Receptor-type phosphotyrosine phosphatase-.beta.

TABLE OF CONTENTS
1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
2.1. PTKases
2.2. PTPases
3. SUMMARY OF THE INVENTION
4. DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
6. EXAMPLE: ISOLATION AND CHARACTERIZATION OF HUMAN RPTP.beta. cDNA
6.1. MATERIALS
6.2. METHODS
6.3. RESULTS
7. EXAMPLE: CHROMOSOMAL LOCALIZATION OF THE HUMAN RPTP.beta. GENE
7.1. METHODS
7.2. RESULTS
8. EXAMPLE: EXPRESSION OF RPTP.alpha. RNA
8.1. ISOLATION OF MOUSE SEQUENCES HOMOLOGOUS TO HUMAN RPTP.beta.
8.4. CELL LABELING AND IMMUNOPRECIPITATION
8.5. RESULTS: DETECTION OF RPTP.beta. EXPRESSION IN A HUMAN NEUROBLASTOMA CELL LINE, Lan 5
9. EXAMPLE: IDENTIFICATION OF A VARIANT FORM OF RPTP.beta.
10. EXAMPLE: TISSUE SPECIFIC EXPRESSION OF RPTP.beta.
10.1. METHODS: IN SITU HYBRIDIZATION ANALYSIS
10.2. RESULTS
10.2.1. Tissue Specific Expression of RPTP.beta.
10.2.2. Localization of RPTP.beta. Expression in the Brain
11. GENERAL DISCUSSION FOR SECTIONS 6-10
1. INTRODUCTION
The invention in the field of biochemistry and cell and molecular biology relates to novel receptor-type protein tyrosine phosphatase proteins or glycoproteins, termed RPTP.alpha., .beta. and .gamma., DNA coding therefor, methods for production and identification of the proteins, and methods for screening compounds capable of binding to and inhibiting or stimulating PTPase enzymatic activity.
2. BACKGROUND OF THE INVENTION
The identification of several growth factor receptors and retroviral oncogenes as tyrosine-specific protein kinases indicated that protein phosphorylation on tyrosine residues plays a key role in cellular growth control. This notion has recently received support by the observation that the level of tyrosine phosphorylation of enzymes thought to play an important role in signal transduction (such as phospholipase C) correlates with their increased activity upon growth factor stimulation, thus establishing a functional role for tyrosine phosphorylation (Ullrich, A., et al., Cell 61:203-212 (1990)).
The degree and pattern of phosphorylation of tyrosine residues on cellular proteins are regulated by the opposing activities of protein-tyrosine kinases (PTKases; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (PTPases; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). The structural characteristics and evolution of PTKases as well as their role in the regulation of cell growth have been reviewed (Hunter, T., et al., Annu. Rev. Biochem. 54:897-930 (1985); Ullrich, A., et al., supra).
2.1. PTKases
Tyrosine kinases comprise a discrete family of enzymes having common ancestry with, but major differences from, serine/threonine-specific protein kinases (Hanks, S. K. et al., (1988) Science 241, 42-52). The mechanisms leading to changes in activity of tyrosine kinases are best understood for receptor-type tyrosine kinases which have a transmembrane topology (Ullrich, A. et al., supra). With such kinases, the binding of specific ligands to the extracellular domain of these enzymes is thought to induce their oligomerization leading to an increase in tyrosine kinase activity and activation of the signal transduction pathways (Ullrich, A. et al., supra). The importance of this activity is supported by the knowledge that dysregulation of kinase activity through mutation or over-expression is a mechanism for oncogenic transformation (Hunter, T et al., supra; Ullrich, A. et al., 1990, supra).
2.2. PTPases
The protein phosphatases are composed of at least two separate and distinct families (Hunter, T. Cell, 58:1013-1016 (1989)), the protein serine/threonine phosphatases and the protein tyrosine phosphatases. This is in contrast to protein kinases, which show clear sequence similarity between serine/threonine-specific and tyrosine-specific enzymes.
There appear to be two varieties of PTPase molecules. The first group is comprised of small, soluble enzymes that contain a single conserved phosphatase catalytic domain, and include (1) placental PTPase 1B (Charbonneau, H. et al., Proc. Natl. Acad. Sci. 86:5252-5256 (1989); Chernoff, J. et al., Proc. Natl. Acad. Sci. USA 87:2735-2789 (1990)), (2) T-cell PTPase (Cool, D. E. et al., Proc. Natl. Acad. Sci. USA 86:5257-5261 (1989)), and (3) rat brain PTPase (Guan, K., et al., Proc. Natl. Acad. Sci. USA, 87:1501-1505 (1990).
The second group is made up of the more complex, receptor-linked PTPases, termed RPTPs, which are of high molecular weight and contain two tandemly repeated conserved domains separated by 56-57 amino acids. One example of RPTPs are the leukocyte common antigens (LCA) (Ralph, S. J., EMBO J., 6:1251-1257 (1987); Charbonneau, H., et al., Proc. Natl. Acad. Sci. USA, 85:7182-7186 (1988)). LCA, also known as CD45, T200 and Ly-5 (reviewed in Thomas, M. L., Ann. Rev. Immunol. 7:339-369 (1989)) comprises a group of membrane glycoproteins expressed exclusively in hemopoietic (except late erythroid) cells, derived from a common gene by alternative splicing events involving the amino terminus of the proteins. Whereas the precise function of CD45 is unknown, many studies have implicated these antigens in a number of processes, including the activity of cytotoxic T lymphocytes and natural killer cells, IL-2 receptor expression, B-cell differentiation, and T lymphocyte proliferation (Pingel, J. T. et al., Cell 58:1055-1065 (1989)).
Other examples of RPTPs are the LCA-related protein, LAR (Streuli, M., et al., J. Exp. Med., 168:1523-1530 (1988)), and the LAR-related Drosophila proteins DLAR and DPTP (Streuli, M., et al., Proc. Natl. Acad. Sci. USA, 86:8698-8702 (1989)). Jirik et al. screened a cDNA library derived from the human hepatoblastoma cell line, HepG2, with a probe encoding the two PTPase domains of LCA (FASEB J. 4: A2082 (1990), abstr. 2253) and discovered a cDNA clone encoding a new RPTP, named He-PTP. The HePTP gene appeared to be expressed in a variety of human and murine cell lines and tissues.
While we are beginning to understand more about the structure and diversity of the PTPases, much remains to be learned about their cellular functions. It has been suggested (Tonks, N. K., et al., Biochemistry, 27:8695-8701 (1988)) that the small, soluble PTPase enzymes may have a "housekeeping" function. On the other hand, the RPTPs would be expected to be more restricted in their activities because of their location in the cell membrane and their potential regulation by extracellular ligands. Regarding the role of LCA (CD45) in T cells, it was found that T cell clones deficient in the expression of LCA failed to proliferate when stimulated by a specific antigen or by cross-linking of CD3 (Pingel, J. T., et al., supra). PTPase cross-linking inhibits T cell receptor CD3-mediated activation in human T cells (Kiener, P. A. et al., J. Immunol. 143:23-28 (1989)). The PTPase activity of LCA plays a role in the activation of pp56.sup.lck, a lymphocyte-specific PTKase (Mustelin, T., et al., Proc. Natl. Acad. Sci. USA, 86:6302-6306 (1989); Ostergaard, H. L., et al., Proc. Natl. Acad. Sci. USA, 86:8959-8963 (1989)). These authors hypothesized that the phosphatase activity of LCA activates pp56.sup.lck by dephosphorylation of a C-terminal tyrosine residue, which may, in turn, be related to T-cell activation.
Using site-directed mutagenesis to determine which of four conserved cysteines in LCA (two per phosphatase domain) was required for enzyme activity toward artificial substrates, Streuli et al. (1989, supra) found that only one cysteine residue (residue 177 of LCA phosphatase domain-1) of LCA was essential for activity, indicating that, most likely, only the first phosphatase domain has enzymatic activity. However, the possibility that the second domain can dephosphorylate a different substrate was not excluded. More recently, Streuli et. al. (EMBO J., 9:2399-2407 (1990)) determined that the second conserved domain of LCA (and of LAR) lacked detectable phosphatase activity but sequences within the domain could influence substrate specificity.
In order to better understand and to be able to control phosphotyrosine metabolism, one must comprehend not only the role of kinase activity, but also the action of phosphatase enzymes as well. Elevation of cellular phosphotyrosine may occur through mechanisms not involving the activation of a tyrosine kinase itself. For instance, expression of the v-crk oncogene, though not a tyrosine kinase itself, induces the phosphorylation of tyrosine residues through a poorly understood mechanism (Mayer, B. J. et al. (1988) Nature 332, 272-275). Potentially, such an outcome could result from either mutation of the substrate or through a general decrease in cellular phosphatase activity, especially in view of the normally high turnover rate of cellular tyrosine-phosphate (Sefton, B. M. et al. (1980) Cell 20, 807-816). The latter possibility is suggested by the demonstration that tyrosine phosphatase inhibitors can "reversibly transform" cells (Klarlund, J. K. Cell 41: 707-717 (1985)). PTPases could therefore be viewed as potential recessive oncogenes.
It is becoming clear that dephosphorylation of tyrosine can by itself function as an important regulatory mechanism. Dephosphorylation of a C-terminal tyrosine residue stimulates tyrosine kinase activity in the src-family of tyrosine kinases (Hunter, T. (1987) Cell 49, 1-4). Tyrosine dephosphorylation has been suggested to be an obligatory step in the mitotic activation of the MPF (maturation promoting factor) kinase (Morla, A. O. et al. (1989) Cell 58, 193-203). Lastly, mutant analysis of primitive eukaryotes has established crucial roles for serine phosphatase in cellular physiology (Cyert, M. S. et al. (1989) Cell 57, 891-893). These observations point out the need in the art for increasing our understanding of the mechanisms that regulate tyrosine phosphatase activity.
It is clear in the art that further analysis of structure-function relationships among these membrane receptors are needed to gain important understanding of the mechanisms of cell growth, differentiation, and oncogenesis.
3. SUMMARY OF THE INVENTION
The present inventor has conceived of a role for RPTPs in cellular control mechanisms, both as potential anti-oncogenes, and as effectors in a newly discovered mechanism of transmembrane signalling. They therefore undertook a search for individual RPTP genes and proteins potentially involved in such processes, and describe herein the identification of a novel, member of the RPTP family, RPTP.beta., which has a transmembrane topology. The extracellular domains of members of this RPTP family are unrelated to any other RPTPs previously described. The novel RPTP.beta., in a manner analogous to receptor tyrosine kinases, is subject to direct regulation by extracellular ligands which bind to the extracellular portion.
The present invention thus provides a human receptor-type protein tyrosine phosphatase-.beta. (RPTP.beta.) protein or glycoprotein molecule, a functional derivative of the human RPTP.beta. or a homolog of human RPTP.beta. in another mammalian species. When the RPTP.beta. molecule is of natural origin, it is substantially free of other proteins or glycoproteins with which it is natively associated. RPTP.beta. is naturally expressed in mammalian brain and is developmentally and anatomically regulated. The RPTP.beta. molecule of the present invention may not be of natural origin, and, may be prepared by chemical or recombinant means. Thus, the substantially pure RPTP.beta. protein or glycoprotein of the present invention may be produced by biochemical purification of the protein or glycoprotein of natural origin; alternatively, the RPTP.beta. may be produced by recombinant means in prokaryotic or eukaryotic hosts.
In particular, the invention is directed to a RPTP.beta. having the amino acid sequence SEQ ID NO:1 of human RPTP.beta., shown in FIGS. 1A-1L and 2A-2B, or a functional derivative thereof.
The invention is further directed to a nucleic acid molecule, preferably, DNA, consisting essentially of a nucleotide sequence encoding RPTP.beta., preferably of human origin, or encoding a functional derivative thereof. The nucleic acid molecule, preferably comprises the sequence SEQ ID NO:2 (see FIGS. 1A-1L). The DNA molecule is preferably cDNA or genomic DNA. The invention is further directed to the DNA molecule in the form of an expression vehicle, as well as prokaryotic and eukaryotic hosts transformed or transfected with the DNA molecule.
Also included in the present invention is a process for preparing an RPTP.beta. protein or glycoprotein, or a functional derivative thereof, comprising:
(a) culturing a host capable of expressing the protein, glycoprotein or functional derivative under culturing conditions;
(b) expressing the protein, glycoprotein or functional derivative; and
(c) recovering the protein, glycoprotein or functional derivative from the culture.
The invention is directed to an antibody, either polyclonal, monoclonal, or chimeric, which is specific for the RPTP.beta. protein or glycoprotein.
The invention is also directed to a method for detecting the presence of nucleic acid encoding a normal or mutant RPTP.beta. in a subject comprising:
(a) contacting a cell or an extract thereof from the subject with an oligonucleotide probe encoding at least a portion of the normal or mutant RPTP.beta. under hybridizing conditions; and
(b) measuring the hybridization of the probe to the nucleic acid of the cell, thereby detecting the presence of the nucleic acid.
The DNA can be selectively amplified, using the polymerase chain reaction, prior to assay.
The invention is further directed to a method for detecting the presence, or measuring the quantity of RPTP.beta. in a cell or cells, comprising:
(a) contacting said cell or an extract thereof with an antibody specific for an epitope of the RPTP.beta.; and
(b) detecting the binding of the antibody to the cell or extract thereof, or measuring the quantity of antibody bound,
thereby detecting the presence or measuring the quantity of the RPTP.beta..
The present invention is also directed to methods for identifying and isolating a compound capable of binding to RPTP.beta. from a chemical or biological preparation comprising:
(a) attaching the RPTP.beta., or the ligand-binding portion thereof, to a solid phase matrix;
(b) contacting the chemical or biological preparation with the solid phase matrix allowing the compound to bind, and washing away any unbound material;
(c) detecting the presence of the compound bound to the solid phase; and, for purposes of isolation,
(d) eluting the bound compound, thereby isolating the compound.
Finally, the invention includes a method for identifying an agent capable of stimulating or inhibiting the phosphatase enzymatic activity of RPTP.beta., comprising:
(a) contacting the agent with RPTP.beta. in pure form, in a membrane preparation, or in a whole live or fixed cell;
(b) incubating the mixture in step (a) for a sufficient interval;
(c) measuring the enzymatic activity of RPTP.beta.;
(d) comparing the enzymatic activity to that of RPTP.beta. incubated without the agent,
thereby determining whether the agent stimulates or inhibits the activity.

4. DESCRIPTION OF THE FIGURES
FIGS. 1(1)-1(8) show the nucleotide sequence (SEQ ID NO:2) and predicted amino acid sequence (SEQ ID NO:1) of human RPTP.beta..
FIGS. 2A-2B presents the amino acid sequence of RPTP.beta.. The hydrophobic signal peptide is underlined and the transmembrane peptide is designated in bold. The 21 potential N-glycosylation sites are indicated by filled arrows. The CAH-related domain and the two phosphatase domains, DI and DII, are indicated by the boxes. The open arrows represent the boundaries of the deletion in the variant RPTP.beta. clones.
FIGS. 3A-3D describes the identification of a CAH-related domain in the extracellular region of RPTP.beta.. FIGS. 3A-3C shows an alignment of the amino acid sequence of the CAH-related domain of RPTP.beta. with the corresponding domain of RPTP.gamma. and six different isoforms of CAH (I-VII). The amino acid sequences that are boxed in black are those that are identical in all six isoforms of CAH. The sequences boxed in the gray hatches are those that are identical between the CAH-related domains of RPTP.beta. and RPTP.gamma.. FIG. 3D is a table showing the percent similarity (taking into account conservative amino acid substitutions) between the CAH-related domains of RPTP.beta., RPTP.gamma. and the six isoforms of CAH.
FIGS. 4A-4B shows the chromosomal localization of human RPTP.beta.. A completely stippled box indicates that the hybrid designated in the left column contains the chromosome indicated in the upper row; lower-right stippling indicates the presence of the long arm (or a part of the long arm, indicated by small fraction of stippling) of the chromosome indicated; upper left stippling indicates the presence of the short arm (or partial short arm) of the chromosome indicated; an open box indicates absence of the chromosome indicated; the column for chromosome 7 is boldly outlined and stippled to highlight the correlation of the presence of this chromosome with the presence of the RPTP.beta. gene. The pattern of retention of the RPTP.beta. sequences in the hybrids is shown on the right where the presence of the gene is indicated by a "+" sign in a stippled box and absence of the gene is indicated by a "-" sign in an open box. FIG. 4B shows a schematic diagram of chromosome 7, indicating that RPTP.beta. maps to 7q31-q33. chromosomal in situ hybridization of a 1.8 kb RPTP.beta. cDNA to normal human metaphase chromosomes confirmed localization of the gene to 7q and revealed a peak of grains centered over the 7q31.3-q32 region, as illustrated. Each dot to the right represents an autoradiographic grain.
FIGS. 5A-5C show the expression of RPTP.beta. mRNA in various human cell lines and murine tissues using Northern blot analysis. In FIG. 5A, 20 .mu.g of total cellular RNA (lanes 1-5) or 1 .mu.g of poly-A.sup.+ RNA (lane 6) isolated from the various indicated glioblastoma and neuroblastoma cell lines were loaded onto a 1% agarose/2.2M formaldehyde RNA gel and probed with a DNA fragment isolated from the human brain stem cDNA clone that begins with sequences just 5' of the region encoding the transmembrane region and extends and includes all of the sequence in the phosphatase domain I. In FIG. 5B, poly-A.sup.+ RNA (1 .mu.g/sample) from the indicated murine tissues were loaded onto an RNA gel and probed with the PCR-amplified murine DNA fragment, pBSMBDII. FIG. 5C shows the blot from FIG. 5B which was stripped of the probe and rehybridized with a .sup.32 P-labeled rat actin probe.
FIG. 6 is a gel pattern showing the identification of endogenous RPTP.beta. protein expressed in Lan 5cells. RPTP.beta. was immunoprecipitated with normal rabbit serum (NRS, lane 1) or immune anti-RPTP.beta. antiserum (.alpha.PTP.beta., lanes 2 and 3) from lysates of �.sup.35 S!methionine-labeled Lan 5 cells in the absence (lanes 1 and 2) or presence (lane 3) of tunicamycin. Immunoprecipitation of the EGF receptor with RK2 antibody (.alpha.EGFR, lanes 4 and 5) from lysates of �.sup.35 S!methionine-labeled Lan 5 cells labeled in the absence (lane 4) or presence (lane 5) of tunicamycin.
FIGS. 7A and 7B (FIG. 6) shows the identification of variant RPTP.beta. using Northern blots. FIG. 7A is a schematic diagram of the protein encoded by the full length RPTP.beta. cDNA compared to the putative protein encoded by the two independently isolated cDNA clones that carry an identical deletion of 2577 bp in the extracellular region of the protein. The position of the deletion is indicated by the dotted line with the number of amino acids remaining at both the 5' and 3' end of the deletion indicated. The location of the two probes using in Northern analysis (probes 1 and 2) are indicated. TM, transmembrane peptide; DI, phosphatase domain I; DII, phosphatase domain II. FIG. 7B shows the results of Northern analysis. poly-A.sup.+ RNA (1 .mu.g) isolated from the Lan 5 neuroblastoma cell line was separated on a RNA formaldehyde gel and probed with human probe 1 (P1) that contains 1.3 kb of sequences derived from the extreme 5' end of the cDNA clone and human probe 29P2) that contains 1.6 kb of sequences derived from the portion of the full length cDNA clone that is deleted in the variant cDNA clones.
FIGS. 8A and 8B show the results of an in situ hybridization analysis of RPTP.beta. expression in developing and adult mouse brain. FIG. 8A shows a sagittal section through an embryonic day 20 (E2) mouse, and indicates that RPTP.beta. was expressed in the developing nervous system. The highest level of expression was observed in the ventricular and subventricular zones (VZ). FIG. 8B is a sagittal section through an adult mouse brain and shows discrete bands of expression in the Purkinje cells of the cerebellum (PK), the dentate gyrus (DG), and the subependymal layer of the anterior horn of the lateral ventricle (AH).

5. DETAILED DESCRIPTION OF THE INVENTION
Through the use of recombinant DNA methods, the present inventor has identified novel mammalian receptor-type (transmembrane) protein tyrosine phosphatases (PTPase; EC 3.1.3.48). In view of their receptor-like structure, and the likelihood that they are part of a family, the inventor has termed these protein, RPTP.alpha., RPTP.beta., RPTP.gamma., etc. (receptor protein tyrosine phosphatase-alpha, beta, gamma, etc.). The family is designated herein as the "RPTPs" (and is also referred to as R-PTPases)
Human RPTP.beta. is a protein or glycoprotein having 2307 amino acids. In contrast, human RPTP.alpha. has 802 amino acids and human RPTP.gamma. has 1445 amino acids. RPTP.beta. has an extracellular domain, a single transmembrane domain and a cytoplasmic portion with two tandem catalytic phosphatase domains. The extracellular domain contains a stretch of 266 amino acids with striking homology to the zinc-containing enzyme carbonic anhydrase (CAH) indicating that RPTP.beta. together with RPTP.gamma. (HPTP.zeta.) represent a subfamily of RPTPs.
The gene encoding RPTP.beta., denoted RPTP.beta. (or PTP.zeta.) has been mapped by the present inventor to human chromosome 7.sub.q 31-.sub.q 33, a site rather distinct from the site to which RPTP.gamma. was mapped (3.sub.p 14.2-.sub.p 21).
The cDNA cloning of human RPTP.beta., and the complete DNA and amino acid sequence of human RPTP.beta. are described herein. Northern analysis has been used to identify the natural expression of the protein in various cells and tissues. RPTP.gamma. was shown to be expressed in anatomically distinct regions of rat brain and its expression was found to be developmentally regulated.
Remarkably, in addition to being composed of intracellular domains having enzymatic activity, the receptor family to which RPTPs belong includes transmembrane proteins having and N-terminal extracellular domains, analogous to the tyrosine kinase enzyme family (Tonks, N. K. et al. (1988) Biochemistry 27:8695-8701; Charbonneau, H. et al. (1988) Proc. Natl. Acad. Sci. USA 85:7182-7186; Streuli, M. et al. (1988) J. Exp. Med. 168:1523-2530; Streuli, M. et al. (1989) Proc. Natl. Acad. Sci. USA 86:8698-8702). The present inventor has therefore concluded that ligands in the extracellular environment can control the activity of this membrane-associated subclass of PTPases.
The present inventor has further produced a polyclonal antibody to RPTP.beta. by immunization or rabbits with a 15 amino acid synthetic peptide of RPTP.beta. conjugated to a carrier.
RPTP.beta. is useful in methods for screening drugs and other agents which are capable of activating or inhibiting the PTPase enzymatic activity, and thereby affecting major pathways of cellular metabolism. By attaching an intact RPTP.beta., or the ligand-binding portion thereof, to a solid phase matrix, an affinity probe is created which can be used to screen biological products or chemical agents for their capacity to interact with the receptor on the basis of their binding activity. Bound material can then be eluted from the affinity probe in purified form.
Methods for coupling proteins and peptides to the solid phase, the solid phase substances useful in these methods, and means for elution, are well known to those of skill in the art.
The RPTP.beta. protein or derivatives thereof having enzymatic activity can be used for testing of compounds capable of enhancing or inhibiting the phosphatase activity. The ability of a compound under test to modify phosphatase activity can be tested in an in vitro system wherein the test compound is added to purified RPTP.beta. protein, or an enzymatically active derivative thereof, and the affects on enzyme activity measured using standard enzymological procedures well known to those of skill in the art.
Alternatively, the action of a compound on RPTP.beta. enzymatic activity can be measured in a whole cell preparation using live or fixed cells, or a membrane fraction derived from live or fixed cells. This method is useful for screening compounds acting via the extracellular receptor portion of the protein, as well as compounds acting directly on the enzymatic portion of the protein. A test compound is incubated with cells, or with a membrane preparation derived therefrom, which express high amounts of the RPTP.beta., such as transfected COS or NIH-3T3 cells. The amount of cellular phosphotyrosine is then measured, using methods well-known in the art (Honegger, A. M. et al., Cell 51:199-209 (1987); Margolis, B. et al., Cell 57:1101-1107 (1989)). The results are compared to results obtained in the absence of the test compound, or in the absence or presence of a known activator of RPTP.beta. enzymatic. In such studies, the action of the test compound in the presence of an activator of tyrosine kinase can also be measured. A compound which stimulates RPTP.beta. enzymatic activity will result in a net decrease in the amount of phosphotyrosine, whereas a compound which inhibits RPTP.beta. enzymatic activity will result in a net increase in the amount of phosphotyrosine.
In the case of growth factor receptors which are tyrosine kinases, such as the receptors for epidermal growth factor (EGF) and for platelet-derived growth factor (PDGF), tyrosine phosphorylation is linked to cell growth and to oncogenic transformation. Activation of a PTPase, leading to dephosphorylation, would serve as a counterregulatory mechanism to prevent or inhibit growth, and might serve as an endogenous regulatory mechanism against cancer. Thus, mutation or dysregulation of this receptor/enzyme system may promote susceptibility to cancer.
The insulin receptor is also a tyrosine kinase, and phosphorylation of tyrosine in cells bearing insulin receptors would be associated with normal physiological function. In contrast to the case of cell growth and cancer, activation of an RPTP would counteract insulin effects. Subnormal RPTP levels or enzymatic activity would act to remove a normal counterregulatory mechanisms. Perhaps more important, though, over-activity, or inappropriate activation, of an RPTP such as RPTP.beta. would be expected to partially or totally inhibit the action of insulin on cells, leading to diabetes (of an insulin-resistant variety). Thus, susceptibility to diabetes may be associated with RPTP.beta. dysregulation.
Therefore, the methods of the present invention for identifying normal or mutant RPTP.beta. genes, or for measuring the amount or activity of RPTP.beta. associated with a cell or tissue, can serve as methods for identifying susceptibility to cancer, diabetes, or other diseases associated with alterations in cellular phosphotyrosine metabolism.
The present invention provides methods for evaluating the presence of, and the level of, normal or mutant RPTP.beta. in a cell or in a subject. Absence, or more typically, low expression of RPTP.beta., or presence of a mutant RPTP.beta., in an individual may serve as an important predictor of susceptibility to oncogenic transformation and the development of cancer. Alternatively, over-expression of RPTP.beta., possibly due to a mutant receptor/enzyme system insensitive to negative regulation, or due to overabundance of a stimulatory ligand in the body, may serve as an important predictor of susceptibility to diabetes.
An oligonucleotide probe encoding a part of the RPTP.beta. sequence (see below) is used to test cells from a subject for the presence DNA or RNA sequences encoding the RPTP.beta.. A preferred probe would be one directed to the nucleic acid sequence encoding at least 4 amino acid residues, and preferably at least 5 amino acid residues, of the RPTP.beta.. Qualitative or quantitative assays can be performed using such probes. For example, Northern analysis (see Sections 8 and 10 below) is used to measure expression of an RPTP.beta. mRNA in a cell or tissue preparation.
Such methods can be used even with very small amounts of DNA obtained from an individual, following use of selective amplification techniques. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Pat. No. 4,237,224), Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which references are herein incorporated by reference).
An in vitro, enzymatic method which is capable of increasing the concentration of such desired nucleic acid molecules is called the "polymerase chain reaction or "PCR" (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich, H. et al., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis, K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194).
The PCR provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.
The precise nature of the two oligonucleotide probes of the PCR method is critical to the success of the method. Polymerase dependent amplification of a nucleic acid molecule proceeds by the addition of a 5' nucleotide triphosphate to the 3' hydroxyl end of a nucleic acid molecule. Thus, the action of a polymerase extends the 3' end of a nucleic acid molecule. These inherent properties are exploited in the selection of the oligonucleotide probes of the PCR. The oligonucleotide sequences of the probes of the PCR method are selected such that they contain sequences identical to, or complementary to, sequences which flank the particular nucleic acid sequence whose amplification is desired. More specifically, the oligonucleotide sequences of the "first" probe is selected such that it is capable of hybridizing to an oligonucleotide sequence located 3' to the desired sequence, whereas the oligonucleotide sequence of the "second" probe is selected such that it contains an oligonucleotide sequence identical to one present 5' to the desired region. Both probes possess 3' hydroxy groups, and therefore can serve as primers for nucleic acid synthesis.
PCR reaction conditions are cycled between (a) those conducive to hybridization and nucleic acid polymerization, and (b) those which result in the denaturation of duplex molecules. In the first step of the reaction, the nucleic acids of the sample are transiently heated, and then cooled, in order to denature any double-stranded molecules which may be present. The "first" and "second" probes are then added to the sample at a concentration which greatly exceeds that of the desired nucleic acid molecule. When the sample is incubated under conditions conducive to hybridization and polymerization, the "first" probe will hybridize to the nucleic acid molecule of the sample at a position 3' to the sequence to be amplified. If the nucleic acid molecule of the sample was initially double-stranded, the "second" probe will hybridize to the complementary strand of the nucleic acid molecule at a position 3' to the sequence which is the complement of the sequence whose amplification is desired. Upon addition of a polymerase, the 3' ends of the "first" and (if the nucleic acid molecule was double-stranded) "second" probes will be extended. The extension of the "first" probe will result in the synthesis of an oligonucleotide having the exact sequence of the desired nucleic acid. Extension of the "second" probe will result in the synthesis of an oligonucleotide having the exact sequence of the complement of the desired nucleic acid.
The PCR reaction is capable of exponential amplification of specific nucleic acid sequences because the extension product of the "first" probe, of necessity, contains a sequence which is complementary to a sequence of the "second" probe, and thus can serve as a template for the production of an extension product of the "second" probe. Similarly, the extension product of the "second" probe, of necessity, contains a sequence which is complementary to a sequence of the "first" probe, and thus can serve as a template for the production of an extension product of the "first" probe. Thus, by permitting cycles of polymerization, and denaturation, a geometric increase in the concentration of the desired nucleic acid molecule can be achieved. Reviews of the PCR are provided by Mullis, K. B. (Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986)); Saiki, R. K., et al. (Bio/Technology 3:1008-1012 (1985)); and Mullis, K. B., et al. (Meth. Enzymol. 155:335-350 (1987)).
In one embodiment, the present invention is directed to a naturally occurring mammalian RPTP.beta.. In another embodiment, the invention is directed to a recombinant mammalian RPTP.beta.. The preferred mammalian RPTP.beta. of the present invention is of human origin. The invention provides the naturally occurring molecule substantially free of other proteins with which it is natively associated. "Substantially free of other proteins or glycoproteins" indicates that the protein has been purified away from at least 90 percent (on a weight basis), and from even at least 99 percent if desired, of other proteins and glycoproteins with which it is natively associated, and is therefore substantially free of them. That can be achieved by subjecting the cells, tissue or fluid containing the RPTP.beta. to standard protein purification techniques such as immunoabsorbent columns bearing an antibody specific for the protein. Other forms of affinity purification can utilize solid-phase substrates which can bind the PTPase domain, or a ligand that will bind to the receptor domain. Alternatively, the purification can be achieved by a combination of standard methods, such as ammonium sulfate precipitation, molecular sieve chromatography, and ion exchange chromatography.
It will be understood that the RPTP.beta. of the present invention can be biochemically purified from a variety of cell or tissue sources. For preparation of naturally occurring RPTP.beta., tissues such as mammalian brain, especially of human origin, are preferred.
Alternatively, because the gene for the RPTP.beta. can be isolated or synthesized, the polypeptide can be synthesized substantially free of other proteins or glycoproteins of mammalian origin in a prokaryotic organism or in a non-mammalian eukaryotic organism, if desired. As intended by the present invention, a recombinant RPTP.beta. molecule produced in mammalian cells, such as transfected COS, NIH-3T3, or CHO cells, for example, is a protein with a naturally occurring amino acid sequence or is a functional derivative thereof. Where a naturally occurring protein or glycoprotein is produced by recombinant means, it is provided substantially free of the other proteins and glycoproteins with which it is natively associated.
Alternatively, methods are well known for the synthesis of polypeptides of desired sequence on solid phase supports and their subsequent separation from the support.
In a further embodiment, the invention provides "functional derivatives" of the RPTP.beta.. By "functional derivative" is meant a "fragment," "variant," "analog," or "chemical derivative" of RPTP.beta., which terms are defined below. A function al derivative retains at least a portion of the function of the RPTP.beta., such as (a) binding to a specific antibody, (b) phosphatase enzymatic activity or (c) binding of the extracellular "receptor" domain to a ligand, which permits its utility in accordance with the present invention.
A "fragment" of the RPTP.beta. refers to any subset of the molecule, that is, a shorter peptide.
A "variant" of the RPTP.beta. refers to a molecule substantially similar to either the entire peptide or a fragment thereof. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methods well-known in the art.
Alternatively, amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synthesized peptide. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Obviously, the mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see European Patent Publication No. EP 75,444).
At the genetic level, these variants ordinarily are prepared by site-directed mutagenesis (as exemplified by Adelman et al., DNA 2:183 (1983)) of nucleotides in the DNA encoding the peptide molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. The variants typically exhibit the same qualitative biological activity as the nonvariant peptide.
An "analog" of the RPTP.beta. refers to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
A "chemical derivative" of the RPTP.beta. contains additional chemical moieties not normally a part of the peptide. Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, .alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with diethylprocarbonate, pH 5.5-7.0, because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0.
Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing .alpha.-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high PK.sub.a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine .epsilon.-amino group.
The specific modification of tyrosyl residues per se has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'--N--C--N--R') such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues, under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
Derivatization with bifunctional agents is useful for cross-linking the protein or peptide to a water-insoluble support matrix or to other macromolecular carriers. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidyl-propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-�(p-azidophenyl)dithio!propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the X-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecule Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl groups.
Such derivatized moieties may improve the solubility, absorption, biological half life, and the like. The moieties may alternatively eliminate or attenuate any undesirable side effect of the protein and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980)
This invention is also directed to an antibody specific for an epitope of RPTP.beta., preferably, of human RPTP.beta., and the use of such antibody to detect the presence of, or measure the quantity or concentration of, the RPTP.beta. in a cell, a cell or tissue extract, or a biological fluid.
The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, and anti-idiotypic (anti-Id) antibodies.
Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigens. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. The hybridoma producing the mAbs of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo production makes this the presently preferred method of production. Briefly, cells from the individual hybridomas are injected intraperitoneally into pristane-primed BALB/c mice to produce ascites fluid containing high concentrations of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies and methods for their production are known in the art (Cabilly et al, Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984); Neuberger et al., Nature 314:268-270 (1985); Taniguchi et al., European Patent Application 171496 (published Feb. 19, 1985); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Neuberger et al., PCT Application WO 86/01533 (published Mar. 13, 1986); Kudo et al., European Patent Application 184187 (published Jun. 11, 1986); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Sahagan et al., J. Immunol. 137:1066-1074 (1986); Robinson et al., International Patent Publication #PCT/US86/02269 (published May 7, 1987); Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988)). These references are hereby incorporated by reference.
An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An anti-Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). The anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other hybrid clones expressing antibodies of identical specificity.
Accordingly, mAbs generated against RPTP.beta. may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for an RPTP.beta. epitope.
The anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as an epitope of RPTP.beta..
The term "antibody" is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab').sub.2, which are capable of binding antigen. Fab and F(ab').sub.2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
It will be appreciated that Fab and F(ab').sub.2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of RPTP.beta. according to the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab').sub.2 fragments).
An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one, or more than one epitope.
An antibody is said to be specific for an antigen because it reacts in a highly selective manner, with that antigen and not with the multitude of other antigens which are structurally distinct.
The antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the presence of cells which express the RPTP.beta. protein. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection. For such methods, the antibody is preferably specific for an extracellular epitopic of RPTP.beta..
The antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of RPTP. In situ detection may be accomplished by removing a histological specimen from a patient, and providing the a labeled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the RPTP.beta. but also its distribution on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection. Such assays for RPTP.beta. typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leucocytes, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody specific for RPTP.beta., and detecting the antibody by any of a number of techniques well-known in the art.
The biological sample may be treated with a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled RPTP.beta.-specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support may then be detected by conventional means.
By "solid phase support" is intended any support capable of binding antigen or antibodies. Well-known supports, or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
The binding activity of a given lot of anti-RPTP.beta. antibody may be determined according to well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
One of the ways in which the RPTP.beta.-specific antibody can be detectably labeled is by linking the antibody, or linking a second antibody which binds to the anti-RPTP.gamma. antibody, to an enzyme and use in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by calorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect R-PTPase through the use of a radioimmunoassay (RIA) (see, for example, Work, T. S. et al., Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, New York, 1978, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
The antibody can also be detectably labeled using fluorescence emitting metals such as .sup.152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
The antibody molecules of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody.
In another type of "sandwich" assay, which may also be useful with the antigens of the present invention, the so-called "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncompleted labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.
The presence of normally functioning RPTP.beta. in a subject can also be tested using direct enzymatic assays, for the tyrosine phosphatase activity. Such biochemical measurements can be performed in vitro, using purified enzymes, allowing precise measurements of enzyme activity, or with membrane preparations, or whole cells, where the net phosphotyrosine level is determined.
In additional embodiments of the present invention, a nucleic acid molecule, preferably DNA, comprising a sequence encoding an RPTP.beta. protein molecule and methods for expressing the DNA sequence are provided. One of ordinary skill in the art will know how to identify and clone additional PTPase molecules, of human or other mammalian species, which have sequence homology to the RPTP.beta. molecules described herein, using the genetic sequences and oligonucleotides of the present invention without undue experimentation. Furthermore, manipulation of the genetic constructs of the present invention allow the grafting of a particular ligand-binding receptor domain onto the transmembrane and catalytic portions of the RPTP.beta. resulting in chimeric molecules. Non-limiting examples of such chimeric molecules include the RPTP.beta. wherein the receptor is an epidermal growth factor receptor, a fibroblast growth factor receptor, and the like. Genetically engineered chimeric receptors are known in the art (see, for example, Riedel, H. et al., Nature 324:628-670 (1986)).
Genetic constructs encoding RPTP.beta., functional derivative thereof, and chimeric molecules such as those described above, can be used in gene therapy. An abnormal or dysfunctional RPTP.beta., which results in disease, may be replaced by infusion of cells of the desired lineage (such as hemopoietic cells, neurons, etc.) transfected with DNA encoding normal RPTP.beta.. Alternatively, or additionally, cells carrying a chimeric RPTP.beta. having a receptor to a ligand of choice (e.g., EGF) can be used for such gene therapy.
The recombinant DNA molecules of the present invention can be produced through any of a variety of means, such as, for example, DNA or RNA synthesis, or more preferably, by application of recombinant DNA techniques. Techniques for synthesizing such molecules are disclosed by, for example, Wu, R., et al. (Prog. Nucl. Acid. Res. Molec. Biol. 21:101-141 (1978)). Procedures for constructing recombinant molecules in accordance with the above-described method are disclosed by Sambrook et al. (supra).
Oligonucleotides representing a portion of an RPTP.beta. are useful for screening for the presence of genes encoding such proteins and for the cloning of an RPTP.beta. gene. Techniques for synthesizing such oligonucleotides are disclosed by, for example, Wu et al., supra).
Protein molecules are fragmented as with cyanogen bromide, or with proteases such as papain, chymotrypsin, trypsin, etc. (Oike, Y., et al., J. Biol. Chem. 257:9751-9758 (1982); Liu, C., et al., Int. J. Pept. Protein Res. 21:209-215 (1983)). Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J. D., In: Molecular Biology of the Gene, 4th Ed., Benjamin/Cummings Publishing Co., Inc., Menlo Park, Calif. (1987)). Using the genetic code, one or more different oligonucleotides can be identified, each of which would be capable of encoding the amino acid. The probability that a particular oligonucleotide will, in fact, constitute the actual XXX-encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Such "codon usage rules" are disclosed by Lathe, R., et al., J. Molec. Biol. 183:1-12 (1985). Using such "codon usage rules", a single oligonucleotide, or a set of oligonucleotides, that contains a theoretical "most probable" nucleotide sequence capable of encoding RPTP.beta. is identified.
Although occasionally an amino acid sequence may be encoded by only a single oligonucleotide, frequently the amino acid sequence may be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of this set contain oligonucleotides which are capable of encoding the peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the nucleotide sequence of the gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the RPTP.beta..
The oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence capable of encoding the RPTP.beta. fragment is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probable" sequence, or set of sequences. An oligonucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the RPTP.beta. gene (Sambrook et al., supra).
A suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the RPTP.beta. gene (or complementary to such an oligonucleotide) is identified as above and synthesized, using procedures well known in the art (Belagaje, R., et al., J. Biol. Chem. 254:5765-5780 (1979); Maniatis, T., et al., In: Molecular Mechanisms in the Control of Gene Expression, Nierlich, D. P., et al., Eds., Acad. Press, N.Y. (1976); Wu, R., et al., Prog. Nucl. Acid Res. Molec. Biol. 21:101-141 (1978); Khorana, R. G., Science 203:614-625 (1979)). DNA synthesis may be achieved using an automated synthesizers. The oligonucleotide probe or set is hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from cells which are capable of expressing the RPTP.beta. gene. Techniques of nucleic acid hybridization are disclosed by Sambrook et al. (supra), and by, S Haymes, B. D., et al. (In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985)), which references are herein incorporated by reference. Techniques such as, or similar to, those described above have successfully enabled the cloning of genes for human aldehyde dehydrogenases (Hsu, L. C., et al., Proc. Natl. Acad. Sci. USA 82:3771-3775 (1985)), fibronectin (Suzuki, S., et al., EMBO J. 4:2519-2524 (1985)), the human estrogen receptor gene (Walter, P., et al., Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)), tissue-type plasminogen activator (Pennica, D., et al., Nature 301:214-221 (1983)) and human term placental alkaline phosphatase complementary DNA (Kam, W., et al., Proc. Natl. Acad. Sci. USA 82: (715-8719 (1985)).
In a alternative way of cloning the RPTP.beta. gene, a library of expression vectors is prepared by cloning DNA or, more preferably, cDNA (from a cell capable of expressing RPTP.beta.) into an expression vector. The library is then screened for members capable of expressing a protein which binds to anti-RPTP.beta. antibody, and which has a nucleotide sequence that is capable of encoding a polypeptide that has the same amino acid sequence as all or part of RPTP.beta.. In this embodiment, DNA, or more preferably cDNA, is extracted and purified from a cell which is capable of expressing RPTP.beta. protein. The purified cDNA is fragmented (by shearing, endonuclease digestion, etc.) to produce a pool of DNA or cDNA fragments. DNA or cDNA fragments from this pool are then cloned into an expression vector in order to produce a genomic library of expression vectors whose members each contain a unique cloned DNA or cDNA fragment.
An "expression vector" is a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) is capable of expressing a DNA molecule which has been cloned into the vector and of thereby producing a peptide or protein. Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. If a eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequences. Importantly, since eukaryotic DNA may contain intervening sequences, and since such sequences cannot be correctly processed in prokaryotic cells, it is preferable to employ cDNA from a cell which is capable of expressing RPTP.gamma. in order to produce a prokaryotic genomic expression vector library. Procedures for preparing cDNA and for producing a genomic library are disclosed by Sambrook et al. (supra).
A DNA sequence encoding the RPTP.beta. of the present invention, or its functional derivatives, may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Sambrook et al., supra, and are well known in the art.
A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to a polypeptide coding sequence. An operable linkage is a linkage in which the regulatory DNA sequences and coding sequence are connected in such a way as to permit gene expression. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis. Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
If desired, the non-coding region 3' to the coding sequence may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3'-region naturally contiguous to the DNA coding sequence, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the desired host cell, then a 3' region functional in the host cell may be substituted.
Two DNA sequences (such as a promoter region sequence and a RPTP.beta. coding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter to regulate direct the transcription of the RPTP.beta. coding sequence. A promoter region is operably linked to a DNA coding sequence if the promoter is capable of effecting transcription of the coding sequence. Thus, to express the protein, transcriptional and translational signals recognized by an appropriate host are necessary. In order to be "operably linked" it is not necessary that two sequences be immediately adjacent to one another.
A promoter is a double-stranded DNA (or RNA) molecule which is capable of binding to RNA polymerase and promoting the transcription of an "operably linked" nucleic acid coding sequence. As used herein, a "promoter sequence" is the sequence of the promoter which is found on that strand of the DNA (or RNA) which is transcribed by the RNA polymerase. A "promoter sequence complement" has a sequence which is the complement of the "promoter sequence." Hence, upon extension of a primer DNA or RNA adjacent to a single-stranded "promoter sequence complement" or, of a "promoter sequence," a double-stranded molecule is created which will contain a functional promoter, if that extension proceeds towards the "promoter sequence" or the "promoter sequence complement." This functional promoter will direct the transcription of a nucleic acid molecule which is operably linked to that strand of the double-stranded molecule which contains the "promoter sequence" (and not that strand of the molecule which contains the "promoter sequence complement").
Certain RNA polymerases exhibit a high specificity for such promoters. The RNA polymerases of the bacteriophages T7, T3, and SP-6 are especially well characterized, and exhibit high promoter specificity. The promoter sequences which are specific for each of these RNA polymerases also direct the polymerase to transcribe from only one strand of a duplex DNA template. Strand selection is determined by the orientation of the promoter sequence, and determines the direction of transcription since RNA is only polymerized enzymatically by the addition of a nucleotide 5' phosphate to a 3' hydroxyl terminus.
The promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral. Suitable promoters are repressible, or, more preferably, constitutive. Examples of suitable prokaryotic promoters include promoters capable of recognizing the T4 (Malik, S. et al., J. Biol. Chem. 263:1174-1181 (1984); Rosenberg, A. H. et al., Gene 59:191-200 (1987); Shinedling, S. et al., J. Molec. Biol. 195:471-480 (1987); Hu, M. et al., Gene 42:21-30 (1986)), T3, Sp6, and T7 (Chamberlin, M. et al., Nature 228:227-231 (1970); Bailey, J. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 80:2814-2818 (1983); Davanloo, P. et al., Proc. Natl. Acad. Sci. (U.S.A.) 81:2035-2039 (1984)) polymerases; the P.sub.R and P.sub.L promoters of bacteriophage .lambda. (The Bacteriophage Lambda, Hershey, A. D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1973); Lambda II, Hendrix, R. W., Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1980)); the trD, recA, heat shock, and lacZ promoters of E. coli; the .alpha.-amylase (Ulmanen, I., et al., J. Bacteriol. 162:176-182 (1985)) and the .sigma.-28-specific promoters of B. subtilis (Gilman, M. Z., et al., Gene 32:11-20 (1984)); the promoters of the bacteriophages of Bacillus (Gryczan, T. J., In: The Molecular Biology of the Bacilli, Academic Press, Inc., N.Y. (1982)); Streptomyces promoters (Ward, J. M., et al., Mol. Gen. Genet. 203:468-478 (1986)); the int promoter of bacteriophage .lambda.; the bla promoter of the .beta.-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pPR325, etc. Prokaryotic promoters are reviewed by Glick, B. R. (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); Watson, J. D. et al. (In: Molecular Biology of the Gene, Fourth Edition, Benjamin Cummins, Menlo Park, Calif. (1987)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)). Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C., et al., Nature (London) 290:304-310 (1981)); and the yeast gal4 gene promoter (Johnston, S. A., et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P. A., et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)). All of the above listed references are incorporated by reference herein.
Strong promoters are preferred. Examples of such preferred promoters are those which recognize the T3, SP6 and T7 polymerases, the P.sub.L promoter of bacteriophage .lambda., the recA promoter and the promoter of the mouse metallothionein I gene. A most preferred promoter for eukaryotic expression of RPTP is an SV40 promoter such as that driving transcription in the pLSV vector (Livneh, E., et al., (1986) J. Biol. Chem. 261, 12490-12497). The sequences of such polymerase recognition sites are disclosed by Watson, J. D. et al. (In: Molecular Biology of the Gene, Fourth Edition, Benjamin/Cummings Publishing Co., Inc., Menlo Park, Calif., (1987)).
Having now generally described the invention, the same will be more readily understood through reference to the following example which is provided by way of illustration, and is not intended to be limiting of the present invention, unless specified.
6. EXAMPLE
ISOLATION AND CHARACTERIZATION OF HUMAN RPTP.beta. cDNA
6.1. MATERIALS
Restriction endonucleases and modifying enzymes were purchases from Boehringer-Mannheim or New England Biolabs. Taq DNA polymerase was from Perkin-Elmer/Cetus. The .lambda.gt11 forward and reverse primers (24-mers) used in the polymerase chain reactions as well as all sequencing primers, were synthesized on an automated DNA synthesizer (Applied Biosystems, model 380A) using either methoxy or .beta.-cyanoethyl phosphoramidites (House, C., et al., J. Biol. Chem., 262:772-777 (1987)). The .lambda.gt11 human brainstem cDNA library was obtained form the American Type Culture Collection (no. 37432). The LCA (CD45) clone used as a probe for screening the library was received from E. H. Fischer (University of Washington, Seattle). All sequencing reactions were performed using the Sequenase kit (United States Biochemical).
6.2. METHODS
A cDNA clone containing a portion of the coding sequences for RPTP.beta. was isolated after screening a .lambda.gt11 human infant brain stem cDNA library under conditions of reduced stringency with a nick translated LCA probe that included both phosphatase domains (Kaplan et al., Proc. Natl. Acad. Sci. USA 87:7000-7004 (1990)). Since the 5' end of this gene was not present in the original clone, the library was rescreened with a DNA fragment that was generated from the 5' end of the original clone. The probe was labeled with .sup.32 P-dCTP utilizing the random prime method (USB) and hybridization was performed under moderately stringent conditions at 42.degree. C. in a buffer containing 50% formamide, 5.times.SSC, 20 mM Tris-HCl pH 7.6, 1.times. Denhardt's solution, 0.1% SDS and 100 .mu.g/ml of sheared and denatured salmon sperm DNA. After hybridization, phage filters were washed three times for 20 min at 50.degree. C. in a buffer containing 0.1.times.SSC/0.1% SDS and then were processed for autoradiography. The brainstem library was rescreened a total of three times in order to isolate overlapping cDNA clones that contained the entire coding sequence for RPTP.beta..
cDNA inserts from positive recombinant plaque-purified phage were subcloned into the plasmid vector, BlueScript (Stratagene, La Jolla, Calif.), and sequenced by the dideoxy chain termination method using the Sequenase Version 2.0 Kit (USB).
6.3. RESULTS
The present inventors previously isolated a portion of a novel RPTP denoted RPTP.beta. (co-pending commonly assigned U.S. patent application Ser. No. 07/654,188, filed Feb. 26, 1991, from which the present application claims priority; Kaplan et al., 1990, supra). A similar phosphatase was independently cloned by another group and was termed called PTP.zeta. (Krueger et al., 1990, supra; Krueger et al., 1992, supra). The reasons for the sequence differences between RPTP.beta. disclosed herein and PTP.zeta. are not yet clear. Four overlapping cDNA clones containing the entire coding sequence for RPTP.beta. were isolated from a human brain stem library. The deduced amino acid sequence reveals an open reading frame of 2307 amino acids (FIGS. 2A-2B). RPTP.beta. belongs to the high molecular weight, transmembrane class of PTPases. The sequence contains a signal peptide (underlined in FIGS. 2A-2B) followed by a long extracellular domain of 1611 amino acids containing 21 potential N-glycosylation sites (indicated by arrows in FIG. 2A-2B). A hydrophobic, transmembrane peptide (bold sequences in FIG. 2A-2B) joins the extracellular portion of the protein to two tandemly repeated and conserved phosphatase domains (designated DI and DII). One distinguishing feature of this phosphatase is the homology it shares with different isoforms of carbonic anhydrase (CAH) over a stretch of 283 amino acids located at the extreme amino terminus of the protein (designated CAH in FIGS. 2A-2B). In addition to RPTP.beta., the extracellular domain of a related RPTP, RPTP.gamma., shares homology with CAH (co-pending commonly assigned U.S. patent application Ser. No. 07/654,188, filed Feb. 26, 1991, from which the present application claims priority; co-pending commonly assigned U.S. patent application Ser. No. 08/015,986, filed May 10, 1993, titled "Novel Receptor-Type Phosphotyrosine Phosphatase-Gamma).
Alignment of the CAH-related domains of RPTP.beta. and RPTP.gamma. with the six known isoforms of CAH is shown in FIGS. 3A-3C. FIG. 3D shows the percent similarity, taking into account conservative amino acid substitutions, between the CAH-related domain of RPTP.beta., the corresponding domain of RPTP.beta. and the six CAH enzymes. The amino acid sequence similarity of the CAH-related domain of RPTP.beta. to the six CAH isoforms ranges from 45-50%. The highest degree of similarity (58%) exists between the CAH-related sequences of RPTP.beta. and RPTP.gamma.. Therefore, RPTP.beta. and RPTP.gamma. represent a new subgroup of RPTPs characterized by the presence of CAH-related regions in the amino terminal portions of their extracellular domains.
7. EXAMPLE
CHROMOSOMAL LOCALIZATION OF THE HUMAN RPTP.beta. GENE
7.1. METHODS
Isolation, propagation and characterization of parental and somatic cell hybrids used in this study have been described (Durst et al., Proc. Natl. Acad. Sci. USA 84:1070-1074 (1987); Huebner et al., Am. J. Hum. Genet. 48:726-740 (1991)). Presence of specific human chromosomes or regions of chromosomes has been confirmed by DNA hybridization using probes for genes assigned to specific chromosome regions. FIG. 4A depicts diagrammatically the chromosomes or partial chromosomes retained in most of the hybrids used.
Chromosomal in situ hybridization was performed as described (Cannizzarro et al., Cancer Res. 51:3818-3820 (1991)). Slides containing metaphase chromosomes from normal male (46 XY) peripheral blood lymphocytes were aged at 4.degree. C. for 7-10 days and pretreated with ribonuclease A (Sigma) for 1 h at 37.degree. C. The chromosomal DNA was denatured in a hybridization mixture containing 50% formamide, 2.times. SSC and 10% dextran sulfate (pH 7.0). Hybridization was carried out at 37.degree. C. overnight. After rinsing at 39.degree. C. in three changes of 50% formamide and 2.times. SSC, and five changes of 2.times. SSC, slides were dehydrated, air dried, subjected to autoradiography and banded with Wright's-Giemsa stain solution mixed with 1-3 parts of ph 9.2 borate buffer (Cannizzaro et al., supra).
7.2. RESULTS
The chromosomal localization of the human RPTP.beta. gene was initially determined utilizing a panel of rodent-human hybrids carrying defined human chromosomes or chromosome regions. The results from screening the rodent-human hybrids which are summarized-in FIG. 4A, correlates the presence of the human RPTP.beta. locus in hybrid cells with human chromosome 7. A more precise localization of the RPTP.beta.f gene was determined by chromosomal in situ hybridization to metaphase chromosomes of normal human lymphocytes. This technique places the RPTP.beta. gene at 7q31-33 with the most likely position at 7q31.3-q32 which is diagrammatically shown to the right of the chromosome sketch in FIG. 4B.
8. EXAMPLE
EXPRESSION OF RPTP.alpha. RNA
8.1. ISOLATION OF MOUSE SEQUENCES HOMOLOGOUS TO HUMAN RPTP.beta.
Two oligonucleotides in conserved phosphatase domain II were synthesized according to the nucleotide sequence of human RPTP.beta.. These oligonucleotides in conjunction with phage DNA from a mouse brain cDNA library (Clonetech, Palo Alto, Calif.) were used in the PCR with Taq polymerase (Perkin-Elmer) to amplify homologous mouse RPTP.beta. sequences. The amplified product was purified and cloned into the BlueScript plasmid vector (Stratagene, La Jolla, Calif.). Homology was confirmed by DNA sequence analysis as described above. This subsoned fragment is called pBSMBDII.
8.2. NORTHERN ANALYSIS
Total cellular RNA was prepared with the Stratagene RNA isolation kit. Poly A.sup.+ RNA was further selected utilizing oligo-dt cellulose chromatography (Stratagene). For Northern analysis, the RNA was separated on a 1.0% agarose/2.2M formaldehyde gel and transferred to a Nytran membrane (Schleicher and Schuell) by capillary action. The membrane was prehybridized and hybridized in 0.5M sodium phosphate ph 7.2, 7% SDS, 1 mM EDTA, 100 .mu.g/ml salmon sperm DNA and then washed in 40 mM sodium phosphate ph 7.2, 1% SDS, 1 mM EDTA at 65.degree. C. For the blot containing RNA isolated from various mouse tissues, a .sup.32 P-labeled probe was made utilizing PBSMBDII as template in the random primer labeling reaction (US Biochemicals). The human glioblastoma and neuroblastoma RNA blots were probed with labeled restriction fragments isolated from different parts of the human RPTP.beta. cDNA clones.
8.3. ANTIBODIES
A peptide derived from the carboxy-terminal 15 amino acids of human RPTP.beta. was synthesized and coupled to keyhole limpet hemocyanin according to standard procedures. Two rabbits were inoculated to produce polyclonal antisera against RPTP.gamma.. Anti-EGF receptor immunoprecipitates were performed with RK2 antibody which recognizes the EGF receptor (Kris et al., Cell 40:619-625 (1985)).
8.4. CELL LABELING AND IMMUNOPRECIPITATION
The human neuroblastoma cell line, Lan 5, was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Cultured cells were incubated with 10 .mu.g/ml tunicamycin (Sigma Chemical Co.) for 1 hour prior to �.sup.35 S!-methionine labeling. Treated and untreated cells were washed twice with methionine free DMEM and labeled for 4 hours with 0.15 mCi/ml �.sup.35 S!-methionine (NEN, DuPont) in DMEM lacking methionine and supplemented with 1% dialyzed FBS. During the labeling period, 10 .mu.g/ml tunicamycin-was added to the medium of the treated cells. Cells were then washed with ice cold phosphate buffered saline (PBS) and solubilized in a lysis buffer containing 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pH 7.5), 150 mM NaCl, 1.0% Triton X-100, 10% glycerol, 1.5 mM MgCl.sub.2, 1 mM ethylene glycol-bis �B-aminoethyl ether!-N,N,N', N'-tetraacetic acid (EGTA), 10 .mu.g of leupeptin per ml, 1 mM phenylmethylsulfonyl fluoride, and 10 .mu.g of aprotinin per ml. Cell lysates were clarified and then immunoprecipitated with normal rabbit serum, rabbit anti-RPTP.beta. antiserum or RK2 antiserum for 2 hour at 4.degree. C. The immune complexes were precipitated with Protein A-Sepharose (Sigma Chemical Co.) for 45 min at 4.degree. C. and washed 10 times with RIPA buffer (20 mM Tris-HCl, pH 7.6, 300 mM NaCl, 2 mM EDTA, 1.0% Triton X-100, 1.0% sodium deoxycholate and 0.1% SDS). The immunoprecipitated material was analyzed on a 7.5% SDS-polyacrylamide gel and fluorography.
5 8.5. RESULTS: DETECTION OF RPTP.beta. EXPRESSION IN A HUMAN NEUROBLASTOMA CELL LINE, Lan 5
Since all of the clones encoding the entire sequence for RPTP.beta. were isolated from a human brain stem library, the expression of RPTP.beta. mRNA was examined in different human glioblastoma cell lines and a human neuroblastoma cell line, Lan 5 (Sonnenfield et al., J. Neurosci. Res. 8:375-391 (1982)). A human RPTP.beta. probe hybridized to three major transcripts of 8.8, 7.5 and 6.4 kb, respectively (FIG. 5A). These transcripts were only detected in RNA isolated from the Lan 5 neuroblastoma cell line and were absent in the RNA isolated from the four glioblastoma cell lines even though similar amounts of total cellular RNA were loaded as revealed by ethidium bromide staining of the 28S and 18S ribosomal RNAs.
In order to obtain further insights into the nature of the three RPTP.beta. transcripts, performed an additional Northern analysis was performed on RNA isolated from Lan 5 cells with probes derived from nonconserved sequences in the extracellular domain of RPTP.beta.. An identical pattern of transcripts was revealed utilizing these probes.
The Lan 5 neuroblastoma cell line was used to study the endogenous expression of RPTP.beta. protein. Cell lysates prepared from cultures labeled with �.sup.35 S!methionine for 4 hours were immunoprecipitated with normal rabbit serum or anti-RPTP.beta. antiserum (FIG. 6). A protein with apparent molecular weight of approximately 300 kDa was recognized by the anti-RPTP.beta. antiserum but not by normal rabbit serum (lanes 1 and 2). Since there are 21 potential N-glycosylation sites, tunicamycin was employed to examine the possibility that the 300 kDa protein immunoprecipitated by the anti-RPTP.beta. antiserum was a glycosylated form of RPTP.beta.. The effects of tunicamycin treatment on the mobility of RPTP.beta. immunoprecipitated from �.sup.35 S!methionine-labeled cells was compared to its ability to inhibit the glycosylation of the EGF receptor which is also expressed in this cell line. Untreated cell lysates and lysates prepared from cells treated with tunicamycin were immunoprecipitated with an anti EGF receptor antibody (RK2) that recognizes the 170 kDa glycosylated and the 135 kDa nonglycosylated forms of the EGF receptor (Kris et al., supra). These results are also shown in FIG. 6, lanes 4 and 5). The protein immunoprecipitated with anti-RPTP.beta. antiserum from Lan 5 cells that had been metabolically labeled in the presence of tunicamycin (FIG. 6, lane 3) migrated with an apparent molecular weight of 250 kDa. This result was consistent with the predicted molecular weight of 254 kDa deduced from the amino acid sequence of RPTP.beta..
9. EXAMPLE
IDENTIFICATION OF A VARIANT FORM OF RPTP.beta.
The overlapping human cDNA clones collectively contained approximately 8.1 kb of coding and noncoding sequences and appeared to represent the largest transcript that is 8.8 kb in length. In screening the human brain stem library and a human caudate library (Stratagene, La Jolla, Calif.), the present inventors isolated two independent cDNA clones that each contained an identical deletion of 2577 nucleotides from the extracellular domain of RPTP.beta.. This in-frame deletion joined amino acid 754 to amino acid 1614 (shown by the open arrows in FIGS. 2A-2B and in FIG. 7A) thereby maintaining the transmembrane domain and the two phosphatase domains. A deletion of this size could account for the difference between the 8.8 kb and 6.4 kb transcripts expressed in the Lan 5 neuroblastoma cell line (FIG. 5A). Hence, duplicate blots were made from RNA isolated from the Lan 5 cell line. One blot was analyzed with a probe that should hybridize to both the full length and deleted forms of RPTP.gamma. (P1). The other blot was analyzed with a probe that should only hybridized to the full length form (P2). The location of probes 1 and 2 in the full length RPTP.beta. cDNA is shown in FIG. 7A. Northern analysis with the two probes shown in FIG. 7B revealed that probe 1 hybridized to the three distinct transcripts (P1) whereas probe 2 hybridized only to the 7.5 and 8.8 kb transcripts (P2). This result suggested that the 6.4 kb transcript represented a deleted variant form of RPTP.beta. which could be the result of alternative mRNA splicing. However, only the high molecular weight form of RPTP.beta. was detected following immunoprecipitation with anti RPTP.beta. antibodies and SDS-PAGE analysis (FIG. 6). The reason for the inability to detect the low molecular weight form in lysates from of RPTP.beta. in Lan5 cells is not clear.
10. EXAMPLE
TISSUE SPECIFIC EXPRESSION OF RPTP.beta.
10.1. METHODS: IN SITU HYBRIDIZATION ANALYSIS
Fresh frozen tissue was cut on a cryostat into 20 .mu.m thick sections and thaw mounted onto gelatin coated slides. The sections were fixed in 4% paraformaldehyde in 0.1M sodium phosphate (pH 7.4) for 30 min and rinsed three time for 5 min each in 0. 1M sodium phosphate and once for 10 min in 2.times. SSC. Two probes were used in the hybridization analysis; (1) a 49 base oligonucleotide complementary to phosphatase domain II, and (2) a 50 base oligonucleotide complementary to the carbonic anhydrase-like domain. The oligonucleotides was labeled with �(.alpha.-.sup.35 S!dATP (NEN, DuPont) using terminal deoxynucleotidyl transferase (Boehringer Mannheim) and purified using Sephadex G25 quick spin columns (Boehringer Mannheim). The specific activity of the labeled probes was between 5.times.10.sup.8 and 1.times.10.sup.9 cpm/.mu.g. Prehybridizations and hybridizations were carried out in a buffer containing 50% deionized formamide, 4.times. SSC, 1.times. Denhardt's, 500 .mu.g/ml denatured salmon sperm DNA, 250 .mu.g/ml yeast tRNA and 10% dextran sulfate. The tissue was incubated for 12 h at 45.degree. C. in hybridization solution containing the labeled probe (1.times.10.sup.6 cpm/section) and 10 mM dithiothreitol (DTT). Controls for specificity were performed on adjacent sections by diluting the labeled oligonucleotides with a 30-fold concentration of the appropriate unlabeled oligonucleotide and by hybridization with a sense probe. After hybridization, the sections were washed in 2 changes of 2.times. SSC at room temperature for 1 h, 1.times. SSC at 55.degree. C. for 30 min, 0.5.times. SSC at 55.degree. C. for 30 min, 0.5.times. SSC at room temperature for 15 min and dehydrated in 60%, 80%, and 100% ethanol. After air drying, the sections were exposed to X-ray film for 5-10 d.
10.2. RESULTS
10.2.1. Tissue Specific Expression of RPTP.beta.
Northern analysis of various tissue RNAs was performed to determine the tissue-specific expression of RPTP.beta.. The probe used in this analysis was a portion of the murine homolog of RPTP.beta. that was amplified in the PCR (described above) and contains 405 nucleotides encoding 135 amino acids of Domain II. Based on a nucleotide sequence comparison to the equivalent region of the human cDNA clone, the murine and human clones are 88% identical at the nucleotide level in this region of Domain II of RPTP.beta.. The results of this Northern analysis (FIG. 5B) indicated the presence of two major transcripts of 8.8 and 6.4 kb, respectively. These two transcripts are similar in size to the largest and smallest transcripts observed in Lan 5 RNA (see FIG. 5A). However, no band corresponding to the 7.5 kb transcript was detected. A minor transcript of approximately 9.4 kb was sometimes observed in RNA prepared from mouse brain tissue and may represent cross-reaction to a highly related phosphatase.
RPTP.beta. transcripts were not detected in the lung, heart, liver, spleen, kidney, muscle, testes and thymus. The quality of the RNA isolated from the various tissues was compared by parallel hybridization with an actin probe of the same blots (FIG. 5C). Hence, Northern hybridization analysis indicates that RPTP.beta. mRNA is strictly expressed in the brain of the adult mouse.
10.2.2. Localization of RPTP.beta. Expression in the Brain
In order to more precisely localize the expression of RPTP.alpha. in the brain, in situ hybridization was performed on brain tissue from the adult and the embryonic mouse. The results of this analysis confirmed that RPTP.beta. was expressed in the central nervous system. In a day 20 embryonic mouse (E20), a high level of expression was observed in the ventricular and subventricular zones of the brain (FIG. 8A), and in the spinal cord.
The level of expression was lower in the adult brain, and was localized to the Purkinje cell layer of the cerebellum, the dentate gyrus, and the subependymal layer of the anterior horn of lateral ventricle (FIG. 8B). Both probes (complementary to the CAH-like domain and the second phosphatase domain) gave identical results. The addition of a 30-fold excess of unlabeled oligonucleotide completely blocked the labeling in all areas. Furthermore, no signal was observed in adjacent sections hybridized with the sense probe, indicating that the probes hybridize to mRNA in a sequence specific manner.
These results demonstrated that RPTP.beta. has a restricted tissue specificity to specific regions of the nervous system.
11. GENERAL DISCUSSION FOR SECTIONS 6-10
The present inventor has cloned and characterized a human RPTP that is expressed in the central nervous system and cannot be detected in other murine tissues. Amino acid sequence analysis revealed that RPTP.beta. has a striking degree of sequence homology at it N-terminal end with various isoforms of the enzyme CAH over a stretch of 283 amino acids. RPTP.gamma. was also found to contain a CAH-related sequence near the amino terminus in the extracellular domain. Therefore, RPTP.beta. and RPTP.gamma. are concluded to be members of a new subgroup of transmembrane phosphatases that can be classified on the basis of a CAH-related domain present in the amino terminal regions of their extracellular domain.
A three dimensional model of the CAH-related domain of RPTP.gamma. based upon the known crystal structure of CAH and a detailed description of this model was provided elsewhere by the present inventors laboratory (see, for example, co-pending commonly assigned U.S. patent application Ser. No. .sub.------, filed May 10, 1993, titled "Novel Receptor-Type Phosphotyrosine Phosphatase-Gamma). Since key residues known to participate in the catalytic activity of CAH are missing from both RPTP.beta. (as well as from RPTP.gamma.), it is likely that the CAH domains in these two phosphatases do not possess classical carbonic anhydrase activity.
It has been postulated that under-expression or inactivation of PTPases might lead to oncogenesis suggesting that PTPases may function as tumor suppressors. This finds support in the findings from the present inventor's laboratory that RPTP.gamma. maps to human chromosome region 3p21, a region is frequently deleted in both renal cell and lung carcinomas (LaForgia et al., supra). The presently reported localization of the human RPTP.beta. gene to human chromosome 7q31.3-q32 renders it important to investigate loss of, or mutations in, the RPTP.beta. gene in certain tumor types, especially those exhibiting deletions of 7q.
In the case of the PTPase termed CD45, alternative mRNA splicing was shown to produce six distinct isoforms, as a result of the differential usage of three exons encoding sequences in the extracellular domain (Streuli et al., J. Exp. Med. 166:1548-1566 (1987); Streuli et al., EMBO J. 8:787-796 (1989)). Alternative splicing within the first catalytic domain of RPTP.alpha. has also been described (Matthews et al., Proc. Natl. Acad. Sci. USA 87:4444-4448 (1990)). The results of Northern blot analysis reveal the presence of multiple RPTP.beta. transcripts in both mouse brain and a human neuroblastoma cell line, Lan 5, that appear to either result from alternatively spliced transcripts or from highly related genes. The Northern blot analyses indicate that the 8.8 kb and the 6.4 kb transcripts are strictly expressed in the brain. Northern blot analysis described in FIGS. 5A-5C in conjunction with the analysis of two independently isolated cDNA clones suggest that the smallest transcript of 6.4 kb may result from a deletion of approximately 2.6 kb encoding a large portion of the extracellular domain of RPTP.beta..
The importance of tyrosine phosphorylation in the control and regulation of specific neuronal processes is under intense investigation. It was demonstrated that tyrosine phosphorylation is enhanced at the time of synaptogenesis during development (Cudmore et al., J. Neurochem. 57:1240-1248 (1991); Girault et al., Proc. Natl. Acad. Sci. USA 89:2769-2773 (1992); Qu et al., Neuron 2:367-378 (1990)) and may play a role in synaptic plasticity in the adult (Girault et al., J. Neurochem. 58:518-528 (1992)). In addition, NGF and other neurotrophic factors mediate their effects on neuronal survival by binding to and activating receptors with tyrosine kinase activity (Cordon-Cardo et al., Cell 66:1-20 (1991); Kaplan et al., Science 252:554-557 (1991); Klein et al., Cell 65:189-197 (1991); Schlessinger, J. et al., Neuron 9:383-391 (1992). Stimulation of other growth factor receptors with tyrosine kinase activities have profound effects on survival and differentiation of cultured neurons (Aizenman et al., Brain Res. 406:32-42 (1987); Morrison et al., Science 238:72-75 (1987)) suggesting a potential role in normal neuronal development. Finally, several cytoplasmic tyrosine kinases including the products of the proto-oncogenes c-src and c-yes are expressed at high levels in specific regions of neurons of the adult brain (Pang et al., Proc. Natl. Acad. Sci. USA 85:762-7661988a; Pang et al., Soc. Neurosci. Abstr. 14:44.6 (1988); Maness et al., Proc. Natl. Acad. Sci. USA 85:5001-5005 (1988); Sudol et al., Molec. Cell. Biol. 9:4545-4549 (1989)).
RPTP.beta. represents the first cloned mammalian PTPase whose tissue specific expression is restricted to the nervous system. Several Drosophila RPTPs have been identified that are selectively expressed on central nervous system axons in the embryo (Tian et al., Cell 67:675-685 (1991); Yang et al., Cell 67:661-673 1991). The identification of brain-specific PTPases, such as RPTP.beta., is an important initial step towards understanding the regulation of tyrosine phosphorylation by the PTPases in mammalian neural tissues. The relatively high level of RPTP.beta. expression in the embryonic central nervous system CNS suggests that it plays a role in the development of the nervous system. It is of note that the ventricular and subventricular zones are the primary sites of cell division in the developing brain (Altman et al., Exp. Neurol. 107:23-35 (1990). Interestingly, RPTP.beta. is expressed in the few regions of the adult brain that continue to show mitotic activity such as the dentate gyrus and the subependymal layer of the anterior horn of the lateral ventricle. Immunocytochemical techniques are being applied to determine the precise cellular distribution of RPTP.beta. protein and to help elucidate its role in the regulation of specific developmental and neuronal processes.
The sequences of the conserved phosphatase domains of the human RPTP.beta., as described in detail above, is compared in Table I, below, with RPTP.alpha. and RPTP.gamma. and with the sequences of LCA, LAR, and two soluble PTPases, placental phosphatase 1B and T-cell PTPase. The two soluble enzymes have a sequence identity of 70%; however, when each is compared with the RPTPs (Phosphatase domains PD1 or PD2, also referred to above as PDI and PDII), this number drops to 29-42%. In all cases, the soluble PTPases showed a greater identity with PD1 than with PD2 of the RPTPs. RPTP.alpha. appears to be most related to LAR, since their PD1 sequences are 56% identical and their PD2 sequences are 52% identical. The conserved domains of RPTP.beta. and RPTP.gamma. are most related to each other, even more so than are the two soluble PTPases, .beta. and .gamma. being 75% identical in both PD1 and PD2. It is interesting that, in general, the sequence relationship between PD1 and PD2 within any RPTP appears to be no closer than that seen between different members of the family, i.e., the identities between PD1 and PD2 range from a high of 47% for LAR to a low of 29% for RPTP .gamma..
While the cytoplasmic domains of RPTP.alpha., .beta., and .gamma. are highly conserved, the extracellular domains of these receptors are unrelated to one another as well as to those of LAR and LCA. This suggests that each of these receptors has its own distinct ligand. It is likely that the binding of such ligands to the RPTPs plays a crucial role, together with growth factor receptors exhibiting PTKase activity, in the regulation of the level of tyrosine phosphorylation of targets proteins involved in signal transduction. The diversity of the RPTPs described herein reveals the existence of a multigene family. Greater understanding of structure-function relationships among these membrane receptors will provide important insights into the mechanisms involved in cell growth, differentiation, and oncogenesis.
Although the inventor does not intend to be bound by any particular theory, the high interspecies conservation of the catalytic domains of the various RPTPs indicates an important role for these receptors in cell growth control.
TABLE 4__________________________________________________________________________Identities Between Conserved Phosphatase Domains (Percent) PTPase T-cell LCA LAR RPTPase-.alpha. RPTPase-.beta. RPTPAse-.gamma. 1B PTPase PD1 PD1 PD1 PD2 PD1 PD2 PD1 PD2 PD1 PD2__________________________________________________________________________PTPase 1B 100 -- -- -- -- -- -- -- -- -- -- --T-cell PTPase 70 100 -- -- -- -- -- -- -- -- -- --LCA PD1 37 36 100 -- -- -- -- -- -- -- -- --LCA PD2 30 26 31 100 -- -- -- -- -- -- -- --LAR PD1 39 42 50 28 100 -- -- -- -- -- -- --LAR PD2 29 33 42 34 45 100 -- -- -- -- -- --R-PTPase-.alpha. PD1 36 38 50 32 56 45 100 -- -- -- -- --R-PTPase-.alpha. PD2 33 34 40 32 41 52 43 100 -- -- -- --R-PTPase-.beta. PD1 35 39 41 31 33 41 47 33 100 -- -- --R-PTPase-.beta. PD2 29 30 31 30 31 34 31 37 30 100 -- --R-PTPase-.gamma. PD1 35 34 32 29 39 36 34 32 75 27 100 --R-PTPase-.gamma. PD2 29 29 30 28 32 36 31 34 33 75 29 100__________________________________________________________________________ Alignments of the conserved phosphatase domains were carried out as described above. The regions compared are designated in FIG. 3C and FIGS. 5A-5C. PD = phosphatase domain.
The references cited above are all incorporated by reference herein, whether specifically incorporated or not.
Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 9- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 2308 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- Met Arg Ile Leu Lys Arg Phe Leu Ala Cys Il - #e Gln Leu Leu Cys Val# 15- Cys Arg Leu Asp Trp Ala Asn Gly Tyr Tyr Ar - #g Gln Gln Arg Lys Leu# 30- Val Glu Glu Ile Gly Trp Ser Tyr Thr Gly Al - #a Leu Asn Gln Lys Asn# 45- Trp Gly Lys Lys Tyr Pro Thr Cys Asn Ser Pr - #o Lys Gln Ser Pro Ile# 60- Asn Ile Asp Glu Asp Leu Thr Gln Val Asn Va - #l Asn Leu Lys Lys Leu# 80- Lys Phe Gln Gly Trp Asp Lys Thr Ser Leu Gl - #u Asn Thr Phe Ile His# 95- Asn Thr Gly Lys Thr Val Glu Ile Asn Leu Th - #r Asn Asp Tyr Arg Val# 110- Ser Gly Gly Val Ser Glu Met Val Phe Lys Al - #a Ser Lys Ile Thr Phe# 125- His Trp Gly Lys Cys Asn Met Ser Ser Asp Gl - #y Ser Glu His Ser Leu# 140- Glu Gly Gln Lys Phe Pro Leu Glu Met Gln Il - #e Tyr Cys Phe Asp Ala145 1 - #50 1 - #55 1 -#60- Asp Arg Phe Ser Ser Phe Glu Glu Ala Val Ly - #s Gly Lys Gly Lys Leu# 175- Arg Ala Leu Ser Ile Leu Phe Glu Val Gly Th - #r Glu Glu Asn Leu Asp# 190- Phe Lys Ala Ile Ile Asp Gly Val Glu Ser Va - #l Ser Arg Phe Gly Lys# 205- Gln Ala Ala Leu Asp Pro Phe Ile Leu Leu As - #n Leu Leu Pro Asn Ser# 220- Thr Asp Lys Tyr Tyr Ile Tyr Asn Gly Ser Le - #u Thr Ser Pro Pro Cys225 2 - #30 2 - #35 2 -#40- Thr Asp Thr Val Asp Trp Ile Val Phe Lys As - #p Thr Val Ser Ile Ser# 255- Glu Ser Gln Leu Ala Val Phe Cys Glu Val Le - #u Thr Met Gln Gln Ser# 270- Gly Tyr Val Met Leu Met Asp Tyr Leu Gln As - #n Asn Phe Arg Glu Gln# 285- Gln Tyr Lys Phe Ser Arg Gln Val Phe Ser Se - #r Tyr Thr Gly Lys Glu# 300- Glu Ile His Glu Ala Val Cys Ser Ser Glu Pr - #o Glu Asn Val Gln Ala305 3 - #10 3 - #15 3 -#20- Asp Pro Glu Asn Tyr Thr Ser Leu Leu Val Th - #r Trp Glu Arg Pro Arg# 335- Val Val Tyr Asp Thr Met Ile Glu Lys Phe Al - #a Val Leu Tyr Gln Gln# 350- Leu Asp Gly Glu Asp Gln Thr Lys His Glu Ph - #e Leu Thr Asp Gly Tyr# 365- Gln Asp Leu Gly Ala Ile Leu Asn Asn Leu Le - #u Pro Asn Met Ser Tyr# 380- Val Leu Gln Ile Val Ala Ile Cys Thr Asn Gl - #y Leu Tyr Gly Lys Tyr385 3 - #90 3 - #95 4 -#00- Ser Asp Gln Leu Ile Val Asp Met Pro Thr As - #p Asn Pro Glu Leu Asp# 415- Leu Phe Pro Glu Leu Ile Gly Thr Glu Glu Il - #e Ile Lys Glu Glu Glu# 430- Glu Gly Lys Asp Ile Glu Glu Gly Ala Ile Va - #l Asn Pro Gly Arg Asp# 445- Ser Ala Thr Asn Gln Ile Arg Lys Lys Glu Pr - #o Gln Ile Ser Thr Thr# 460- Thr His Tyr Asn Arg Ile Gly Thr Lys Tyr As - #n Glu Ala Lys Thr Asn465 4 - #70 4 - #75 4 -#80- Arg Ser Pro Thr Arg Gly Ser Glu Phe Ser Gl - #y Lys Gly Asp Val Pro# 495- Asn Thr Ser Leu Asn Ser Thr Ser Gln Pro Va - #l Thr Lys Leu Ala Thr# 510- Glu Lys Asp Ile Ser Leu Thr Ser Gln Thr Va - #l Thr Glu Leu Pro Pro# 525- His Thr Val Glu Gly Thr Ser Ala Ser Leu As - #n Asp Gly Ser Lys Thr# 540- Val Leu Arg Ser Pro His Met Asn Leu Ser Gl - #y Thr Ala Glu Ser Leu545 5 - #50 5 - #55 5 -#60- Asn Thr Val Ser Ile Thr Glu Tyr Glu Glu Gl - #u Ser Leu Leu Thr Ser# 575- Phe Lys Leu Asp Thr Gly Ala Glu Asp Ser Se - #r Gly Ser Ser Pro Ala# 590- Thr Ser Ala Ile Pro Phe Ile Ser Glu Asn Il - #e Ser Gln Gly Tyr Ile# 605- Phe Ser Ser Glu Asn Pro Glu Thr Ile Thr Ty - #r Asp Val Leu Ile Pro# 620- Glu Ser Ala Arg Asn Ala Ser Glu Asp Ser Th - #r Ser Ser Gly Ser Glu625 6 - #30 6 - #35 6 -#40- Glu Ser Leu Lys Asp Pro Ser Met Glu Gly As - #n Val Trp Phe Pro Ser# 655- Ser Thr Asp Ile Thr Ala Gln Pro Asp Val Gl - #y Ser Gly Arg Glu Ser# 670- Phe Leu Gln Thr Asn Tyr Thr Glu Ile Arg Va - #l Asp Glu Ser Glu Lys# 685- Thr Thr Lys Ser Phe Ser Ala Gly Pro Val Me - #t Ser Gln Gly Pro Ser# 700- Val Thr Asp Leu Glu Met Pro His Tyr Ser Th - #r Phe Ala Tyr Phe Pro705 7 - #10 7 - #15 7 -#20- Thr Glu Val Thr Pro His Ala Phe Thr Pro Se - #r Ser Arg Gln Gln Asp# 735- Leu Val Ser Thr Val Asn Val Val Tyr Ser Gl - #n Thr Thr Gln Pro Val# 750- Tyr Asn Gly Glu Thr Pro Leu Gln Pro Ser Ty - #r Ser Ser Glu Val Phe# 765- Pro Leu Val Thr Pro Leu Leu Leu Asp Asn Gl - #n Ile Leu Asn Thr Thr# 780- Pro Ala Ala Ser Ser Ser Asp Ser Ala Leu Hi - #s Ala Thr Pro Val Phe785 7 - #90 7 - #95 8 -#00- Pro Ser Val Asp Val Ser Phe Glu Ser Ile Le - #u Ser Ser Tyr Asp Gly# 815- Ala Pro Leu Leu Pro Phe Ser Ser Ala Ser Ph - #e Ser Ser Glu Leu Phe# 830- Arg His Leu His Thr Val Ser Gln Ile Leu Pr - #o Gln Val Thr Ser Ala# 845- Thr Glu Ser Asp Lys Val Pro Leu His Ala Se - #r Leu Pro Val Ala Gly# 860- Gly Asp Leu Leu Leu Glu Pro Ser Leu Ala Gl - #n Tyr Ser Asp Val Leu865 8 - #70 8 - #75 8 -#80- Ser Thr Thr His Ala Ala Ser Lys Thr Leu Gl - #u Phe Gly Ser Glu Ser# 895- Gly Val Leu Tyr Lys Thr Leu Met Phe Ser Gl - #n Val Glu Pro Pro Ser# 910- Ser Asp Ala Met Met His Ala Arg Ser Ser Gl - #y Pro Glu Pro Ser Tyr# 925- Ala Leu Ser Asp Asn Glu Gly Ser Gln His Il - #e Phe Thr Val Ser Tyr# 940- Ser Ser Ala Ile Pro Val His Asp Ser Val Gl - #y Val Thr Tyr Gln Gly945 9 - #50 9 - #55 9 -#60- Ser Leu Phe Ser Gly Pro Ser His Ile Pro Il - #e Pro Lys Ser Ser Leu# 975- Ile Thr Pro Thr Ala Ser Leu Leu Gln Pro Th - #r His Ala Leu Ser Gly# 990- Asp Gly Glu Trp Ser Gly Ala Ser Ser Asp Se - #r Glu Phe Leu Leu Pro# 10050- Asp Thr Asp Gly Leu Thr Ala Leu Asn Ile Se - #r Ser Pro Val Ser Val# 10205- Ala Glu Phe Thr Tyr Thr Thr Ser Val Phe Gl - #y Asp Asp Asn Lys Ala# 10401030 - # 1035- Leu Ser Lys Ser Glu Ile Ile Tyr Gly Asn Gl - #u Thr Glu Leu Gln Ile# 10550- Pro Ser Phe Asn Glu Met Val Tyr Pro Ser Gl - #u Ser Thr Val Met Pro# 10705- Asn Met Tyr Asp Asn Val Asn Lys Leu Asn Al - #a Ser Leu Gln Glu Thr# 10850- Ser Val Ser Ile Ser Ser Thr Lys Gly Met Ph - #e Pro Gly Ser Leu Ala# 11005- His Thr Thr Thr Lys Val Phe Asp His Glu Il - #e Ser Gln Val Pro Glu# 11201110 - # 1115- Asn Asn Phe Ser Val Gln Pro Thr His Thr Va - #l Ser Gln Ala Ser Gly# 11350- Asp Thr Ser Leu Lys Pro Val Leu Ser Ala As - #n Ser Glu Pro Ala Ser# 11505- Ser Asp Pro Ala Ser Ser Glu Met Leu Ser Pr - #o Ser Thr Gln Leu Leu# 11650- Phe Tyr Glu Thr Ser Ala Ser Phe Ser Thr Gl - #u Val Leu Leu Gln Pro# 11805- Ser Phe Gln Ala Ser Asp Val Asp Thr Leu Le - #u Lys Thr Val Leu Pro# 12001190 - # 1195- Ala Val Pro Ser Asp Pro Ile Leu Val Glu Th - #r Pro Lys Val Asp Lys# 12150- Ile Ser Ser Thr Met Leu His Leu Ile Val Se - #r Asn Ser Ala Ser Ser# 12305- Glu Asn Met Leu His Ser Thr Ser Val Pro Va - #l Phe Asp Val Ser Pro# 12450- Thr Ser His Met His Ser Ala Ser Leu Gln Gl - #y Leu Thr Ile Ser Tyr# 12605- Ala Ser Glu Lys Tyr Glu Pro Val Leu Leu Ly - #s Ser Glu Ser Ser His# 12801270 - # 1275- Gln Val Val Pro Ser Leu Tyr Ser Asn Asp Gl - #u Leu Phe Gln Thr Ala# 12950- Asn Leu Glu Ile Asn Gln Ala His Pro Pro Ly - #s Gly Arg His Val Phe# 13105- Ala Thr Pro Val Leu Ser Ile Asp Glu Pro Le - #u Asn Thr Leu Ile Asn# 13250- Lys Leu Ile His Ser Asp Glu Ile Leu Thr Se - #r Thr Lys Ser Ser Val# 13405- Thr Gly Lys Val Phe Ala Gly Ile Pro Thr Va - #l Ala Ser Asp Thr Phe# 13601350 - # 1355- Val Ser Thr Asp His Ser Val Pro Ile Gly As - #n Gly His Val Ala Ile# 13750- Thr Ala Val Ser Pro His Arg Asp Gly Ser Va - #l Thr Ser Thr Lys Leu# 13905- Leu Phe Pro Ser Lys Ala Thr Ser Glu Leu Se - #r His Ser Ala Lys Ser# 14050- Asp Ala Gly Leu Val Gly Gly Gly Glu Asp Gl - #y Asp Thr Asp Asp Asp# 14205- Gly Asp Asp Asp Asp Asp Asp Arg Gly Ser As - #p Gly Leu Ser Ile His# 14401430 - # 1435- Lys Cys Met Ser Cys Ser Ser Tyr Arg Glu Se - #r Gln Glu Lys Val Met# 14550- Asn Asp Ser Asp Thr His Glu Asn Ser Leu Me - #t Asp Gln Asn Asn Pro# 14705- Ile Ser Tyr Ser Leu Ser Glu Asn Ser Glu Gl - #u Asp Asn Arg Val Thr# 14850- Ser Val Ser Ser Asp Ser Gln Thr Gly Met As - #p Arg Ser Pro Gly Lys# 15005- Ser Pro Ser Ala Asn Gly Leu Ser Gln Lys Hi - #s Asn Asp Gly Lys Glu# 15201510 - # 1515- Glu Asn Asp Ile Gln Thr Gly Ser Ala Leu Le - #u Pro Leu Ser Pro Glu# 15350- Ser Lys Ala Trp Ala Val Leu Thr Ser Asp Gl - #u Glu Ser Gly Ser Gly# 15505- Gln Gly Thr Ser Asp Ser Leu Asn Glu Asn Gl - #u Thr Ser Thr Asp Phe# 15650- Ser Phe Ala Asp Thr Asn Glu Lys Asp Ala As - #p Gly Ile Leu Ala Ala# 15805- Gly Asp Ser Glu Ile Thr Pro Gly Phe Pro Gl - #n Ser Pro Thr Ser Ser# 16001590 - # 1595- Val Thr Ser Glu Asn Ser Glu Val Phe His Va - #l Ser Glu Ala Glu Ala# 16150- Ser Asn Ser Ser His Glu Ser Arg Ile Gly Le - #u Ala Glu Gly Leu Glu# 16305- Ser Glu Lys Lys Ala Val Ile Pro Leu Val Il - #e Val Ser Ala Leu Thr# 16450- Phe Ile Cys Leu Val Val Leu Val Gly Ile Le - #u Ile Tyr Trp Arg Lys# 16605- Cys Phe Gln Thr Ala His Phe Tyr Leu Glu As - #p Ser Thr Ser Pro Arg# 16801670 - # 1675- Val Ile Ser Thr Pro Pro Thr Pro Ile Phe Pr - #o Ile Ser Asp Asp Val# 16950- Gly Ala Ile Pro Ile Lys His Phe Pro Lys Hi - #s Val Ala Asp Leu His# 17105- Ala Ser Ser Gly Phe Thr Glu Glu Phe Glu Gl - #u Val Gln Ser Cys Thr# 17250- Val Asp Leu Gly Ile Thr Ala Asp Ser Ser As - #n His Pro Asp Asn Lys# 17405- His Lys Asn Arg Tyr Ile Asn Ile Val Ala Ty - #r Asp His Ser Arg Val# 17601750 - # 1755- Lys Leu Ala Gln Leu Ala Glu Lys Asp Gly Ly - #s Leu Thr Asp Tyr Ile# 17750- Asn Ala Asn Tyr Val Asp Gly Tyr Asn Arg Pr - #o Lys Ala Tyr Ile Ala# 17905- Ala Gln Gly Pro Leu Lys Ser Thr Ala Glu As - #p Phe Trp Arg Met Ile# 18050- Trp Glu His Asn Val Glu Val Ile Val Met Il - #e Thr Asn Leu Val Glu# 18205- Lys Gly Arg Arg Lys Cys Asp Gln Tyr Trp Pr - #o Ala Asp Gly Ser Glu# 18401830 - # 1835- Glu Tyr Gly Asn Phe Leu Val Thr Gln Lys Se - #r Val Gln Val Leu Ala# 18550- Tyr Tyr Thr Val Arg Asn Phe Thr Leu Arg As - #n Thr Lys Ile Lys Lys# 18705- Gly Ser Gln Lys Gly Arg Pro Ser Gly Arg Va - #l Val Thr Gln Tyr His# 18850- Tyr Thr Gln Trp Pro Asp Met Gly Val Pro Gl - #u Tyr Ser Leu Pro Val# 19005- Leu Thr Phe Val Arg Lys Ala Ala Tyr Ala Ly - #s Arg His Ala Val Gly# 19201910 - # 1915- Pro Val Val Val His Cys Ser Ala Gly Val Gl - #y Arg Thr Gly Thr Tyr# 19350- Ile Val Leu Asp Ser Met Leu Gln Gln Ile Gl - #n His Glu Gly Thr Val# 19505- Asn Ile Phe Gly Phe Leu Lys His Ile Arg Se - #r Gln Arg Asn Tyr Leu# 19650- Val Gln Thr Glu Glu Gln Tyr Val Phe Ile Hi - #s Asp Thr Leu Val Glu# 19805- Ala Ile Leu Ser Lys Glu Thr Glu Val Leu As - #p Ser His Ile His Ala# 20001990 - # 1995- Tyr Val Asn Ala Leu Leu Ile Pro Gly Pro Al - #a Gly Lys Thr Lys Leu# 20150- Glu Lys Gln Phe Gln Leu Leu Ser Gln Ser As - #n Ile Gln Gln Ser Asp# 20305- Tyr Ser Ala Ala Leu Lys Gln Cys Asn Arg Gl - #u Lys Asn Arg Thr Ser# 20450- Ser Ile Ile Pro Val Glu Arg Ser Arg Val Gl - #y Ile Ser Ser Leu Ser# 20605- Gly Glu Gly Thr Asp Tyr Ile Asn Ala Ser Ty - #r Ile Met Gly Tyr Tyr# 20802070 - # 2075- Gln Ser Asn Glu Phe Ile Ile Thr Gln His Pr - #o Leu Leu His Thr Ile# 20950- Lys Asp Phe Trp Arg Met Ile Trp Asp His As - #n Ala Gln Leu Val Val# 21105- Met Ile Pro Asp Gly Gln Asn Met Ala Glu As - #p Glu Phe Val Tyr Trp# 21250- Pro Asn Lys Asp Glu Pro Ile Asn Cys Glu Se - #r Phe Lys Val Thr Leu# 21405- Met Ala Glu Glu His Lys Cys Leu Ser Asn Gl - #u Glu Lys Leu Ile Ile# 21602150 - # 2155- Gln Asp Phe Ile Leu Glu Ala Thr Gln Asp As - #p Tyr Val Leu Glu Val# 21750- Arg His Phe Gln Cys Pro Lys Trp Pro Asn Pr - #o Asp Ser Pro Ile Ser# 21905- Lys Thr Phe Glu Leu Ile Ser Val Ile Lys Gl - #u Glu Ala Ala Asn Arg# 22050- Asp Gly Pro Met Ile Val His Asp Glu His Gl - #y Gly Val Thr Ala Gly# 22205- Thr Phe Cys Ala Leu Thr Thr Leu Met His Gl - #n Leu Glu Lys Glu Asn# 22402230 - # 2235- Ser Val Asp Val Tyr Gln Val Ala Lys Met Il - #e Asn Leu Met Arg Pro# 22550- Gly Val Phe Ala Asp Ile Glu Gln Tyr Gln Ph - #e Leu Tyr Lys Val Ile# 22705- Leu Ser Leu Val Ser Thr Arg Gln Glu Glu As - #n Pro Ser Thr Ser Leu# 22850- Asp Ser Asn Gly Ala Ala Leu Pro Asp Gly As - #n Ile Ala Glu Ser Leu# 23005- Glu Ser Leu Val2305- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 6924 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown- (ii) MOLECULE TYPE: cDNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..6924- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- ATG CGA ATC CTA AAG CGT TTC CTC GCT TGC AT - #T CAG CTC CTC TGT GTT 48Met Arg Ile Leu Lys Arg Phe Leu Ala Cys Il - #e Gln Leu Leu Cys Val# 15- TGC CGC CTG GAT TGG GCT AAT GGA TAC TAC AG - #A CAA CAG AGA AAA CTT 96Cys Arg Leu Asp Trp Ala Asn Gly Tyr Tyr Ar - #g Gln Gln Arg Lys Leu# 30- GTT GAA GAG ATT GGC TGG TCC TAT ACA GGA GC - #A CTG AAT CAA AAA AAT 144Val Glu Glu Ile Gly Trp Ser Tyr Thr Gly Al - #a Leu Asn Gln Lys Asn# 45- TGG GGA AAG AAA TAT CCA ACA TGT AAT AGC CC - #A AAA CAA TCT CCT ATC 192Trp Gly Lys Lys Tyr Pro Thr Cys Asn Ser Pr - #o Lys Gln Ser Pro Ile# 60- AAT ATT GAT GAA GAT CTT ACA CAA GTA AAT GT - #G AAT CTT AAG AAA CTT 240Asn Ile Asp Glu Asp Leu Thr Gln Val Asn Va - #l Asn Leu Lys Lys Leu# 80- AAA TTT CAG GGT TGG GAT AAA ACA TCA TTG GA - #A AAC ACA TTC ATT CAT 288Lys Phe Gln Gly Trp Asp Lys Thr Ser Leu Gl - #u Asn Thr Phe Ile His# 95- AAC ACT GGG AAA ACA GTG GAA ATT AAT CTC AC - #T AAT GAC TAC CGT GTC 336Asn Thr Gly Lys Thr Val Glu Ile Asn Leu Th - #r Asn Asp Tyr Arg Val# 110- AGC GGA GGA GTT TCA GAA ATG GTG TTT AAA GC - #A AGC AAG ATA ACT TTT 384Ser Gly Gly Val Ser Glu Met Val Phe Lys Al - #a Ser Lys Ile Thr Phe# 125- CAC TGG GGA AAA TGC AAT ATG TCA TCT GAT GG - #A TCA GAG CAC AGT TTA 432His Trp Gly Lys Cys Asn Met Ser Ser Asp Gl - #y Ser Glu His Ser Leu# 140- GAA GGA CAA AAA TTT CCA CTT GAG ATG CAA AT - #C TAC TGC TTT GAT GCA 480Glu Gly Gln Lys Phe Pro Leu Glu Met Gln Il - #e Tyr Cys Phe Asp Ala145 1 - #50 1 - #55 1 -#60- GAC CGA TTT TCA AGT TTT GAG GAA GCA GTC AA - #A GGA AAA GGG AAG TTA 528Asp Arg Phe Ser Ser Phe Glu Glu Ala Val Ly - #s Gly Lys Gly Lys Leu# 175- AGA GCT TTA TCC ATT TTG TTT GAG GTT GGG AC - #A GAA GAA AAT TTG GAT 576Arg Ala Leu Ser Ile Leu Phe Glu Val Gly Th - #r Glu Glu Asn Leu Asp# 190- TTC AAA GCG ATT ATT GAT GGA GTC GAA AGT GT - #T AGT CGT TTT GGG AAG 624Phe Lys Ala Ile Ile Asp Gly Val Glu Ser Va - #l Ser Arg Phe Gly Lys# 205- CAG GCT GCT TTA GAT CCA TTC ATA CTG TTG AA - #C CTT CTG CCA AAC TCA 672Gln Ala Ala Leu Asp Pro Phe Ile Leu Leu As - #n Leu Leu Pro Asn Ser# 220- ACT GAC AAG TAT TAC ATT TAC AAT GGC TCA TT - #G ACA TCT CCT CCC TGC 720Thr Asp Lys Tyr Tyr Ile Tyr Asn Gly Ser Le - #u Thr Ser Pro Pro Cys225 2 - #30 2 - #35 2 -#40- ACA GAC ACA GTT GAC TGG ATT GTT TTT AAA GA - #T ACA GTT AGC ATC TCT 768Thr Asp Thr Val Asp Trp Ile Val Phe Lys As - #p Thr Val Ser Ile Ser# 255- GAA AGC CAG TTG GCT GTT TTT TGT GAA GTT CT - #T ACA ATG CAA CAA TCT 816Glu Ser Gln Leu Ala Val Phe Cys Glu Val Le - #u Thr Met Gln Gln Ser# 270- GGT TAT GTC ATG CTG ATG GAC TAC TTA CAA AA - #C AAT TTT CGA GAG CAA 864Gly Tyr Val Met Leu Met Asp Tyr Leu Gln As - #n Asn Phe Arg Glu Gln# 285- CAG TAC AAG TTC TCT AGA CAG GTG TTT TCC TC - #A TAC ACT GGA AAG GAA 912Gln Tyr Lys Phe Ser Arg Gln Val Phe Ser Se - #r Tyr Thr Gly Lys Glu# 300- GAG ATT CAT GAA GCA GTT TGT AGT TCA GAA CC - #A GAA AAT GTT CAG GCT 960Glu Ile His Glu Ala Val Cys Ser Ser Glu Pr - #o Glu Asn Val Gln Ala305 3 - #10 3 - #15 3 -#20- GAC CCA GAG AAT TAT ACC AGC CTT CTT GTT AC - #A TGG GAA AGA CCT CGA1008Asp Pro Glu Asn Tyr Thr Ser Leu Leu Val Th - #r Trp Glu Arg Pro Arg# 335- GTC GTT TAT GAT ACC ATG ATT GAG AAG TTT GC - #A GTT TTG TAC CAG CAG1056Val Val Tyr Asp Thr Met Ile Glu Lys Phe Al - #a Val Leu Tyr Gln Gln# 350- TTG GAT GGA GAG GAC CAA ACC AAG CAT GAA TT - #T TTG ACA GAT GGC TAT1104Leu Asp Gly Glu Asp Gln Thr Lys His Glu Ph - #e Leu Thr Asp Gly Tyr# 365- CAA GAC TTG GGT GCT ATT CTC AAT AAT TTG CT - #A CCC AAT ATG AGT TAT1152Gln Asp Leu Gly Ala Ile Leu Asn Asn Leu Le - #u Pro Asn Met Ser Tyr# 380- GTT CTT CAG ATA GTA GCC ATA TGC ACT AAT GG - #C TTA TAT GGA AAA TAC1200Val Leu Gln Ile Val Ala Ile Cys Thr Asn Gl - #y Leu Tyr Gly Lys Tyr385 3 - #90 3 - #95 4 -#00- AGC GAC CAA CTG ATT GTC GAC ATG CCT ACT GA - #T AAT CCT GAA CTT GAT1248Ser Asp Gln Leu Ile Val Asp Met Pro Thr As - #p Asn Pro Glu Leu Asp# 415- CTT TTC CCT GAA TTA ATT GGA ACT GAA GAA AT - #A ATC AAG GAG GAG GAA1296Leu Phe Pro Glu Leu Ile Gly Thr Glu Glu Il - #e Ile Lys Glu Glu Glu# 430- GAG GGA AAA GAC ATT GAA GAA GGC GCT ATT GT - #G AAT CCT GGT AGA GAC1344Glu Gly Lys Asp Ile Glu Glu Gly Ala Ile Va - #l Asn Pro Gly Arg Asp# 445- AGT GCT ACA AAC CAA ATC AGG AAA AAG GAA CC - #C CAG ATT TCT ACC ACA1392Ser Ala Thr Asn Gln Ile Arg Lys Lys Glu Pr - #o Gln Ile Ser Thr Thr# 460- ACA CAC TAC AAT CGC ATA GGG ACG AAA TAC AA - #T GAA GCC AAG ACT AAC1440Thr His Tyr Asn Arg Ile Gly Thr Lys Tyr As - #n Glu Ala Lys Thr Asn465 4 - #70 4 - #75 4 -#80- CGA TCC CCA ACA AGA GGA AGT GAA TTC TCT GG - #A AAG GGT GAT GTT CCC1488Arg Ser Pro Thr Arg Gly Ser Glu Phe Ser Gl - #y Lys Gly Asp Val Pro# 495- AAT ACA TCT TTA AAT TCC ACT TCC CAA CCA GT - #C ACT AAA TTA GCC ACA1536Asn Thr Ser Leu Asn Ser Thr Ser Gln Pro Va - #l Thr Lys Leu Ala Thr# 510- GAA AAA GAT ATT TCC TTG ACT TCT CAG ACT GT - #G ACT GAA CTG CCA CCT1584Glu Lys Asp Ile Ser Leu Thr Ser Gln Thr Va - #l Thr Glu Leu Pro Pro# 525- CAC ACT GTG GAA GGT ACT TCA GCC TCT TTA AA - #T GAT GGC TCT AAA ACT1632His Thr Val Glu Gly Thr Ser Ala Ser Leu As - #n Asp Gly Ser Lys Thr# 540- GTT CTT AGA TCT CCA CAT ATG AAC TTG TCG GG - #G ACT GCA GAA TCC TTA1680Val Leu Arg Ser Pro His Met Asn Leu Ser Gl - #y Thr Ala Glu Ser Leu545 5 - #50 5 - #55 5 -#60- AAT ACA GTT TCT ATA ACA GAA TAT GAG GAG GA - #G AGT TTA TTG ACC AGT1728Asn Thr Val Ser Ile Thr Glu Tyr Glu Glu Gl - #u Ser Leu Leu Thr Ser# 575- TTC AAG CTT GAT ACT GGA GCT GAA GAT TCT TC - #A GGC TCC AGT CCC GCA1776Phe Lys Leu Asp Thr Gly Ala Glu Asp Ser Se - #r Gly Ser Ser Pro Ala# 590- ACT TCT GCT ATC CCA TTC ATC TCT GAG AAC AT - #A TCC CAA GGG TAT ATA1824Thr Ser Ala Ile Pro Phe Ile Ser Glu Asn Il - #e Ser Gln Gly Tyr Ile# 605- TTT TCC TCC GAA AAC CCA GAG ACA ATA ACA TA - #T GAT GTC CTT ATA CCA1872Phe Ser Ser Glu Asn Pro Glu Thr Ile Thr Ty - #r Asp Val Leu Ile Pro# 620- GAA TCT GCT AGA AAT GCT TCC GAA GAT TCA AC - #T TCA TCA GGT TCA GAA1920Glu Ser Ala Arg Asn Ala Ser Glu Asp Ser Th - #r Ser Ser Gly Ser Glu625 6 - #30 6 - #35 6 -#40- GAA TCA CTA AAG GAT CCT TCT ATG GAG GGA AA - #T GTG TGG TTT CCT AGC1968Glu Ser Leu Lys Asp Pro Ser Met Glu Gly As - #n Val Trp Phe Pro Ser# 655- TCT ACA GAC ATA ACA GCA CAG CCC GAT GTT GG - #A TCA GGC AGA GAG AGC2016Ser Thr Asp Ile Thr Ala Gln Pro Asp Val Gl - #y Ser Gly Arg Glu Ser# 670- TTT CTC CAG ACT AAT TAC ACT GAG ATA CGT GT - #T GAT GAA TCT GAG AAG2064Phe Leu Gln Thr Asn Tyr Thr Glu Ile Arg Va - #l Asp Glu Ser Glu Lys# 685- ACA ACC AAG TCC TTT TCT GCA GGC CCA GTG AT - #G TCA CAG GGT CCC TCA2112Thr Thr Lys Ser Phe Ser Ala Gly Pro Val Me - #t Ser Gln Gly Pro Ser# 700- GTT ACA GAT CTG GAA ATG CCA CAT TAT TCT AC - #C TTT GCC TAC TTC CCA2160Val Thr Asp Leu Glu Met Pro His Tyr Ser Th - #r Phe Ala Tyr Phe Pro705 7 - #10 7 - #15 7 -#20- ACT GAG GTA ACA CCT CAT GCT TTT ACC CCA TC - #C TCC AGA CAA CAG GAT2208Thr Glu Val Thr Pro His Ala Phe Thr Pro Se - #r Ser Arg Gln Gln Asp# 735- TTG GTC TCC ACG GTC AAC GTG GTA TAC TCG CA - #G ACA ACC CAA CCG GTA2256Leu Val Ser Thr Val Asn Val Val Tyr Ser Gl - #n Thr Thr Gln Pro Val# 750- TAC AAT GGT GAG ACA CCT CTT CAA CCT TCC TA - #C AGT AGT GAA GTC TTT2304Tyr Asn Gly Glu Thr Pro Leu Gln Pro Ser Ty - #r Ser Ser Glu Val Phe# 765- CCT CTA GTC ACC CCT TTG TTG CTT GAC AAT CA - #G ATC CTC AAC ACT ACC2352Pro Leu Val Thr Pro Leu Leu Leu Asp Asn Gl - #n Ile Leu Asn Thr Thr# 780- CCT GCT GCT TCA AGT AGT GAT TCG GCC TTG CA - #T GCT ACG CCT GTA TTT2400Pro Ala Ala Ser Ser Ser Asp Ser Ala Leu Hi - #s Ala Thr Pro Val Phe785 7 - #90 7 - #95 8 -#00- CCC AGT GTC GAT GTG TCA TTT GAA TCC ATC CT - #G TCT TCC TAT GAT GGT2448Pro Ser Val Asp Val Ser Phe Glu Ser Ile Le - #u Ser Ser Tyr Asp Gly# 815- GCA CCT TTG CTT CCA TTT TCC TCT GCT TCC TT - #C AGT AGT GAA TTG TTT2496Ala Pro Leu Leu Pro Phe Ser Ser Ala Ser Ph - #e Ser Ser Glu Leu Phe# 830- CGC CAT CTG CAT ACA GTT TCT CAA ATC CTT CC - #A CAA GTT ACT TCA GCT2544Arg His Leu His Thr Val Ser Gln Ile Leu Pr - #o Gln Val Thr Ser Ala# 845- ACC GAG AGT GAT AAG GTG CCC TTG CAT GCT TC - #T CTG CCA GTG GCT GGG2592Thr Glu Ser Asp Lys Val Pro Leu His Ala Se - #r Leu Pro Val Ala Gly# 860- GGT GAT TTG CTA TTA GAG CCC AGC CTT GCT CA - #G TAT TCT GAT GTG CTG2640Gly Asp Leu Leu Leu Glu Pro Ser Leu Ala Gl - #n Tyr Ser Asp Val Leu865 8 - #70 8 - #75 8 -#80- TCC ACT ACT CAT GCT GCT TCA AAG ACG CTG GA - #A TTT GGT AGT GAA TCT2688Ser Thr Thr His Ala Ala Ser Lys Thr Leu Gl - #u Phe Gly Ser Glu Ser# 895- GGT GTT CTT TAT AAA ACG CTT ATG TTT TCT CA - #A GTT GAA CCA CCC AGC2736Gly Val Leu Tyr Lys Thr Leu Met Phe Ser Gl - #n Val Glu Pro Pro Ser# 910- AGT GAT GCC ATG ATG CAT GCA CGT TCT TCA GG - #G CCT GAA CCT TCT TAT2784Ser Asp Ala Met Met His Ala Arg Ser Ser Gl - #y Pro Glu Pro Ser Tyr# 925- GCC TTG TCT GAT AAT GAG GGC TCC CAA CAC AT - #C TTC ACT GTT TCT TAC2832Ala Leu Ser Asp Asn Glu Gly Ser Gln His Il - #e Phe Thr Val Ser Tyr# 940- AGT TCT GCA ATA CCT GTG CAT GAT TCT GTG GG - #T GTA ACT TAT CAG GGT2880Ser Ser Ala Ile Pro Val His Asp Ser Val Gl - #y Val Thr Tyr Gln Gly945 9 - #50 9 - #55 9 -#60- TCC TTA TTT AGC GGC CCT AGC CAT ATA CCA AT - #A CCT AAG TCT TCG TTA2928Ser Leu Phe Ser Gly Pro Ser His Ile Pro Il - #e Pro Lys Ser Ser Leu# 975- ATA ACC CCA ACT GCA TCA TTA CTG CAG CCT AC - #T CAT GCC CTC TCT GGT2976Ile Thr Pro Thr Ala Ser Leu Leu Gln Pro Th - #r His Ala Leu Ser Gly# 990- GAT GGG GAA TGG TCT GGA GCC TCT TCT GAT AG - #T GAA TTT CTT TTA CCT3024Asp Gly Glu Trp Ser Gly Ala Ser Ser Asp Se - #r Glu Phe Leu Leu Pro# 10050- GAC ACA GAT GGG CTG ACA GCC CTT AAC ATT TC - #T TCA CCT GTT TCT GTA3072Asp Thr Asp Gly Leu Thr Ala Leu Asn Ile Se - #r Ser Pro Val Ser Val# 10205- GCT GAA TTT ACA TAT ACA ACA TCT GTG TTT GG - #T GAT GAT AAT AAG GCG3120Ala Glu Phe Thr Tyr Thr Thr Ser Val Phe Gl - #y Asp Asp Asn Lys Ala# 10401030 - # 1035- CTT TCT AAA AGT GAA ATA ATA TAT GGA AAT GA - #G ACT GAA CTG CAA ATT3168Leu Ser Lys Ser Glu Ile Ile Tyr Gly Asn Gl - #u Thr Glu Leu Gln Ile# 10550- CCT TCT TTC AAT GAG ATG GTT TAC CCT TCT GA - #A AGC ACA GTC ATG CCC3216Pro Ser Phe Asn Glu Met Val Tyr Pro Ser Gl - #u Ser Thr Val Met Pro# 10705- AAC ATG TAT GAT AAT GTA AAT AAG TTG AAT GC - #G TCT TTA CAA GAA ACC3264Asn Met Tyr Asp Asn Val Asn Lys Leu Asn Al - #a Ser Leu Gln Glu Thr# 10850- TCT GTT TCC ATT TCT AGC ACC AAG GGC ATG TT - #T CCA GGG TCC CTT GCT3312Ser Val Ser Ile Ser Ser Thr Lys Gly Met Ph - #e Pro Gly Ser Leu Ala# 11005- CAT ACC ACC ACT AAG GTT TTT GAT CAT GAG AT - #T AGT CAA GTT CCA GAA3360His Thr Thr Thr Lys Val Phe Asp His Glu Il - #e Ser Gln Val Pro Glu# 11201110 - # 1115- AAT AAC TTT TCA GTT CAA CCT ACA CAT ACT GT - #C TCT CAA GCA TCT GGT3408Asn Asn Phe Ser Val Gln Pro Thr His Thr Va - #l Ser Gln Ala Ser Gly# 11350- GAC ACT TCG CTT AAA CCT GTG CTT AGT GCA AA - #C TCA GAG CCA GCA TCC3456Asp Thr Ser Leu Lys Pro Val Leu Ser Ala As - #n Ser Glu Pro Ala Ser# 11505- TCT GAC CCT GCT TCT AGT GAA ATG TTA TCT CC - #T TCA ACT CAG CTC TTA3504Ser Asp Pro Ala Ser Ser Glu Met Leu Ser Pr - #o Ser Thr Gln Leu Leu# 11650- TTT TAT GAG ACC TCA GCT TCT TTT AGT ACT GA - #A GTA TTG CTA CAA CCT3552Phe Tyr Glu Thr Ser Ala Ser Phe Ser Thr Gl - #u Val Leu Leu Gln Pro# 11805- TCC TTT CAG GCT TCT GAT GTT GAC ACC TTG CT - #T AAA ACT GTT CTT CCA3600Ser Phe Gln Ala Ser Asp Val Asp Thr Leu Le - #u Lys Thr Val Leu Pro# 12001190 - # 1195- GCT GTG CCC AGT GAT CCA ATA TTG GTT GAA AC - #C CCC AAA GTT GAT AAA3648Ala Val Pro Ser Asp Pro Ile Leu Val Glu Th - #r Pro Lys Val Asp Lys# 12150- ATT AGT TCT ACA ATG TTG CAT CTC ATT GTA TC - #A AAT TCT GCT TCA AGT3696Ile Ser Ser Thr Met Leu His Leu Ile Val Se - #r Asn Ser Ala Ser Ser# 12305- GAA AAC ATG CTG CAC TCT ACA TCT GTA CCA GT - #T TTT GAT GTG TCG CCT3744Glu Asn Met Leu His Ser Thr Ser Val Pro Va - #l Phe Asp Val Ser Pro# 12450- ACT TCT CAT ATG CAC TCT GCT TCA CTT CAA GG - #T TTG ACC ATT TCC TAT3792Thr Ser His Met His Ser Ala Ser Leu Gln Gl - #y Leu Thr Ile Ser Tyr# 12605- GCA AGT GAG AAA TAT GAA CCA GTT TTG TTA AA - #A AGT GAA AGT TCC CAC3840Ala Ser Glu Lys Tyr Glu Pro Val Leu Leu Ly - #s Ser Glu Ser Ser His# 12801270 - # 1275- CAA GTG GTA CCT TCT TTG TAC AGT AAT GAT GA - #G TTG TTC CAA ACG GCC3888Gln Val Val Pro Ser Leu Tyr Ser Asn Asp Gl - #u Leu Phe Gln Thr Ala# 12950- AAT TTG GAG ATT AAC CAG GCC CAT CCC CCA AA - #A GGA AGG CAT GTA TTT3936Asn Leu Glu Ile Asn Gln Ala His Pro Pro Ly - #s Gly Arg His Val Phe# 13105- GCT ACA CCT GTT TTA TCA ATT GAT GAA CCA TT - #A AAT ACA CTA ATA AAT3984Ala Thr Pro Val Leu Ser Ile Asp Glu Pro Le - #u Asn Thr Leu Ile Asn# 13250- AAG CTT ATA CAT TCC GAT GAA ATT TTA ACC TC - #C ACC AAA AGT TCT GTT4032Lys Leu Ile His Ser Asp Glu Ile Leu Thr Se - #r Thr Lys Ser Ser Val# 13405- ACT GGT AAG GTA TTT GCT GGT ATT CCA ACA GT - #T GCT TCT GAT ACA TTT4080Thr Gly Lys Val Phe Ala Gly Ile Pro Thr Va - #l Ala Ser Asp Thr Phe# 13601350 - # 1355- GTA TCT ACT GAT CAT TCT GTT CCT ATA GGA AA - #T GGG CAT GTT GCC ATT4128Val Ser Thr Asp His Ser Val Pro Ile Gly As - #n Gly His Val Ala Ile# 13750- ACA GCT GTT TCT CCC CAC AGA GAT GGT TCT GT - #A ACC TCA ACA AAG TTG4176Thr Ala Val Ser Pro His Arg Asp Gly Ser Va - #l Thr Ser Thr Lys Leu# 13905- CTG TTT CCT TCT AAG GCA ACT TCT GAG CTG AG - #T CAT AGT GCC AAA TCT4224Leu Phe Pro Ser Lys Ala Thr Ser Glu Leu Se - #r His Ser Ala Lys Ser# 14050- GAT GCC GGT TTA GTG GGT GGT GGT GAA GAT GG - #T GAC ACT GAT GAT GAT4272Asp Ala Gly Leu Val Gly Gly Gly Glu Asp Gl - #y Asp Thr Asp Asp Asp# 14205- GGT GAT GAT GAT GAT GAT GAC AGA GGT AGT GA - #T GGC TTA TCC ATT CAT4320Gly Asp Asp Asp Asp Asp Asp Arg Gly Ser As - #p Gly Leu Ser Ile His# 14401430 - # 1435- AAG TGT ATG TCA TGC TCA TCC TAT AGA GAA TC - #A CAG GAA AAG GTA ATG4368Lys Cys Met Ser Cys Ser Ser Tyr Arg Glu Se - #r Gln Glu Lys Val Met# 14550- AAT GAT TCA GAC ACC CAC GAA AAC AGT CTT AT - #G GAT CAG AAT AAT CCA4416Asn Asp Ser Asp Thr His Glu Asn Ser Leu Me - #t Asp Gln Asn Asn Pro# 14705- ATC TCA TAC TCA CTA TCT GAG AAT TCT GAA GA - #A GAT AAT AGA GTC ACA4464Ile Ser Tyr Ser Leu Ser Glu Asn Ser Glu Gl - #u Asp Asn Arg Val Thr# 14850- AGT GTA TCC TCA GAC AGT CAA ACT GGT ATG GA - #C AGA AGT CCT GGT AAA4512Ser Val Ser Ser Asp Ser Gln Thr Gly Met As - #p Arg Ser Pro Gly Lys# 15005- TCA CCA TCA GCA AAT GGG CTA TCC CAA AAG CA - #C AAT GAT GGA AAA GAG4560Ser Pro Ser Ala Asn Gly Leu Ser Gln Lys Hi - #s Asn Asp Gly Lys Glu# 15201510 - # 1515- GAA AAT GAC ATT CAG ACT GGT AGT GCT CTG CT - #T CCT CTC AGC CCT GAA4608Glu Asn Asp Ile Gln Thr Gly Ser Ala Leu Le - #u Pro Leu Ser Pro Glu# 15350- TCT AAA GCA TGG GCA GTT CTG ACA AGT GAT GA - #A GAA AGT GGA TCA GGG4656Ser Lys Ala Trp Ala Val Leu Thr Ser Asp Gl - #u Glu Ser Gly Ser Gly# 15505- CAA GGT ACC TCA GAT AGC CTT AAT GAG AAT GA - #G ACT TCC ACA GAT TTC4704Gln Gly Thr Ser Asp Ser Leu Asn Glu Asn Gl - #u Thr Ser Thr Asp Phe# 15650- AGT TTT GCA GAC ACT AAT GAA AAA GAT GCT GA - #T GGG ATC CTG GCA GCA4752Ser Phe Ala Asp Thr Asn Glu Lys Asp Ala As - #p Gly Ile Leu Ala Ala# 15805- GGT GAC TCA GAA ATA ACT CCT GGA TTC CCA CA - #G TCC CCA ACA TCA TCT4800Gly Asp Ser Glu Ile Thr Pro Gly Phe Pro Gl - #n Ser Pro Thr Ser Ser# 16001590 - # 1595- GTT ACT AGC GAG AAC TCA GAA GTG TTC CAC GT - #T TCA GAG GCA GAG GCC4848Val Thr Ser Glu Asn Ser Glu Val Phe His Va - #l Ser Glu Ala Glu Ala# 16150- AGT AAT AGT AGC CAT GAG TCT CGT ATT GGT CT - #A GCT GAG GGG TTG GAA4896Ser Asn Ser Ser His Glu Ser Arg Ile Gly Le - #u Ala Glu Gly Leu Glu# 16305- TCC GAG AAG AAG GCA GTT ATA CCC CTT GTG AT - #C GTG TCA GCC CTG ACT4944Ser Glu Lys Lys Ala Val Ile Pro Leu Val Il - #e Val Ser Ala Leu Thr# 16450- TTT ATC TGT CTA GTG GTT CTT GTG GGT ATT CT - #C ATC TAC TGG AGG AAA4992Phe Ile Cys Leu Val Val Leu Val Gly Ile Le - #u Ile Tyr Trp Arg Lys# 16605- TGC TTC CAG ACT GCA CAC TTT TAC TTA GAG GA - #C AGT ACA TCC CCT AGA5040Cys Phe Gln Thr Ala His Phe Tyr Leu Glu As - #p Ser Thr Ser Pro Arg# 16801670 - # 1675- GTT ATA TCC ACA CCT CCA ACA CCT ATC TTT CC - #A ATT TCA GAT GAT GTC5088Val Ile Ser Thr Pro Pro Thr Pro Ile Phe Pr - #o Ile Ser Asp Asp Val# 16950- GGA GCA ATT CCA ATA AAG CAC TTT CCA AAG CA - #T GTT GCA GAT TTA CAT5136Gly Ala Ile Pro Ile Lys His Phe Pro Lys Hi - #s Val Ala Asp Leu His# 17105- GCA AGT AGT GGG TTT ACT GAA GAA TTT GAG GA - #A GTG CAG AGC TGT ACT5184Ala Ser Ser Gly Phe Thr Glu Glu Phe Glu Gl - #u Val Gln Ser Cys Thr# 17250- GTT GAC TTA GGT ATT ACA GCA GAC AGC TCC AA - #C CAC CCA GAC AAC AAG5232Val Asp Leu Gly Ile Thr Ala Asp Ser Ser As - #n His Pro Asp Asn Lys# 17405- CAC AAG AAT CGA TAC ATA AAT ATC GTT GCC TA - #T GAT CAT AGC AGG GTT5280His Lys Asn Arg Tyr Ile Asn Ile Val Ala Ty - #r Asp His Ser Arg Val# 17601750 - # 1755- AAG CTA GCA CAG CTT GCT GAA AAG GAT GGC AA - #A CTG ACT GAT TAT ATC5328Lys Leu Ala Gln Leu Ala Glu Lys Asp Gly Ly - #s Leu Thr Asp Tyr Ile# 17750- AAT GCC AAT TAT GTT GAT GGC TAC AAC AGA CC - #A AAA GCT TAT ATT GCT5376Asn Ala Asn Tyr Val Asp Gly Tyr Asn Arg Pr - #o Lys Ala Tyr Ile Ala# 17905- GCC CAA GGC CCA CTG AAA TCC ACA GCT GAA GA - #T TTC TGG AGA ATG ATA5424Ala Gln Gly Pro Leu Lys Ser Thr Ala Glu As - #p Phe Trp Arg Met Ile# 18050- TGG GAA CAT AAT GTG GAA GTT ATT GTC ATG AT - #A ACA AAC CTC GTG GAG5472Trp Glu His Asn Val Glu Val Ile Val Met Il - #e Thr Asn Leu Val Glu# 18205- AAA GGA AGG AGA AAA TGT GAT CAG TAC TGG CC - #T GCC GAT GGG AGT GAG5520Lys Gly Arg Arg Lys Cys Asp Gln Tyr Trp Pr - #o Ala Asp Gly Ser Glu# 18401830 - # 1835- GAG TAC GGG AAC TTT CTG GTC ACT CAG AAG AG - #T GTG CAA GTG CTT GCC5568Glu Tyr Gly Asn Phe Leu Val Thr Gln Lys Se - #r Val Gln Val Leu Ala# 18550- TAT TAT ACT GTG AGG AAT TTT ACT CTA AGA AA - #C ACA AAA ATA AAA AAG5616Tyr Tyr Thr Val Arg Asn Phe Thr Leu Arg As - #n Thr Lys Ile Lys Lys# 18705- GGC TCC CAG AAA GGA AGA CCC AGT GGA CGT GT - #G GTC ACA CAG TAT CAC5664Gly Ser Gln Lys Gly Arg Pro Ser Gly Arg Va - #l Val Thr Gln Tyr His# 18850- TAC ACG CAG TGG CCT GAC ATG GGA GTA CCA GA - #G TAC TCC CTG CCA GTG5712Tyr Thr Gln Trp Pro Asp Met Gly Val Pro Gl - #u Tyr Ser Leu Pro Val# 19005- CTG ACC TTT GTG AGA AAG GCA GCC TAT GCC AA - #G CGC CAT GCA GTG GGG5760Leu Thr Phe Val Arg Lys Ala Ala Tyr Ala Ly - #s Arg His Ala Val Gly# 19201910 - # 1915- CCT GTT GTC GTC CAC TGC AGT GCT GGA GTT GG - #A AGA ACA GGC ACA TAT5808Pro Val Val Val His Cys Ser Ala Gly Val Gl - #y Arg Thr Gly Thr Tyr# 19350- ATT GTG CTA GAC AGT ATG TTG CAG CAG ATT CA - #A CAC GAA GGA ACT GTC5856Ile Val Leu Asp Ser Met Leu Gln Gln Ile Gl - #n His Glu Gly Thr Val# 19505- AAC ATA TTT GGC TTC TTA AAA CAC ATC CGT TC - #A CAA AGA AAT TAT TTG5904Asn Ile Phe Gly Phe Leu Lys His Ile Arg Se - #r Gln Arg Asn Tyr Leu# 19650- GTA CAA ACT GAG GAG CAA TAT GTC TTC ATT CA - #T GAT ACA CTG GTT GAG5952Val Gln Thr Glu Glu Gln Tyr Val Phe Ile Hi - #s Asp Thr Leu Val Glu# 19805- GCC ATA CTT AGT AAA GAA ACT GAG GTG CTG GA - #C AGT CAT ATT CAT GCC6000Ala Ile Leu Ser Lys Glu Thr Glu Val Leu As - #p Ser His Ile His Ala# 20001990 - # 1995- TAT GTT AAT GCA CTC CTC ATT CCT GGA CCA GC - #A GGC AAA ACA AAG CTA6048Tyr Val Asn Ala Leu Leu Ile Pro Gly Pro Al - #a Gly Lys Thr Lys Leu# 20150- GAG AAA CAA TTC CAG CTC CTG AGC CAG TCA AA - #T ATA CAG CAG AGT GAC6096Glu Lys Gln Phe Gln Leu Leu Ser Gln Ser As - #n Ile Gln Gln Ser Asp# 20305- TAT TCT GCA GCC CTA AAG CAA TGC AAC AGG GA - #A AAG AAT CGA ACT TCT6144Tyr Ser Ala Ala Leu Lys Gln Cys Asn Arg Gl - #u Lys Asn Arg Thr Ser# 20450- TCT ATC ATC CCT GTG GAA AGA TCA AGG GTT GG - #C ATT TCA TCC CTG AGT6192Ser Ile Ile Pro Val Glu Arg Ser Arg Val Gl - #y Ile Ser Ser Leu Ser# 20605- GGA GAA GGC ACA GAC TAC ATC AAT GCC TCC TA - #T ATC ATG GGC TAT TAC6240Gly Glu Gly Thr Asp Tyr Ile Asn Ala Ser Ty - #r Ile Met Gly Tyr Tyr# 20802070 - # 2075- CAG AGC AAT GAA TTC ATC ATT ACC CAG CAC CC - #T CTC CTT CAT ACC ATC6288Gln Ser Asn Glu Phe Ile Ile Thr Gln His Pr - #o Leu Leu His Thr Ile# 20950- AAG GAT TTC TGG AGG ATG ATA TGG GAC CAT AA - #T GCC CAA CTG GTG GTT6336Lys Asp Phe Trp Arg Met Ile Trp Asp His As - #n Ala Gln Leu Val Val# 21105- ATG ATT CCT GAT GGC CAA AAC ATG GCA GAA GA - #T GAA TTT GTT TAC TGG6384Met Ile Pro Asp Gly Gln Asn Met Ala Glu As - #p Glu Phe Val Tyr Trp# 21250- CCA AAT AAA GAT GAG CCT ATA AAT TGT GAG AG - #C TTT AAG GTC ACT CTT6432Pro Asn Lys Asp Glu Pro Ile Asn Cys Glu Se - #r Phe Lys Val Thr Leu# 21405- ATG GCT GAA GAA CAC AAA TGT CTA TCT AAT GA - #G GAA AAA CTT ATA ATT6480Met Ala Glu Glu His Lys Cys Leu Ser Asn Gl - #u Glu Lys Leu Ile Ile# 21602150 - # 2155- CAG GAC TTT ATC TTA GAA GCT ACA CAG GAT GA - #T TAT GTA CTT GAA GTG6528Gln Asp Phe Ile Leu Glu Ala Thr Gln Asp As - #p Tyr Val Leu Glu Val# 21750- AGG CAC TTT CAG TGT CCT AAA TGG CCA AAT CC - #A GAT AGC CCC ATT AGT6576Arg His Phe Gln Cys Pro Lys Trp Pro Asn Pr - #o Asp Ser Pro Ile Ser# 21905- AAA ACT TTT GAA CTT ATA AGT GTT ATA AAA GA - #A GAA GCT GCC AAT AGG6624Lys Thr Phe Glu Leu Ile Ser Val Ile Lys Gl - #u Glu Ala Ala Asn Arg# 22050- GAT GGG CCT ATG ATT GTT CAT GAT GAG CAT GG - #A GGA GTG ACG GCA GGA6672Asp Gly Pro Met Ile Val His Asp Glu His Gl - #y Gly Val Thr Ala Gly# 22205- ACT TTC TGT GCT CTG ACA ACC CTT ATG CAC CA - #A CTA GAA AAA GAA AAT6720Thr Phe Cys Ala Leu Thr Thr Leu Met His Gl - #n Leu Glu Lys Glu Asn# 22402230 - # 2235- TCC GTG GAT GTT TAC CAG GTA GCC AAG ATG AT - #C AAT CTG ATG AGG CCA6768Ser Val Asp Val Tyr Gln Val Ala Lys Met Il - #e Asn Leu Met Arg Pro# 22550- GGA GTC TTT GCT GAC ATT GAG CAG TAT CAG TT - #T CTC TAC AAA GTG ATC6816Gly Val Phe Ala Asp Ile Glu Gln Tyr Gln Ph - #e Leu Tyr Lys Val Ile# 22705- CTC AGC CTT GTG AGC ACA AGG CAG GAA GAG AA - #T CCA TCC ACC TCT CTG6864Leu Ser Leu Val Ser Thr Arg Gln Glu Glu As - #n Pro Ser Thr Ser Leu# 22850- GAC AGT AAT GGT GCA GCA TTG CCT GAT GGA AA - #T ATA GCT GAG AGC TTA6912Asp Ser Asn Gly Ala Ala Leu Pro Asp Gly As - #n Ile Ala Glu Ser Leu# 23005# 6924Glu Ser Leu Val2305- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 267 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:- Gly Asp Pro Tyr Trp Ala Tyr Ser - # Gly Ala Tyr Gly Pro Glu HisTrp# 15- Val Thr Ser Ser Val Ser Cys Gly - # Gly Arg His Gln Ser Pro IleAsp# 30- Ile Leu Asp Gln Tyr Ala Arg Val - # Gly Glu Glu Tyr Gln Glu LeuGln# 45- Leu Asp Gly Phe Asp Asn Glu Ser - # Ser Asn Lys Thr Trp Met LysAsn# 60- Thr Gly Lys Thr Val Ala Ile Leu - # Leu Lys Asp Asp Tyr Phe ValSer# 80- Gly Ala Gly Leu Pro Gly Arg Phe - # Lys Ala Glu Lys Val Glu PheHis# 95- Trp Gly His Ser Asn Gly Ser Ala - # Gly Ser Glu His Ser Ile AsnGly# 110- Arg Arg Phe Pro Val Glu Met Gln - # Ile Phe Phe Tyr Asn Pro AspAsp# 125- Phe Asp Ser Phe Gln Thr Ala Ile - # Ser Glu Asn Arg Ile Ile GlyAla# 140- Met Ala Ile Phe Phe Gln Val Ser - # Pro Arg Asp Asn Ser Ala LeuAsp# 160- Pro Ile Ile His Gly Leu Lys Gly - # Val Val His His Glu Lys GluThr# 175- Phe Leu Asp Pro Phe Val Leu Arg - # Asp Leu Leu Pro Ala Ser LeuGly# 190- Ser Tyr Tyr Arg Tyr Thr Gly Ser - # Leu Thr Thr Pro Pro Cys SerGlu# 205- Ile Val Glu Trp Ile Val Phe Arg - # Arg Pro Val Pro Ile Ser TyrHis# 220- Gln Leu Glu Ala Phe Tyr Ser Ile - # Phe Thr Thr Glu Gln Gln AspHis# 240- Val Lys Ser Val Glu Tyr Leu Arg - # Asn Asn Phe Arg Pro Gln GlnArg# 255- Leu His Asp Arg Val Val Ser Lys - # Ser Ala Val# 265- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 260 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:- Ala Ser Pro Asp Trp Gly Tyr Asp - # Asp Lys Asn Gly Pro Glu GlnTrp# 15- Ser Lys Leu Tyr Pro Ile Ala Asn - # Gly Asn Asn Gln Ser Pro ValAsp# 30- Ile Lys Thr Ser Glu Thr Lys His - # Asp Thr Ser Leu Lys Pro IleSer# 45- Val Ser Tyr Asn Pro Ala Thr Ala - # Lys Glu Ile Ile Asn Val GlyHis# 60- Ser Phe His Val Asn Phe Glu Asp - # Asn Asp Asn Arg Ser Val LeuLys# 80- Gly Gly Pro Phe Ser Asp Ser Tyr - # Arg Leu Phe Gln Phe His PheHis# 95- Trp Gly Ser Thr Asn Glu His Gly - # Ser Glu His Thr Val Asp GlyVal# 110- Lys Tyr Ser Ala Glu Leu His Val - # Ala His Trp Asn Ser Ala LysTyr# 125- Ser Ser Leu Ala Glu Ala Ala Ser - # Lys Ala Asp Gly Leu Ala ValIle# 140- Gly Val Leu Met Lys Val Gly Glu - # Ala Asn Pro Lys Leu Gln LysVal# 160- Leu Asp Ala Leu Gln Ala Ile Lys - # Thr Lys Gly Lys Arg Ala ProPhe# 175- Thr Asn Phe Asp Pro Ser Thr Leu - # Leu Pro Ser Ser Leu Asp PheTrp# 190- Thr Tyr Pro Gly Ser Leu Thr His - # Pro Pro Leu Tyr Glu Ser ValThr# 205- Trp Ile Ile Cys Lys Glu Ser Ile - # Ser Val Ser Ser Glu Gln LeuAla# 220- Gln Phe Arg Ser Leu Leu Ser Asn - # Val Glu Gly Asp Asn Ala ValPro# 240- Met Gln His Asn Asn Arg Pro Thr - # Gln Pro Leu Lys Gly Arg ThrVal# 255- Arg Ala Ser Phe 260- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 259 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- Ser His His Trp Gly Tyr Gly Lys - # His Asn Gly Pro Glu His TrpHis# 15- Lys Asp Phe Pro Ile Ala Lys Gly - # Glu Arg Gln Ser Pro Val AspIle# 30- Asp Thr His Thr Ala Lys Tyr Asp - # Pro Ser Leu Lys Pro Leu SerVal# 45- Ser Tyr Asp Gln Ala Thr Ser Leu - # Arg Ile Leu Asn Asn Gly HisAla# 60- Phe Asn Val Glu Phe Asp Asp Ser - # Gln Asp Lys Ala Val Leu LysGly# 80- Gly Pro Leu Asp Gly Thr Tyr Arg - # Leu Ile Gln Phe His Phe HisTrp# 95- Gly Ser Leu Asp Gly Gln Gly Ser - # Glu His Thr Val Asp Lys LysLys# 110- Tyr Ala Ala Glu Leu His Leu Val - # His Trp Asn Thr Lys Tyr GlyAsp# 125- Phe Gly Lys Ala Val Gln Gln Pro - # Asp Gly Leu Ala Val Leu GlyIle# 140- Phe Leu Lys Val Gly Ser Ala Lys - # Pro Gly Leu Gln Lys Val ValAsp# 160- Val Leu Asp Ser Ile Lys Thr Lys - # Gly Lys Ser Ala Asp Phe ThrAsn# 175- Phe Asp Pro Arg Gly Leu Leu Pro - # Glu Ser Leu Asp Tyr Trp ThrTyr# 190- Pro Gly Ser Leu Thr Thr Pro Pro - # Leu Leu Glu Cys Val Thr TrpIle# 205- Val Leu Lys Glu Pro Ile Ser Val - # Ser Ser Glu Gln Val Leu LysPhe# 220- Arg Lys Leu Asn Phe Asn Gly Glu - # Gly Glu Pro Glu Glu Leu MetVal# 240- Asp Asn Trp Arg Pro Ala Gln Pro - # Leu Lys Asn Arg Gln Ile LysAla# 255- Ser Phe Lys- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 259 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:- Ala Lys Glu Trp Gly Tyr Ala Ser - # His Asn Gly Pro Asp His TrpHis# 15- Glu Leu Phe Pro Asn Ala Lys Gly - # Glu Asn Gln Ser Pro Ile GluLeu# 30- His Thr Lys Asp Ile Arg His Asp - # Pro Ser Leu Gln Pro Trp SerVal# 45- Ser Tyr Asp Gly Gly Ser Ala Lys - # Thr Ile Leu Asn Asn Gly LysThr# 60- Cys Arg Val Val Phe Asp Asp Thr - # Tyr Asp Arg Ser Met Leu ArgGly# 80- Gly Pro Leu Pro Gly Pro Tyr Arg - # Leu Arg Gln Phe His Leu HisTrp# 95- Gly Ser Ser Asp Asp His Gly Ser - # Glu His Thr Val Asp Gly ValLys# 110- Tyr Ala Ala Glu Leu His Leu Val - # His Trp Asn Pro Lys Tyr AsnThr# 125- Phe Lys Glu Ala Leu Lys Gln Arg - # Asp Gly Ile Ala Val Ile GlyIle# 140- Phe Leu Lys Ile Gly His Glu Asn - # Gly Glu Phe Gln Ile Phe LeuAsp# 160- Ala Leu Asp Lys Ile Lys Thr Lys - # Gly Lys Glu Ala Pro Phe ThrLys# 175- Phe Asp Pro Ser Cys Leu Phe Pro - # Ala Cys Arg Asp Tyr Trp ThrTyr# 190- Gln Gly Ser Phe Thr Thr Pro Pro - # Cys Glu Glu Cys Ile Val TrpLeu# 205- Leu Leu Lys Glu Pro Met Thr Val - # Ser Ser Asp Gln Met Ala LysLeu# 220- Arg Ser Leu Leu Ser Ser Ala Glu - # Asn Glu Pro Pro Val Pro LeuVal# 240- Ser Asn Trp Arg Pro Pro Gln Pro - # Ile Asn Asn Arg Val Val ArgAla# 255- Ser Phe Lys- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 268 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:- Ala Glu Ser His Trp Cys Tyr Glu - # Val Gln Ala Glu Ser Ser AsnTyr# 15- Pro Cys Leu Val Pro Val Lys Trp - # Gly Gly Asn Cys Gln Lys AspArg# 30- Gln Ser Pro Ile Asn Ile Val Thr - # Thr Lys Ala Lys Val Asp LysLys# 45- Leu Gly Arg Phe Phe Phe Ser Gly - # Tyr Asp Lys Lys Gln Thr TrpThr# 60- Val Gln Asn Asn Gly His Ser Val - # Met Met Leu Leu Glu Asn LysAla# 80- Ser Ile Ser Gly Gly Gly Leu Pro - # Ala Pro Tyr Gln Ala Lys GlnLeu# 95- His Leu His Trp Ser Asp Leu Pro - # Tyr Lys Gly Ser Glu His SerLeu# 110- Asp Gly Glu His Phe Ala Met Glu - # Met His Ile Val His Glu LysGlu# 125- Lys Gly Thr Ser Arg Asn Val Lys - # Glu Ala Gln Asp Pro Glu AspGlu# 140- Ile Ala Val Leu Ala Phe Leu Val - # Glu Ala Gly Thr Gln Val AsnGlu# 160- Gly Phe Gln Pro Leu Val Glu Ala - # Leu Ser Asn Ile Pro Lys ProGlu# 175- Met Ser Thr Thr Met Ala Glu Ser - # Ser Leu Leu Asp Leu Leu ProLys# 190- Glu Glu Lys Leu Arg His Tyr Phe - # Arg Tyr Leu Gly Ser Leu ThrThr# 205- Pro Thr Cys Asp Glu Lys Val Val - # Trp Thr Val Phe Arg Glu ProIle# 220- Gln Leu His Arg Glu Gln Ile Leu - # Ala Phe Ser Gln Lys Leu TyrTyr# 240- Asp Lys Glu Gln Thr Val Ser Met - # Lys Asp Asn Val Arg Pro LeuGln# 255- Gln Leu Gly Gln Arg Thr Val Ile - # Lys Ser Gly Ala# 265- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 262 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:- Gln His Val Ser Asp Trp Thr Tyr - # Ser Glu Gly Ala Leu Asp GluAla# 15- His Trp Pro Gln His Tyr Pro Ala - # Cys Gly Gly Gln Arg Gln SerPro# 30- Ile Asn Leu Gln Arg Thr Lys Val - # Arg Tyr Asn Pro Ser Leu LysGly# 45- Leu Asn Met Thr Gly Tyr Glu Thr - # Gln Ala Gly Glu Phe Pro MetVal# 60- Asn Asn Gly His Thr Val Gln Ile - # Gly Leu Pro Ser Thr Met ArgMet# 80- Thr Val Ala Asp Gly Ile Val Tyr - # Ile Ala Gln Gln Met His PheHis# 95- Trp Gly Gly Ala Ser Ser Glu Ile - # Ser Gly Ser Glu His Thr ValAsp# 110- Gly Ile Arg His Val Ile Glu Ile - # His Ile Val His Tyr Asn SerLys# 125- Tyr Lys Thr Tyr Asp Ile Ala Gln - # Asp Ala Pro Asp Gly Leu AlaVal# 140- Leu Ala Ala Phe Val Glu Val Lys - # Asn Tyr Pro Glu Asn Thr TyrTyr# 160- Ser Asn Phe Ile Ser His Leu Ala - # Asn Ile Lys Tyr Pro Gly GlnArg# 175- Thr Thr Leu Thr Gly Leu Asp Val - # Gln Asp Met Leu Pro Arg AsnLeu# 190- Gln His Tyr Tyr Thr Tyr His Gly - # Ser Leu Thr Thr Pro Pro CysThr# 205- Glu Asn Val His Trp Phe Val Leu - # Ala Asp Phe Val Lys Leu SerArg# 220- Thr Gln Val Trp Lys Leu Glu Asn - # Ser Leu Leu Asp His Arg AsnLys# 240- Thr Ile His Asn Asp Tyr Arg Arg - # Thr Gln Pro Leu Asn His ArgVal# 255- Val Glu Ser Asn Phe Pro 260- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 261 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:- Gly His His Gly Trp Gly Tyr Gly - # Gln Asp Asp Gly Pro Ala SerHis# 15- Trp His Lys Leu Tyr Pro Ile Ala - # Gln Gly Asp Arg Gln Ser ProIle# 30- Asn Ile Ile Ser Ser Gln Ala Val - # Tyr Ser Pro Ser Leu Gln ProLeu# 45- Glu Leu Ser Tyr Glu Ala Cys Met - # Ser Leu Ser Ile Thr Asn AsnGly# 60- His Ser Val Gln Val Asp Phe Asn - # Asp Ser Asp Asp Arg Thr ValVal# 80- Thr Gly Gly Pro Leu Glu Gly Pro - # Tyr Arg Leu Lys Gln Phe HisPhe# 95- His Trp Gly Lys Lys His Asp Val - # Gly Ser Glu His Thr Val AspGly# 110- Lys Ser Phe Pro Ser Glu Leu His - # Leu Val His Trp Asn Ala LysLys# 125- Tyr Ser Thr Phe Gly Glu Ala Ala - # Ser Ala Pro Asp Gly Leu AlaVal# 140- Gly Val Phe Leu Glu Thr Gly Asp - # Glu His Pro Ser Met Asn ArgLeu# 160- Thr Asp Ala Leu Tyr Met Val Arg - # Phe Lys Gly Thr Lys Ala GlnPhe# 175- Ser Cys Phe Asn Pro Lys Cys Leu - # Leu Pro Ala Ser Arg His TyrTrp# 190- Thr Tyr Pro Gly Ser Leu Thr Thr - # Pro Pro Leu Ser Glu Ser ValThr# 205- Trp Ile Val Leu Arg Glu Pro Ile - # Cys Ile Ser Glu Arg Gln MetGly# 220- Lys Phe Arg Ser Leu Leu Phe Thr - # Ser Glu Asp Asp Glu Arg IleHis# 240- Met Val Asn Asn Phe Arg Pro Pro - # Gln Pro Leu Lys Gly Arg ValVal# 255- Lys Ala Ser Phe Arg 260__________________________________________________________________________

	Number	Date	Country
Parent	654188	Feb 1991
Parent	551270	Jul 1990

Receptor-type phosphotyrosine phosphatase-.beta.

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

Parent Case Info

Foreign Referenced Citations (1)

Non-Patent Literature Citations (52)

Divisions (1)

Continuation in Parts (2)