PROTEIN M0T12 AND DIAGNOSIS OF INSULIN-DEPENDENT

DIABETES

Field of the Invention The present invention relates to the novel protein termed MOT12, nucleotide sequences encoding OT12, as well as various products and methods useful for the diagnosis and treatment of various M0T12 related diseases and conditions, such as insulin-dependent diabetes .

Background of the Invention The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to be prior art to the invention.

Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins, which enables regulation of the activity of mature proteins by altering their structure and function. The best characterized protein kinases in eukaryotes phosphorylate proteins on the alcohol moiety of serine, threonine and tyrosine residues. These kinases largely fall into two groups, those specific for phosphorylating serines and threonines, and those

specific for phosphorylating tyrosines.

The phosphorylation state of a given substrate is also regulated by a class of proteins responsible for removal of the phosphate group added to a given substrate by a protein kinase. The protein phosphatases can also be classified as being specific for either serine/threonine or tyrosine. The known enzymes can be divided into two groups - receptor and non-receptor type proteins. Most receptor-type protein tyrosine phosphatases (RPTPs) contain two conserved catalytic tyrosine phosphatase domains each of which encompasses a segment of 240 amino acid residues (Saito et al, Cell Growth and Diff. 2:59-65, 1991) . The RPTPs can be subclassified further based upon the amino acid sequence diversity of their extracellular domains (Saito, et al, supra; Krueger, et al, Proc. Natl. Acad. Sci. USA 89:7417-7421, 1992) .

Summary of the invention The present invention relates to MOT12 polypeptides, nucleic acids encoding such polypeptides, cells containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. The present invention is based upon the isolation and characterization of a new protein which we have designated MOT12. The polypeptides and nucleic acids may be produced using well known and standard synthesis techniques when given the sequences presented herein. MOT12 is a receptor-type protein tyrosine

phosphatase previously identified RPTP IA-2 (approximately 70% homology in the incracellular domain and less than 30% homology in the extracellular domain) (see Lan, M. et al, DNA and Cell. Bio. 13:505-513, 1994; Kambayashi, Y. et al, Biochem. J. 305:331-335, 1995; Lu, J. et al, BBRC 204 (2) :930-936, 1994; 094/03610 all of which are incorporated herein by reference in their entirety, including any drawings) . M0T12 has an unusual extracellular domain that is structurally related to a secretory protein of neuroendocrine cells, Resplδ

(Bloomquist, B. et al, J. Biol. Chem. 269 (12) :9113-9122, 1994) . Unlike most RPTPs it has but a single intracellular catalytic domain. It also has an unusually long juxtamembrane domain. The expression of M0T12 is highly restricted as it is found only in adult brain and pancreas in neurosecretory cell types. Furthermore, in situ hybridization studies have shown expression is further restricted in the brain to cells in the cerebriform plexus, habencular nucleus and a defined region of the hypocampus. This restricted expression pattern suggests a role for MOT12 in growth, differentiation and survival of neurons in the adult, possibly by regulation of a specific protein tyrosine kinase such as, for example Trk, which is the predominant receptor-type protein tyrosine kinase expressed in neurons.

Of particular interest is the expression of MOT12 in the pancreas. Insulin-dependent diabetes mellitus (IDDM) is a chronic autoimmune disease characterized by progressive destruction of insulin-

secreting islet cells of the pancreas. This destruction leads to an impairment of glucose metabolism. IDDM, which usually occurs in children and young adults, affects almost one million Americans. Without daily insulin injections, a person with this form of diabetes will quickly die. By the time diabetes becomes evident, 80-90% of the insulin-producing cells will have been destroyed. Early identification of patients that are likely to develop IDDM would allow treatment with, for example, immunosuppressive drugs before massive destruction of the islet cells has occurred and the need for added insulin has become acute.

Progression to IDDM can be determined before clinical onset of diabetes, and serological markers found at clinical onset are also detectable in the preclinical period. Autoantibodies reacting with islets on frozen sections of human pancreas are the most commonly used marker for diagnosis and prediction of IDDM (Bottazzo, et al Lancet ii :1279-1283 , 1974; Bonifacio, E. et al, Lancet 335:147-149, 1990) . The precise molecular targets of islet cell antibodies are still not known. Immunoprecipitation of detergent lysates of metabolically labeled islet cells with sera from IDDM patients resulted in the identification of a 64-kDa autoantigen (Baekkeskov, S. et al. Nature

298:167-169, 1982) . This antigen was subsequently identified as glutamic acid decarboxylase (GAD) (Baekkeskov, S., et al, Nature 347:151-156, 1990) . Anti-GAD antibodies are present in over 70% of newly diagnosed IDDM patients and have been detected up to 7

years before clinical onset of IDDM. However, anti-GAD autoantibodies are also present in nondiabetic patients with autoimmunity to other endocrine organs and thus this antigen cannot be used to specifically identify persons at risk for developing IDDM (Christie, M et al, Diabetes 43:1254-1259, 1994) .

Analysis of sera from IDDM patients showed that three major fragments (50, 40 and 37 kDa) from a trypsin-digested islet homogenates can be detected. Antibodies reacting with the 50 kDa fragment also react with GAD (Christie, M. et al, J. Exp. Med. 172:789-795, 1990) . One study of identical twins showed that antibodies to the 40 and 37 kDa fragments were both specific and sensitive to predicting IDDM (Christie, M. et al, Diabetes 41:782-787, 1992) . Furthermore, antibodies to these antigens identify rapid progressors to IDDM in the absence of anti-GAD antibodies (Christi, M. et al, Diabetes 43:1254-1259, 1994; Bonifacio, E. et al, Dibetologia 1995; Bingley, P. et al, Diabetes 43:1304-1310, 1994) . Recently the 40 kDa fragment was identified by two groups as the PTP-like molecule IA-2 (Payton, M. et al, J. Clin. Invest. 96:1506-1511, 1995; Passini, N. et al, Proc. Natl. Acad. Aci . USA 92:9412-9416, 1995, see also Arden S. et al. , J. Clin. Invest. 97 (2) :551-561, 1996, all of which are incorporated herein by reference in their entirety, including any drawings) .

Surprisingly, the MOT12 protein of the present invention appears to encompasses the 37 kDa fragment described above. The protein of present invention is

useful for developing a method for screening the sera of individuals at risk for developing IDDM for the presence of diagnostic autoantibodies .

Thus, in a first aspect the invention features an isolated, enriched, or purified nucleic acid encoding a M0T12 polypeptide. By "M0T12" it is meant an amino acid sequence substantially similar to the sequence show in Figure 1, or fragments thereof. A sequence that is substantially similar will preferably have at least 90% identity (more preferably at least 95% and most preferably 99-100%) to the sequence of Figure 1.

By "identity" is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.

By "isolated" in reference to nucleic acid is meant a polymer of 6 (preferably 21, more preferably 39, most preferably 75) or more nucleotides conjugated to each other, including DNA or RNA that is isolated from a natural source or that is synthesized. In certain embodiments of the invention longer nucleic acids are preferred, for example those of 300, 600, 900 or more nucleotides and/or those having at least 50%, 60%, 75%,

90%, 95% or 99% identity to the full length sequence shown in Figure 1. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term "isolated" indicates that a naturally occurring sequence has been removed from its normal cellular (i.e.. chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment . The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90 - 95% pure at least) of non-nucleotide material naturally associated with it and thus is meant to distinguished from isolated chromosomes. By the use of the term "enriched" in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2 - 5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an

increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.

It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term

"purified" in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation) ; instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml) . Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA) . The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure

individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10 ⁶ -fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.

By "a M0T12 polypeptide" is meant 30 (preferably 35, more preferably 40) or more contiguous amino acids set forth in the full length amino acid sequence of Figure 1, or a functional derivative thereof as described herein. In certain aspects, polypeptides of 100, 200, 300 or more are preferred. The M0T12 polypeptide can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained.

In preferred embodiments the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in the full length amino acid sequence of Figure 1, a functional derivative thereof, or encodes at least 30, 35, 40, 5, 100, 200, or 300 contiguous amino acids thereof; the M0T12 polypeptide comprises, consists essentially of, or consists of at least 30, 35 or 40 contiguous amino acids of a MOT12 polypeptide. The nucleic acid may be isolated from a natural source by cDNA cloning or

subtractive hybridization; the natural source may be mammalian (human) blood, semen, or tissue and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer. In preferred embodiments the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in the full length amino acid sequence of Figure 1, a functional derivative thereof, or encodes at least 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids thereof; the MOT12 polypeptide comprises, consists essentially of, or consists of at least 30, 35, or 40 contiguous amino acids of a M0T12 polypeptide. The nucleic acid may be isolated from a natural source by cDNA cloning or subtractive hybridization; the natural source may be mammalian (human) blood, semen, or tissue and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer. In yet other preferred embodiments the nucleic acid is a conserved or unique region, for example those useful for the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, and obtaining antibodies to polypeptide regions. Examples of amino acid sequences of the present invention include the following amino acid sequences (the isolated, purified or enriched nucleic acids encoding them are also within the scope of the present invention) . By "conserved nucleic acid regions", are meant

regions present on two or more nucleic acids encoding a M0T12 polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acid encoding M0T12 polypeptides are provided in Abe, et al . J. Biol. Chem.. 19:13361 (1992) (hereby incorporated by reference herein in its entirety, including any drawings) . Preferably, conserved regions differ by no more than 5 out of 20 nucleotides.

By "unique nucleic acid region" is meant a sequence present in a full length nucleic acid coding for a MOT12 polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide. Such regions preferably comprise 30 or 45 contiguous nucleotides present in the full length nucleic acid encoding a M0T12 polypeptide. In particular, a unique nucleic acid region is preferably of mammalian origin. The invention also features a nucleic acid probe for the detection of a M0T12 polypeptide or nucleic acid encoding a M0T12 polypeptide in a sample. The nucleic acid probe contains nucleic acid that will hybridize to a sequence set forth in Figure 1 or a functional derivative thereof.

In preferred embodiments the nucleic acid probe hybridizes to nucleic acid encoding at least 30, 50, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the full-length sequence set forth in Figure 1 or a functional derivative thereof.

Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.

Methods for using the probes include detecting the presence or amount of MOT12 RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to M0T12 RNA. The nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a MOT12 polypeptide may be used in the identification of the sequence of the nucleic acid detected (for example see, Nelson et al . , in Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego (Kricka, ed. , 1992) hereby incorporated by reference herein in its entirety, including any drawings) . Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe.

The invention also features recombinant nucleic acid, preferably in a cell or an organism. The recombinant nucleic acid may contain a sequence set forth in Figure 1 or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell. The recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence

complimentary to an RNA sequence encoding a M0T12 polypeptide and a transcriptional termination region functional in a cell.

In another aspect the invention features an isolated, enriched, or purified M0T12 polypeptide.

By "isolated" in reference to a polypeptide is meant a polymer of 30 or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. In certain aspects longer polypeptides are preferred, such as those with 50, 100, 300, 500, 750, or more contiguous amino acids set forth in Figure 1. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term "isolated" indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment . The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90 - 95% pure at least) of non-amino acid material naturally associated with it .

By the use of the term "enriched" in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2 - 5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential

reduction in the amount of other amino acids present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no amino acid from other sources. The other source amino acid may, for example, comprise amino acid encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to elevate the proportion of the desired nucleic acid. It is also advantageous for some purposes that an amino acid sequence be in purified form. The term "purified" in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation) ; instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e . g . , in terms of mg/ml) . Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly

contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.

In preferred embodiments the MOT12 polypeptide contains at least 30, 35, 40, 50, 100, 150, 200, 250, 300, or 350 contiguous amino acids of the full-length sequence set forth in Figure 1, or a functional derivative thereof.

In yet another aspect the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity to a MOT12 polypeptide. The antibody contains a sequence of amino acids that is able to specifically bind to MOT12 polypeptide. By "specific binding affinity" is meant that the antibody binds to MOT12 polypeptides with greater affinity than it binds to other polypeptides under specified conditions.

Antibodies having specific binding affinity to a MOT12 polypeptide may be used in methods for detecting the presence and/or amount of a MOT12 polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the MOT12 polypeptide. Diagnostic kits for performing such methods may be constructed to include a first container means containing the antibody and a second container means having a conjugate of a binding partner of the antibody and a label.

In another aspect the invention features a hybridoma which produces an antibody having specific

binding affinity to a M0T12 polypeptide. By "hybridoma" is meant an immortalized cell line which is capable of secreting an antibody, for example a M0T12 antibody. In preferred embodiments the M0T12 antibody comprises a sequence of amino acids that is able to specifically bind a M0T12 polypeptide.

In another aspect, the invention describes a polypeptide comprising a recombinant M0T12 polypeptide or a unique fragment thereof . By "unique fragment , " is meant an amino acid sequence present in a full-length MOT12 polypeptide that is not present in any other naturally occurring polypeptide. Preferably, such a sequence comprises at least 6 contiguous amino acids present in the full sequence. More preferably, such a sequence comprises 12 contiguous amino acids present in the full sequence. Even more preferably, such a sequence comprises 18 contiguous amino acids present in the full sequence.

By "recombinant MOT12 polypeptide" is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g.. present in a different cell or tissue than found in nature) , purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature. In another aspect, the invention describes a recombinant cell or tissue containing a purified nucleic acid coding for a MOT12 polypeptide. In such cells, the nucleic acid may be under the control of its genomic

regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter. By "exogenous" it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the M0T12 polypeptide.

In another aspect, the invention features a M0T12 polypeptide binding agent able to bind to a M0T12 polypeptide. The binding agent is preferably a purified antibody which recognizes an epitope present on a M0T12 polypeptide. Other binding agents include molecules which bind to the MOT12 polypeptide and analogous molecules which bind to a MOT12 polypeptide. Such binding agents may be identified by using assays that measure MOT12 binding partner activity. By "purified" in reference to an antibody is meant that the antibody is distinct from naturally occurring antibody, such as in a purified form. Preferably, the antibody is provided as a homogeneous preparation by standard techniques. Uses of antibodies to the cloned polypeptide include those to be used as therapeutics, or as diagnostic tools.

The invention features a method for screening for human cells containing a MOT12 polypeptide or an equivalent sequence. The method involves identifying the novel polypeptide in human cells using techniques that are routine and standard in the art, such as those described herein for identifying MOT12 (e.g.. cloning, Southern or Northern blot analysis, __ situ hybridization, PCR amplification, etc . ) . The invention also features methods of

screening human cells for binding partners of M0T12 polypeptides and screening other organisms for M0T12 or the corresponding binding partner. The present invention also features the purified, isolated or enriched versions of the peptides identified by the methods described above .

Another aspect of the invention features an isolated, enriched, or purified nucleic acid molecule comprising a nucleotide sequence that: (a) encodes a polypeptide having the full length amino acid sequence set forth Fig. 1; (b) the complement of the nucleotide sequence of (a) ; (c) hybridizes under highly stringent conditions to the nucleotide molecule of (a) and encodes a naturally occurring M0T12 protein; (d) a MOT12 polypeptide having the full length amino acid sequence of sequence set forth in Fig. 1 except that it lacks one or more of the following segments of amino acid residues 1-614, 615-706, 707-1082; (e) the complement of the nucleotide sequence of (d) ; (f) a polypeptide having the amino acid sequence set forth in Fig 1 from amino acid residues 1-614, 615-706, 707-1082; (g) the complement of the nucleotide sequence of (f) ; or (h) encodes a polypeptide having the full length amino acid sequence set forth in Fig 1 except that it lacks one or more of the regions selected from the group consisting of an extracellular region, a transmembrane region, and an intracellular region, or (i) the complement of the nucleotide sequence of (h) .

The term "extracellular region" refers to a portion of the full length M0T12 amino acid sequence

that exists outside of the cell. This region often binds a ligand which activates the catalytic activity of the phosphatase.

The term "transmembrane region" refers to a portion of the M0T12 amino acid molecule that spans the plasma membrane of cells. The transmembrane region exists between the extracellular region and the intracellular region.

The term "intracellular region" refers to a portion of M0T12 that exists withing the cell. This region contains one or more catalytic regions in receptor protein phosphatases.

Functional regions of M0T12 may be identified by aligning the amino acid sequence of MOT12 with amino acid sequences of other polypeptides with known functional regions. If regions of MOT12 share high amino acid identity with the amino acid sequences of known functional regions, then M0T12 can be determined to contain these functional regions by those skilled in the art. The functional regions can be determined, for example, by using computer programs and sequence information available to those skilled in the art.

Other functional regions of signal transduction molecules that may exist in the MOT12 amino acid sequence include, but are not limited to, proline- rich regions or phosphoryl tyrosine regions . These regions can interact with natural binding partners such as SH2 or SH3 domains of other signal transduction molecules . Another aspect of the invention includes a

nucleic acid vector containing a nucleic acid molecule described in the last three aspects of the invention.

Another aspect of the invention relates to a recombinant cell or tissue that contains a nucleic acid molecule described in the last three aspects of the invention.

In another aspect, the invention provides an assay to identify agents capable of interfering with the interaction between MOT12 and a MOT12 binding partner. Such assays may be performed in vitro or in vivo and are described in detail herein or can be obtained by modifying existing assays, such as the growth assay described in Serial No. 08/487,088, filed June 7, 1995, (incorporated herein by reference including any drawings) or the assays described in Serial No.

60/005,167, filed October 13, 1995 (incorporated herein by reference including any drawings) . Another assay which could be modified to use the genes of the present invention are described in International Application No. WO 94/23039, published October 13, 1994. Other possibilities include detecting kinase activity in an autophosphorylation assay or testing for kinase activity on standard substrates such as histones, myelin basic protein, gamma tubulin, or centrosomal proteins. Binding partners may be identified by putting the N- terminal portion of the protein into a two-hybrid screen or detecting phosphotyrosine of a dual specificity kinase. Fields and Song, U.S. Patent No. 5,283,173, issued February 1, 1994 and is incorporated by reference herein.

Hence, the invention relates to a method of identifying compounds that modulate the function of a M0T12 polypeptide. The method comprises the following steps: (a) expressing a M0T12 polypeptide in cells; (b) adding a compound to the cells; and (c) monitoring a change or an absence of a change in a cellular event . The event is selected from the group consisting of insulin receptor function, glucose uptake, cell phenotype, cell proliferation, catalytic activity of the M0T12 polypeptide, or the interaction between a M0T12 polypeptide and a natural binding partner.

The term "compound" as used herein preferably refers to non-peptide molecules. The term "compound" can also refer to peptidomimetic molecules as well as peptide molecules.

The term "function" as used herein refers to the cellular role of a non-receptor protein phosphatase. The protein phosphatase family includes members that regulate many steps in signaling cascades, including cascades controlling cell growth, migration, differentiation, gene expression, muscle contraction, glucose metabolism, cellular protein synthesis, and regulation of the cell cycle.

The term "insulin receptor function" refers to the ability of the insulin receptor to bind insulin, regulate glucose uptake by a cell, autophosphorylate, and bind natural binding partners. These functions are readily measured by those skilled in the art. A method of identifying compounds that modulate the function insulin receptor is reported herein by example with

respect to the insulin receptor. This method monitors autophosphorylation of the insulin receptor.

The term "modulates" refers to the ability of a compound to alter the function of a protein phosphatase. A modulator preferably activates the catalytic activity of a protein phosphatase, more preferably activates or inhibits the catalytic activity of a protein phosphatase depending on the concentration of the compound exposed to the protein phosphatase, or most preferably inhibits the catalytic activity of a protein phosphatase.

The term "catalytic activity", in the context of the invention, defines the rate at which a protein phosphatase dephosphorylates a substrate. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a product as a function of time. Dehosphorylation of a substrate occurs at the active-site of a protein phosphatase. The active-site is normally a cavity in which the substrate binds to the protein phosphatase and is dephosphorylated.

The term "substrate" as used herein refers to a molecule dephosphorylated by a protein phosphatase. The substrate is preferably a peptide and more preferably a small organic molecule. A preferred substrate is p-nitorphenyl phosphate which is converted to p-nitrophenol upon reaction with the phosphatase of the invention.

The term "activates" refers to increasing the cellular function of a protein phosphatase. The protein

phosphatase function is preferably the interaction with a natural binding partner or catalytic activity.

The term "inhibit" refers to decreasing the cellular function of a protein phosphatase. The protein phosphatase function is preferably the interaction with a natural binding partner or catalytic activity.

The term "modulates" also refers to altering the function of a protein phosphatase by increasing or decreasing the probability that a complex forms between a protein phosphatase and a natural binding partner. A modulator preferably increases the probability that such a complex forms between the protein phosphatase and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the protein phosphatase and the natural binding partner depending on the concentration of the compound exposed to the protein phosphatase, and most preferably decreases the probability that a complex forms between the protein phosphatase and the natural binding partner. The term "complex" refers to an assembly of at least two molecules bound to one another. Signal transduction complexes often contain at least two protein molecules bound to one another.

The term "expressing" as used herein refers to the production of a M0T12 polypeptide from a nucleic acid vector containing a M0T12 gene within a cell. The nucleic acid vector is transfected into cells using well known techniques in the art as described herein.

The term "adding" as used herein refers to administering a solution comprising a compound to the

medium bathing cells. The solution comprising the compound can also comprise an agent, such as dimethyl sulfoxide, which facilitates the uptake of the compound into the cells. The term "monitoring" refers to observing the effect of adding the compound to the cells of the method. The effect can be manifested in a change in cell phenotype, cell proliferation, protein phosphatase catalytic activity, or in the interaction between a protein phosphatase and a natural binding partner. The term "glucose uptake" as used herein refers to the rate at which cells absorb glucose in the medium bathing the cells. The rate of glucose uptake by cells is known to those skilled in the art. These methods include detecting the time-dependent concentration of glucose in the medium bathing the cells. Glucose concentrations, for example, can be determined enzymatically by techniques that are commercially available and well known to those skilled in the art.

The term "cell phenotype" refers to the outward appearance of a cell or tissue or the function of the cell or tissue. Examples of cell or tissue phenotype are cell size (reduction or enlargement) , cell proliferation (increased or decreased numbers of cells) , cell differentiation (a change or absence of a change in cell shape) , cell survival, apoptosis (cell death) , or the utilization of a metabolic nutrient (e.g., glucose uptake) . Changes or the absence of changes in cell phenotype are readily measured by techniques known in

the art .

The term "cell proliferation" refers to the rate at which a group of cells divides. The number of cells growing in a vessel can be quantitated by a person skilled in the art when that person visually counts the number of cells in a defined area using a common light microscope. Alternatively, cell proliferation rates can be quantitated by laboratory apparatae that optically measure the density of cells in an appropriate medium. The method can utilize any of the molecules disclosed in the invention. These molecules include nucleic acid molecules encoding MOT12 polypeptides, nucleic acid vectors, recombinant cells, polypeptides, or antibodies of the invention. A preferred embodiment is that the MOT12 polypeptide refered to in the method of identifying compounds that modulate MOT12 function comprises the full-length amino acid sequence set forth in Fig. 1. In another aspect, the invention features a method of diagnosing an abnormal cell proliferative condition in an organism. The method relates to abnormal cell proliferative conditions associated with an aberration in a signal transduction pathway, where the pathway is characterized by an interaction between a M0T12 polypeptide and a natural binding partner. The method comprises the step of detecting the abnormal interaction.

The term "abnormal cell proliferative condition" refers to a function in an organism's cells or tissue that deviate from a normal function in the

cells or tissue of that organism. Cell proliferative disorders include cancers, fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, slow wound healing rates, psoriasis, and inflammation. The abnormal condition can be diagnosed when the organism' s cells exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, and injection applications. For cells external to the patient, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques, and carrier techniques.

The term "signal transduction pathway" refers to the molecules that propagate an extracellular signal through the cell membrane to become an intracellular signal. This signal can stimulate a cellular response. The polypeptide molecules involved in signal transduction processes are typically receptor and non- receptor protein kinases, receptor and non-receptor protein phosphatases, and transcription factors. The term "aberration" , in conjunction with a signal transduction process, refers to a MOT12 polypeptide that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type MOT12, mutated such that it can no longer interact with a binding partner, is no

longer modified by another protein kinase or protein phosphatase, or no longer interacts with a natural binding partner.

The term "interaction" defines the complex formed between a M0T12 polypeptide and a natural binding partner. Compounds can bind to either the M0T12 polypeptide or the natural binding partner and disrupt the interaction between the two molecules.

Methods of detecting the ability of a compound to disrupt or enhance an interaction between MOT12 and a natural binding partner exist in the ar . These methods include, but are not limited to, determining the effect of the compound upon the catalytic activity of a MOT12 polypeptide, the phosphorylation state of the MOT12 polypeptide or a natural binding partner, the ability of MOT12 to bind a natural binding partner, or a difference in a cell morphology. Differences in cell morphology include growth rates and differentiation rates of cells . These phenomena are simply measured by methods in the art. These methods typically involve observing the number of cells or the appearance of cells under a microscope with respect to time (days) .

The method can be performed in vi tro as well as in vivo. In vivo applications include introducing a group of cells to an organism and then determining the effect of a compound administered to the organism on the state of the organism as well as the introduced cells. The art contains multiple methods of introducing a group of cells to an organism as well as methods of administering a compounds to an organism. The organism

is preferably an animal such as a frog, mouse, rat, rabbit, monkey, or ape, and also a human.

The term "detecting an abnormal interaction" defines a method of identifying a M0T12 molecule with an aberration in its activity. Detection is accomplished by using an antibody or antibody fragment of the invention, a nucleic acid probe of the invention, or a compound identified by the invention.

Techniques used in the art that incorporate this method include in vi tro, in vivo, and in si tu hybridization techniques. These techniques utilize nucleic acid probes of the invention.

A preferred embodiment of the invention is that the diagnosis method relates to an organism that is a mammal.

Another preferred embodiment of the invention is that the diagnosis method relates to abnormal conditions selected from the group consisting of cancer, diabetes, and immune disorder. The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims .

Brief Description of the Drawings

Figure 1 shows the full length nucleic acid and amino acid sequences of M0T12.

Figure 2 shows the full length sequence of

M0T12 aligned with the IA-2 protein sequence.

Description of the Preferred Embodiments

The present invention relates to M0T12 polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing.

The human M0T12 gene encodes proteins that are potential drug targets for treating patients afflicted with diabetes, specifically non-insulin-dependent diabetes mellitus (NIDDM) . NIDDM occurs in patients who retain some endogenous insulin secretory capacity, however the great majority of them are both insulin deficient and insulin resistant. As a result, many patients afflicted with NIDDM are resistant to the primary mode of therapy, intravenous insulin administration. In addition, approximately 10-20% of patients become resistant to a secondary treatment of diabetes, oral administration of hypoglycemic agents such as sulfonylureas. Because insulin resistance can be caused by an abnormality in the insulin signalling pathway (Olefsky, 1988, in Cecil Textbook of Medicine , 18th Ed., 2: 1360-1381) , treatments that enhance the activity of a PTP that activates IR or decrease the activity of a PTP that inactivates IR would likely alleviate the symptoms of NIDDM.

Hence, MOT12 provides a target for identifying therapeutics for NIDDM. If MOT12 or PTPs structurally related to it modulate IR function, then compounds that inhibit or activate the activity of MOT12 or its

homologs would serve as potential therapeutics for NIDDM. Even if MOT12 does not regulate IR function, it is still likely that comounds which inhibit or activate this PTP or its homologs will modulate the activity of PTPs that regulate IR function. Because under-active IR causes NIDDM, compounds that specifically modulate the function of M0T12 or related phosphatases are novel potential therapeutics for patients afflicted with NIDDM. Compounds that modulate the function of MOT12 and related PTPs are also potentially useful therapeutics for treating or preventing cancers and immune disorders. For example, ectopic expression of RPTP produces a trasformed phenotype in embryonic fibroblasts. Zheng et al. , Nature 359 : 336-339. In addition, the gene for human RPTPy has been localized to chromosome 3p21 which is a segment frequently altered in renal and small lung carcinoma. Furthermore, mutations in the gene encoding PTP1C (also known as HCP or SHP) are the cause of the othea ten phenotype in mice, which suffer severe immunodeficiency and systemic autoimmune disease accompanied by hyperproliferation of macrophages. Schultz et al., 1993, Cell 13 : 1445-1454.

Methods of Detecting Patients at Risk for IDDM

Methods for detecting antibodies in patients sera are well known in the art. One method is an

Enzyme-linked Immunosorbent Assay or ELISA. Briefly, an antigen such as MOT12 is bound to a solid surface, such as for example the plastic of a 96-well microtitre plate. Blood is taken from a test patient and

centrifuged to remove cells and other particulate matter. The remaining serum is diluted in a buffer such as phosphate buffered saline and applied to the wells containing the antigen. After a time sufficient for the antibodies to bind to the antigen, the serum is removed and the wells briefly washed. Bound antibody is detected using a detecting antibody tagged with an enzyme such as horseradish peroxidase, which detecting antibody recognizes the constant portion of the antigen bound antibody. In the final step, a colourmetric substrate for the enzyme is added and the amount of colour produced detected by eye or by spectrophotometer. The antigen used can be purified or semipurified or expressed on the surface of whole cells. Details for performing these types and other relevant assays are found in, for example, Current Protocols in Immunology, Volume 1, Chapter 2, 1994, ed. Coligan, J.E. et al. A modified ELISA protocol used to detect GAD autoantibodies is described in Mehta, H. et al. , Clin. Chem. 41 (2) :263-269, 1996. Another technique is radioassay, as described in Gianans, R. et al. Diabetes, 44 (11) :1340-1344, (1995) , incorporated herein by reference in its entirety including any drawings.

Various other features and aspects of the invention are: nucleic acid molecules encoding a MOT12 polypeptide; nucleic acid probes for the detection of MOT12; a probe-based method and kit for detecting MOT12 messages or related PTPs in organisms; DNA constructs comprising a MOT12 nucleic acid molecule and cells containing these constructs; purified MOT12

polypeptides; M0T12 antibodies and hybridomas; antibody- based methods and kits for detecting M0T12; identification of agents; isolation of compounds which interact with a M0T12 polypeptide; compositions of compounds that interact with M0T12 and M0T12 molecules; pharmaceutical formulations and modes of administration; derivatives of complexes; antibodies to complexes; disruption of M0T12 protein complexes; purification and production of complexes; transgenic animals containing MOT12 nucleic acid constructs; antisense and ribozyme approaches, gene therapy; and evaluation of disorders. One skilled in the art appreciates that any modifications made to a complex can be manifested in a modification of any of the molecules in that complex. Thus, the invention includes any modifications to nucleic acid molecules, polypeptides, antibodies, or compounds in a complex. All of these aspects and features are explained in detail with respect to PYK-2 in PCT publication WO 96/18738, which is incorporated herein by reference in its entirety, including any drawings. Disclosure for the assay is set forth in 08/488,156 (filed 06/07/95), incorporated herein by reference in its entirety, including any drawings. Those skilled in the art will readily appreciate that such descriptions can be easily adapted to M0T12 polypeptides and nucleic acid molecules as well, and is therefore equally applicable to the present invention.

EXAMPLES

The examples below are non-limiting and are

merely representative of various aspects and features of the present invention. The examples below demonstrate the isolation, and characterization of the novel protein M0T12.

MATERIALS AND METHODS

CELL LINES and CULTURE CONDITIONS

All cell lines were obtained from the American Type Culture Collection (ATCC) and were grown according to ATCC recommendations.

MOLECULAR CLONING

Total RNAs were isolated using the Guanidine Salts/Phenol extraction protocol of Chomczynski and Sacchi (P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987) from primary neonatal rat sympathetic, motor, or sensory neuronal cells. These RNAs were used as templates to generate single-stranded cDNAs using the Superscript Preamplification System for First Strand Synthesis kit purchased from GibcoBRL (Life Technologies, U.S.A.; Gerard, GF et al. (1989) , FOCUS 11, 66) under conditions recommended by manufacturer. A typical reaction used 10 ug total RNA or 2 ug poly(A) + RNA with 1.5 ug oligo (dT) 12-18 in a reaction volume of 60 ul . The product was treated with RNaseH and diluted to 100 ul with H20. For subsequent PCR amplification, 1-4 ul of these sscDNAs were used in each reaction. Oligonucleotides were synthesized on an Applied Biosystems 394 DNA synthesizer using established

phosphoramidite chemistry and were used unpurified after precipitation with ethanol. The degenerate oligonucleotide primers are: A GAYTTYTGGVRNATGRTNTGGGA (sense) and B CGGCCSAYNCCNGCNSWRCARTG (antisense)

These primers were derived from the peptide sequences DFW$M(I/V/M)W(D/E) (sense strand catalytic domain) where $=SKNRQHREDGC and HC(R/S/Y/F)AG(V/I/M)GR (antisense strand catalytic domain) , respectively. Degenerate nucleotide residue designations are: N = A, C, G, or T; R = A or G; and Y = C or T. These primers produce a product of approximately 350 bp on all known tyrosine phosphatases .

A PCR reaction was performed using Primers A and B applied to the single-stranded primary rat neuronal cDNAs listed above. The primers were added at a final concentration of 5 uM each to a mixture containing 10 mM Tris.HCl (pH8.3), 50 mM KCl, 1.5 mM MgC12, 200 uM each deoxynucleoside triphosphate, 0.001% gelatin, and 1.5 U AmpliTaq DNA Polymerase (Perkin- Elmer/Cetus) , and 1-4 ul cDNA. Following 3 min denaturation at 95°C, the cycling conditions were 94°C for 30 s, 37°C for 1 min, a 2 min ramp to 72°C, and 72°C for 1 min for the first 3 cycles, followed by 9 °C for 30 s, 50°C for 1 min, and 72°C for 1 min 45 s for 35 cycles. PCR fragments migrating at ~350bp were isolated from 2% agarose gels using GeneClean, and cloned into the pBluescript vector. Colonies were selected for mini plasmid DNA-preparations using Qiagen columns and the plasmid DNAs were sequenced using cycle sequencing dye-

terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City, CA) . Sequencing reaction products were run on an ABI Prism 377 DNA Sequencer, and analyzed using the BLAST alignment algorithm (Altschul, S.F. et al . , J. Mol. Biol .215 :403-10) . Multiple copies of a novel clone (M0T12) were isolated by PCR with primers A and B on single-stranded cDNA from rat primary motor neurons as a template. This clone was subsequently designated as rM0T12. An adult human brain (caudate nucleus) cDNA library in lambda gtlO (Clontech, Palo Alto, CA) was identified by PCR to contain MOT12. Phage were screened on nitrocellulose filters with the random primed 32P- labeled insert from the human MOT12 clone. This probe was used at 2x106 cpm/ml in hybridization buffer containing 6xSSC, lx Denhardt ' s reagent, 0.1% SDS, with 0. lmg/ml denatured, fragmented salmon sperm DNA. After overnight hybridization at 65°C, filters were washed at medium stringency in lxSSC, 0.1%SDS at 42°C. Full length cDNA clones were sequenced on both strands using manual sequencing with T7 polymerase and oligonucleotide primers (Tabor and Richardson, 1987, Proc. Natl. Acad. Sci. U.S.A. 84: 4767-71) . Additional clones were isolated by PCR and by screening a human pancreatic cDNA library.

NORTHERN BLOT ANALYSIS

Northern blots containing 2 ug poly A+RNA per lane from eight different adult human tissues (spleen, thymus, prostate, testis, ovary, small intestine,

colonic mucosa, and peripheral blood leukocytes) and four different human fetal tissues (brain, lung, liver, and kidney) on a charge-modified nylon membrane were obtained from Clontech (Palo Alto, CA) . Filters were hybridized with random prime [a32P] dCTP-labeled probes synthesized from the 350 bp insert from human M0T12 clone. Hybridization was performed at 60°C overnight in 6XSSC, 0.1% SDS, IX Denhardt' s solution, 100 mg/ml denatured herring sperm DNA with 1-2 x 106 cpm/ml of 32P-labeled DNA probes. The filters were washed in

O.lXSSC/0.1% SDS, 65°C, and exposed overnight on Kodak XAR-2 film.

SEMI-QUANTITATIVE PCR DETECTION OF M0T12

RNA was isolated from a variety of human cell lines, fresh frozen tissues, and primary tumors. Single stranded cDNA was synthesized from 10 mg of each RNA as described above using the Superscript Preamplification System (GibcoBRL) . These single strand templates were then used in a 35 cycle PCR reaction with M0T12-specific oligonucleotides. Reaction products were electrophoresed on 2% agarose gels, stained with ethidium bromide and photographed on a UV light box. The relative intensity of the MOT12-specific bands were estimated for each sample.

RESULTS

SEQUENCE ANALYSIS OF cDNA CLONES ENCODING HUMAN MOT12

We designed degenerate primers A and B based on conserved residues within the catalytic domain of all

known protein tyrosine phosphatases (PTPs) , to use for identification of novel PTPs using polymerase chain reaction (PCR) . When applied to primary rat motor neuron sscDNA as a template, multiple copies of a novel PTP termed M0T12 were isolated. The novel sequence was most similar to IA2/PTP (GeneBank Accesion #L18983) and the clone was designated rat M0T12. Using this PCR strategy, M0T12 was also found to be abundant in other purified primary neuronal populations, comprising -15% of all PTPs in the motor neuron population. The rat MOT12 probe was also used to screen a cDNA library constructed from human caudate nucleus and pancreas mRNA to isolate overlapping clones spanning the complete open reading frame of human MOT12. The complete sequence of human MOT12 was determined from full length clones isolated from the human caudate nucleus library and of the partial human MOT12 isolated from human pancreas.

The 4,136 bp human MOT12 (MOT12_h) nucleotide sequence is shown in FIG. 1 and contains a single open reading frame encoding a polypeptide of 1015 amino acids. The MOT12-h coding region is flanked by a 41 nucleotide 5 ' -untranslated region and a 1048 nucleotide 3 ' -untranslated region ending with a poly(A) tail. Only two ambiguities were seen between all clones, one G to A substitution at nucleotide 1180 in the clone isolated from human pancreas resulting in a Gly to Glu amino acid change, and a silent C to T change at nucleotide 2375 of one of the caudate nucleus clones.

MOT12 has a single predicted hydrophobic transmembrane domain dividing the protein into a 614

amino acid extracelluar domain and a 376 amino acid cytoplasmic domain. The intracellular portion contains a single predicted catalytic domain displaying significant homology to the catalytic domain of other tyrosine phosphatases . Unlike most tyrosine phosphatases, M0T12 has an atypically long juxtame brane region of 92 amino acids. A search of the public domain databases reveals MOT12 is novel, with highest homology to IA2/PTP, a PTP originally isolated from a human insulinoma, and subsequently found to be a major early autoantigen for insulin-dependent diabetes mellitus (IDDM) . IA2/PTP has an overall structure quite similar to MOT12 with a 575 amino acid extracellular domain sharing 22% sequence identity to MOT12, a single transmembrane domain, and a 378 amino acid cytoplasmic domain with 71% identity to MOT12. IA2/PTP also has an extended cytoplasmic juxtamembrane region of 96 amino acids. Overall these features place MOT12 and IA2/PTP in a distinct family of receptor PTPs. The nucleotide sequence of the extracellular domain is GC-rich (63%) and translates into a protein sequence rich in aliphatic (51 G, 58 A, 34 V, 72 L, 5 I) and based 40 D, 58 E) amino acids and is rich if Pro (51) and Ser{50) . Limited (-30% identity) internal repeats are present in two regions spanning -50-100 amino acids each. The region surrounding the N-terminal signal sequence has high identity (32%) to RESP18 (PIR A53523) , an endocrine secretory protein that is produced in light concentrations in neuroendocrine cells. This homology suggests MOT12 might also utilize an

alternative secretory pathway, similar to that found used for secretion of neuroendocrine hormones.

Several features in the catalytic domains of IA2/PTP and M0T12 distinguish them from other PTPs. The highly conserved VHCSAG in the predicted phosphotyrosine binding pocket of all PTPs, is changed to VHCSDG in these two PTPs . This cysteine residue has been shown to be absolutely required for activity and is conserved in M0T12. Within this motif is an Ala to Asp change in M0T12 and IA2/PTP compared with most other PTPs. The crystal structure of human and bovine PTP1B and a yerninia PTP, implicates this Ala to Asp change to involve one of the nonpolar residues that makes contact with the phosphotyrosine. The other contact residues are A217, 1219, F182, and Q262 in PTP1B. The corresponding residues of M0T12 are D947, A949, R914, and Q990. Two of these residues place charged residues at predicted phosphotyrosine contact sites (D947 and R914) of M0T12. The predicted binding pocket of M0T12 also has some unique features. While conserving R231 (MOT12 R951) at the base of the binding pocket, the residues on the predicted rim of the binding pocket (just following the highly conserved KNRY motif) are atypical for MOT12 (Ser-Leu-Ala-Val at positions 776- 779) . Since these residues interact with the acid side chains of the phosphopeptide, it may effect the substrate specificity of the MOT12 PTP. Finally, the predicted surface loop of MOT12 lies between amino acids 911-919 (WYDRGVPSS) . Typically this sequence is WPDHGVPxS in most PTPs and the alterations in MOT12 are

more typical of the regulatory, noncatalytic second PTP motif in the class III family of receptor PTPs. Class III PTPs are transmembrane proteins that contain two catalytic PTP motifs in their cytoplasmic domain, and include PTPd, LAR, OST, PTPs, PTPu, PTPk, KKP2, PTPz, PTPg, CD 5, and PTPe.

Therefore, several atypical residues in MOT12 at highly conserved positions suggest it may have altered catalytic activity or specificity compared to other PTPs. These changes include the presence of two charged residues at the predicted phosphotyrosine contact points, nonconserved residues on the predicted rim of the binding pocket and in the predicted surface loop of the catalytic domain. A 40 kDa fragment from the catalytic domain of

IA2/PTP has previously been shown to serve as an important autoantigen of IDDM (Lu, J, et al, PNAS, 1995:93, 2307-2311) . Furthermore, a 37 kDa autoantigen has been suggested to be immunologically related to IA2/PTP (Payton, M et al., J. Clin. Invest. 1995; 86, 1506-1511) . Owing to the high degree of sequence identity between MOT12 and IA2/PTP it is likely that the cross-reactive 37 kDa IDDM autoantigen is directed against the related MOT12.

EXPRESSION PROFILE of HUMAN MOT12

Northern blots of poly(A) + mRNA from adult human tissue samples were hybridized with DNA probes specific to human MOT12. A M0T12 mRNA transcript of approximately 5.4 kb was identified in adult human brain

and pancreas whereas pancreas also had an additional band at 3.5 kb. A very weak 5.4 kb transcript was found in adult spleen. No detectable signal was seen in heart, placenta, lung, liver, skeletal muscle, kidney, thymus, prostate, testis, ovary, small intestine, colon, or in peripheral blood leukocytes. (Table I) . M0T12 was also detected by PCR analysis in several neuroblastoma, astrocytoma, and meningioma cell lines, but was absent from 3 glioblastomas tested. M0T12 as also present in several metastatic pancreatic adenocarcinoma cell lines, yet absent from a nonmetastic epithelioid pancreatic tumor. (Table I) .

Table I

PTP-MOT12 Expression (PCR on sscDNA)

IMR32 +++ Neuroblastoma

SY5Y +++ Neuroblastoma

SW1088 ++ Astrocytoma

SW1783 + Astrocytoma

Meningioma + Primary meningioma

LANS + Neuroblastoma?

U-138 mG - Glioblastoma

U87 MF - Glioblastoma, astrocytoma, grade III

SK-N-SH - Neuroblastoma, metastasis to bone marrow

SF763 - Glioblastoma

ASPC + Pancreatic adenocarcinoma- metastatic

ASPC + TPA + Pancreatic adenocarcmoma- metastatic

CAPAN-l + Pancreatic adenocarcinoma-liver metastasis

HS766T + Pancreatic carcinoma - LN metastasis

PANC-1 - Pancreatic epithelioid carcinoma

PANC-1 + TPA - Pancreatic epithelioid carcinoma

Motor neurons

Sensory neurons

Sympathetic neurons

Brain ++ (Amygdala, Caudate, Hippocampus, Sub. nig. , Subthalamus, Thalamus)

Pancreas ++

Heart -

Placenta -

Lung -

Skeletal Muscle -

Kidney •-

Spleen -

Thymus -

Prostate -

Testis -

Ovary -

Small intestine -

Colon -

PBS -

In Situ EXPRESSION PROFILE of HUMAN M0T12

The developmental expression of M0T12 was assessed by in situ analysis in 14-day old mouse embryos

using a fragment of the rat M0T12 as a probe. Expression confined to neural tissue along ventral portion of neural tube near the spinal vertebrae. Heart, liver, gastrointestinal tract, muscle and connective tissue were all negative.

Neuronal specific expression of M0T12 was assessed in the adult sagittal and coronal sections from adult rat brain. Expression was strong and confined to neurons with localization to the specific regions of the hippocampus, cortex piritormis, nucleus lateralis habenulae, and portions of the lateal cerebral cortex and ventral brainstem. A similar anatomic pattern of hybridization was seen with both mid-sagittal sections and multiple coronal levels, supporting the specificity of the hybridization.

Example 2 : Recombinant Expression of M0T12 MATERIALS AND METHODS EXPRESSION VECTOR CONSTRUCTION

Expression constructs were generated by PCR- assisted mutagenesis in which the entire coding domains of Aurora! and Aurora2 were tagged on their carboxy- terminai ends with the hemophilus influenza hemaglutinin (HA) epitope YPYDVPDYAS (Pati, 1992) . These constructs were introduced into two mammalian expression vectors : pLXSN (Miller, A.D. et al, Biotechniques 7, 980-988, 2989) for the generation of virus producing lines; and pRK5 for transient expression analysis. Inserts were designed to be flanked by unique BamHI and NotI sites and cloned into pLXSN at

Expression constructs were generated by PCR- assisted mutagenesis in which the entire coding domain of M0T12-h was tagged on its carboxy-terminai end with the hemophilus influenza hemaglutinin (HA) epitope YPYDVPDYAS (Pati, 1992) . This construct was introduced into pRK5 for transient expression analysis.

The entire 375 amino acid cytoplasmic domain and the 91-amino acid cytoplasmic juxtamembrane domain of M0T12 were generated by PCR and ligated into pGEX vector for bacterial production of GST-fusion proteins for immunization of rabbits for antibody production.

GENERATION OF M0T12-SPECIFIC IMMUNOREAGENTS

M0T12-specific immunoreagents were raised in rabbits against KLH-conjugated synthetic peptides corresponding to either the C-terminal region of M0T12 ( ¹⁰⁰⁴ EEVNAILKALPQ ^101E ) within the juxtamembrane region of the cytoplasmic domain ( ⁶⁴³ SQHRLKEKLSGLGGDPG ⁶⁵⁹ ) ( ⁶⁷⁷ RPPDRPEGPHTS ^68B ) or within the surface loop in the catalytic domain ( ⁹¹¹ WYDRGVPSSSRS ⁹²² ) . All peptides were synthesized with a N-terminal cysteine for conjugation of KLH. Additional immunoreagents were generated by immunizing rabbits with the bacterially expressed GST- fusion proteins containing the complete (GEX-M0Tl2cyto) or partial cytoplasmic (GEX-M0T12jm) domain of M0T12.

TRANSIENT EXPRESSION OF AURORAS IN MAMMALIAN CELLS

The pRK5 expression plasmids (10 ug DNA/100 mm plate) containing the HA-tagged M0T12 gene was introduced into COS and 293 cells with lipofectamine

(Gibco BRL) . After 72 hours, the cells were harvested in 0.5 ml solubilization buffer (20 mM HEPES pH7.35, 150 mM Nacl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl ₂ , 1 mM EGTA, 2 mM pheylmethylsulfonyl fluoride, 1 μg/ml aprotinin) . Sample aliquots were resolved by SDS polyacrylamide gel electrophoresis (PAGE) on 8% acrylamide/0.5% bis-acrylamide gels and electrophoretically transferred to nitrocellulose. Non¬ specific binding was blocked by preincubating blots in Blotto (phosphate buffered saline containing 5% w/v non¬ fat dried milk and 0.2% v/v NP-40 (Sigma)) , and recombinant protein was detected using a murine Mab to the HA decapeptide tag. Alternatively, recombinant protein can be detected using various MOT12-specific antisera.

RESULTS

Recombinant HA-tagged M0T12 expressed in COS cells was detected using an HA Mab or MOT12 specific antisera. The MOT12 protein migrated with apparent Mr of 150,000. This is substantially larger than the predicted 114 kDa of the unprocessed M0T12 precursor. While MOT12 lacks any N-linked glycosylation sites, its mobility could be affected by other post-translational modifications, or its hydrophobic acid nature could result in abberant migration on SDS-PAGE. Nonetheless, this analysis confirms the recombinant protein can be stabley produced in mammalian cells. Dephosphorylation assays to determine activity and target specificity of this putative phosphatase is ongoing using PNPP, a specific

phosphopeptide substrate and in vitro phosphorylated TrkB as substrates. Furthermore, we can use such reagent to localize expression of endogenous and recombinant M0T12 within cells. Furthermore, these reagents can be used in an effort to identify substrates for the M0T12 in order to better understand their normal biologic role.

Future work will address the catalytic activity, and specificity of M0T12, to identify associated proteins, to determine if a fragment of this abundant pancreatic receptor is the 37 kD autoantigen seen in IDDM patients, and to address its role as an abundantly expressed PTP in very select neuronal populations. We will also assess of M0T12 interacts with any of the neurotrophin receptors (TrkA, TrkB,

TrkC) since these are some of the most abundant tyrosine kinases in the neuronal cells which express M0T12.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising" , "consisting essentially of" and "consisting of" may be

replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Other embodiments are within the following claims.