EBNERIN CLONING OF A SECRETED VON EBNER'S GLAND PROTEIN

Abstract: The present invention is directed to a novel von Ebner's gland secreted protein, designated Ebnerin, which is formed in the ducts of von Ebner's gland and secreted into fluid bathing the taste buds contained in the taste papillae. Ebnerin has been identified by isolation of a rat cDNA encoding a novel secreted protein of 1290 amino acids.

Description: EBNERIN CLONING OF A SECRETED VON EBNER'S GLAND PROTEIN TECHNICAL FIELD OF THE INVENTION The present invention is directed to a novel protein secreted by von Ebner's glands. BACKGROUND OF THE INVENTION Saliva is the first digestive fluid secreted by the gastro-intestinal pathway and performs a variety of functions. It is essential in the formation of small boluses of food, provides lubrication for swallowing and speech, dissolves a number of chemicals in food substances, and provides digestive enzymes such as amylase and lipase (Hamosh & Scow, 1973; Field & Hand, 1987). About 90% of saliva is produced by three major glands, the parotid, the submaxillary and the sublingual glands whose secretions drain into the oral cavity. Von Ebner's glands (VEG) are unique salivary glands contained within the tongue and which drain directly into the clefts of the circumvallate and foliate papillae which contain the major taste buds. Secretions of von Ebner's gland directly modulate taste perception (Gurkan & Bradley, 1988). While von Ebner's gland is known to secrete certain proteins, such as lipase, very little is known of the protein composition of the gland's secretions or the molecular mechanisms that may influence taste perception. SUMMARY OF THE INVENTION The present invention is directed to a novel von Ebner's gland secreted protein, designated Ebnerin, which is formed in the ducts of von Ebner's gland and secreted into fluid bathing the taste buds contained in the taste papillae. Ebnerin has been identified by isolation of a rat cDNA encoding a novel secreted protein of 1290 amino acids. The present invention is directed to a substantially pure polypeptide having the sequence shown in or corresponding to SEQ ID NO:2. The present invention is also directed to a substantially pure polypeptide having a sequence corresponding to SEQ ID NO:2, the sequence of said polypeptide selected from the group consisting of: SEQ ID NO:2, muteins of SEQ ID NO:2, truncations of SEQ ID NO: 2, and fusion proteins thereof. The present invention is also directed to a DNA molecule comprising a sequence that encodes a polypeptide having a sequence corresponding to SEQ ID NO:2, the sequence of said polypeptide selected form the group consisting of SEQ ID NO:2, muteins of SEQ ID NO:2, truncations of SEQ ID NO:2, and fusion proteins thereof. Preferably, the DNA molecule is substantially free of other DNA molecules. Preferably the sequence encoding the polypeptide is an intron- free DNA sequence. The present invention is also directed to a cell population transformed with the DNA molecule. The DNA molecule corresponds to SEQ ID NO: l. The present invention provides a single stranded DNA molecule comprising at least 20 contiguous nucleotides of a subsequence found in SEQ ID NO: 1 or a DNA sequence complementary thereto. Another embodiment of the present invention is a method for determining the presence of a polynucleotide encoding a polypeptide which is substantially homologous to a polypeptide according to SEQ ID NO:2, wherein said polypeptide is secreted from von Ebner's gland. The method comprises providing a sample suspected of comprising said polynucleotide; incubating the sample with a nucleotide probe having a sequence complementary to a single stranded DNA containing at least 20 contiguous nucleotides of a subsequence found in SEQ ID NO:l, under conditions where said probe will form hybrids with nucleic acids from the sample; and detecting nucleic acid hybrids. The present invention is also directed to an antibody specifically immuno- reactive with a polypeptide according to SEQ ID NO: 1, wherein said polypeptide is secreted from von Ebner's gland. Preferably the antibody is monoclonal. The present invention is also directed to a method for determining the presence of Ebnerin in a tissue. The method includes incubating a tissue sample suspected of containing Ebnerin with an antibody which is specifically reactive with a polypeptide according to SEQ ID NO:l; and detecting the formation of antibody-antigen complexes involving said antibody. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1. Structure and deduced amino acid sequence of Ebnerin. Fig. 1 (a) Ebnerin contains a putative transmembranε domain (23 amino acids) and cytoplasmic region of 17 amino acids at the C-terminus. The N-terminus and major portion of Ebnerin are extracellular and contain a number of repeated domains. Rl, R2, R3 and R6 represent the repeated domains with sequence similarity to the scavenger receptor cysteine rich domain (SRCR) while R4, R5 and R7 are repeated domains homologous to the repeat domain in bone morphogenetic protein- 1 (BMP-1). F3 is a region of Ebnerin that shows homology to the zona pellucida region (ZP) of related proteins. Fig. 1 (b) cDNA for Ebnerin isolated from the taste cDNA library encodes 1290 amino acids with 15 N-linked glycosylation sites that are indicated by black dots above. A putative transmembrane domain is underlined Fig. 2. Alignment of amino acid sequence of Ebnerin compared to other proteins. Fig. 2 (a) Repeated domains of Ebnerin (Rl, R2, R3 and R6) show 72% amino acid sequence identity to the scavenger receptor cysteine rich domain (SRCR) in WC1 (Wijngaard et al., 1992), 64% to CD6 (Aruffo et al., 1991) and 47% to the macrophage scavenger receptor (Kodama et al., 1990). Fig. 2(b) The repeated domains of R4, R5 and R7 show 30% amino acid identity to bone morphogenetic protein type-1 (BMP-1) (Fukagawa et al., 1994), 32% to metalloendopeptidase (MEPD) (Elaroussi et al., 1994), 33% to serum inducible protein (PS4E) (Feng & Liau, 1993) and 34% to enteropeptidase precursor (ENPR) (Matsushima et al., 1994). Fig. 2 (c) The extracellular region near the transmembrane domain of Ebnerin (F3) shows 12.3% and 22% amino acid identity to the zona pellucida (ZP) region of the glycoprotein in pancreatic secretory granule membranes (GP-2) (Fukuoka et al., 1991) and uromodulin (UROM) (Pennica et al., 1987), respectively. Amino acids in Ebnerin that are identical to other proteins are shaded and conserved cysteine residues are indicated by asterisks above. Fig. 3. Tissue distribution of Ebnerin mRNA analyzed by Northern blot. Twenty mg total RNA from various rat tissues were loaded on each lane and hybridized with Ebnerin cDNA probe under high stringency. The blot was exposed to X-ray film for 24- hr. VEG: von Ebner's gland; CV: circumvallate papillae, PG: parotid gland; SG: submaxillary gland, LNG: lateral nasal gland, Olf: olfactory epithelial tissues. An mRNA species of approximately 5 kb was detected in VEG, CV, LNG and Olf. Fig. 4 (a) In situ hybridization of 33 P-Ebnerin cRNA probe on tongue in saggital section (10X). Hybridization signals are located in the VEG, von Ebner's gland. Fig. 4 (b) Bright field image of coronal section of circumvallate papillae (200X) . TB: taste buds, TE: tongue epithelium, DO: duct opening of von Ebner's gland, VEG: von Ebner's gland. Fig. 4 (c) Dark image of same section as in Fig 4 (b). Note that the hybridization signals are confined to DO in VEG. Fig 5. Western Blot analysis of Ebnerin. Protein (100 mg/lane) extracted from a variety of tissues was separated by electrophoresis on a 4-12% polyacrylamide gel and transferred to a nitrocellulose filter. The blot was incubated with affinity purified antiserum to Ebnerin (1: 100) and immunostained proteins were visualized by the ECL method. TE: the front region of tongue epithelium, VEG: von Ebner's gland, sup: supernatant, pellet: membrane preparations of VEG that were separated from supernatant by centrifugation at 30000g for 30 min. -fpeptide: VEG pellet preparations was incubated with the antibodies preabsorbed with peptide antigen (20 mg/ml at 4°C for 24 hr). Fig 6. Immunohistochemical localization of Ebnerin in rat tongue. Fig. 6(a) Antibodies (1:200) and Fig. 6(b) Antibodies preabsorbed with peptide antigen (20 mg/ml) were incubated with rat tongue tissue sections containing circumvallate papillae (CV), von Ebner's gland (VEG). Note in (a) the intense immunoreactivity in the VEG, its duct opening (DO), the cleft of CV and the surface of tongue epithelium (TE). Taste buds (TB) are located near the duct openings of VEG. (100X) Fig. 6(c) Higher magnification of CV section immunostained for Ebnerin antibodies. The intense immunoreactivity is in the cleft of CV where taste buds are located. X400. Fig. 6(d) Higher magnification of VEG section immunostained for Ebnerin. Note that intense immunoreactivity is confined to the epithelial cells of secretory ducts (SD) and is absent from secretory acini (SA). X400. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is directed to a novel von Ebner's gland secreted protein, designated Ebnerin, which is formed in the ducts of von Ebner's gland and secreted into fluid bathing the taste buds contained in the taste papillae. Ebnerin has been identified by isolation of a rat cDNA encoding a novel secretory protein of 1290 amino acids that is specifically expressed in von Ebner's gland (VEG) and secreted onto the tongue surface along the apical region of taste buds in the clefts of circumvallate papillae. Ebnerin possesses a putative single transmembrane domain at the C-terminus with 17 amino acids in the cytoplasmic area. The extracellular region of Ebnerin contains a number of repeated domains with homology to the scavenger receptor cysteine-rich (SRCR) domain and to a repeated domain of bone-morphogenetic protein-I and other related proteins. Western blot analysis reveals that Ebnerin exists as both paniculate and soluble forms. In situ hybridization and immunohistochemistry demonstrate that Ebnerin is located in secretory duct epithelial cells of von Ebner's gland (VEG) and is secreted onto the tongue surface along the apical region of taste buds in the clefts of circumvallate papillae. The unique structure and localization of Ebnerin suggest that it is a binding protein in saliva for regulation of taste sensation. Ebnerin displays substantial homology to a number of proteins. The greatest homology lies in four repeated domains which correspond to the scavenger receptor cysteine rich (SRCR) domain of a variety of proteins. Some of these proteins are expressed on the surfaces of cells involved in host defense mechanisms of the immune system, such as T cells, B cells and macrophages and exemplified by the macrophage scavenger receptor proteins. Others are secreted and appear to participate in host defense, such as complement factor I Clr and Cls (Journet and Tosi, 1986; Mackinnon et al., 1987), cyclophilin C binding protein (Friedman et al., 1993) or MAC II binding protein (Koths et al., 1993). Members of this family are expressed in a wide range of organisms from mammals to invertebrates. A prominent member of the family is a speract receptor which occurs in sea urchin sperm and binds speract, the sperm activating peptide secreted by eggs and involved in the chemotaxis of sea urchin eggs and sperm (Bleil & Wassarman, 1980; Kinloch et al, 1992; Lepage et al., 1992). The SRCR family resembles the immunoglobulin superfamily of proteins with multiple copies of cysteine rich domains occurring both in secreted and membrane associated proteins. Like other members of the SRCR family, Ebnerin occurs both in a paniculate and a secreted form. Presumably the secreted form is derived by proteolytic cleavage close to the transmembrane area, analogous to the secretion of the Alzheimer's precursor protein (Meier-Ruge et al. , 1994) or glycosylphosphatidylinositol (GPI)-linked proteins such as uromodulin and GP-2 proteins (Rindler et al., 1990; Fukuoka et al., 1991; Hoops et al., 1991). Since members of the SRCR family bind proteins and other ligands, it is likely that Ebnerin possesses a similar function. It is unclear whether the SRCR domains would be involved in ligand binding. They do not appear to participate in ligand binding of the macrophage scavenger receptor (Rohrer et al, 1990). However, almost the entire extracellular domain of the speract receptor comprises SRCR domains which presumably mediate binding to speract secreted by eggs (Kinloch et al, 1992; Lepage et al., 1992). Interestingly, the C-terminal region of Ebnerin displays modest homology to the zona pellucida protein which is released by eggs and interacts with the sperm outer membrane. It is unclear whether the coincidence of homology with egg and sperm signaling proteins in the Ebnerin sequence has physiological relevance. Ebnerin also possesses three repeated domains which display 30-35 % amino acid identity to domains that are held in common by bone morphogenetic protein-I (BP-1), complement I receptor and the Drosophila protein Tolloid which determines dorsal-ventral patterning (Wozney et al., 1988; Schimell et al., 1991). The repeated domain that is common to these proteins and Ebnerin is thought to play a role in ligand binding (Schimell et al., 1991). Accordingly, this area of Ebnerin may mediate ligand interactions. The existence of Ebnerin in both soluble and paniculate fractions and its localization to extracellular fluid surrounding taste buds indicates that it is secreted onto the tongue's surface along the clefts of the circumvallate and foliate papillae. Presumably the soluble form of Ebnerin arises by cleavage of its transmembrane domain in analogy to other membrane anchored secretory proteins such as granule secretory proteins and uromodulin (Rindler et al., 1990; Fukuoka et al., 1991; Hoops & Rindler, 1991 ) . The cleavage might occur through glycophosphatidylinositol anchored regions or at a lysine-lysine bond. In such a cleavage the very short cytoplasmic and transmembranes domains would be removed giving rise to a soluble protein whose molecular weight would be indistinguishable from the membrane associated protein, consistent with a similar mobility in Western blots of the paniculate and soluble forms of Ebnerin. The selective localization of Ebnerin in cells of the ducts of VEG but not in the acinar cells, distinguishes Ebnerin from lipase, mucin and other proteins secreted by acinar cells of salivary glands (Hamosh & Scow, 1973; Field & Hand, 1987). Images of Ebnerin at high magnification immunohistochemistry suggest that it may be an aggregated protein or associated with other proteins when secreted. Ebnerin may primarily bind to soluble proteins or other tastants or preferentially bind directly to surface proteins of the taste buds. Secretion of growth hormone has been established for parotid and submaxillary salivary glands, (Amano et al., 1993; Humphreys-beher et al., 1994), but has not been investigated for VEG. The structural similarity of Ebnerin to binding proteins for growth hormone such as BMP-1 (Fukagawa et al., 1994) and TGF-/S receptor III (Lopez-Casillas, 1991; Wang et al., 1991; Moren et al., 1992) suggests that it might serve as a carrier for putative growth factors produced by VEG. One protein that might interact with Ebnerin is the von Ebner's gland protein (VEGP) whose sequence resembles that of the odorant binding protein (OBP) (Schmale et al., 1990; Pevsner et al., 1988a). VEGP is also a secretory protein specifically expressed in von Ebner's gland, although its cellular localization and function has not been ascertained. Other members of the lipocalin family of small soluble carrier proteins have been identified (Nagata, 1992). OBP binds a wide range of odorants and is thought to serve as a carrier to deliver them to odorant receptors or possibly to remove odorants from odorant receptors (Pevsner et al., 1985; 1988b, 1990). Conceivably VEGP binds tastants and transports them to or away from taste receptors. It is tempting to suggest that VEGP is a ligand bound by Ebnerin. To investigate such possibility, we examined possible binding of a glutathione-fusion protein for VEGP to 35 S-labeled Ebnerin but have failed to detect direct binding. Despite the absence of direct evidence, participation of Ebnerin in modulating effects of tastants on taste buds would be consistent with studies demonstrating that VEG secretions alter the sensitivity of taste buds to applied tastants (Gurkan & Bradley, 1988). The possible analogy of functions of VEG proteins including VEGP and Ebnerin to similar proteins in olfactory tissues fits with our observations that Ebnerin or an Ebnerin-like transcript is also expressed in olfactory tissues. In describing the present invention, the following terminology is used in accordance with the definitions set out below. Two DNA sequences are "substantially homologous" when at least about 85% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See e.g., Maniatis et al., supra; DNA Cloning, vols. I and II supra; Nucleic Acid Hybridization, supra. Stringent conditions are used for detecting close homologs. Non-stringent conditions are used to detect more distantly related sequences. A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) in to chromosomal DNA making up the genome of the cell. Transformed cells can be selected or screened for by standard genetic techniques. A population of transformed cells is generally a pure culture of cells in which all members of the population contain the exogenous DNA. In procaryotes and yeast, for example, the exogenous DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the exogenous DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA. Polypeptides are polymers made up of a sequence of amino acids linked by peptide bonds, containing at least 10 and usually 50 or more amino acids in the sequence. Proteins are polypeptides which usually have 35 or more amino acids and form a characteristic three dimensional structure (tertiary structure). One amino acid sequence "corresponds" to another amino acid sequence if at least 75 % of the amino acid positions in the first sequence are occupied by the same amino acid residues in the second sequence. Preferably 90% of the amino acid positions are identical, and most preferably 95% of the amino acid positions are identical. "Conservative amino acid substitutions" are the substitution of one amino acid residue in a sequence by another residue of similar properties, such that the secondary and tertiary structure of the resultant peptides are substantially the same. Conservative amino acid substitutions occur when an amino acid has substantially the same charge as the amino acid for which it is substituted and the substitution has no significant effect on the local conformation of the protein. Amino acid pairs which may be conservatively substituted for one another are well-known to those of ordinary skill in the art. For the purposes of defining the present invention, two proteins are homologous if 70% of the amino acids in their respective sequences are the same; usually the amino acid sequences of homologous proteins are 80% identical. The sequences of substantially homologous proteins will be 85% identical, preferably the identity will be 90%, most preferably 95%. Two proteins are similar if the majority of the differences between their respective amino acid sequences involve conservative substitutions. The polypeptides of this invention encompass Ebnerin and Ebnerin analogs. Ebnerin is a naturally occurring, mature protein secreted from von Ebner's gland, and further encompasses all precursors and allelic variations of Ebnerin, as well as including forms of heterogeneous molecular weight that may result from inconsistent processing in vivo. An example of the Ebnerin sequence is shown in Figure 1. "Ebnerin analogs" are a class of peptides which includes: 1) "Ebnerin muteins," which are polypeptides which are substantially homologous to Ebnerin. Preferably the amino acid sequence of the "mutein" differs from that of Ebnerin by 8 or fewer amino acid residues, more preferably, 7 or fewer residues, even more preferably about 5 or fewer residues and most preferably about 2 or fewer residues. It is sometimes preferred that any differences in the amino acid sequences of the two proteins involve only conservative amino acid substitutions. Alternatively, changes such as the elimination of cysteine which alter the activity or stability of the protein may be preferred. 2) "Truncated Ebnerin peptides," which include fragments of either "Ebnerin" or "Ebnerin muteins" that preferably retain either (i) an amino acid sequence unique to Ebnerin, or (ii) an epitope unique to Ebnerin. 3) "Ebnerin fusion proteins" include heterologous polypeptides which are made up of one of the above polypeptides (Ebnerin, Ebnerin muteins or truncated Ebnerin peptides) fused to any heterologous amino acid sequence. Preferably such heterologous sequences are fused to the N-terminal end of the Ebnerin sequence and comprise a leader sequence to direct secretion. A composition comprising a selected protein A is "substantially pure" when protein A makes up at least about 75% by weight of the total protein. Preferably, protein A comprises at least about 90% by weight of the total weight, most preferably at least about 99% by weight of the total weight. In the case of a composition comprising a selected biologically active protein, which is substan¬ tially free of contaminating proteins, it is sometimes preferred that the composition having the activity of the protein of interest contain species with only a single molecular weight (i.e., a "homogeneous" composition). As used herein, a "biological sample" refers to a sample of tissue or fluid isolated from a individual, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, virally infected cells, recombinant cells, and cell components). "Human tissue" is an aggregate of human cells which may constitute a solid mass. This term also encompasses a suspension of human cells, such as blood cells, or a human cell line. The term "binding partner" as used herein refers to a molecule capable of binding a ligand molecule with high specificity, as for example an antigen and an antibody specific therefor. In general, the specific binding partners must bind with sufficient affinity to immobilize the analyte copy/complementary strand duplex (in the case of capture probes) under the isolation conditions. Specific binding partners are known in the art, and include, for example, biotin and avidin or streptavidin, IgG and protein A, the numerous known receptor-ligand couples, and complementary polynucleotide strands. In the case of complementary polynucleotide binding partners, the partners are normally at least about 15 bases in length, and may be least 40 bases in length. The polynucleotides may be composed of DNA, RNA, or synthetic nucleotide analogs. The term "coupled" as used herein refers to attachment by covalent bonds or by strong non-covalent interactions (e.g., hydrophobic interactions, hydrogen bonds, etc.). Covalent bonds may be, for example, ester, ether, phosphoester, amide, peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds, and the like. An "epitope" is a structure, usually made up of a short peptide sequence or oligosaccharide, that is specifically recognized or specifically bound by a component of the immune system. T-cell epitopes have generally been shown to be linear oligopeptides. Two epitopes correspond to each other if they can be specifically bound by the same antibody. Two antibodies correspond to each other if both are capable of binding to the same epitope, and binding of one antibody to its epitope prevents binding by the other antibody. The term "immunoglobulin molecule" encompasses whole antibodies made up of four immunoglobulin peptide chains, two heavy chains and two light chains, as well as immunoglobulin fragments. "Immunoglobulin fragments" are protein molecules related to antibodies, which are known to retain the epitopic binding specificity of the original antibody, such as Fab, F(ab)' 2 , Fv, etc. A "single stranded DNA molecule" comprises at least 20 nucleotides which can be used as a hybridization probe for RNA or DNA in or extracted from cells. In the present invention, the nucleotides comprise all or a part of SEQ ID NO: 1 or a DNA sequence complementary thereto. Typically 20 nucleotides will provide a sufficient specificity and uniqueness; however, 25, 30, 35, or 40 contiguous nucleotides of the disclosed sequence, for example, may be desirable for a particular application. Diagnostic Assays Detection of sequences cross-reactive with Ebnerin and their expression, may be on the nucleotide or peptide level. Antibodies can be prepared by immunizing mammals with peptides expressed from nucleic acid sequences corresponding to cross-reactive polypeptides, as indicated above, and selecting those antibodies specific to the Ebnerin using techniques that are well known to those skilled in the art. The antibodies may be polyclonal or monoclonal, as is known in the art. These antibodies can detect the presence of cross-reactive protein by a variety of immunoassay techniques. The nucleotide probe sequences provided herein can be used to detect expression of mRNA conesponding to cross- reactive proteins in accordance with any of the standard techniques. Expression may be detected either by in situ hybridization or by extraction and detection of mRNA. The particular procedures for gene probe assays and immunoassays are well-known to those skilled in the art. General Methods The practice of the present invention employs, unless otherwise indicated, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are well known to the skilled worker and are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover, ed. , 1985); "Oligonucleotide Synthesis" (MJ. Gait, ed., 1984); "Nucleic Acid Hybridization" (B.D. Hames & S J. Higgins, eds., 1985); "Transcription and Translation" (B.D. Hames & SJ. Higgins, eds., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1986); "Immobilized Cells and Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide to Molecular Cloning" (1984), and Sambrook, et al., "Molecular Cloning: a Laboratory Manual" (1989). DNA segments or oligonucleotides having specific sequences can be syn¬ thesized chemically or isolated by one of several approaches. The basic strategies for identifying, amplifying and isolating desired DNA sequences as well as assembling them into larger DNA molecules containing the desired sequence domains in the desired order, are well known to those of ordinary skill in the art. See, e.g., Sambrook, et al., (1989); B. Perbal, (1984). Preferably, DNA segments corresponding to Ebnerin may be isolated individually using the polymerase chain reaction (M.A. Innis, et al., "PCR Protocols: A Guide To Methods and Applications," Academic Press, 1990). A complete sequence may be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g.. Edge (1981) Nature 292:756; Nambair, et al. (1984) Science 223:1299; Jay, et al. (1984) J. Biol. Chem., 259:6311. The assembled sequence can be cloned into any suitable vector or replicon and maintained there in a composition which is substantially free of vectors that do not contain the assembled sequence. This provides a reservoir of the assembled sequence, and segments or the entire sequence can be extracted from the reservoir by excising from DNA in the reservoir material with restriction enzymes or by PCR amplification. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice (see, e.g., Sambrook, et al., incorporated herein by reference). The construction of vectors containing desired DNA segments linked by appropriate DNA sequences is accomplished by techniques similar to those used to construct the segments. These vectors may be constructed to contain additional DNA segments, such as bacterial origins of replication to make shuttle vectors (for shuttling between prokaryotic hosts and mammalian hosts), etc. Procedures for construction and expression of mutant proteins of defined sequence are well known in the art. A DNA sequence encoding a mutant form of Ebnerin can be synthesized chemically or prepared from the wild-type sequence by one of several approaches, including primer extension, linker insertion and PCR (see, e.g., Sambrook, et al.). Mutants can be prepared by these techniques having additions, deletions and substitutions in the wild-type sequence. It is preferable to test the mutants to confirm that they are the desired sequence by sequence analysis and/or the assays described below. Mutant protein for testing may be prepared by placing the coding sequence for the polypeptide in a vector under the control of a promoter, so that the DNA sequence is transcribed into RNA and translated into protein in a host cell transformed by this (expression) vector. The mutant protein may be produced by growing host cells transfected by an expression vector containing the coding sequence for the mutant under conditions whereby the polypeptide is expressed. The selection of the appropriate growth conditions is within the skill of the art. The presence of Ebnerin in a tissue may be determined by incubating a tissue sample suspected of containing Ebnerin with an antibody which is specifically reactive with a polypeptide according to SEQ ID NO: l and then detecting the formation of antibody-antigen complexes involving said antibody. Any immunoassay format known in the art may be employed. EXAMPLES METHODS Polymerase Chain Reaction (PCR) and Northern analysis Circumvallate papillae containing taste buds and tongue epithelium were dissected as previously described (Hwang et al., 1990 ), and RNA was extracted from the circumvallate papillae. Ten μg total RNA were reverse transcribed using 1 μg of oligo-dT as primer. One μ\ of the reverse transcription mix was used in a 25 μ\ PCR reaction containing 1.5 mM MgCl 2 , 400 nM primers, 200 μM dNTP and 0.5 unit of Taq Polymerase (Boehringer Mannheim). To search for genes associated with salty taste, degenerate primers based on the published sequences of the amiloride sensitive sodium channel (Canessa et al., 1993) were used to amplify mRNA from rat circumvallate papillae. PCR conditions were 35 cycles of 1 min at 95°C, 2 min at 45°C or 65°C and 1 min at 72°C. PCR products were then subcloned into Bluescript vector and sequenced. The PCR primers u s e d i n t h i s s t u d y w e r e t h e f o r w a r d p r i m e r (GGACAGAATTCGNGGNAA(T/C)TA(T/C)GGNGA(T/C)TG) and the reverse primer (GATCCACTCGAGNGA(T/C)TTNACNGANGGCCA) , which respectively correspond to 372 SLGGNYGDC 380 and 520 WPSVKSQ 526 of the amiloride sensitive sodium channel (Canessa et al., 1993). Total RNA (20 μg) was prepared from various rat tissues, fractionated on 1 % agarose/formaldehyde gels and blotted onto a nitrocellulose membrane. [ 32 P]dCTP labeled PCR product (400 bp) was used as a probe. The blot was hybridized in 50% formamide, 5XSSC hybridization buffer at 42°C and washed with 0. IXSSC at 65°C. The blot was then exposed to X-ray film for 1 day at -70°C. Constructing and screening of a rat circumvallate papillae cDNA library. A rat taste cDNA library was constructed as previously described (Li et al. , 1994). Total RNA was extracted from the circumvallate papillae of 500 rats. Poly (A + )RNA was prepared by passing RNA through an oligo-dT column twice. About 5 μg poly (A + )RNA were converted to cDNA for construction of the cDNA library, using a Lambda Zap vector cDNA library synthesis Kit (Stratagene). The library consists of 1.5X 10 6 independent clones with an average insert size about 1.2 kb. The cDNA clone generated by PCR was labeled using a random primer kit and [ 32 P]dCTP, and was used to screen a rat circumvallate papillae cDNA library under high stringency (50% formamide, 5XSSC 42°C for hybridization and IXSSC 55°C for a final wash). Positive clones were purified and cDNA was prepared for restriction mapping and sequence analysis. In situ hybridization cRNA probes were made for in situ hybridization on rat tongue tissue sections. A PCR clone in a Bluescript vector containing a 400 bp insert was used to generate antisense RNA probes with [ 33 P]UTP labeling (NEN). Sense RNA probes provided negative controls. Tissue sections (16 μm) were fixed in 4% paraformaldehyde, rinsed in phosphate buffered saline and digested with 10 μg/ml proteinase K at 37°C for 30 min. Sections were then rinsed in 0.1 M triethanolamine, acetylated in 0.25% acetic anhydride for 10 min, and dehydrated in a graded series of ethanol solutions. Hybridization was performed with 10 6 cpm/100 μ\ of probe in 50% formamide, 10% dextran sulfate, 0.3M MgCl 2 , lOmM Tris (pH 8), lXDenhardt's solution, 0.5mg/ml tRNA and 10 mM DTT overnight at 55 °C. Excess cRNA probe was removed by digestion with RNase A (20 g/ml) for 30 min and washed at a final stringency of 0. IXSSC at 60°C for 30 min. Slides were hand-dipped in Kodak NTB2 emulsion, exposed for 3-4 days at 4°C, developed and stained with Giemsa stain (Sigma). Immunoblot and unmunohistochemistry A peptide corresponding to a partial amino sequences of Ebnerin ( 'FTTDHSNTRRGFRADYYS 946 ) was synthesized with the addition of a lysine on the N-terminus to facilitate conjugating to bovine serum albumin with glutaraldehyde. More specifically, a solution of 5mg/ml of the synthetic peptide in PBS was mixed well with carrier protein (BSA) 50 mg and the pH was adjusted to 7.5. An equal volume of glutaraldehyde was slowly added to the peptide/BSA solution with constant agitation and then incubated at room temperature for 1 hour. Glycine was added from a 1 M stock in PBS (pH 7.2) to a final concentration of 200mM. The solution was incubated with stirring for 1 hour. Dialysis was performed on the BSA conjugated peptides against PBS overnight with more than three changes. The conjugated antigen was injected into rabbits. Rabbits were injected subdermal with 500 μl peptide-BSA (250 mg antigen) + 500 μl Freund's Complete Adjuvant. Three weeks later, the rabbits were boosted by injecting subcutaneously 500 μl peptide-BSA and 500 μl Freund's incomplete Adjuvant. The rabbits were reboosted with 250 μl every four weeks. The rabbits were bled ten days after the boosts. Antisera were purified by an Affι-gel-15 (Bio-Rad) affinity column following standard procedures (Antibodies Lab Manual, 1988). The purified antiserum contains 12 mg/ml antibody. In Western blot analysis 1:100 dilution of purified antiserum were used. Protein samples were prepared from various tissues homogenized in PBS buffer containing proteinase inhibitors (1 mM pepstain A, 0.1 mM aprotinin, 0.1 mM phenylmethylsulfonyl fluoride and 10 mM leupeptin). Protein samples (100 mg) were denatured in SDS sample buffer at 100°C for 5 min before loading onto a 4-12% gradient SDS gel. Fractionated proteins were transferred onto nitrocellulose membranes and blots were blocked in PBS with 5% non-fat dry milk. Blots were then incubated with 1: 200 dilution antibody in the same buffer overnight at 4°C. ECL assay was used to detect stained proteins following instructions from the kit (Amersham). Tissue sections for immunohistochemical studies were fixed with 4% paraformadehyde at room temperature for 15 min, 0.4 % Triton in PBS 30 min and 0.3% H 2 O 2 in methanol for 15 min with washes in PBS following each treatment. Sections were then blocked in 5% normal goat serum in PBS for 1 hr and incubated with 1:200 diluted antibody in PBS for 24 hr at 4°C. ABC and DAB detecting kits (Vector) were used to visualize immunoreactivity signals in the tissue sections. RESULTS Molecular Cloning of Ebnerin: In a search for taste bud associated proteins linked to salty taste perception, PCR analysis was conducted employing primers based on the sequence of the amiloride sensitive sodium channel, which is involved in salty taste perception (Heck et al., 1984; Schiffman et al., 1983 ). A novel PCR product was identified unrelated to the amiloride-sensitive channel. Initial Northern blots of this PCR product revealed selective enrichment in von Ebner's gland. The PCR product was employed to screen a cDNA library derived from circumvallate papillae containing taste buds and obtained a cDNA of 4267 bp which encodes a protein of 1289 amino acids that is designated Ebnerin (Fig 1). Ebnerin displays sequence homology to a variety of proteins (Fig. 1 A & Fig. 2). It possesses four repeat domains with 50-70% amino acid identity to various members of the scavenger receptor protein family which binds a variety of proteins and peptides (Kodama et al., 1990; Rohrer et al., 1990). There also are three repeat domains with about 30-35% amino acid identity to bone morphogenetic type-I protein (BMP-1) (Fukagawa et al., 1994), TGF-jS receptor type HI (Lopez-Casillas et al., 1991; Wang et al., 1991; Moren et al., 1992) and related proteins (Wozney et al., 1988; Elaroussi & DeLuca, 1994; Feng & Liau, 1993; Fukagawa et al., 1994). In the C-terminal area of Ebnerin, a 305 amino acid domain displays 12-22*% amino acid identity to the zona pellucida (ZP) region in a sperm binding protein of eggs ( Lepage et al., 1992) and in uromodulin (UROM) (Pennica et al., 1987) and to the glycoprotein of zymogen granule membranes (Fukuoka et al., 1991). Ebnerin possesses a single 23 amino acid transmembrane region with a very short, 17 amino acid presumed intracellular C-terminal area. There are 15 N-linked glycosylation sites in the putative extracellular domain (Fig. IB). Localization of Ebnerin mRNA: Northern blot analysis of Ebnerin was conducted in a variety of tissues (Fig. 3). The most prominent expression of Ebnerin mRNA occurs in von Ebner's glands (VEG) with an intense band at about 4.5-5 kb. Circumvallate papillae tissue displays a similarly intense band of the same size. Since circumvallate papillae tissue contains VEG tissue, this band probably derives from contaminating VEG material. Tissue of the olfactory epithelium and the lateral nasal gland displays less intense bands which are slightly smaller than those in VEG. mRNA expression was not detected in any other tissue examined including epithelium from the frontal region of the tongue, tongue tissue that is devoid of VEG, or in parotid gland, submaxillary gland, liver, lung, kidney, colon, testes, brain and spleen. In situ hybridization reveals discrete localizations of Ebnerin mRNA (Fig. 4). Autoradiographic grains are highly localized to VEG located immediately beneath the circumvallate papillae. Higher power magnification reveals very high levels of grains associated with the openings of the ducts of VEG. Localization of Ebnerin Protein: An antiserum was developed to an 18 amino acid peptide in the repeat domain that is homologous to BP-1 protein. Western blot analysis reveals a single immunoreactive band of 210 kDa which occurs with similar intensity in paniculate and supernatant preparations of VEG. HEK293 cells transfected with cDNA for Ebnerin display a band of about 170 kDa, while untransfected cells have no immunoreactivity (Fig. 5). The larger size of native Ebnerin than transfected protein could be due to N-glycosylation of protein in VEG or an additional N-terminal peptide not revealed by cDNA cloning. No immunoreactivity was detected in tongue tissue lacking VEG or in kidney, lung, brain, or colon. Preabsorption with the immunizing peptide eliminates immunoreactivity . Immunohistochemistry with Ebnerin antiserum revealed selective staining associated with VEG (Fig. 6). Staining was also evident in the clefts in apical portions of the circumvallate papillae. Preabsorption with the immunizing peptide abolishes immunoreactivity. High magnification revealed staining selectively localized to the apical region of the taste buds. Staining was also evident at the openings of the ducts of VEG. In the VEG itself intense staining overlied the epithelium of the ducts of the gland, while the acinar tissue did not stain. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Li, Xiao Snyder, Solomon H. (ii) TITLE OF INVENTION: Ebnerin: Molecular Cloning of a Secreted von Ebner's Gland Protein Associated with Taste Buds (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Banner & Allegretti, Ltd. (B) STREET: 1001 G Street, N.W. (C) CITY: Washington (D) STATE: D.C. (E) COUNTRY: USA (F) ZIP: 20001 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS <D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Wolffe, Susan A. (B) REGISTRATION NUMBER: 33,568 (C) REFERENCE/DOCKET NUMBER: 01107.48790 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 202-508-9100 (B) TELEFAX: 202-508-9299 (C) TELEX: 197430 BBMB UT (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4360 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus rattus (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 94..3963 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GGAACAGATT CTGGTTTGGC TGTGAGGCTG GTGAATGGAG GAGACAGGTG TCGGGGTCGC 60 GTGGAGATCC TTTACCAGGG TTCCTGGGGC ACC ATG TGT GAT GAC AGC TGG GAC 114 Met Cys Asp Asp Ser Trp Asp 1 5 ATC AAT GAT GCC AAC GTG GTG TGC AGG CAG CTG GGC TGT GGC TGG GCC 162 lie Asn Asp Ala Asn Val Val Cys Arg Gin Leu Gly Cys Gly Trp Ala 10 15 20 TTG TCT GCC CCA GGA AGT GCC CAG TTT GGA CAG GGT CTG GGT CCC ATT 210 Leu Ser Ala Pro Gly Ser Ala Gin Phe Gly Gin Gly Leu Gly Pro lie 25 30 35 GTT CTG GAT GAC GTG GCC TGT AGA GGA CAT GAG GCC TAT CTG TGG AGC 258 Val Leu Asp Asp Val Ala Cys Arg Gly His Glu Ala Tyr Leu Trp Ser 40 45 50 55 TGC TCC CAC CGA GGC TGG CTC TCT CAT AAC TGT GGA CAT CAG GAG GAT 306 Cys Ser His Arg Gly Trp Leu Ser His Asn Cys Gly His Gin Glu Asp 60 65 70 GCT GGA GTG ATC TGC TCA GAT TCT CAA ACA AGC AGT CCC ACA CCC GGT 354 Ala Gly Val lie Cys Ser Asp Ser Gin Thr Ser Ser Pro Thr Pro Gly 75 80 85 TGG TGG AAC CCC GGG GGC ACA AAT AAC GAT GTG ATC TAC GAC ACT CAA 402 Trp Trp Asn Pro Gly Gly Thr Asn Asn Asp Val lie Tyr Asp Thr Gin 90 95 100 GAA ACC ACA GAA ACT TCT CAA ACA AGC AGT CCC ACA CCT GAT TGG TGG 450 Glu Thr Thr Glu Thr Ser Gin Thr Ser Ser Pro Thr Pro Asp Trp Trp 105 110 115 AAC CAT GGG GGC ACA ATT AAT GAT GTG ATC TAT GAC ACT CAA GAA ACC 498 Asn His Gly Gly Thr lie Asn Asp Val lie Tyr Asp Thr Gin Glu Thr 120 125 130 135 ACA GAA GGA ACA GAT TCT GGT TTG GCT GTG AGG CTG GTG AAT GGA GGA 546 Thr Glu Gly Thr Asp Ser Gly Leu Ala Val Arg Leu Val Asn Gly Gly 140 145 150 GAC AGG TGT CGG GGT CGT GTG GAG ATC CTT TAC CAG GGT TCC TGG GGC 594 Asp Arg Cys Arg Gly Arg Val Glu lie Leu Tyr Gin Gly Ser Trp Gly 155 160 165 ACC GTG TGT GAC GAC AGC TGG GAC ATC AAT GAT GCC AAC GTG GTG TGC 642 Thr Val Cys Asp Asp Ser Trp Asp lie Asn Asp Ala Asn Val Val Cys 170 175 180 AGG CAG CTG GGC TGT GGC TGG GCC TTG TCT GCC CCA GGA AGT GCC CAG 690 Arg Gin Leu Gly Cys Gly Trp Ala Leu Ser Ala Pro Gly Ser Ala Gin 185 190 195 TTT GGA CAG GGC TCT GGG TCC ATT GTT CTG GAT GAC GTG GCC TGT AGA 738 Phe Gly Gin Gly Ser Gly Ser lie Val Leu Asp Asp Val Ala Cys Arg 200 205 210 215 GGA CAT GAG GCC TAT CTG TGG AGC TGC TCC CAC CGA GGC TGG CTC TCT 786 Gly His Glu Ala Tyr Leu Trp Ser Cys Ser His Arg Gly Trp Leu Ser 220 225 230 CAT AAC TGT GGA CAT CAG GAG GAT GCT GGA GTC ATC TGT TCA TAT TCT 834 His Asn Cys Gly His Gin Glu Asp Ala Gly Val lie Cys Ser Tyr Ser 235 240 245 CAA ACA AGC AGT CCC ACA CCC GAT TCT CAA ACA AGC AGT CCC ACA CCC 882 Gin Thr Ser Ser Pro Thr Pro Asp Ser Gin Thr Ser Ser Pro Thr Pro 250 255 260 GGT TGG TGG AAC CCC GGG GGC ACA AAT AAC GAT GTG TCC TAT GGA CCC 930 Gly Trp Trp Asn Pro Gly Gly Thr Asn Asn Asp Val Ser Tyr Gly Pro 265 270 275 GAA CAG ACC ACA GAC GCA ACA GAT TCT GGT TTG GCT GTG AGG CTG GTG 978 Glu Gin Thr Thr Asp Ala Thr Asp Ser Gly Leu Ala Val Arg Leu Val 280 285 290 295 AAT GGA GGA GAC AGG TGT CAG GGT CGT GTG GAG ATC CTT TAC CAG GGT 1026 Asn Gly Gly Asp Arg Cys Gin Gly Arg Val Glu lie Leu Tyr Gin Gly 300 305 310 TCC TGG GGT ACC GTG TGT GAC GAC AGC TGG GAC ACC AAG GAT GCC AAC 1074 Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Thr Lys Asp Ala Asn 315 320 325 GTG GTG TGC AGG CAG CTG GTC TGT GGC TGG GCC TTG TCT GCC CCA GGA 1122 Val Val Cys Arg Gin Leu Val Cys Gly Trp Ala Leu Ser Ala Pro Gly 330 335 340 AGT GCC CAC TTT GGA CAA GGC TCT GGA TCC ATT GTT CTG GAT GAC GTG 1170 Ser Ala His Phe Gly Gin Gly Ser Gly Ser lie Val Leu Asp Asp Val 345 350 355 GCC TGT ACA GGA CAT GAG GCC TAT CTG TGG AGC TGC TCC CAC CGA GGC 1218 Ala Cys Thr Gly His Glu Ala Tyr Leu Trp Ser Cys Ser His Arg Gly 360 365 370 375 TGG CTC TCT CAT AAC TGT GGC CAC CAT GAG GAT GCT GGA GTC ATC TGT 1266 Trp Leu Ser His Asn Cys Gly His His Glu Asp Ala Gly Val lie Cys 380 385 390 TCA GAT GCC CAA ACC CAG AGC ACA ACC TGG CCA GAT ATG TGG CCT ACT 1314 Ser Asp Ala Gin Thr Gin Ser Thr Thr Trp Pro Asp Met Trp Pro Thr 395 400 405 ACC ACT CCA GAA ACT ACA ACA GAT TGG TGG ACT ACA AAA TAT TCT TCC 1362 Thr Thr Pro Glu Thr Thr Thr Asp Trp Trp Thr Thr Lys Tyr Ser Ser 410 415 420 TCT GTT CCT ACA ACA CAA TTC CCC ACC ATA GCC GAT TGG TGG ACA ACT 1410 Ser Val Pro Thr Thr Gin Phe Pro Thr lie Ala Asp Trp Trp Thr Thr 425 430 435 CCT TCT CCG GAA TAC ACC TGT GGA GGT TTA CTG ACC CTA CCC TAT GGG 1458 Pro Ser Pro Glu Tyr Thr Cys Gly Gly Leu Leu Thr Leu Pro Tyr Gly 440 445 450 455 CAG TTT TCC AGC CCA TAC TAC CCT GGA AGC TAT CCT AAC AAT GCC AGA 1506 Gin Phe Ser Ser Pro Tyr Tyr Pro Gly Ser Tyr Pro Asn Asn Ala Arg 460 465 470 TGT TTG TGG AAA ATT TTC GTC TCC AGC ATG AAC CGT GTG ACA GTG GTC 1554 Cys Leu Trp Lys lie Phe Val Ser Ser Met Asn Arg Val Thr Val Val 475 480 485 TTC ACA GAT GTG CAG CTT GAA GGA GGT TGC AAC TAT GAC TAC ATC CTG 1602 Phe Thr Asp Val Gin Leu Glu Gly Gly Cys Asn Tyr Asp Tyr lie Leu 490 495 500 GTT TTT GAT GGC CCT GAA AAC AAT TCT TCT CTC ATT GCT CGG GTT TGT 1650 Val Phe Asp Gly Pro Glu Asn Asn Ser Ser Leu lie Ala Arg Val Cys 505 510 515 GAT GGG TTC AAT GGA TCT TTC ACC TCA ACC CAG AAC TTC ATG TCT GTA 1698 Asp Gly Phe Asn Gly Ser Phe Thr Ser Thr Gin Asn Phe Met Ser Val 520 525 530 535 GTC TTT ATC ACG GAT GGC AGT GTC ACG AGG AGA GGG TTC CAA GCT GAC 1746 Val Phe lie Thr Asp Gly Ser Val Thr Arg Arg Gly Phe Gin Ala Asp 540 545 550 TAC TAC TCC ACT CCT ATC AGC ACC AGC ACA ACC TCT CCA ACG ACG TTC 1794 Tyr Tyr Ser Thr Pro lie Ser Thr Ser Thr Thr Ser Pro Thr Thr Phe 555 560 565 CCG ATC GTT ACT GAT TGG TGG ACA ACT CCT TCT CCG GAA TAC ACC TGT 1842 Pro lie Val Thr Asp Trp Trp Thr Thr Pro Ser Pro Glu Tyr Thr Cys 570 " 575 580 GGA GGT TTA CTG ACC CTA CCC TAT GGG CAG TTT TCC AGC CCA TAC TAC 1890 Gly Gly Leu Leu Thr Leu Pro Tyr Gly Gin Phe Ser Ser Pro Tyr Tyr 585 590 595 CCT GGA AGC TAT CCT AAC AAT GCC AGA TGT TTG TGG AAA ATT TTC GTC 1938 Pro Gly Ser Tyr Pro Asn Asn Ala Arg Cys Leu Trp Lys lie Phe Val 600 605 610 615 CCC AGC ATG AAC CGT GTG ACA GTG GTC TTC ACA GAT GTG CAG CTT GAA 1986 Pro Ser Met Asn Arg Val Thr Val Val Phe Thr Asp Val Gin Leu Glu 620 625 630 GGA GGT TGC AAC TAT GAC TAC ATC CTG GGT TTT GAT GGT CCT GAA TAC 2034 Gly Gly Cys Asn Tyr Asp Tyr lie Leu Gly Phe Asp Gly Pro Glu Tyr 635 640 645 AAT TCT TCT CTC ATT GCT CGG GTT TGT GAT GGG TCC AAT GGA TCT TTC 2082 Asn Ser Ser Leu lie Ala Arg Val Cys Asp Gly Ser Asn Gly Ser Phe 650 655 660 ACC TCA ACC CAG AAC TTC ATG TCT GTA GTC TTT ATC ACG GAT GGC AGT 2130 Thr Ser Thr Gin Asn Phe Met Ser Val Val Phe lie Thr Asp Gly Ser 665 670 675 GTC ACG AGG AGA GGG TTC CAA GCT GAC TAC TAC TCC ACT CCT ATC AGG 2178 Val Thr Arg Arg Gly Phe Gin Ala Asp Tyr Tyr Ser Thr Pro lie Arg 680 685 690 695 ACC AGC ACA ACT CCT CCA ACG ACG TTC CCG ATC ATT ACT GGA AAT GAT 2226 Thr Ser Thr Thr Pro Pro Thr Thr Phe Pro lie lie Thr Gly Asn Asp 700 705 710 TCT TCA TTG GTG CTG AGG CTG GTA AAT GGA ACA AAC CGG TGT GAG GGC 2274 Ser Ser Leu Val Leu Arg Leu Val Asn Gly Thr Asn Arg Cys Glu Gly 715 720 725 CGA GTG GAG ATC TTG TAC AGA GGC TCT TGG GTA CCG TGT GCC GAC GAC 2322 Arg Val Glu lie Leu Tyr Arg Gly Ser Trp Val Pro Cys Ala Asp Asp 730 735 740 AGC TGG GAC ATC AAT GAT GCC AAT GTG GTC TGC AGA CAG CTC GGT TGT 2370 Ser Trp Asp lie Asn Asp Ala Asn Val Val Cys Arg Gin Leu Gly Cys 745 750 755 GGC TCT GCT CTG TCT GCT CCA GGA AAT GCT TGG TTT GGT CAG GGT TCA 2418 Gly Ser Ala Leu Ser Ala Pro Gly Asn Ala Trp Phe Gly Gin Gly Ser 760 765 770 775 GGG CTC ATT GTC CTG GAT GAT GTG TCT TGC TCT GGG TAT GAG TCC CAC 2466 Gly Leu lie Val Leu Asp Asp Val Ser Cys Ser Gly Tyr Glu Ser His 780 785 790 CTG TGG AAT TGT CGT CAC CCT GGC TGG CTT GTT CAT AAT TGT CGT CAT 2514 Leu Trp Asn Cys Arg His Pro Gly Trp Leu Val His Asn Cys Arg His 795 800 805 GTT GAG GAT GCA GGA GTC ATT TGC TCA CTC CCT GAT CCG ACT CCC TCT 2562 Val Glu Asp Ala Gly Val lie Cys Ser Leu Pro Asp Pro Thr Pro Ser 810 815 820 CCT GGT CCA GTT TGG ACA AGT CCT CCT TTT GTA AAC TAT ACT TGT GGA 2610 Pro Gly Pro Val Trp Thr Ser Pro Pro Phe Val Asn Tyr Thr Cys Gly 825 830 835 GGT TTC CTG ACT GGA CTC TCT GGG CAA TTT TCT AGC CCA TAC TAC CCT 2658 Gly Phe Leu Thr Gly Leu Ser Gly Gin Phe Ser Ser Pro Tyr Tyr Pro 840 845 850 855 GGG AGC TAT CCT AAT AAT GCC AGA TGT TTG TGG AAC ATT GAA GTC CCA 2706 Gly Ser Tyr Pro Asn Asn Ala Arg Cys Leu Trp Asn lie Glu Val Pro 860 865 870 AAC AAC TAC CGC GTG ACT GTG GTC TTC AGA GAT GTG CAG CTG GAA GGG 2754 Asn Asn Tyr Arg Val Thr Val Val Phe Arg Asp Val Gin Leu Glu Gly 875 880 885 GGC TGC AAC TAT GAC TAT ATA GAG ATT TTT GAT GGC CCC CAC CAC AGT 2802 Gly Cys Asn Tyr Asp Tyr lie Glu lie Phe Asp Gly Pro His His Ser 890 895 900 TCA CCT CTC ATT GCC CGG GTT TGT GAT GGG GCC ATG GGC TCT TTC ACT 2850 Ser Pro Leu lie Ala Arg Val Cys Asp Gly Ala Met Gly Ser Phe Thr 905 910 915 TCA ACA TCC AAC TTC ATG TCA GTT CGC TTC ACC ACT GAT CAC AGT GTT 2898 Ser Thr Ser Asn Phe Met Ser Val Arg Phe Thr Thr Asp His Ser Val 920 925 930 935 ACT CGA AGA GGG TTC CGG GCT GAC TAC TAC TCA GAC TTT GAC AAT AAT 2946 Thr Arg Arg Gly Phe Arg Ala Asp Tyr Tyr Ser Asp Phe Asp Asn Asn 940 945 950 ACC ACC AAT CTC CTT TGT CTG TCA AAT CAC ATG AGA GCC AGT GTG AGC 2994 Thr Thr Asn Leu Leu Cys Leu Ser Asn His Met Arg Ala Ser Val Ser 955 960 965 AGG AGC TAC CTT CAG TCC ATG GGC TAC TCC TCC AGG GAT CTT GTC ATT 3042 Arg Ser Tyr Leu Gin Ser Met Gly Tyr Ser Ser Arg Asp Leu Val lie 970 975 980 CCT GGT TGG AAC GTG AGT TAC CAG TGT CAG CCT CAG ATA ACA CAA AGG 3090 Pro Gly Trp Asn Val Ser Tyr Gin Cys Gin Pro Gin lie Thr Gin Arg 985 990 995 GAG GTC ATA TTC ACA ATT CCC TAC ACA GGC TGC GGT ACT ACC AAA CAG 3138 Glu Val lie Phe Thr lie Pro Tyr Thr Gly Cys Gly Thr Thr Lys Gin 1000 1005 1010 1015 GCT GAC AAC GAG ACC ATC AAC TAC TCC AAC TTC CTC AAA GCG GCT GTT 3186 Ala Asp Asn Glu Thr lie Asn Tyr Ser Asn Phe Leu Lys Ala Ala Val 1020 1025 1030 TCA AAT GGC ATC ATC AAA AGG AGA AAG GAT CTC CAC ATC CAT GTC AGC 3234 Ser Asn Gly lie lie Lys Arg Arg Lys Asp Leu His lie His Val Ser 1035 1040 1045 TGC AAG ATG CTT CAG AAC ACC TGG GTC AAC ACC ATG TAC ATC ACC AAC 3282 Cys Lys Met Leu Gin Asn Thr Trp Val Asn Thr Met Tyr lie Thr Asn 1050 1055 1060 AAC ACA GTC GAG ATC CAG GAA GTC CAG TAT GGC AAT TTT GAC GTG AAT 3330 Asn Thr Val Glu lie Gin Glu Val Gin Tyr Gly Asn Phe Asp Val Asn 1065 1070 1075 ATT TCC TTT TAT ACA TCC TCC TCC TTC TTG TAT CCA GTG ACC AGC AGC 3378 lie Ser Phe Tyr Thr Ser Ser Ser Phe Leu Tyr Pro Val Thr Ser Ser 1080 1085 1090 1095 CCA TAT TAT GTG GAT CTG GAC CAG AAT TTG TAC CTT CAG GCC GAA GTC 3426 Pro Tyr Tyr Val Asp Leu Asp Gin Asn Leu Tyr Leu Gin Ala Glu Val 1100 1105 1110 CTC CAT TCG GAT ACC TCT TTG GCT CTG TTT GTG GAC ACC TGT GTG GCT 3474 Leu His Ser Asp Thr Ser Leu Ala Leu Phe Val Asp Thr Cys Val Ala 1115 1120 1125 TCG CCA CAT CCC AAT GAC TTC TCG TCT TTG ACA TAT GAT CTC ATC AGG 3522 Ser Pro His Pro Asn Asp Phe Ser Ser Leu Thr Tyr Asp Leu lie Arg 1130 1135 1140 AGT GGA TGC ATA CGA GAT GAA ACT TAC CAA TCT TAC TCC TCG CCC TCA 3570 Ser Gly Cys lie Arg Asp Glu Thr Tyr Gin Ser Tyr Ser Ser Pro Ser 1145 1150 1155 CCA CGC ATC ACC CGC TTT AAA TTC AGT TCT TTC CAC TTC CTG AAC CGC 3618 Pro Arg lie Thr Arg Phe Lys Phe Ser Ser Phe His Phe Leu Asn Arg 1160 1165 1170 1175 TTC CCC TCA GTA TAC CTA CAG TGT AAA CTG GTG GTT TGT CGA GCA AAC 3666 Phe Pro Ser Val Tyr Leu Gin Cys Lys Leu Val Val Cys Arg Ala Asn 1180 1185 1190 GAT GTC TCC TCA CGG TGC TAC AGA GGA TGT GTA GTA AGG TCC AAG AGG 3714 Asp Val Ser Ser Arg Cys Tyr Arg Gly Cys Val Val Arg Ser Lys Arg 1195 1200 1205 GAT GTA GGC TCC TAC CAA GAA AAG GTG GAT GTT GTT CTG GGA CCC ATC 3762 Asp Val Gly Ser Tyr Gin Glu Lys Val Asp Val Val Leu Gly Pro lie 1210 1215 1220 CAG TTG CAA TCT CCC AGC AAA GAA AAG AGG AGT CTC GAC TTG GCA GTG 3810 Gin Leu Gin Ser Pro Ser Lys Glu Lys Arg Ser Leu Asp Leu Ala Val 1225 1230 1235 GCA GAT GTG GAG AAG CCA GCC AGC TCC CAG GAG GTC TAT CCC ACT GCA 3858 Ala Asp Val Glu Lys Pro Ala Ser Ser Gin Glu Val Tyr Pro Thr Ala 1240 1245 1250 1255 GCC ATC TTT GGT GGA GTC TTC CTG GCC CTG GTT GTA GCT GTG GCA GCC 3906 Ala lie Phe Gly Gly Val Phe Leu Ala Leu Val Val Ala Val Ala Ala 1260 1265 1270 TTC ACA CTG GGA AGG AAG ACA CGC ACT GCC CGT GGT CAA CCT CCA AGT 3954 Phe Thr Leu Gly Arg Lys Thr Arg Thr Ala Arg Gly Gin Pro Pro Ser 1275 1280 1285 ACT AAG ATG TGAAGCAAAA CAACCCAGAC ATTGGTCCCA AATGCATAGA 4003 Thr Lys Met 1290 TTCCCAGAAA AGATGGAAGT CAGGAGTGTC TAATGCCTGG CACCCAGATA CACGATGACT 4063 AGGCTTCCCT TAGCACAAAT GTGTGGCCGA GTATGATCAG ATGGTAAAGA AGAAAGGTGG 4123 GGGCCAAGTT TTCCCAGGGT CTAGAGGCTG AAGGCTGGGA AGAATGTCAT AGGAGAATGA 4183 GATCAGTGTC TACAATAACA GGCAACTGTG AGCCAAACAT TGGCATCACC ATCCTTTCTC 4243 TAGCTAGAAT TTCCCTTTCC CCCTTTTATA CTGACTTTTT TGAACTGTAG TGTTAAATGG 4303 ACCTTTCCGT ACAACAAACT AAAATAAAGA ATCTTTTTCC AAAAAAAAAA AAAAAAA 4360 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1290 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Cys Asp Asp Ser Trp Asp lie Asn Asp Ala Asn Val Val Cys Arg 1 5 10 15 Gin Leu Gly Cys Gly Trp Ala Leu Ser Ala Pro Gly Ser Ala Gin Phe 20 25 30 Gly Gin Gly Leu Gly Pro lie Val Leu Asp Asp Val Ala Cys Arg Gly 35 40 45 His Glu Ala Tyr Leu Trp Ser Cys Ser His Arg Gly Trp Leu Ser His 50 55 60 Asn Cys Gly His Gin Glu Asp Ala Gly Val lie Cys Ser Asp Ser Gin 65 70 75 80 Thr Ser Ser Pro Thr Pro Gly Trp Trp Asn Pro Gly Gly Thr Asn Asn 85 90 95 Asp Val lie Tyr Asp Thr Gin Glu Thr Thr Glu Thr Ser Gin Thr Ser 100 105 110 Ser Pro Thr Pro Asp Trp Trp Asn His Gly Gly Thr lie Asn Asp Val 115 120 125 lie Tyr Asp Thr Gin Glu Thr Thr Glu Gly Thr Asp Ser Gly Leu Ala 130 135 140 Val Arg Leu Val Asn Gly Gly Asp Arg Cys Arg Gly Arg Val Glu lie 145 150 155 160 Leu Tyr Gin Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp lie 165 170 175 Asn Asp Ala Asn Val Val Cys Arg Gin Leu Gly Cys Gly Trp Ala Leu 180 185 190 Ser Ala Pro Gly Ser Ala Gin Phe Gly Gin Gly Ser Gly Ser lie Val 195 200 205 Leu Asp Asp Val Ala Cys Arg Gly His Glu Ala Tyr Leu Trp Ser Cys 210 215 220 Ser His Arg Gly Trp Leu Ser His Asn Cys Gly His Gin Glu Asp Ala 225 230 235 240 Gly Val lie Cys Ser Tyr Ser Gin Thr Ser Ser Pro Thr Pro Asp Ser 245 250 255 Gin Thr Ser Ser Pro Thr Pro Gly Trp Trp Asn Pro Gly Gly Thr Asn 260 265 270 Asn Asp Val Ser Tyr Gly Pre Glu Gin Thr Thr Asr> Ala Thr Asp Ser 275 280 * 285 Gly Leu Ala Val Arg Leu Val Asn Gly Gly Asp Arg Cys Gin Gly Arg 290 295 300 Val Glu lie Leu Tyr Gin Glv Ser Trp Gly Thr Val Cys Asp Asp Ser 305 310 " 315 320 Trp Asp Thr Lys Asp Ala Asn Val Val Cys Arg Gin Leu Val Cys Gly 325 330 335 Trp Ala Leu Ser Ala Pro Gly Ser Ala His Phe Gly Gin Gly Ser Gly 340 345 350 Ser lie Val Leu Asp Asp Val Ala Cys Thr Gly His Glu Ala Tyr Leu 355 360 365 Trp Ser Cys Ser His Arg Gly Trp Leu Ser His Asn Cys Gly His His 370 375 380 Glu Asp Ala Gly Val lie Cys Ser Asp Ala Gin Thr Gin Ser Thr Thr 385 390 395 400 Trp Pro Asp Met Trp Pro Thr Thr Thr Pro Glu Thr Thr Thr Asp Trp 405 410 415 Trp Thr Thr Lys Tyr Ser Ser Ser Val Pro Thr Thr Gin Phe Pro Thr 420 425 430 lie Ala Asp Trp Trp Thr Thr Pro Ser Pro Glu Tyr Thr Cys Gly Gly 435 440 445 Leu Leu Thr Leu Pro Tyr Gly Gin Phe Ser Ser Pro Tyr Tyr Pro Gly 450 455 460 Ser Tyr Pro Asn Asn Ala Arg Cys Leu Trp Lys lie Phe Val Ser Ser 465 470 475 480 Met Asn Arg Val Thr Val Val Phe Thr Asp Val Gin Leu Glu Gly Gly 485 490 495 Cys Asn Tyr Asp Tyr lie Leu Val Phe Asp Gly Pro Glu Asn Asn Ser 500 505 510 Ser Leu lie Ala Arg Val Cys Asp Gly Phe Asn Gly Ser Phe Thr Ser 515 520 525 Thr Gin Asn Phe Met Ser Val Val Phe He Thr Asp Gly Ser Val Thr 530 535 540 Arg Arg Gly Phe Gin Ala Asp Tyr Tyr Ser Thr Pro He Ser Thr Ser 545 550 555 560 Thr Thr Ser Pro Thr Thr Phe Pro He Val Thr Asp Trp Trp Thr Thr 565 570 575 Pro Ser Pro Glu Tyr Thr Cys Gly Gly Leu Leu Thr Leu Pro Tyr Gly 580 585 590 Gin Phe Ser Ser Pro Tyr Tyr Pro Gly Ser Tyr Pro Asn Asn Ala Arg 595 600 605 Cys Leu Trp Lys He Phe Val Pro Ser Met Asn Arg Val Thr Val Val 610 615 620 Phe Thr Asp Val Gin Leu Glu Gly Gly Cys Asn Tyr Asp Tvr He Leu 625 630 635 " 640 Gly Phe Asp Gly Pro Glu Tyr Asn Ser Ser Leu He Ala Arg Val Cys 645 650 655 Asp Gly Ser Asn Gly Ser Phe Thr Ser Thr Gin Asn Phe Met Ser Val 660 665 670 Val Phe He Thr Asp Gly Ser Val Thr Arg Arg Gly Phe Gin Ala Asp 675 680 685 Tyr Tyr Ser Thr Pro He Arg Thr Ser Thr Thr Pro Pro Thr Thr Phe 690 695 700 Pro He He Thr Gly Asn Asp Ser Ser Leu Val Leu Arg Leu Val Asn 705 710 715 720 Gly Thr Asn Arg Cys Glu Gly Arg Val Glu He Leu Tyr Arg Gly Ser 725 730 735 Trp Val Pro Cys Ala Asp Asp Ser Trp Asp He Asn Asp Ala Asn Val 740 745 750 Val Cys Arg Gin Leu Gly Cys Gly Ser Ala Leu Ser Ala Pro Gly Asn 755 760 765 Ala Trp Phe Gly Gin Gly Ser Gly Leu He Val Leu Asp Asp Val Ser 770 775 780 Cys Ser Gly Tyr Glu Ser His Leu Trp Asn Cys Arg His Pro Gly Trp 785 790 795 800 Leu Val His Asn Cys Arg His Val Glu Asp Ala Gly Val He Cys Ser 805 810 815 Leu Pro Asp Pro Thr Pro Ser Pro Gly Pro Val Trp Thr Ser Pro Pro 820 825 830 Phe Val Asn Tyr Thr Cys Gly Gly Phe Leu Thr Gly Leu Ser Gly Gin 835 840 845 Phe Ser Ser Pro Tyr Tyr Pro Gly Ser Tyr Pro Asn Asn Ala Arg Cys 850 855 860 Leu Trp Asn He Glu Val Pro Asn Asn Tyr Arg Val Thr Val Val Phe 865 870 875 880 Arg Asp Val Gin Leu Glu Gly Gly Cys Asn Tyr Asp Tyr He Glu He 885 890 895 Phe Asp Gly Pro His His Ser Ser Pro Leu He Ala Arg Val Cys Asp 900 905 910 Gly Ala Met Gly Ser Phe Thr Ser Thr Ser Asn Phe Met Ser Val Arg 915 920 925 Phe Thr Thr Asp His Ser Val Thr Arg Arg Gly Phe Arg Ala Asp Tyr 930 935 940 Tyr Ser Asp Phe Asp Asn Asn Thr Thr Asn Leu Leu Cys Leu Ser Asn 945 950 955 960 His Met Arg Ala Ser Val Ser Arg Ser Tyr Leu Gin Ser Met Gly Tyr 965 970 975 Ser Ser Arg Asp Leu Val He Pro Gly Trp Asn Val Ser Tyr Gin Cys 980 985 990 Gin Pro Gin He Thr Gin Arg Glu Val He Phe Thr He Pro Tyr Thr 995 1000 1005 Gly Cys Gly Thr Thr Lys Gin Ala Asp Asn Glu Thr He Asn Tyr Ser 1010 1015 1020 Asn Phe Leu Lys Ala Ala Val Ser Asn Gly He He Lys Arg Arg Lys 1025 1030 1035 1040 Asp Leu His He His Val Ser Cys Lys Met Leu Gin Asn Thr Trp Val 1045 1050 1055 Asn Thr Met Tyr He Thr Asn Asn Thr Val Glu He Gin Glu Val Gin 1060 1065 1070 Tyr Gly Asn Phe Asp Val Asn He Ser Phe Tyr Thr Ser Ser Ser Phe 1075 1080 1085 Leu Tyr Pro Val Thr Ser Ser Pro Tyr Tyr Val Asp Leu Asp Gin Asn 1090 1095 1100 Leu Tyr Leu Gin Ala Glu Val Leu His Ser Asp Thr Ser Leu Ala Leu 1105 1110 1115 1120 Phe Val Asp Thr Cys Val Ala Ser Pro His Pro Asn Asp Phe Ser Ser 1125 1130 1135 Leu Thr Tyr Asp Leu He Arg Ser Gly Cys He Arg Asp Glu Thr Tyr 1140 1145 1150 Gin Ser Tyr Ser Ser Pro Ser Pro Arg He Thr Arg Phe Lys Phe Ser 1155 1160 1165 Ser Phe His Phe Leu Asn Arg Phe Pro Ser Val Tyr Leu Gin Cys Lys 1170 1175 1180 Leu Val Val Cys Arg Ala Asn Asp Val Ser Ser Arg Cys Tyr Arg Gly 1185 1190 1195 1200 Cys Val Val Arg Ser Lys Arg Asp Val Gly Ser Tyr Gin Glu Lys Val 1205 1210 1215 Asp Val Val Leu Gly Pro He Gin Leu Gin Ser Pro Ser Lys Glu Lys 1220 1225 1230 Arg Ser Leu Asp Leu Ala Val Ala Asp Val Glu Lys Pro Ala Ser Ser 1235 1240 1245 Gin Glu Val Tyr Pro Thr Ala Ala He Phe Gly Gly Val Phe Leu Ala 1250 1255 1260 Leu Val Val Ala Val Ala Ala Phe Thr Leu Gly Arg Lys Thr Arg Thr 1265 1270 1275 1280 Ala Arg Gly Gin Pro Pro Ser Thr Lys Met 1285 1290 References: Amano, O., Yoshino, Y., Nishikawa, K. & Iseki, S. (1993) Cell Tissue Res. 273, 467-474. Aruffo, A., Melnick, M. B., Linsley, P. S., Seed, B. (1991) J. Exp. Med. 174, 949-52. Bleil, J.D. & Wassarman, P.M. (1980) Cell 20, 873-882. Canessa, C. M., Horisberger, J. D. & Rossier, B. C. (1993) Nature 361, 467-470. Elaroussi, M. A. & DeLuca, H. F. (1994) Biochim. Biophys. Acta. 1217, 1-8. Feng, P. & Liau, G. (1993) J. Biol. Chem. 268, 21453-21457 Field, R.B. & Hand, A.R. (1987) Am. J. Physiol. 253, G217-25. Friedman, j., Trahey, M. & Weissman, I. (1993) Proc. Natl. Acad. Sci. USA 90, 6815-6819. Fukagawa, M., Suzuki, N., Hogan, B.L. & Jones, C. M. ( 1994) Dev. Biol. 163, 175-83. Fukuoka, S., Freedman, S.D.& Scheele, G.A. (1991) Proc. Natl. Acad. Sci. USA 88, 2898-902. Gurkan, S. & Bradley, R.M. (1988) Chem. Senses 13, 655-61. Hamosh, M., Scow, R.O. (1973) J. Clin. Invest. 52, 88-95. Harlow, E. & Lane, D. (1988J Antibodies: a laboratory manual, pp. 313-315, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Heck, G. L., Mierson, S. & DeSimone, J. A. (1984) Science 223, 403-405. Hoops, T. C. & Rindler, M. J. (1991) J. Biol. Chem. 266, 4257-4236. Humphreys-beher, M.G., Macauley, S.P., Chegini, N., van Setten, G., Purushotham, K., Stewart, C, Wheeler, T.T. & Schultz, G.S. (1994) Endocrinology 134, 963-970. Hwang, P. M., Verma, A., Bredt, D.S. & Snyder, S.H. (1990) Proc. Natl. Acad. Sci. USA 87, 7395-9. Journet, A. & Tosi, M. (1986) Biochem. J. 240, 783-87. Kinloch, R. A., Roller, R. J., Fimiani, C. M., Wassarman, D. A. & Wassarman, P. M. (1992) Proc. Natl. Acad. Sci. USA 85, 6409-13. Kock, K., Blaker, M. & Schmale, H. (1992) Cell Tissue Res. 267, 313-20. Kodama, T., Freeman, M., Rohrer, L., Zabrecky, J., Matsudaira, P. & Krieger, M. (1990) Nature 343, 531-5. Koths, K., Taylor, E., Casipit, C. & Wang, A. (1993) J. Biol. Chem. 268, 14245-49 Lepage, T. Ghiglione, C. & Gache, C. (1992) Development 114, 147-163. Li, X.J., Blackshaw, S. & Snyder, S.H.(1994) Proc. Natl. Acad. Sci. USA 91, 1814-1818. Lopez-Casillas, F., Cheifetz, S. Doody, J., Andres, J.L., Lane, W.S. & Massague, J. (1991) Cell 67, 785-795. Mackinnon, CM., Carter, P.E., Smyth, S.J., Dunbar, B. & Fothergill. J.E. (1987) Eur. J. Biochem. 169, 547-53. Matsushima, M., Ichinose, M., Yahagi, N., Kakei, N., Tsukada, S., Miki, K., Kurokawa, K., Tashiro, K., Shiokawa, K., Shinomiya, K. et al. (1994) J. Biol. Chem. 269, 19976-82. Meier-Ruge, W., Iwangoff, P. & Bertoni-Freddari, C. (1994) Ann. New York Acad. Sci. 719, 230-7. Moren, A. Ichijo, H. & Miyazono, K. (1992) Biochem. Biophysic. Res. Comm. 189, 356-362. Nagata, A. (1992) Proc. Natl. Acad. Sci. USA 88, 4020-24. Pevsner, J., Trifiletti, R.R., Strittmatter, S.M. & Snyder, S.H. (1985) Proc. Natl. Acad. Sci. USA 82, 3050-54. Pennica, D. et al (1987) Science 236, 83-88. Pevsner, J., Reed, R.R., Feinstein, P.G. & Snyder, S.H. (1988a) Science 241, 336-339. Pevsner, J. Hwang, P.M., Sklar, P.B., Venable, J.C. & Snyder, S.H. (1988b) Proc. Natl. Acad. Sci. USA 85, 2383-87. Pevsner, J., Hou, V., Snowman, A.M. & Snyder, S.H. (1990) J. Biol. Chem. 265, 6118-6125. Rindler, M.J., Naik, S.S., Li, N., Hoops, T.C. & Peraldi, M.N. (1990) J. Biol. Chem. 265, 20784-9. Rohrer, L., Freeman, M., Kodama, T., Penman, M. & Krieger-M. (1990) Nature 343, 570-2. Schiffman, S. S., Lockhead, E. & Maes, F. W. (1983) Proc. Natl. Acad. Sci. USA 80, 6136-6140. Schmale, H., Holtgreve, Grez, H. & Christiansen, H., (1990) Nature 343, 366-9. Shimεll, M.J., Ferguson, E.L., Childs, S.R. & O'Connor, M.B. (1991) Cell 67, 469-481. Wang, X-F., Lin, H.Y. Ng-Eaton, E., Downward, J., Lodish, H.F. & Weinberg, R.A. (1991) Cell 67, 797-805. Wijngaard, P.L., Metzelaar, M.J., MacHugh, N.D., Morrison, W. I. & Clevers, H. C. (1992) J. Immunol. 149, 3273-7. Wozney, J.M., Rosen, V., Celeste, A.J., Mitsock, L.M., Whitters, M.J., Kriz, R.W., Hewick, R.M. & Wang, E.A. (1988) Science 242, 1528-1534.

Claims: CLAIMS 1. A substantially pure polypeptide having the sequence shown in SEQ ID NO:2. 2. A substantially pure polypeptide having a sequence corresponding to the sequence of SEQ ID NO:2. 3. The polypeptide of claim 2 wherein the sequence of said polypeptide is selected from the group consisting of: SEQ ID NO:2, muteins of SEQ ID NO:2, truncations of SEQ ID NO:2, and fusion proteins thereof. 4. The polypeptide of claim 3 wherein the polypeptide is soluble. 5. The polypeptide of claim 3 which is devoid of the transmembrane region and the cytoplasmic region. 6. The polypeptide of claim 3 which is devoid of the cytoplasmic region. 7. An isolated and purified DNA molecule comprising a sequence that encodes a polypeptide having a sequence corresponding to SEQ ID NO:2, the sequence of said polypeptide selected from the group consisting of SEQ ED NO:2, muteins of SEQ ID NO:2, truncations of SEQ ID NO:2, and fusion proteins of said polypeptide. 8. The DNA molecule of claim 7 which is free of other DNA molecules. 9. The DNA molecule of claim 7, wherein said sequence encoding said polypeptide is an intron-free DNA sequence. 10. An intron-free DNA molecule encoding the amino acid sequence of SEQ ID NO:2. 11. The DNA molecule according to claim 7, corresponding to SEQ K> NO: l. 12. A cell population transformed with the DNA molecule of claim 7. 35 13. A single stranded DNA molecule comprising at least 20 nucleotides, wherein said nucleotides consist of a subsequence found in SEQ ID NO: 1 or a DNA sequence complementary thereto. 14. A method for determining the presence of a polynucleotide encoding a polypeptide substantially homologous to a polypeptide according to SEQ ID NO:2, wherein said polypeptide is secreted from von Ebner's gland, comprising: incubating a sample suspected of containing said polynucleotide with a nucleotide probe having a sequence complementary to a single stranded DNA containing at least 20 contiguous nucleotides of a subsequence found in SEQ ID NO:l, under conditions where said probe will form hybrids with nucleic acids from the sample; and detecting nucleic acid hybrids. 15. A preparation of an antibody wherein said preparation is specifically reactive with a polypeptide according to SEQ ID NO: l, wherein said polypeptide is secreted from von Ebner's gland. 16. The preparation of claim 15 wherein said antibody is monoclonal. 17. A method for determining the presence of Ebnerin in a tissue comprising: incubating a tissue sample suspected of containing Ebnerin with an antibody which is specifically reactive with a polypeptide according to SEQ ID NO: l; detecting the formation of antibody-antigen complexes containing said antibody.

Inventors: LI XIAO-JIANG, SNYDER SOLOMON H, LI, XIAO-JIANG, SNYDER, SOLOMON, H.
Applicants: UNIV JOHNS HOPKINS MED, THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE
Application year: 1996
Main classification: C12N15
IPC codes: C07K14, C07K16, C12N15, C12P21, C12Q1, G01N33