WO2001044479A1 - Galactosyltransferase homolog, znssp8 - Google Patents

Abstract

Novel soluble, and membrane bound, Beta 1,3 galactosyltransferase polypeptides, polynucleotides encoding the polypeptides, antibodies and related compositions and methods are disclosed. The polypeptides may be used for detecting anti-complementary molecules, agonists and antagonists. The polypeptides, polynucleotides and antibodies may also be used in methods that modulate cell-cell interactions, extracellular matrix interactions and glycoprotein and glycolipid modifications.

Description

Description GALACTOSYLTRANSFERASE HOMOLOG, ZNSSP8

BACKGROUND OF THE INVENTION

Beta-l,3-galactosyltransferase molecules are classified in the family of glycosyltransferases. In addition to transferring carbohydrate molecules to glycoproteins during biosynthesis, members of this family have also been detected on the cell surface where they are thought to be involved in varying aspects of cell-cell interactions. This family includes carbohydrate transferring enzymes, such as sialyltransferases and fucosyltransferases, and galactosyltransferases. During the formation of C inked glycoproteins and the modification of N-linked ones, each sugar transfer is catalyzed by a different type of glycosyltransferase. Each glycosyltransferase enzyme is specific for both the donor sugar nucleotide and the acceptor molecule.

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. The Beta- 1,3-galactosyl-transferases are characterized by the elongation of type I oligosaccharide chains, and the Beta-l,4-galactosyl-transferases are characterized by the elongation of type II oligosaccharide chains. Both types of carbohydrate structures are present in soluble oligosaccharides of human milk, and are also found on glycoproteins and glycolipids, and are important precursors of blood group antigens. Both galactosyltransferases require a divalent cation (Mn²⁺) to function. Beta- 1,4- galactosyltransferases are expressed in various cell types and tissues, while the Beta- 1,3-galactosyltransferases seem to have more restricted tissue distributions.

Some galactosyltransferases are found in the Golgi apparatus. These Golgi-localized enzymes have structure similarity: a short Ν-terminal domain that faces the cytosol, a single transmembrane a helix, and a large C-terminal domain that faces the Golgi lumen and that contains the catalytic site. The transmembrane a helix is necessary and sufficient to restrict the enzyme to the Golgi. Of the Beta- 1,3- galactosyltransferase family two members (See Amado, M. et al., J. Biol. Chem. 273, 21: 12770-12778, 1998) have been predicted to have two potentially different initiation codons, resulting in two different N-terminal cytoplasmic domains.

Additionally, galactosyltransferases have been shown to be expressed on the cell surface, where their function is theorized to participate in cellular interactions, perhaps as receptors, or receptor-like complementary molecules. 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 Biol. 61:143-158, 1984.

The failure of tumor cell-tumor cell adhesion is believed to be a contributing factor to 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 are therefore sought for the study, diagnosis, prevention or treatment of metastases. βl — >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, is involved in contact and adhesion between germ-line and follicle cells (Amado, M. et al., J. Biol. Chem. 273, 21: 12770-12778,

1998). 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 imcomplete or disordered gylcan biosynthesis has been recognized as a cancer-associated phenomenon. Tn and sialosyl-Tn antigens are among the most investigated cancer- associated carbohydrates 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

Within one aspect the invention provides an isolated polypeptide comprising residues 83 to 343 of SEQ ID NO:2. Within an embodiment, the isolated polypeptide comprises residues 33 to 378 of SEQ ID NO:2 is provided. Within additional embodiments, the isolated polypeptides comprise residues 15 to 343 of SEQ ID NO:2, residues 15 to 378 of SEQ ID NO:2; residues 33 to 343 of SEQ ED NO:2, and residues 1 to 378 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 14 of SEQ ID NO:2; a polypeptide comprising residues 15 to 32 of SEQ ID NO:2; a polypeptide comprising residues 33 to 82 of SEQ ID NO:2; a polypeptide comprising residues 83 to 343 of SEQ ED NO:2; a polypeptide comprising residues 344 to 378 of SEQ ID NO:2; a polypeptide comprising residues 33 to 343 of SEQ ED NO:2; a polypeptide comprising residues 15 to 343 of SEQ ED NO:2; a polypeptide comprising residues 1 to 343 of SEQ DD NO:2; a polypeptide comprising residues 83 to 378 of SEQ

ED NO:2; a polypeptide comprising residues 33 to 378 of SEQ ED NO:2; a polypeptide comprising residues 15 to 378 of SEQ ED NO:2; a polypeptide comprising residues 1 to 378 of SEQ ED NO:2; a polypeptide comprising residues 15 to 82 of SEQ ED NO:2; a polypeptide comprising residues 1 to 32 of SEQ ED NO:2; and a polypeptide comprising residues 1 to 82 of SEQ ED NO:2. Within another aspect, the invention provides an isolated polynucleotide encoding a polypeptide wherein the polypeptide comprises residues 83 to 343 of SEQ ED NO:2. Within an embodiment, the isolated polynucleotide comprises residues 33 to 343 of SEQ ED NO:2. Within additional embodiments, the isolated polynucleotide comprises residues 15 to 343 of SEQ ED NO:2, residues 15 to 378 of SEQ ED NO:2; and residues 1 to 378 of SEQ ED 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 14 of SEQ ED NO:2; a polypeptide comprising residues 15 to 32 of SEQ ED NO:2; a polypeptide comprising residues 33 to 82 of SEQ ED NO:2; a polypeptide comprising residues 83 to 343 of SEQ ED NO:2; a polypeptide comprising residues 344 to 378 of SEQ ED NO:2; a polypeptide comprising residues 33 to 343 of SEQ ED NO:2; a polypeptide comprising residues 15 to 343 of SEQ ED NO:2 a polypeptide comprising residues 1 to 343 of SEQ ED NO:2; a polypeptide comprising residues 83 to 378 of SEQ ED NO:2; a polypeptide comprising residues 33 to 378 of SEQ ED NO:2; a polypeptide comprising residues 15 to 378 of SEQ ED NO:2; a polypeptide comprising residues 1 to 378 of SEQ ED NO:2; a polypeptide comprising residues 15 to 82 of SEQ ED NO:2; a polypeptide comprising residues 1 to 32 of SEQ ED NO:2; and a polypeptide comprising residues 1 to 82 of SEQ ED NO:2. Within another aspect, the invention provides an expression vector comprising the following operably linked elements: a) a transcription promoter; b) a DNA segment wherein the DNA segment is a polynucleotide encoding the polypeptide comprising residues 83 to 343 of SEQ ED NO:2; 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 another embodiment, is provided a method of producing a polypeptide comprising culturing said 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 14 of SEQ ED NO:2; a polypeptide comprising residues 15 to 32 of SEQ ED NO:2; a polypeptide comprising residues 33 to 82 of SEQ ED NO:2; a polypeptide comprising residues 83 to 343 of SEQ ED NO:2; a polypeptide comprising residues 344 to 378 of SEQ ED NO:2; a polypeptide comprising residues 33 to 343 of SEQ ED NO:2; a polypeptide comprising residues 15 to 343 of SEQ ED NO:2 a polypeptide comprising residues 1 to 343 of SEQ ED NO:2; a polypeptide comprising residues 83 to 378 of SEQ ED NO:2; a polypeptide comprising residues 33 to 378 of SEQ ED NO:2; a polypeptide comprising residues 15 to 378 of SEQ ED NO:2; a polypeptide comprising residues 1 to 378 of SEQ ED NO:2; a polypeptide comprising residues 15 to 82 of SEQ ED NO:2; a polypeptide comprising residues 1 to 32 of SEQ ED NO:2; and a polypeptide comprising residues 1 to 82 of SEQ ED NO:., 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 378 of SEQ ED NO:2. Within another embodiment, the antibody is a monoclonal antibody. Within another embodiment, the invention provides an antibody which specifically binds to a polypeptide of residues 83 to 343 of SEQ ED NO:2.

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 5 to 12 of SEQ ED NO:2; a polypeptide consisting of residues 43 to 48 of SEQ ED NO:2; a polypeptide consisting of residues 66 to 74 of SEQ ED NO:2; a polypeptide consisting of residues 66 to 85 of SEQ ED NO:2; a polypeptide consisting of residues 78 to 85 of SEQ ED NO:2; a polypeptide consisting of residues 96 to 121 of SEQ ED NO:2; a polypeptide consisting of residues 133 to 144 of SEQ ED NO:2; a polypeptide consisting of residues 133 to 155 of SEQ ED NO:2; a polypeptide consisting of residues 146 to 155 of SEQ ED NO:2; a polypeptide consisting of residues 223 to 237 of SEQ ED NO:2; a polypeptide consisting of residues 304 to 311 of SEQ ED NO:2; a polypeptide consisting of residues 324 to 331 of SEQ ED NO:2; a polypeptide consisting of residues 324 to 345 of SEQ ED NO:2; and a polypeptide consisting of residues 333 to 345 SEQ ED 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 binds to residues 1 to 378 of SEQ DD NO:2. Within another embodiment, the antibody is a monoclonal antibody. Within another aspect the invention provides an epitope-bearing polypeptide selected from the group consisting of: a polypeptide comprising residues 33 to 82 of SEQ ED NO:2; a polypeptide comprising residues 344 to 378 of SEQ ED NO:2; a polypeptide comprising residues 33 to 343 of SEQ ED NO:2; a polypeptide comprising residues 15 to 343 of SEQ ED NO:2; a polypeptide comprising residues 1 to 343 of SEQ ED NO:2; a polypeptide comprising residues 83 to 378 of SEQ ED NO:2; a polypeptide comprising residues 15 to 378 of SEQ ED NO:2; a polypeptide comprising residues 1 to 378 of SEQ ED NO:2; a polypeptide comprising residues 15 to 82 of SEQ ED NO:2; a polypeptide comprising residues 1 to 32 of SEQ ED NO:2; and a polypeptide comprising residues 1 to 82 of SEQ ID NO:2. Within another aspect, the invention provides a method of forming a reversible complementary molecule-anti-complementary molecule complex comprising; providing an complementary molecule comprising residues 83 to 343 of SEQ ED NO:2; and contacting the complementary molecule with a anti-complementary molecule; whereby the complementary molecule binds the anti-complementary molecule. Within an embodiment, the anti-complementary molecule is selected from the group consisting of: glycoproteins; glycolipids; and antibodies.

These and other aspects of the invention will become evident upon reference to the following detailed description of the invention.

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.

A "complementary molecule-anti-complementary molecule complex" is formed when an anti-complementary molecule (i.e., substrate, ligand, or peptide) binds to a complementary molecule (i.e., enzyme, receptor, or receptor-like molecule) resulting in a change in the properties of the complementary molecule. This change can result in an initiation of a cascade of reactions leading to a change in cellular function, or the inability of the complementary molecule to bind additional peptides. The forming of a complementary molecule-anti-complementary molecule complex can be reversible. 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 "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 polypeptide" is a polypeptide that is not bound to a cell membrane. Soluble polypeptides lack a transmembrane and cytoplasmic domains. Soluble polypeptides 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 polyeptides 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 polypeptide may have intracellular 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 mRN A 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 upon the discovery of a novel cDNA sequence (SEQ ED NO:l) and corresponding polypeptide (SEQ ED NO:2) having homology to a family of proteins, the βl— 3galactosyltransferases (βl→3GalTases). βl→3GalTases are the β3 subfamily of human galactosyltransferases (β3Gal-T family) which includes HSY15014 (Kolbinger, F. et al., Journal of Biol. Chem. 273: 433-440,

1998), HSGALT3, HSGALT4, (Amado, M. et al., ibid) and E07739 (Katsutoshi, S. et al., Japanese patent, JP 1994181759-A/l). βl→3GalTases are responsible for transferring galactose to carbohydrate chains during biosynthesis. It has been predicted that βl— >3GalTases are in the alpha/beta barrel (TIM barrel) folding class of enzymes, similar to other glycosyltransferases such as the alpha-amylases and beta-glycanases (Yuan, Y. et al., Cell 88:9-11, 1997). Also in the β3Gal-T family is the Drosophila melanogaster Brainiac, (BRN) (Goode, S. et al., Devel. Biol. 178:35-50, 1996), known as "putative neurogenic secreted signaling protein" or NSSP. BRN is required for epithelial development. This activity may be due to possible cell interactions between the membrane bound glycosyltransferase and oligosaccharide substrates on adjacent cell surfaces (Shur, ibid). Thus, β3Gal-T family members are also known as neurogenic secreted signal peptides. See, for example, Shur, B.D., ibid, and Amado, M. et al., ibid. This novel polypeptide and its polynucleotides have been designated ZNSSP8.

The β3Gal-Ts are predicted to be Type U transmembrane proteins. An ortholog to E07739, is AF029790 (Hennet, T. et al., Journal of Biol. Chem. 273:58-65, 1998), which is claimed to be a Type π transmembrane domain based on hydrophobicity analysis. However, due to the close proximity of this domain to the initiating methionine and lack of positively charged residues preceding the domain it is possible that AF029790 is not membrane bound but rather a extracellular secreted protein. The novel ZNSSP8 polypeptide-encoding polynucleotides of the present invention were identified as a homolog of BRN. A full-length clone comprising nucleotide 1 to 1311 of SEQ ED NO:l was isolated from a bone marrow cDNA library. Analysis of the DNA encoding a ZNSSP8 polypeptide revealed an open reading frame encoding 378 amino acids (SEQ ED NO: 2). ZNSSP8 shares homology with β3Gal-T's. Amino acid residues 33 to 82 of SEQ ID NO:2 form a "stem" domain, and amino acid residues 83 to 343 of SEQ ED NO:2 form a "catalytic" domain.

Due to the close proximity of the hydrophobic domain (residues 15 to 32 of SEQ ED NO:2) to the initiation methionine, and the lack of positively charged residues preceding this domain, ZNSSP8 can be a secreted protein, or a membrane- bound protein. As a secreted protein, the mature polypeptide comprises residues 33 to 343 of SEQ ED NO:2. As a membrane bound protein, ZNSSP8 comprises residues 15 to 343 of SEQ ED NO:2. Conserved negatively charged amino acid residues 149, 155, 160, 220, 282, and 283 of SEQ ED NO:2 are contained within the catalytic domain. Additionally, the sequence of amino acid residues from residue 282 to 287 is representative of a peptide motif of this family. This motif is further described by the following amino acid residue profile: [D,E] [D] [V] [F,Y] [L,T,V,M] [G]. Those skilled in the art will recognize that predicted domain boundaries are approximations based on primary sequence content, and may vary slightly; however, such estimates are generally accurate to within ±5 amino acid residues. The present invention also provides post translationally modified polypeptides or polypeptide fragments. A potential N-linked glycosylation site can be found at amino acid residue 167 of SEQ ED NO: 2. Other potential N-linked glycosylation sites are at residues 59, 276, and 335. Post translational modifications in^' members of the β3Gal-T family may regulate whether the protein is expressed in the Golgi or on the surface of the cell. Other examples of post translational modifications include proteolytic cleavage, disulfide bonding and hydroxylation.

The highly conserved, negatively charged residues at positions 149, 155, 160, 220, 282, and 283 of SEQ ED NO:2 and the amino acid sequence between 282 and 287 of ZNSSP8 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 ZNSSP8 polynucleotide from RNA obtained from a variety of tissue sources or cell lines. In particular, highly degenerate primers designed from the ZNSSP8 sequences are useful for this purpose.

Analysis of the tissue distribution of ZNSSP8 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. Additionally, portions of the polynucleotides of the present invention can be identified by querying sequence databases and identifying the tissues from, which the sequences are derived. Portions of the polynucleotides of the present invention have been identified in a prostate, bone marrow, and umbilical cord cDNA libraries as well as a cDNA library made from the following cell lines: a muti-potentional, hematopoetic malignant cell line (K562, ATCC #CCL243), two Burkitt lymphoma cell lines (Daudi, ATCC #CCL213; and Raji, ATCC #CCL 86), a promyelocytic leukemia cell line (HL-60, ATCC #CCL240) which is predominantly neutrophilic promyelocytes, and an acute lymphoblastic (peripheral blood) cell line (MOLT4, ATCC #CRL1582). Additionally, portions of the znsspδ polynucleotide have been observed in cDNA prepared from the following tissues: small intestine tumor, ulcerative colitis, microvascular, dermal endothelial cells, dendritic cells, glioblastoma, peripheral blood eosinophils, 8-9 week old fetus. Similarly, tissue expression of the znsspδ molecules of the present invention can be identified by polymerase chain reaction analysis of various tissues and cell lines. This analysis shows that znsspδ cDNA has been identified in a fetal brain cDNA library, a cDNA library prepared from peripheral blood cells that were selected for CD3 expression and activated with PMA and ionomycin, a bone marrow cDNA library, and cDNA prepared from a human prostate epithelium cell line that had been transformed with human papillomavirus.

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 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 ZNSSP8 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 ZNSSP8 proteins are set forth in SEQ ED NOs:l and 3. DNA sequences encoding other ZNSSP8 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 ZNSSP8 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 ED NO:3 is a degenerate DNA sequence that encompasses all DNAs that encode the ZNSSP8 polypeptide of SEQ ED NO:2. Those skilled in the art will recognize that the degenerate sequence of SEQ ED NO: 3 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus, ZNSSP8 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 1311 of SEQ ED NO:l, and their RNA equivalents are contemplated by the present invention. Table 1 sets forth the one-letter codes used within SEQ ED NO:3 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 NO:3, encompassing all possible codons for a given amino acid, are set forth in Table 2.

o >

> a

O >

X N »

; > >

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 sequence of SEQ ED NO:2. 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 ED NO:3 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 ED NO:l, 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 ZNSSP8 polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ED NO:l (or its complement) 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 ZNSSP8 polypeptide hybridize with a nucleic acid molecule having the nucleotide sequences of SEQ ED NO:l (or its complement) 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 ZNSSP8 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 ED NO:2 (as described below), and a hybridization assay, as described above. Such ZNSSP8 variants include nucleic acid molecules that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ED NO: 1 (or its complement) 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 80%, 90%, 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ED NO:2. Alternatively, ZNSSP8 variants can be characterized as nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ED NO: 1 (or its complement) 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 80%, 90%, 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ED NO:2. The highly conserved amino acids in the catalytic domain of ZNSSP8 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 ZNSSP8 sequences are useful for this purpose.

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 ZNSSP8 RNA. Such tissues and cells can be identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include tissues of bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines.

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 ZNSSP8 polypeptides are then identified and isolated by, for example, hybridization or PCR.

A full-length clone encoding ZNSSP8 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 ZNSSP8 or other specific binding partners.

The invention also provides isolated and purified ZNSSP8 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 ZNSSP8 gene or cDNA. The synthetic oligonucleotides of the present invention have at least 75% identity to a representative ZNSSP8 DNA sequence (SEQ ED NOS:l or 3) 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 or 3 or a sequence complementary to SEQ ED NOs: 1 or 3. Regions from which to construct probes include the 5' and/or 3' coding sequences, anti-complementary molecule 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, IL, 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 ZNSSP8 gene or mRNA transcript in a sample. ZNSSP8 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 ZNSSP8 -like proteins. For example, ZNSSP8 polynucleotides can be used as primers and/or templates in PCR reactions to identify other novel members of the Betal,3 galactosyltransferase family. Such probes can also be used to screen libraries for related sequences encoding novel Betal,3 galactosyltransferases. 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 ZNSSP8 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 ZNSSP8 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. ZNSSP8 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a ZNSSP8 gene. In view of the tissue-specific expression observed for ZNSSP8, this gene region is expected to provide for specific expression in tissues of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma and in malignant and leukemic cell lines. Promoter elements from a ZNSSP8 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 ZNSSP8 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous ZNSSP8 gene in a cell is altered by introducing into the ZNSSP8 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 ZNSSP8 5' non-coding sequence that permits homologous recombination of the construct with the endogenous ZNSSP8 locus, whereby the sequences within the construct become operably linked with the endogenous ZNSSP8 coding sequence. In this way, an endogenous ZNSSP8 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 ZNSSP8 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human ZNSSP8 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 ZNSSP8 as disclosed herein. Such tissues and cell types would include, for example, tissues and cell types of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic tissues and cell types. 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 ZNSSP8-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 ZNSSP8 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 ZNSSP8 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 ID NO:l represents a single allele of human ZNSSP8 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 ED NO:l, 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 ED NO:2. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the ZNSSP8 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. As stated earlier, polynucleotides of SEQ ED NO:l and SEQ ED NO:3 are alternatively spliced variants of the same gene. The present invention also provides isolated ZNSSP8 polypeptides that are substantially similar to the polypeptides of SEQ ED NO:2 and their orthologs. Such polypeptides will be about 80%, 90% identical, or 95% or more identical to SEQ ED NO:2 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]

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1 1 1 1 1

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1 1 1 1 1 1 1 1 1 1 Ω CN CN ro ro CN O CN CN ro ro

1 1 1 1 1 1 1 1 1 1 ω LΠ CN o ro ro rH CN ro H O rH ro CN CN 1 1 1 1 1 1 1 1 1

LD N CN o ro CN H O ro H o H CN H CN

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1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

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1 1 1 1 1 1 1 1 tf LΠ o CN ro H O CN O ro CN CN rH ro CN rH ro CN ro

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LD O LD O CN 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 ZNSSP8. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l 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 ED NO:2) 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. Dlustrative 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 NO:2. 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 an ZNSSP8 gene can be introduced by substituting nucleotides for the nucleotides recited in SEQ ED NO:l. 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 (IRL 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 ZNSSP8 polypeptide can be identified by the ability to specifically bind anti-ZNSSP8 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 complementary molecule- anti-complementary molecule 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 Betal,3 galactosyltransferase-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 ZNSSP8 DNA and polypeptide sequences can be generated through DNA shuffling, as disclosed by Stemmer, Nature 343:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 9J_: 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., anti-complementary molecule 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 ZNSSP8 gene, the gene encodes a polypeptide that is characterized by its anti-complementary molecule binding activity, or by the ability to bind specifically to an anti-ZNSSP8 antibody. More specifically, variant ZNSSP8 genes encode polypeptides, which exhibit greater than 75, 80, or 90%, of the activity of polypeptide encoded by the human ZNSSP8 gene described herein.

Variant ZNSSP8 polypeptides or substantially homologous ZNSSP8 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 288 to 1650 amino acid residues that comprise a sequence that is about 85%, 90%, and 95% or more identical to the corresponding region of SEQ ED NO:2. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the ZNSSP8 polypeptide and the affinity tag. Such sites include thrombin cleavage sites and factor Xa cleavage sites.

For any ZNSSP8 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 ZNSSP8 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 ED NO:l, SEQ ED NO:2, and SEQ ED NO:3. 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 ZIP 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 ZNSSP8 of the present invention with the functionally equivalent domain(s) from another family member, such as HSGALT3, HSGALT4, β3Gal-T2, and β3Gal-T3, or the human species ortholog of Brainiac. Such domains include, but are not limited to, conserved motifs such as the signal, transmembrane, stem, and catalytic 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 Beta-l,3-galactosyltransferase family proteins (e.g.

HSGALT3, HSGALT4, β3Gal-T2, and β3Gal-T3, or the human species ortholog of Brainiac), 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 ZNSSP8 proteins, are constructed using regions or domains of the inventive ZNSSP8 in combination with those of other Beta-l,3-galactosyltransferase molecules (e.g. HSGALT3, HSGALT4, β3Gal-T2, and β3Gal-T3, or the human species ortholog of Brainiac), 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 anti-complementary molecule 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 ZNSSP8 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., tissues of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cells). For example, a protease domain could be targeted to a predetermined cell type (bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cells) by fusing it to the catalytic domain (residues 83 to 343 of SEQ ED NO:2), or a portion thereof. In this way, polypeptides, polypeptide fragments and proteins can be targeted for therapeutic or diagnostic purposes. Such catalytic domain, 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 1,700 amino acid residues, not more than about 1,200 residues, or not more than about 1 ,000 residues, and will in many cases be considerably smaller. For example, residues of ZNSSP8 polypeptide can be fused to E. coli j3-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 ZNSSP8 polypeptide can be fused to maltose binding protein (approximately 343 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.

Some proteins in the β3Gal-T family have been shown to be expressed intracellulary and are involved in intracellular glycoprotein and glycolipid processing.

Other members of this family have been shown to be extracellularly expressed and are involved in glycoprotein and glycolipid processing (such as in the case of the Tn antigen). Other members of the family are expressed extracellularly and are involved in cell-cell interactions and intracellular signaling. Thus, molecules of the present invention can function as an enzyme both intracellularly and extracellulary, in which case its anti-complementary molecule is a substrate. Additionally, molecules of the present invention can function extracellularly and modulate cell-cell interactions. The extracellular binding of ZNSSP8 to its anti-complementary molecule can cause a cellular event in the cell that is expressing it (i.e. ZNSSP8 acts as a receptor or receptorlike molecule), or in the cell expressing the anti-complementary molecule to which it binds (i.e., ZNSSP8 acts as a ligand). Additionally, ZNSSP8 can function extracellularly as a soluble enzyme, ligand, receptor or receptor like molecule. Similarly, as an extracellulary expressed ZNSSP8 enzyme, the processing of its anti- complementary substrate can result in a cellular response (similar to intracellular signaling) in the cell expressing the substrate. Also as an extracellularly expressed protein, ZNSSP8 can function to form a "bridge" between cells maintaining their proximity to each other. Thus, for the purposes of this application, ZNSSP8 is referred to as a complementary molecule and its cognate binding partner is referred to as an anti- complementary molecule.

The invention also provides soluble ZNSSP8 polypeptides, used to form fusion or chimeric proteins with human Ig, as His-tagged proteins, or FLAG™-tagged proteins. One such construct is comprises residues 83 to 343 of SEQ ED NO:2, fused to human Ig. ZNSSP8 or ZNSSP8-Ig chimeric proteins are used, for example, to identify the ZNSSP8 anti-complementary molecules, including the natural anti-complementary molecule, as well as agonists and antagonists of the natural anti-complementary molecule. Using labeled soluble ZNSSP8, cells expressing the anti-complementary molecule are identified by fluorescence immunocytometry or immunohistochemistry. The soluble fusion proteins or soluble Ig fusion protein is useful in studying the distribution of the anti-complementary molecule on tissues or specific cell lineages, and to provide insight into complementary molecule-anti-complementary molecule biology. In an alternative approach, a soluble ZNSSP8 extracellular anti- complementary molecule-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 complementary molecule polypeptides are arrayed in close proximity to each other. Fusions of this type can be used to affinity purify the cognate anti-complementary molecule from solution, as an in vitro assay tool, to block signals in vitro by specifically titrating out anti-complementary molecule, and as antagonists in vivo by administering them to block anti-complementary molecule stimulation. To purify anti-complementary molecule, a ZNSSP8-Ig fusion protein (chimera) is added to a sample containing the anti-complementary molecule under conditions that facilitate complementary molecule-anti-complementary molecule binding (typically near-physiological temperature, pH, and ionic strength). The chimera-anti-complementary molecule complex is then separated by the mixture using protein A, which is immobilized on a solid support (e.g., insoluble resin beads). The anti-complementary molecule 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 ZNSSP8 polypeptide from the host cell, the ZNSSP8 DNA is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide. 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 ZNSSP8 polypeptide.

The present invention also includes "functional fragments" of ZNSSP8 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 an ZNSSP8 polypeptide. As an illustration, DNA molecules having the nucleotide sequence of SEQ ED NO: 1 can be digested with Ba l 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-ZNSSP8 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 an ZNSSP8 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 2-5 A 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. 1, 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 ZNSSP8 gene that has amino acid changes, compared with the amino acid sequence of SEQ ED NO:2. A variant ZNSSP8 gene can be identified on the basis of structure by determining the level of identity with nucleotide and amino acid sequences of SEQ ED NOs: 1 and 2, 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 ZNSSP8 gene can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ED NO: 1 , 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 ED NO:2 or that retain the glycosyltransferase and/or the cell-cell interaction activity of the wild- type ZNSSP8 protein. Such polypeptides may include additional amino acids from, for example, a secretory signal, transmembrane, stem, and catalytic 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 ED NO:2. 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 ZNSSP8, 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 ZNSSP8 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 ZNSSP8 protein (e.g., SEQ ED NO:2). Polypeptides comprising a larger portion of a ZNSSP8 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 signal, transmembrane, stem, and catalytic domains of ZNSSP8 and fragments thereof. Polypeptides in this regard include those comprising residues 1 to 14 of SEQ ED NO:2; residues 15 to 32 of SEQ ED NO:2; residues 33 to 82 of SEQ ED NO:2; residues 83 to 343 of SEQ ED NO:2; residues 344 to 378 of SEQ ED NO:2; residues 83 to 378 of SEQ ED NO:2; residues 33 to 378 of SEQ ED NO:2; residues 15 to 378 of SEQ ED NO:2; residues 15 to 82 of SEQ ED NO:2; residues 33 to 343 of SEQ ED NO:2; residues 15 to 343 of SEQ ED NO:2; residues 1 to 343 of SEQ ED NO:2; residues 1 to 32 of SEQ ED NO:2; residues 1 to 82 of SEQ ED NO:2; and residues 1 to 378 of SEQ ED NO:2.

The present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of an ZNSSP8 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. Nat'l 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 ED NO: 2. Such epitope-bearing peptides and polypeptides can be produced by fragmenting an ZNSSP8 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).

ZNSSP8 polypeptides can also be used to prepare antibodies that specifically bind to ZNSSP8 epitopes, peptides or polypeptides. The ZNSSP8 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 ZNSSP8 polypeptide (e.g., SEQ ED NO:2). Polypeptides comprising a larger portion of a ZNSSP8 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 ZNSSP8 polypeptides encoded by SEQ ED NO:2 from amino acid number 1 to amino acid number 378, or a contiguous 9 to 378 amino acid fragment thereof. As an illustration, potential antigenic sites in ZNSSP8 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 include residue 5 to residue 12 of SEQ ED NO:2 residue 43 to residue 48 of SEQ ED NO:2; residue 66 to residue 74 of SEQ ED NO:2 residue 66 to residue 85 of SEQ ED NO:2; residue 78 residue 85 of SEQ ED NO:2 residue 96 to residue 121 of SEQ ED NO:2; residue 133 to residue 144 of SEQ ID NO:2; residue 133 to residue 155 SEQ ED NO:2; residue 146 to residue 155 of SEQ ED NO:2; residue 223 to residue 237 of SEQ ED NO:2; residue 304 to residue 311 of SEQ ED NO:2; residue 324 residue 331 of SEQ ED NO:2; residue 333 to residue 345 of SEQ

ED NO:2; and residue 324 to residue 345 of SEQ ED NO:2.. Hydrophilic peptides, such as those predicted by one of skill in the art from a hydrophobicity plot are also immonogenic. ZNSSP8 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: residue 5 to residue 12 of SEQ ED NO:2; residue 33 to residue 38 of SEQ ID NO:2; residue 33 to residue 53 of SEQ ED

NO:2; residue 40 to residue 53 of SEQ ED NO:2; residue 67 to residue 72 of SEQ ED NO:2; residue 74 to residue 85 of SEQ ED NO:2; residue 67 to residue 85 of SEQ ED NO:2; residue 97 to residue 123 of SEQ ED NO:2; residue 136 to residue 157 of SEQ ED NO:2; residue 178 to residue 184 of SEQ ED NO:2; residue 220 to residue 254 of SEQ ED NO:2; residue 269 to residue 276 of SEQ ED NO:2; residue 269 to residue 316 of SEQ ED NO:2; residue 306 to residue 316 of SEQ DD NO:2; residue 306 to residue 344 of SEQ ED NO:2; and residue 319 residue 344 of SEQ ED 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 43 to residue 50 of SEQ ED NO:2; residue 65 to residue 74 of SEQ ED NO:2; residue 65 to residue 84 of SEQ ED NO:2; residue 78 to residue 84 of SEQ ED NO:2; residue 96 to residue 107 of SEQ ED NO:2; residue 96 to residue 121 of SEQ ED NO:2; residue 110 to residue 121 of SEQ ED NO:2; residue 133 to residue 138 of SEQ ED NO:2; residue 133 to residue 153 of SEQ ED NO:2; residue 140 to residue 153 of SEQ ED NO:2; residue 229 to residue 239 of SEQ ED NO:2; residue 242 to residue 252 of SEQ ED NO:2; residue 229 to residue 252 of SEQ ED NO:2; residue 269 to residue 275 of SEQ ED NO:2; and residue 332 to residue 341 of SEQ ED NO:2. Antibodies from an immune response generated by inoculation of an animal with these antigens 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 ZNSSP8 polypeptide or a fragment thereof. The immunogenicity of a ZNSSP8 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 of ZNSSP8 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 ZNSSP8 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ZNSSP8 protein or peptide). Genes encoding polypeptides having potential ZNSSP8 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 ZNSSP8 sequences disclosed herein to identify proteins which bind to ZNSSP8. These "binding proteins" which interact with ZNSSP8 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 ZNSSP8 "antagonists" to block ZNSSP8 anti-complementary molecule binding in vitro and in vivo. These anti-ZNSSP8 binding proteins would be useful for modulating, for example, glycoprotein and glycolipid modification, extracellular matrix interactions, and cell-cell interactions, in general.

As used herein, the term "binding proteins" additionally includes antibodies to ZNSSP8 polypeptides, the cognate anti-complementary molecule of ZNSSP8 polypeptides, proteins useful for purification of ZNSSP8 polypeptides, and proteins associated with the catalytic domain (residues 83 to 343 of SEQ ED NO:2).

Antibodies are determined to be specifically binding if they exhibit a threshold level of binding activity (to a ZNSSP8 polypeptide, peptide or epitope) of at least 10-fold greater than the binding affinity to a control (non-ZNSSP8 ) 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 ZNSSP8 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 ZNSSP8 protein or polypeptide. Antibodies to ZNSSP8 may be used for immunohistochemical tagging cells that express ZNSSP8; for isolating ZNSSP8 by affinity purification; for diagnostic assays for determining circulating levels of ZNSSP8 polypeptides; for detecting or quantitating soluble ZNSSP8 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 ZNSSP8 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 ZNSSP8 or fragments thereof may be used in vitro to detect denatured ZNSSP8 or fragments thereof in assays, for example, Western Blots or other assays known in the art. The soluble ZNSSP8 polypeptide (i.e., residues 33 to 378 of SEQ ID

NO:2, or a portion thereof) is useful in studying the distribution of anti-complementary molecules in tissues or specific cell lineages, and to provide insight into complementary molecule/anti -complementary molecule biology. Using labeled ZNSSP8, cells expressing the anti-complementary molecule are identified by fluorescence immunocytometry or immunocytochemistry. Application may also be made of the specificity of Betal,3 galactosyltransferases for their anti-complementary molecules.

Antibodies can be made to soluble, ZNSSP8 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 ZNSSP8 can be used in analysis of tissue distribution of ZNSSP8 by immunohistochemistry on human or primate tissue. These soluble ZNSSP8 polypeptides can also be used to immunize mice in order to produce monoclonal antibodies to a soluble human ZNSSP8 polypeptide. Monoclonal antibodies to a soluble human ZNSSP8 polypeptide can also be used to mimic anti- complementary molecule/complementary molecule coupling, resulting in activation or inactivation of the anti-complementary molecule/complementary molecule 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 ZNSSP8 can be used to determine the distribution, regulation and biological interaction of the ZNSSP8 and its anti-complementary molecule pair on specific cell lineages identified by tissue distribution studies.

Soluble ZNSSP8 proteins or antibodies to the ZNSSP8 protein 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 (anti- complementary molecule or antigen, respectively, for instance). More specifically, ZNSSP8 polypeptides or anti-ZNSSP8 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, transmembrane, stem, or catalytic 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 anti-complementary molecule to ZNSSP8 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, ZNSSP8-cytokine fusion proteins or antibody- cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines), if the ZNSSP8 polypeptide or anti-ZNSSP8 antibody targets hypeφroliferative 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 ZNSSP8 polypeptides or anti-ZNSSP8 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 ZNSSP8 polypeptide or anti-ZNSSP8 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.

ZNSSP8 polynucleotides and/or polypeptides may be useful for regulating the maturation of Betal,3 galactosyltransferase anti-complementary molecule-bearing cells, such as fibroblasts, lymphocytes and hematopoietic cells. ZNSSP8 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 ZNSSP8, 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 modulations, extracellular matrix interactions, and cell-cell interactions are known in the art. Additional studies to determine the enzymatic activity of glycosyltransferases include capillary electrophoresis combined with laser induce fluorescence detection as described by Snow, D. et al. Anal. Biochem. 271:36-42, 1999.

The ZNSSP8 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 ZNSSP8 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 ZNSSP8 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 ZNSSP8 secretory peptide (residues 1 to 14 of SEQ ED NO:2). Alternatively, it can be synthesized de novo. The secretory signal sequence is operably linked to the ZNSSP8 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 catalytic domain of ZNSSP8 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 ZNSSP8 can direct the new catalytic domain to the transmembrane and/or stem domains. This substituted catalytic domain can be chosen from the catalytic domains represented by the Betal,3 galactosyltransferase protein families. Similarly, the catalytic domain of ZNSSP8 protein can be used to substitute the catalytic domain of a different Betal,3 galactosyltransferase. 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, Rockville, Maryland. In general, strong transcription promoters can be usedsuch 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) l_l: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 WJPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica 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 ZNSSP8 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 ZNSSP8 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 ZNSSP8 . 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 Peer, 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 ZNSSP8 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 ZNSSP8 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 ZNSSP8 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 ZNSSP8 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 ZNSSP8 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 B™ (Life Technologies) or ESF 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 (MOI) 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 ZNSSP8 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 POT1 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 ZNSSP8 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 YEPD (2% D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit, MI), 1% Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trαrcs-3-methylproline, 2,4-methanoproline, c/s-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, //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 incoφorating 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 tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA 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; Ellman et al., Methods Enzvmol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., 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 counteφart. 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 ZNSSP8 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 ZNSSP8 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 adsoφtion (IMAC) 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, ρρ.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.

ZNSSP8 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, ZNSSP8 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 ZNSSP8 polypeptides can be measured using a variety of assays that measure, for example, cell-cell interactions, extracellular matrix interactions, glycolipid and glycoprotein modifications, development, and other biological functions associated with Betal,3 galactosyltransferase family members or with Betal,3 galactosyltransferase interactions, such as, differentiation, and proliferation for example. Of particular interest are changes in glycoprotein and glycolipid modifications, extracellular matrix interactions, and in cell-cell interactions in bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines tissue as well as peripheral blood, and bone-marrow and prostate-derived cell lines. 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 moφhogenesis, mesenchyme development, immune recognition, growth control, tumor metastasis and suppression, glycoprotein and glycolipid modifications, extracellular matrix interactions, and cell-cell interactions.

Additional activities likely associated with the polypeptides of the present invention include proliferation of cells of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines 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 modulating, for example, glycoprotein and glycolipid modification, extracellular matrix interactions, and cell-cell interactions, in general, as well as modulating growth and differentiation either working in isolation, or in conjunction with other molecules (growth factors, cytokines, etc.) in bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines. Alternative splicing of ZNSSP8 may be cell-type specific and confer activity to specific anti-complementary molecules.

As a glycosyltransferase, znssp8 polypeptides, including fragments thereof, can activate cells expressing its complementary molecule such that those cells choose a specific differentiation pathway. One example of this activity is the direct action of Fringe, a fucose specific βl,3 N-acetylglucosaminyltransferase, on Notch, a receptor expressed on mouse thymocytes. The glycosyltransferase activity of Fringe represents an example of the modulation of ligand-receptor signaling by differential receptor glycosylation. Thus Fringe prevents the binding of the Notch receptor, by its ligand, Jagged- 1, such that it becomes less competent to signal. Further, the modulation of the Notch receptor in these cells can trigger the cells down a CD8 pathway rather than a CD4 pathway. Specifically, an activated (or under-glycosylated) Notch receptor can bind to 1) a major histocompatability complex I (MHC-I) protein, which directs the thymocyte to a CD8 lineage, or it can bind to 2) a major histocompatability complex II (MHC-II) protein, which directs the thymocyte to a CD4 lineage. Alternatively, a modified (or glcosylated) Notch receptor, upon binding to either a MHC-I or MHC-II protein allows the progression of thymocyte expressing the

Notch receptor down a CD8 lineage. Thus, the glycosylation of the Notch receptor on these cells is related to the specific differentiation of the thymocyte. See Robey, E. et al., Cell 87:483-492, 1996; and Moloney, D.J. et al., Nature 406:369-375. 2000. In this manner znssp8 polypeptides, and fragments thereof, can have the effect of activating cells expressing its anti-complementary molecule such that cells expressing the anti- complementary molecule are directed down a specific differentiation pathway. The anti-complementary molecule can be expressed on the cells, which also express znssp8, or it can be expressed on cells in the immediate environment of the znssp8 expressing cells. Additionally, a soluble znsspδ polypeptide (i.e., a polypeptide that does not contain the transmembrane or hydrophobic domain of znsspδ) can be expressed on cells at a site remote from the anti-complementary molecule, such that the soluble znsspδ molecule acts at a site remote from the cells that express it. The expression of znsspδ in bone marrow and activated CD3 positive peripheral blood cells indicates that molecules of the present invention, including polypeptides, polynucleotides, fragments, and znsspδ binding partners, will be useful in modulating the differentiation of cells in the T cell lineage.

Another assay of interest measures or detects changes in proliferation, differentiation, and development. Additionally, the effects of a ZNSSP8 polypeptides on cell-cell interactions of epithelial cells, extracellular matrix, tumor cells and cells of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines in particular, would be of interest to measure. Yet other assays examines changes in glycoprotein and glycolipid mocifications.

The activity of molecules of the present invention can be measured using a variety of assays that, for example, measure neogenesis or hypeφlasia (i.e., proliferation) of tissues of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines. 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 bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines cells 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 bone marrow and prostate fibroblasts, prostate tumors, diseased and cancerous cells from bone marrow and prostate primary cultures, as well as epidermoid and epithethloid cells of cervical carcinoma. 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), incoφoration of radiolabelled nucleotides (Cook et al., Analytical Biochem. 179: 1-7, 1989), incoφoration 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 ZNSSP8 is a chemotractant in vivo, ZNSSP8 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. Med. 179:881-87, 1994). Release of specific leukocyte cell populations from bone marrow into peripheral blood can also be measured after ZNSSP8 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, ZNSSP8 polypeptides may stimulate inhibition or proliferation of endocrine and exocrine cells of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines.

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 bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, and prostate in 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 moφhological 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 ZNSSP8 polypeptides of the present invention can be used to study proliferation or differentiation in bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines. Such methods of the present invention generally comprise incubating cells derived from these tissues in the presence and absence of ZNSSP8 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 modifications, extracellular matrix interactions, development, fertility and other biological functions associated with Betal,3 galactosyltransferase family members. ZNSSP8 molecules, variants, and fragments can be applied in isolation, or in conjunction with other molecules (growth factors, cytokines, etc.) in bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines.

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 puφose include adenovirus, heφesvirus, 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 incoφorated 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. 1L615-623, 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.

The activity of ZNSSP8 or its anti-complementary molecule can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with such protein interactions 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 enzyme or enzyme 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 ZNSSP8 proteins, their agonists, and antagonists. The microphysiometer can be used to measure responses of a ZNSSP8- responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to ZNSSP8 polypeptide. ZNSSP8-responsive eukaryotic cells comprise cells into which a polynucleotide for ZNSSP8 has been transfected creating a cell that is responsive to ZNSSP8; or cells containing endogenous ZNSSP8 polynucleotides. Differences, measured by a change in the response of cells exposed to ZNSSP8 anti- complementary molecule, relative to a control not exposed to ZNSSP8 anti- complementary molecule, directly measure the ZNSSPδ-modulated cellular responses. Moreover, such ZNSSPδ-modulated responses can be assayed under a variety of stimuli. The present invention provides a method of identifying agonists and antagonists of ZNSSPδ protein, comprising providing cells responsive to a ZNSSPδ 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 measurable change in a cellular response of the second portion of the cells as compared to the first portion of the cells. Moreover, culturing a third portion of the cells in the presence of ZNSSPδ anti-complementary molecule and the absence of a test compound provides a positive control for the ZNSSP8 -responsive cells, and a control to compare the agonist activity of a test compound with that of the ZNSSP8 anti- complementary molecule. Antagonists of ZNSSP8 can be identified by exposing the cells to ZNSSP8 anti-complementary molecule in the presence and absence of the test compound, whereby a reduction in ZNSSPδ-modulated activity is indicative of antagonist activity in the test compound.

Moreover, ZNSSP8 can be used to identify cells, tissues, or cell lines which respond to a ZNSSP8-modulated pathway. The microphysiometer, described above, can be used to rapidly identify cells responsive to ZNSSPδ of the present invention. Cells can be cultured in the presence or absence of ZNSSPδ polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of ZNSSPδ polypeptides are responsive to ZNSSPδ. Such cell lines, can be used to identify anti-complementary molecules, antagonists and agonists of ZNSSPδ polypeptide as described above.

In view of the tissue distribution (bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines) observed for ZNSSPδ expression, agonists (including the native catalytic domain) and antagonists have enormous potential in both in vitro and in vivo applications. Compounds identified as ZNSSPδ agonists and antagonists are useful for studying cell-cell interactions, glycolipid and glycoprotein modifications, extracellular matrix interactions, development, and other biological functions associated with Betal,3 galactosyltransferase family members or with Betal,3 galactosyltransferase interactions in vitro and in vivo. For example, ZNSSPδ 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, ZNSSPδ polypeptides and ZNSSPδ agonists, including small molecules are useful as a research reagent, such as for the expansion, differentiation, and/or cell- cell interactions of bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines. ZNSSPδ polypeptides are added to tissue culture media for these cell types.

The expression of Znsspδ in prostate epithelium transformed with human papillomavirus indicates that the detection of znsspδ polynucleotides, polypeptides, fragments thereof, antibodies and binding partners can be used diagnostically to detect or monitor infection by human papillomavirus in human prostate. Such detection and diagnostic methods are discussed elsewhere in this application.

Compounds identified as ZNSSPδ 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 ZNSSPδ-bearing cells in culture. Antagonists are useful as research reagents for characterizing anti-complementary molecule-complementary molecule interaction.

The invention also provides antagonists, which either bind to ZNSSPδ polypeptides or, alternatively, to a anti-complementary molecule to which ZNSSPδ polypeptides bind, thereby inhibiting or eliminating the function of ZNSSPδ. Such ZNSSPδ antagonists would include antibodies; polypeptides which bind either to the ZNSSPδ polypeptide or to its anti-complementary molecule; natural or synthetic analogs of ZNSSPδ anti-complementary molecules which retain the ability to bind the anti-complementary molecule but do not result in glycoprotein or glycolipid modifications, extracellular matrix interactions, or cell-cell interactions. Such analogs could be peptides or peptide-like compounds. Natural or synthetic small molecules which bind to ZNSSPδ polypeptides and prevent glycoprotein or glycolipid modifications, extracellular matrix interactions, or cell-cell interactions. Also contemplated are soluble ZNSSPδ proteins. As such, ZNSSPδ antagonists would be useful as therapeutics for treating certain disorders where blocking glycosylation or anti-complementary molecule binding would be beneficial. ZNSSPδ polypeptides may be used within diagnostic systems to detect the presence of anti-complementary molecule polypeptides. Antibodies or other agents that specifically bind to ZNSSPδ or its anti-complementary molecule may also be used to detect the presence of circulating complementary molecule or anti-complementary molecule polypeptides. Such detection methods are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay.

Immunohistochemically labeled ZNSSPδ antibodies can be used to detect ZNSSPδ and/or its anti-complementary molecules in tissue samples. ZNSSPδ levels can also be monitored by such methods as RT-PCR, where ZNSSPδ mRNA can be detected and quantified. The information derived from such detection methods would provide insight into the significance of ZNSSP8 polypeptides in various diseases, and as such would serve as diagnostic tools for diseases for which altered levels of ZNSSP8 are significant. Altered levels of ZNSSP8 polypeptides may be indicative of pathological conditions including, for example, cancer and disorders of the bone marrow, brain, prostate, peripheral blood lymphocytes.

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 ZNSSP8 activity (ZNSSPδ antagonists) include anti-ZNSSPδ antibodies and soluble ZNSSPδ polypeptides (such as residues δ3 to 343 in SEQ ID NO:2), as well as other peptidic and non-peptidic agents (including ribozymes). ZNSSPδ 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 ZNSSPδ. In addition to those assays disclosed herein, samples can be tested for inhibition of ZNSSPδ activity within a variety of assays designed to measure complementary molecule/anti-complementary molecule binding or the stimulation/inhibition of ZNSSPδ-dependent cellular responses. For example, ZNSSP8-responsive cell lines can be transfected with a reporter gene construct that is responsive to a ZNSSP8-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 8_.7:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563- 72, 19δ9). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19):9063-6; 19δδ and Habener, Molec. Endocrinol. 4 (δ):10δ7-94; 1990.

Hormone response elements are reviewed in Beato, Cell 56:335-44; 1989. One reporter gene construct would contain a catalytic domain (i.e., residues δ3 to 343 of SEQ ED NO:2) that, upon binding a Betal,3 galactosyltransferase anti-complementary molecule, would signal intracellularly through on of the reporters listed above. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of ZNSSPδ on the target cells, as evidenced by a decrease in ZNSSPδ stimulation of reporter gene expression. Assays of this type will detect compounds that directly block ZNSSPδ binding to a anti-complementary molecule as well as compounds that block processes in the cellular pathway subsequent to anti-complement molecule binding. In the alternative, compounds or other samples can be tested for direct blocking of ZNSSPδ binding to a anti-complementary molecule using ZNSSPδ 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 ZNSSPδ to its anti-complementary molecule is indicative of inhibitory activity, which can be confirmed through secondary assays. Betal,3 galactosyltransferases used within binding assays may be cellular Betal,3 galactosyltransferases, soluble Betal,3 galactosyltransferases, or isolated, immobilized Betal,3 galactosyltransferases.

Also, ZNSSPδ polypeptides, agonists or antagonists thereof may be therapeutically useful for promoting wound healing, for example, in bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines. To verify the presence of this capability in ZNSSPδ polypeptides, agonists or antagonists of the present invention, such ZNSSPδ polypeptides, agonists or antagonists are evaluated with respect to their ability to facilitate wound healing according to procedures known in the art. If desired, ZNSSPδ polypeptide performance in this regard can be compared to growth factors, such as EGF, NGF, TGF-α, TGF-β, insulin, IGF-I, IGF-fl, fibroblast growth factor (FGF) and the like. In addition, ZNSSPδ polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more growth factors to identify synergistic effects.

A ZNSSPδ polypeptide can also be used for purification of anti- complementary molecule. 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 anti-complementary molecules are passed through the column one or more times to allow anti-complementary molecules to bind to the ZNSSPδ molecule polypeptide. The anti-complementary molecule is then eluted using changes in salt concentration, chaotropic agents (guanidine HC1), or pH to disrupt anti-complementary molecule-complementary molecule binding.

An assay system that uses a anti-complementary molecule-binding complementary molecule (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 complementary molecule, 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, 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, and assessment of .

Anti-complementary molecule binding complementary molecule 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 most commonly bind the anti-complementary molecule and 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 anti-complementary molecule, or immunoglobulin constant region sequences. Many cell-surface proteins have naturally occurring, soluble counteφarts 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 ZNSSPδ polypeptides may act as antagonists to or agonists of ZNSSPδ polypeptides, and would be useful to modulate the effects of ZNSSPδ in bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines. The soluble of ZNSSPδ does not contain a transmembrane domain (i.e., the polypeptide of residues 15 to 32 of SEQ ID NO:2) and may act as an agonist or antagonist of ZNSSPδ 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 ZNSSPδ polypeptides can be more wide spread than its membrane- anchored counteφart. Both isoforms would be useful in studying the effects of the present invention in vitro an in vivo.

Molecules of the present invention can be used to identify and isolate Beta 1,3 galactosyltransferases, or members of complement anti -complement pairs involved in glycoprotein and glycolipid modifications, extracellular matrix interactions, and cell-cell interactions. 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. Iδ2, "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, 19δ4) and anti-complementary molecule proteins can be identified.

The molecules of the present invention will be useful in modulating, for example, glycoprotein and glycolipid modification, extracellular matrix interactions, and cell-cell interactions, in general. The polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders associated with tumor growth and metastasis, T cell disorders, epithelial boundary maintenance, development, 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 modifications, extracellular matrix interactions or cell-cell interactions in general or to treat or prevent development of pathological conditions in such diverse tissue as bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines. In particular, certain diseases may be amenable to such diagnosis, treatment or prevention. These diseases include, but are not limited to leukemia, lymphoma, hematopoetic disorders, auto immune disorders, inflammatory bowel disease, diseases associated with defects in glycosyltransferase activities, and cell-cell interactions. The molecules of the present invention can be used to modulate inhibition and proliferation of tissues in the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines

Polynucleotides encoding ZNSSPδ polypeptides are useful within gene therapy applications where it is desired to increase or inhibit ZNSSPδ activity. If a mammal has a mutated or absent ZNSSPδ gene, the ZNSSPδ gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a ZNSSP8 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, 19δ7; Samulski et al., J. Virol. 63:3δ22-δ, 19δ9).

In another embodiment, a ZNSSPδ 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, 198δ; Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/0735δ, published March 16, 1995 by Dougherty et al.; and Kuo et al., Blood δ2:δ45, 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 δ4:7413-7, 19δ7; Mackey et al., Proc. Natl. Acad. Sci. USA 85:δ027-31, 19δδ). 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 ZNSSPδ polynucleotides (SEQ ID NO:l or SEQ ED

NO:3) can be used to target specific tissues such as tissues of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines. 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 ZNSSP8 gene transcription and translation, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of a ZNSSPδ-encoding polynucleotide (e.g., a polynucleotide as set forth in SEQ ED NOs:l or 3) are designed to bind to ZNSSPδ-encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of ZNSSPδ polypeptide-encoding genes in cell culture or in a subject.

Mice engineered to express the ZNSSPδ gene, referred to as "transgenic mice," and mice that exhibit a complete absence of ZNSSPδ gene function, referred to as "knockout mice," may also be generated (Snouwaert et al., Science 257:10δ3, 1992; Lowell et al., Nature 366:740-42, 1993; Capecchi, M.R., Science 244: 12δδ-1292, 19δ9; Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 19δ6). For example, transgenic mice that over-express ZNSSPδ, 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 ZNSSPδ polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which ZNSSPδ expression is functionally relevant and may indicate a therapeutic target for the ZNSSPδ , its agonists or antagonists. For example, a transgenic mouse to engineer is one that over-expresses the soluble ZNSSP8 polypeptide (approximately amino acids 1 to 483 of SEQ ED NO:2), or the membrane-bound complementary molecule, (residues 1 to 523 of SEQ ED NO:2). Moreover, such over-expression may result in a phenotype that shows similarity with human diseases. Similarly, knockout ZNSSP8 mice can be used to determine where ZNSSP8 is absolutely required in vivo. The phenotype of knockout mice is predictive of the in vivo effects of that a ZNSSP8 antagonist, such as those described herein, may have. The human ZNSSPδ cDNA can be used to isolate murine ZNSSPδ mRNA, cDNA and genomic DNA, which are subsequently used to generate knockout mice. These mice may be employed to study the ZNSSPδ 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 ZNSSPδ antisense polynucleotides or ribozymes directed against ZNSSPδ , described herein, can be used analogously to transgenic mice described above.

ZNSSPδ polypeptides, variants, and fragments thereof, may be useful as replacement therapy for disorders associated with Beta-l,3-galactosyltransferase, including disorders related to, for example, tumors and disease states of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and hematopoesis, and in malignant and leukemic cell lines.

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 69:3δ5-3δ7, 1992. As a secreted protein in tissues of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines, ZNSSPδ can play a role in intercellular rearrangement in these and other tissues and cell lines.

ZNSSPδ gene may be useful to as a probe to identify humans who have a defective ZNSSPδ gene. The strong expression of ZNSSP8 in tissues of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines suggests that ZNSSP8 polynucleotides or polypeptides can be used as an indication of aberrant growth in these tissues. Thus, polynucleotides and polypeptides of ZNSSPδ, and mutations to them, can be used as diagnostic indicators of cancer and tumor stage in these tissues.

The ZNSSPδ polypeptide is expressed in tissues of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines. 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 bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, and prostate, cervical carcinoma. Similarly, polynucleotides and polypeptides of ZNSSPδ may be used to replace their defective counteφarts in tumor or diseased tissues. Thus, ZNSSPδ 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.

Moreover, the activity and effect of ZNSSPδ 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. Cell 79: 315-32δ,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 ZNSSPδ, 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 weeks, during which time they may

3 reach a size of 1500 - lδOO 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 well 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., ZNSSPδ, 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 ZNSSP8. Moreover, purified ZNSSP8 or ZNSSP8-conditioned media can be directly injected in to this mouse model, and hence be used in this system. Use of stable ZNSSPδ transfectants as well as use of induceable promoters to activate ZNSSPδ expression in vivo are known in the art and can be used in this system to assess ZNSSPδ induction of metastasis. For general reference see, O'Reilly MS, et al. Ce 79:315-32δ, 1994; and Rusciano D, et al. Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.

Prostatic hypeφlasia is an almost universal phenomenon in 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, 199δ, p. 596 to 5δ8. As a protein that is expressed in prostate tissue, polypeptides, polynucleotides, fragments thereof, antibodies, and binding partners can be used to treat or diagnose prostate related disorders, such as, for example, prostate hypeφlasia. Diagnostically, an increase, or decrease in znsspδ molecules, including polypeptides, polynucleotides and fragments thereof, can signal an enlargement, or shrinkage, respectively, of said tissue. Assays to detect polynucleotides, polypeptides, and fragments 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 enlargement of the prostate. Such methods are also widely known in the art.

Znsspδ is expressed in carcinomas of the cervical epithelium (Example 4). Thus, Znsspδ can be used as a marker for detecting cervical carcinoma. Such detection can include detection by in situ hybridization, or by binding to a znsspδ binding partner. Similarly detection of znsspδ polypeptides, polynucleotides or fragments thereof will find use diagnostically, and/or during surgery or chemotherapy. For example, an antibody, or binding partner, that binds to znsspδ can be used to visualize the diseased tissue during surgical resection, thus minimizing the removal of healthy tissue. Such antibodies, or binding partners can also be used to deliver toxic agents to cervical carcinoma in order to ablate the disease tissue. Such methods are commonly known in the art and are discussed in the application.

Galactosyltransferases 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 Suρρl):63δ-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):lδ75-δ0, 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; 19δ6) 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 znsspδ in tissues and cells derived from bone marrow, prostate, cervical carcinoma, fetal brain, peripheral blood, colon, umbilical cord, and CD3 positive cells, as well as in serum, ascites, milk and saliva can aid in diagnosing, staging, and monitoring such diseases. Such methods of detecting galactosyltransferases include assays that measure the galactosyltransferase molecule by ELISA and are further described by Udagawa, Y., ET al., Eur. J. of Cancer, 34(4):4δ9-495. 199δ.

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 beta- l,3,galactosyltransferase. Expression of the Tn antigen along with the sialosyl-Tn antigen and a TF antigen (characterized by a deficiency in alpha-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: 103-10δ. 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 ZNSSPδ can be useful for studying bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines 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 expression of ZNSSPδ in bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines tissue, a defect in the ZNSSPδ gene may result in defective glycosylation of cell surface carbohydrates of these cells, leading to over sialylation of the Tn antigen. Thus, ZNSSPδ polypeptides would be useful as a bone marrow, fetal brain, prostate, cervical carcinoma, or peripheral blood specific βl,3, galactosyltransferase replacement therapy for pre-cancerous and cancer tissues. To verify the presence of such activity in ZNSSPδ 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 ZNSSPδ, which may cause an auto-immune reaction resulting in an immune attack on the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, and prostate, and cervical carcinoma. In these cases, ZNSSPδ molecules may be used to encourage proper glycosylation and limit the antigenic recognition in tissues over expressing the sialosyl Tn antigen.

Similarly, a defective ZNSSPδ gene may result in improper glycosylation of the surface carbohydrates of the tissues of bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, and prostate, and cervical carcinoma, thus affecting cell-cell interactions and possibly cell cycle regulation. Administering polypeptides of ZNSSPδ to mammals with such a defective gene could treat such cases.

Znsspδ polynucleotides of SEQ ED NO:l map to chromosome 3pl4. Thus, the present invention also provides reagents, which will find use in diagnostic applications. For example, the ZNSSPδ gene, a probe comprising ZNSSPδ DNA or RNA or a subsequence thereof can be used to determine if the ZNSSPδ gene is present on chromosome 3pl4 or if a mutation has occurred. Detectable chromosomal aberrations at the ZNSSP8 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).

Genetic diseases which correspond to aberrations of chromosome 3pl4 include but are not limited to Renal Cell Carcinoma (See Wang, N. and Perkins K. Cancer Genet. Cvtogenet. 11:479-481. 19δ4) and Small Cell Cancer of the Lung (See

Hibi, K., et al., Oncogene 7: 445-449, 1992; Whang-Peng, J., et al., Science 215: 181- 182, 1982; Johnson, B. E. et al„. J. Clin. invest. δ2: 502-507, 19δδ; and Daly, M. C, et al., Genomics 9: 113-119, 1991). Thus polynucleotides and polypeptides of the present invention or fragments thereof can be used both diagnostically and therapeutically for these diseases, as well as other diseases related to an aberration of chromosome 3pl4. 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 10δ: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 l:34-δ, 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 ZNSSPδ 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 ZNSSPδ is an amount sufficient to produce a clinically significant change in extracellular matrix remodeling, scar tissue formation, tumor suppression, platelet aggregation, apoptosis, myogenesis, in tissues of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, and cervical carcinoma. Similarly, a therapeutically effective amount of ZNSSPδ is an amount sufficient to produce a clinically significant change in disorders associated with tissues of the bone marrow, peripheral blood lymphocytes, umbilical cord blood, fetal brain, prostate, cervical carcinoma, and in malignant and leukemic cell lines. 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 Identification of the DNA sequence

The novel ZNSSPδ polypeptide-encoding polynucleotides of the present invention were initially identified by querying a database of partial sequences. The cDNA sequences identified from the query were not full-length. Thus, the full-length cDNA sequence was identified by PCR using oligonucleotide primers ZC23674 (SEQ ED NO:4) and ZC24579 (SEQ ED NO:5), designed to the 5' and 3', untranslated regions of the gene, respectively. The gene was amplified from a bone marrow cDNA library using the following thermalcycler conditions: one cycle at 94°C for 4 minutes; followed by thirty cycles at 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 2 minutes, followed by one cycle at 72°C for 7 minutes, followed by a 4°C hold. The DNA fragment was purified using Qiaex U Agarose Gel Extraction Protocol (Qiagen) and subcloned into pCR2.1 using TOPO TA Cloning Kit (Invitrogen). The polynucleotide sequence of the insert corresponding to the cDNA clone was sequenced resulting in the polynucleotide sequence shown in SEQ ED NO:l. The deduced amino acid sequence of the insert was determined to be full-length and is shown in SEQ ED NO:2. This polypeptide, and the polynucleotides encoding it, were identified as a novel member of the Betal ,3 galactosyltransferase family, ZNSSPδ.

Example 2

Tissue Distribution of Human Znnspδ in Tissue Panels using PCR

A panel of cDNA samples from human tissues was screened for znsspδ 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 into 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 ZC23674 (SEQ ED NO: 6) and ZC24776 (SEQ ED NO: 7), TaKaRa Ex Taq™ (TAKARA Shuzo Co LTD, Biomedicals Group, Japan), and Rediload dye (Research Genetics, Inc., Huntsville, AL). The amplification was carried out as follows: 1 cycle at 94°C for 3 minutes, 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds, followed by 1 cycle at 72°C for 5 minutes. About 10 μl of the PCR reaction product was subjected to standard Agarose gel electrophoresis using a 4% agarose gel. The correct predicted DNA fragment size was observed in fetal brain, CD3+ (T cell) library, bone marrow, and HPV library. The HPV library is a cDNA library prepared from a prostate epithelial cell line (ATCC #CRL-2221, ATCC, Manasas, VA) that has been transformed with human papillomavirus lδ (HPV-lδ). The DNA fragment for fetal brain, CD3+ library, bone marrow, and

HPV library were excised and purified using a Gel Extraction Kit (Qiagen, Chatsworth, CA) according to manufacturer's instructions. Fragments were confirmed by sequencing to show that they were indeed znsspδ.

Table 4

Example 3 Construct for Generating Human znsspδ Transgenic Mice Oligonucleotides were designed to generate a PCR fragment containing a consensus Kozak sequence and the exact human znssp8 coding region. These oligonucleotides were designed with an Fsel site at the 5' end and an Ascl site at the 3' end to facilitate cloning into pTgl2-δ MT.

PCR reactions were carried out using Advantage^® cDNA polymerase (Clontech) to amplify a human znsspδ cDNA fragment. About 200 ng of human znsspδ polynucleotide template (Example 1), and oligonucleotides ZC26479 (SEQ ED NO: 8) and ZC26483 (SEQ ED NO:9) were used in the PCR reaction. PCR reaction conditions were as follows: 95°C for 5 minutes; 15 cycles of 95°C for 60 seconds, 58°C for 60 seconds, and 72°C for 90 seconds; and 72°C for 7 minutes; followed by a 4°C hold. PCR products were separated by agarose gel electrophoresis and purified using a QiaQuick™ (Qiagen) gel extraction kit. The isolated, approximately 1137bp, DNA fragment was digested with Fsel and Ascl (New England BioLabs), ethanol precipitated and ligated into pTgl2-8 MT that was previously digested with Fsel and Ascl. The pTgl2-δ MT plasmid, designed for expression of a gene of interest in transgenic mice, contains an expression cassette flanked by 10 kb of MT-1 5' DNA and 7 kb of MT-1 3' DNA. The expression cassette is comprised of the MT-1 promoter, the rat insulin π intron, a polylinker for the insertion of the desired clone, and the human growth hormone poly A sequence.

About one microliter of the ligation reaction was electroporated into DH10B ElectroMax^® competent cells (GIBCO BRL, Gaithersburg, MD) according to manufacturer's direction and plated onto LB plates containing 100 μg/ml ampicillin, and incubated overnight. Colonies were picked and grown in LB media containing 100 μg/ml ampicillin. Miniprep DNA was prepared from the picked clones and screened for the znsspδ insert by restriction digestion with EcoRI and subsequent agarose gel electrophoresis and analysis. Maxipreps of the correct pTgl2-δ MT znsspδ construct, as verified by sequence analysis, were performed. A Sail fragment containing the 5' and 3' flanking sequences, the MT promoter, the rat insulin II intron, znsspδ cDNA and the human growth hormone poly A sequence was prepared and used for microinjection into fertilized murine oocytes.

Example 4 Chromosomal Assignment and Placement of Znsspδ Znsspδ was mapped to chromosome 3 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 δ3 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 Znsspδ 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 δ5 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 26527 (SEQ ED NO: 10), 1 μl antisense primer, ZC 2652δ (SEQ ED NO: 11), 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 60°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 Znsspδ to the chromosome 3 marker SHGC-34179 with a LOD score of >14 and at a distance of 0 cR_10000 from the marker. The use of surrounding genes and markers positions Znsspδ in the 3pl4 chromosomal region.

Example 4 Detection of ZNSSP8 in Cervical Carcinoma Cell Lines by RT PCR

RT PCR was performed on RNA samples from two cervical carcinoma cell lines samples: an epitheloid cervix carcinoma cell line (HeLa 229, ATCC # CCL- 2.1, ATCC, Manasas, VA) and an epidermoid cervix carcinioma cell line (ME 180, ATCC HTB-33, ATCC, Manasas, VA). The RT-PCR reactions were set up using oligos ZC23674 (SEQ ED NO: 6) and ZC24776 (SEQ ED NO: 7). The protocol for SuperScriptTM One Step RT-PCR System (Life Technologies, Gaithersburg, MD) was followed. The amplification was carried out as follows: 1 cycle at 50 °C for 30 minutes and 94 °C for 2 minutes, 40 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute, followed by 1 cycle at 72°C for 5 minutes. About 10 μl of the PCR reaction product was subjected to standard agarose gel electrophoresis using a 1% agarose gel. The predicted DNA fragment for Znssp8 was observed in both samples.

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 comprising residues 83 to 343 of SEQ ED

NO:2.

2. An expression vector comprising the following operably linked elements: a) a transcription promoter; b) a DNA segment wherein the DNA segment is a polynucleotide encoding the polypeptide of claim 1 ; and c) a transcription terminator.

3. The expression vector according to claim 3 wherein the DNA segment contains an affinity tag.

4. A cultured cell into which has been introduced an expression vector according to claim 3, wherein said cell expresses the polypeptide encoded by the DNA segment.

5. A method of producing a polypeptide comprising culturing a cell according to claim 4, whereby said cell expresses the polypeptide encoded by the DNA segment; and recovering the polypeptide.

6. The polypeptide produced by the method of claim 5.

7. An epitope-bearing polypeptide selected from the group consisting of: a) a polypeptide comprising residues 33 to 82 of SEQ ED NO:2; b) a polypeptide comprising residues 344 to 37δ of SEQ ED NO:2; c) a polypeptide comprising residues 33 to 343 of SEQ ED NO:2; d) a polypeptide comprising residues 15 to 343 of SEQ ED NO:2 e) a polypeptide comprising residues 1 to 343 of SEQ ED NO:2; f) a polypeptide comprising residues δ3 to 37δ of SEQ ED NO:2; g) a polypeptide comprising residues 15 to 37δ of SEQ ED NO:2; h) a polypeptide comprising residues 1 to 37δ of SEQ ED NO:2; i) a polypeptide comprising residues 15 to δ2 of SEQ ED NO:2; j) a polypeptide comprising residues 1 to 32 of SEQ ED NO:2; and k) a polypeptide comprising residues 1 to δ2 of SEQ ED NO:2.

8. An isolated polynucleotide encoding the polypeptide according to claims 1 or 7.

9. A method of producing an antibody comprising the following steps: inoculating an animal with the epitope-bearing polypeptide according to claim 8, wherein the epitope-bearing polypeptide elicits an immune response in the animal; producing the antibody in the animal; and isolating the antibody produced from the animal

10. The antibody produced by the method according to claim 9.