5; and n) a polypeptide comprising residues 23 to 523 of SEQ ID NO:5 wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.

4. An antibody produced by the method of claim 3, which binds to a residues 1 to 523 of SEQ ED NO:2 or to residues 1 to 483 of SEQ ID NO:5.

5. The antibody of claim 4, wherein 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.

6. An antibody which specifically binds to a polypeptide of claim 3.

7. An isolated polynucleotide encoding a polypeptide wherein the polypeptide comprises the polypeptide according to claim 1.

8. An expression vector comprising the following operably linked elements: a) a transcription promoter; b) a DNA segment wherein the DNA segment is chosen from: i) a polynucleotide encoding a polypeptide comprising residues 20 to 483 of SEQ ID NO:2; and ii) a polypeptide comprising residues 23 to 483 of SEQ ED NO: 5; and c) a transcription terminator.

9. A cultured cell into which has been introduced an expression vector according to claim 8, wherein said cell expresses the polypeptide encoded by the DNA segment.

10. A method of producing a polypeptide comprising culturing a cell according to claim 9, whereby said cell expresses the polypeptide encoded by the DNA segment; and recovering the polypeptide.

11. The polypeptide produced by the method according to claim 10.

12. A method of forming a reversible enzyme-substrate complex comprising; providing an enzyme wherein the enzyme is chosen from: a) a polypeptide comprising residues 20 to 483 of SEQ ED NO: 5; and

b) a polypeptide comprising residues 23 to 483 of SEQ JD NO:5; and contacting the enzyme with a substrate; whereby the enzyme binds the substrate.

13. The method of claim 12, wherein the substrate is chosen from: a) glycoproteins; b) glycolipids; and c) antibodies.