Osteoblast-testicular protein tyrosine phosphatase
Açar sözlər
Patent məlumatları
Patent nömrəsi | 5821084 |
Təqdim edildi | 11/20/1994 |
Patent tarixi | 10/12/1998 |
Mücərrəd
İddialar
What is claimed is:
1. An isolated nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:1 from nucleotide 205 to 5337.
2. An isolated nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:1 from nucleotide 259 to 5337.
3. An isolated nucleic acid comprising a nucleotide sequence of at least 15 contiguous bases from the sequence shown in SEQ ID NO:1.
4. An isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence from amino acid 19 to amino acid 1711 of SEQ ID NO:2.
5. An isolated nucleic acid which comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleic acid of any one of claims 1 through 4.
6. A vector comprising the nucleic acid of claim 5.
7. A host cell comprising the nucleic acid of claim 5.
8. A vector comprising the nucleic acid of any one of claims 1 to 4.
9. A host cell comprising the nucleic acid of any one of claims 1 to 4.
10. A process for preparing osteoblast-testicular protein tyrosine phosphatase comprising the steps of:
a) transfecting an expression vector comprising a nucleic acid which encodes osteoblast-testicular protein tyrosine phosphatase, which polypeptide comprises the amino acid sequence of SEQ ID NO:2, into a suitable host cell, and
b) culturing said host cell under conditions which allow expression of said polypeptide.
11. A process according to claim 10, further comprising the step of isolating said polypeptide.
12. An isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence from amino acid 1 to 1068 of SEQ ID No:2,catalytically functional derivatives and variants thereof having OST-PTP catalytic activity.
13. An isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence from amino acid 18 to amino acid 1068 of SEQ ID No:2, catalytically functional derivatives and variants thereof having OST-PTP catalytic activity.
14. An isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence from amino acid 32 to amino acid 921 of SEQ. ID No. 2, catalytically functional derivatives and variants thereof having OST-PTP catalytic activity.
15. An isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID No. 2 from amino acid 1348 to amino acid 1359, catalytically functional derivatives and variants thereof having OST-PTP catalytic activity.
16. An isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID No. 2 from amino acid 1668 to amino acid 1678, catalytically functional derivatives and variants thereof having OST-PTP catalytic activity.
17. An isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID No. 2 from amino acid 1348 to amino acid 1359 and wherein the cysteine at position 1350 is replaced by serine.
18. An isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence from amino acid 32 to 1678 of SEQ ID No. 2, catalytically functional derivatives and variants thereof having OST-PTP catalytic activity.
19. An isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence from amino acid 1 to 1104 of SEQ ID No. 2, catalytically functional derivatives and variants thereof having OST-PTP catalytic activity.
20. An isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence from amino acid 1105 to 1171 of SEQ ID No. 2, catalytically functional derivatives and variants thereof having OST-PTP catalytic activity.
21. An isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID No. 2 from amino acid 1348 to amidino acid 1678, catalytically functional derivatives and variants thereof having OST-PTP catalytic activity.
22. An isolated nucleic acid which comprises a nucleotide sequence which hybridizes under stringent conditions to a nucleic acid of any of claims 12 to 21.
23. A vector comprising the nucleic acid of any of claims 12 to 21.
24. A vector comprising the nucleic acid of claim 22.
25. A host cell comprising the nucleic acid of any of claims 12 to 21.
26. A host cell comprising the nucleic acid of claim 22.
27. A process for preparing a polypeptide comprising:
a) transfecting an expression vector comprising the nucleic acid of any of claims 12 to 21 and which encodes the polypeptide into a suitable host cell; and
b) culturing said host cell under conditions which allow expression of the polypeptide.
28. A process for preparing a polypeptide having OST-PTP tyrosine phosphatase activity comprising:
a) transfecting an expression vector comprising the isolated nucleic acid of claim 22 that encodes the polypeptide having the OST-PTP tyrosine phosphatase activity into a suitable host cell; and
b) culturing said host cell under conditions which allow expression of the polypeptide.
29. A process according to claim 27, further comprising the step of isolating the polypeptide.
30. A process according to claim 28, further comprising the step of isolating the polypeptide.
Təsvir
FIELD OF THE INVENTION
The present invention in the field of biochemistry and cell and molecular biology relates to the discovery of a novel transmembrane receptor-type protein tyrosine phosphatase termed osteotesticular protein tyrosine phosphatase (OST-PTP). The cloning and sequencing of the RNA transcript encoded by the OST-PTP gene is described as weel as the expression and enzymatic characterization of the OST-PTP protein. The invention further relates to methods for screening compounds capable of binding to and inhibiting or stimulating OST-PTP activity and use of this invention to diagnose and treat bone metabolic disorders via targeted conventional treatment or gene therapy.
BACKGROUND OF THE INVENTION
Protein tyrosine phosphorylation is important in the regulation of cell growth, proliferation and differentiation as well as in the homeostasis of metabolic processes. A functional relationship between tyrosine phosphorylation and cell processes has been demonstrated by studies on the activity of enzymes such as phospholipase C (PLC) (Hunter et al., 1985, Ann. Rev. Biochem. 54:897-930, Ullrich et al., 1990, Cell 61:203-212). Growth factor stimulation of certain cells in culture increases the level of tyrosine phosphorylation as well as the activity of PLC. The protein tyrosine kinases are responsible for this phosphorylation of tyrosine residues within cellular proteins. It is important to recognize that the degree and pattern of phosphorylation by these protein tyrosine kinases can be regulated by an opposing dephosphorylation catalyzed by a family of enzymes, the protein tyrosine phosphatases (PTP). In this way, protein tyrosine phosphatases help calibrate the level of tyrosine phosphorylation in a cell and provide another regulatory control system to govern cellular processes.
The protein tyrosine phosphatases are a structurally diverse family of proteins which include transmembrane molecules known as receptor-like PTPs and non-transmembrane molecules known as intracellular PTPS. These proteins all posses a conserved catalytic domain containing the active site sequence, (I/V) HCXAGXXR (S/T)G (SEQ ID NO:3). The cysteine residue within this active site is critical for catalytic activity, forming a phosphocysteine intermediate during the phosphate monoester hydrolysis. Typically, the intracellular PTPs possess a single catalytic domain, whereas the receptor-like PTP often have two cytoplasmic catalytic domains separated by 56-57 amino acids. Fourteen of the sixteen transmembrane receptor-like PTP proteins previously isolated contain two such domains (Walton et al., 1993, Ann. Rev. Biochem. 62:101-120).
One of the best characterized receptor-like tyrosine phosphatases described is CD45 or leukocyte common antigens (Ralph et al., 1987, EMBO J. 6:1251-1257, Charbonneau et al., 1989, Proc. Natl. Acad. Sci. USA, 85:7182-7186), which consists of a single polypeptide comprised of an extracellular domain, a membrane spanning domain and two nearly identical intracellular catalytic domains of approximately 300 amino acids each. CD45 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. Other receptor-like PTP molecules contain one to three tandem Ig-like domains near their N-termini in the extracellular domain often adjacent to multiple fibronectin type III-like repeats (FN-III) resembling the N-CAM family of neural cell adhesion molecules. Recent evidence indicates that PTP molecules with the FN-III motif not only have ligand binding properties but are involved in cell adhesion e.g. receptor-like PTP-.mu. is involved in homophilic binding mediated cell-cell aggregation (Brady-Kalnay et al., 1993, J. Cell. Biol. 122:961-972) .
Accumulating evidence indicates that the process of dephosphorylation of tyrosine can serve as an important regulatory mechanism in cellular function. For example, 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). Also, tyrosine dephosphorylation appears to be an obligatory step in the mitotic activation of the maturation promoting factor (MPF) kinase (Morla et al., 1989, Cell 58:193-203) . However only minimal information regarding the cellular function of particular PTP molecules is presently available. Small, soluble PTP enzymes may have a "housekeeping" function (Tonks et al., 1988, Biochem. 27: 8695-8701). On the other hand, the receptor-like PTP molecules appear to be more specific both in their expression and activity by virtue of their location in the cell membrane and potential regulation by extracellular ligands.
A definitive role for CD45 has been established in T cells where CD45 activated a lymphocyte specific kinase (p56.sup.lck) by dephosphorylation of tyrosine residue 505 leading to T cell activation. CD45 deficient T cell clones failed to proliferate upon receptor stimulation with a specific antigen (Pingel, J. T., 1989, Cell 58:1055-1065). Indirect evidence indicates that a protein tyrosine phosphatase cdc25 was involved in control of the timing of mitosis by regulation of a protein serine/threonine kinase, the cdc2 gene product of p34 cdc2. A mutant form of p34 cdc2, in which tyrosine 15 was changed to phenylalanine, rescued a cdc25 temperature sensitive mutant at the restrictive temperature. Therefore, dephosphorylation of cdc2 by the tyrosine phosphatase cdc25 is necessary for initiation of cell division.
Recent studies have extended the role of protein tyrosine phosphorylation to bone metabolism. Targeted disruption of the mouse c-src proto-oncogene, a protein tyrosine kinase, caused osteopetrosis, a disease characterized by absence of appropriate bone resorption due to impaired osteoclast function (Soriano et al., 1991, Cell 64:693-702). There were no abnormalities detected in brain or platelets despite the fact that these were known to be the sites of highest c-src expression. Congenital ostepetrosis, as exhibited by the op/op mouse, is also due to a disruption of tyrosine phosphorylation. These animals lack biologically active macrophage colony stimulating factor (M-CSF), which serves as the ligand for c-fms, a proto-oncogene encoding a tyrosine kinase which is necessary for osteoclast function. These limited data regarding the dramatic effect of inhibition of protein tyrosine phosphorylation in bone metabolism point out the need in the art to increase our understanding of protein tyrosine phosphatase function in this tissue, particularly since the biological role of PTP is to reduce the cellular level of protein tyrosine phosphorylation.
Two recent abstracts have reported the presence of protein tyrosine phosphatases in bone. In the first, a receptor-like PTP was identified in bone and brain but with maximal expression in growing cells and negligible expression in confluent cultures (Endo et al., 1993, "Identification and Cloning of a Novel Receptor-like Protein Tyrosine Phosphatase from Human Osteoblasts," American Society of Bone and Mineral Research Meeting 8:S5). In a second abstract (Schmidt et al., 1993, "Identification of an Osteoclast Specific Protein Tyrosine Phosphatase and its Potential Role in osteoclast Fusion and Bone Resorption," American Society of Bone and Mineral Research Meeting 8:S144) mouse calvarial cells (osteoblasts) were incubated with bone marrow cells and 1,25 dihydroxyvitamin D3. Multinucleated cells that were tartrate-resistant acid phosphatase (TRAP) positive were enriched. They isolated the previously identified PTP-.epsilon. (Kreuger et al., 1990, EMBO J. 9:3241-3252) as a major PTPase transcript in these cells but reported that it was specific for the osteoclast. The timing of expression and spatial distribution of these protein tyrosine phosphatases as well as their transcript and protein molecular size indicate that they are clearly distinct from the present invention.
The identification of a tissue specific protein tyrosine phosphatase is important not only in understanding cellular functions but also as a basis for targeting therapeutic agents at a specific tissue without the risk of side effects in other cell types. Specific cell targeting has been demonstrated in the treatment of carcinomas. An immunoconjugate of the monoclonal antibody (BR96) and an anticancer agent, doxorubicin, bound to a tumor-associated antigen abundantly expressed on human carcinoma lines (Trail et al., 1993, Science 261:212-215). The BR96-doxorubicin immunoconjugate induced complete regression of xenografted human lung, breast and colon carcinomas growing subcutaneously in athymic mice. Similar principles have recently been applied to induce protein tyrosine phosphorylation and subsequent cell proliferation through the use of chimeric molecules. For example, chimeric molecules which linked the extracellular receptor domain of epidermal growth factor (EGF) to the cytoplasmic domain of the erythropoietin receptor successfully transmitted EGF proliferation signals (ohahi et al., 1994, Proc. Natl. Acad. Sci. USA 91:158-162). In another demonstration of this principle, various human cytokine receptors (interleukin-2, interleukin-4) as well as the murine erythropoietin receptor were separately linked to the cytoplasmic domain of the interleukin-3 receptor .beta. subunit .beta.-Il3 (Sakamaki et al., 1993, J. Biol. Chem. 21:15833-15839). These chimeric receptors were functional in cells in that stimulation by the cytokine matching the receptor domain induced tyrosine phosphorylation and proliferation in the target cells.
Use of antibodies to target drug molecules has also been described previously. Trail described the cure of xenografted human carcinomas by an immunoconjugate of the monoclonal antibody BR96 with doxorubicin (Science 261:212-215 (1993)). BR96 binds to a tumor-associated antigen that is closely related to Lewis Y and is abundantly expressed on human carcinoma lines. Since BR96 binds the majority of human carcinomas of the breast, lung, and colon, it specifically targets the anticancer agent doxorubicin to these carcinomas. BR96-doxorubicin induced complete regressions and cures of xenografted human lung, breast, and colon carcinomas growing subcutaneously in athymic mice and cured 70 percent of mice bearing extensive metastases of a human lung carcinoma.
A similar targeting approach using immunoconjugates or chimeric molecules may be applicable to the important regulatory process of phosphatase induced tyrosine dephosphorylation with the identification of protein tyrosine phosphatase molecules bearing extracellular receptor domains which confer tissue specificity. The invention described herein is a protein tyrosine phosphatase with said requirements, i.e., a novel tissue specific PTP (as well as a truncated receptor transcript) which plays a critical role in the processes of osteogenesis and bone remodeling and shows stage-specific expression in the seminiferous tubules during the process of spermatogenesis.
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes a unique mammalian (rat) receptor-like tyrosine phosphatase protein expressed specifically in bone and testis and termed osteotesticular protein tyrosine phosphatase (OST-PTP). The naturally occurring protein is normally expressed exclusively in bone predominantly during osteoblast proliferation and differentiation; and in the basal seminiferous tubules of the testis concomitant with spermatid maturation during stages I-VII and XII-XIV. The substantially pure OST-PTP protein may be produced by biochemical purification of the naturally occurring protein. Alternatively, the OST-PTP protein may not be of natural origin, such as one prepared by utilizing chemical synthesis technology or produced by recombinant means in prokaryotic or eukaryotic hosts. This invention then provides a molecular, biochemical instrument whereby the dephosphorylation of tyrosine--a critical regulatory step in the mechanism of cell growth, differentiation and oncogenesis--can be specifically addressed in the context of bone maturation and repair and during select stages in the process of spermatogenesis.
The OST-PTP of the present invention is further characterized as a unique 1711 amino acid protein containing an extracellular domain of 1068 amino acids including a 17 amino acid signal sequence associated with ligand binding and 10 fibronectin type III-like domains (FN-III) which mediate cell-cell homophilic binding and cell-matrix heterophilic attachment. The intracellular region consists of a 610 amino acid cytoplasmic domain but is unusual for the protein tyrosine phosphatase (PTP) class of molecules in that two, typical cytoplasmic PTP domains are present but the active site of domain two lacks the invariant cysteine required for catalysis.
The invention is described as a 5455 base nucleotide sequence for a full length cDNA encoding the is OST-PTP protein in a single open reading frame yielding a low abundance RNA transcript of 5800 bases. The nearest homologues to the full-length OST-PTP cDNA are human PTP-.delta., PTP-.gamma. and LAR (leukocyte common antigen related protein) with 27-30% sequence identity. The invention also includes the OST-PTP nucleotide sequence in the form of an expression vehicle as well as prokaryotic and eukaryotic hosts transformed with the DNA encoding the complete OST-PTP molecule or a part of it.
Another aspect of the present invention is a native, synthetic or recombinant molecule of OST-PTP which exhibits all or a portion of the structure and properties exhibited by the native protein such as 1) a consensus PTP catalytic domain and the enzymatic capacity to selectively dephosphorylate a tyrosine phosphorylated substrate, and/or 2) the extracellular FN-III repeat motif associated with intercellular aggregation between osteoblasts and other cell types in the osteogenic matrix and with aggregation of the germ cells with basement membrane and Sertoli cells during spermatogenesis. Thus, functionally equivalent molecules, including truncated forms (analogues, variants, derivatives, and fragments-see Definitions), are considered embodiments of the present OST-PTP invention that exhibit the biological properties, or a portion of those properties, attributed to the parent OST-PTP protein. It has been demonstrated that 1) a glutathione-S-transferase fusion protein incorporating the OST-PTP catalytic region exhibits tyrosine phosphatase activity and substrate specificity, and 2) a truncated, inactive splice variant termed OST retains only the transmembrane and extracellular domains of OST-PTP but is osteoblast specific. Thus, other important embodiments of the present invention include, but are not restricted to, the GST-OST fusion protein and the OST splice variant as derivatives of the parent OST-PTP protein.
Included in the present invention is a method for preparing the OST-PTP protein, or a functional derivative, by culturing a host capable of expressing the protein and from which the protein can be expressed, recovered and purified.
Included in the present invention is any polyclonal, monoclonal or chimeric antibody specific for the OST-PTP protein or a portion of the OST-PTP molecule. This provision extends to immunoconjugates targeted at osteogenic tissue through specificity for an epitope within the OST-PTP molecule.
Also included in this invention is any chimeric molecule which contains a region which will bind to OST, i.e., an antibody or ligand, as well as a region containing a bone-specific or testis-specific agent, for example, an agent which can regulate bone remodeling. Bone-specific therapeutic agents within the present invention include, but are not limited to, any agents which are useful in the bone-related therapies or other treatments including, but not limited to, anti-tumor agents, those agents which stimulate bone growth or which block bone resorption, including inhibitors or activators of PTPases, cytokines and growth factors which are known to work on bone, such as IGF-1 and bone morphogenetic proteins. Testis-specific agents contemplated by the present invention include, but are not limited to, cytokines or activators or inhibitors of PTPases or factors that are steroidogenic agonists or antagonists or anti-tumor agents, such as M-CSF, TNF.alpha. or TGF.beta..
Abnormal levels of the OST-PTP receptor/enzyme system may be detected by methods directed at determining the presence of or measuring the quantity of OST-PTP in a cell or in a subject. The present invention includes use of an antibody specific for an epitope of OST-PTP to contact a target cell or cell extract. The presence of OST-PTP or the quantity of the molecule is detected by antibody binding or quantitative assay of the amount of bound antibody.
The OST-PTP invention contains a receptor-like domain which enables the native protein to function as a transmembrane receptor involved in the process of osteogenesis. Consequently, OST-PTP can serve as a novel affinity probe for the screening of naturally occurring ligands from a biological preparation or synthetic chemical compounds directed at modification of bone growth and differentiation including treatment of metabolic bone disorders such as osteoporosis, osteopetrosis and Paget's Disease. The present invention includes methods for identifying and isolating a compound capable of binding to OST-PTP or a portion thereof such as the OST splice variant. This process involves attaching the OST-PTP molecule, or the ligand-binding domain, to a solid phase matrix and allowing the compound to bind, subsequently washing away unbound material and eluting and isolating the ligand.
Compounds with the ability to stimulate or inhibit OST-PTP activity may serve as therapeutic agents in the treatment of aberrant bone growth as well as metabolic bone diseases. Therefore, the present invention includes methods for identifying compounds capable of altering the enzymatic activity of the OST-PTP molecule. This process involves presentation of the test compound to active OST-PTP in pure form, in a membrane preparation, or in whole cells. After sufficient incubation at appropriate conditions, the enzymatic activity of the test mixture is measured for comparison with the same OST-PTP preparation assayed in the absence of that compound.
OST-PTP expression follows a highly regulated spatial and temporal pattern during osteogenesis and spermatogenesis. Abnormal expression patterns of OST-PTP or the expression of aberrant OST-PTP may be diagnostic of developmental bone disorders or susceptibility to oncogenic transformation. The present invention includes methods for detecting the presence of nucleic acids encoding normal or mutant OST-PTP in a subject. Oligonucleotide probes derived from various portions of OST-PTP can be used to test a subject for the presence of RNA encoding the protein sequence specific to the OST-PTP enzyme. A preferred probe would be one directed to the nucleic acid sequence of at least 5 amino acid residues of the invention. Northern analysis is used to measure OST-PTP mRNA levels in a cell or tissue preparation which is then quantitated by comparison to a loading control such as the RNA for a constitutively expressed message. This technology then provides a basis for comparison of RNA expression levels from normal subjects and patients with potentially altered OST-PTP levels. Moreover, it affords the opportunity to screen for the presence of mutated OST-PTP forms associated with bone disorders which may bind to a panel of preferred OST-PTP probes with different stringencies or at different molecular weights than the OST-PTP of the present invention. Correlation of mismatches with incidence of tumorigenesis may provide an important predictor of bone cancer risk.
The present invention is thus directed at a unique OST-PTP protein and its derivatives in purified, synthetic and recombinant forms. The purview of this invention includes expression restricted to bone tissue and the seminiferous tubules and extends to the properties of this molecule including 1) its catalytic capacity to dephosphorylate a tyrosine phosphorylated substrate and 2) the cell adhesive and communication properties mediated through the extracellular fibronectin-like repeats. The restricted expression pattern of this molecule to bone and testis designates it as a vehicle by which those tissues may be targeted for diagnostic or therapeutic purposes. Moreover, the regulation of OST-PTP by naturally occurring endogenous hormones such as parathyroid hormone suggests that OST-PTP levels can be used directly as an index of normal bone development and remodeling as well as normal spermatogenesis.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 (SEQ ID NO:4 and SEQ ID NO:5) is a schematic depiction of the 1711 amino acid (aa) OST-PTP protein of the present invention containing a 1068 aa extracellular domain. Important structural components include the signal peptide (SP=17 aa), the ten fibronectin type III-like domains (FN-III), the transmembrane domain (TM) and the two intracellular domains associated with phosphatase catalytic activity (PTPase domains I and II). The listed 11 amino acid sequences delineate the characteristic conserved residues at the active site but the substitutions in Domain I--specifically the cysteine to serine substitution--render it inactive (Abbreviations for the amino acid residues are: A, Ala; C, Cys; G. Gly; H. His; K, Kys; L, Leu; N, Asn; Q, Gln; R, Arginine; S, Ser; T, Thr; V, Val.). Invariant residues are indicated with an asterisk.
FIG. 2 presents the results of a basic kinetic analysis of the catalytic portion of the OST-PTP protein expressed as a glutathione-S-transferase fusion protein. Hydrolysis of the artificial substrate para-nitrophenylphosphate (pNPP) was determined by absorbance measurement at a wavelength of 410 nm. Enzyme activity was measured in a 200 .mu.l reaction containing buffer (0.1M imidazole, 0.1 mM EDTA, 0.1% .beta.-mercaptoethanol) 20 mM pNPP and 820 ng of enzyme. Incubation was performed over a range of pH at 30.degree. C. for 10 minutes and maximum activity was detected at pH 5.5.
FIG. 3 is an RNA blot analysis of the expression of the novel OST-PTP transcript in various tissues of the rat and confluent cultures of the UMR 106 osteosarcoma cell line (standard for OST-PTP expression in bone). The Northern blot consisted of 10 ug of polyadenylated RNA per lane (except where indicated that 5 ug of UMR 106 RNA was loaded) fractionated on a 6.6% formaldehyde-1% agarose gel and transferred to a nylon membrane. A riboprobe corresponding to the extracellular domain of the OST-PTP transcript and a cDNA probe for cyclophilin were hybridized to the blot as an indicator of the presence of OST-PTP transcripts normalized for the amount of cyclophilin mRNA which was considered to be standard for all conditions. The results indicated that the OST-PTP transcript was exclusively expressed in RNA derived from bone cells and from testis.
FIG. 4A is an RNA blot analysis as described in FIG. 3 with the addition of a cDNA probe for alkaline phosphatase transcripts. RNA from cultures of primary rat osteoblasts was harvested during cell proliferation (P), extracellular matrix differentiation (D) and extracellular matrix mineralization (M) and from confluent cultures of UMR 106. Note that a smaller 4.8 kb transcript termed OST which contained the extracellular domain was present in the proliferation and differentiation phases in addition to the 5.8 kb OST-PTP transcript.
FIG. 4B is a histogram comparing expression of the OST-PTP transcript during the phases of osteoblast proliferation, differentiation and mineralization. The Northern blot in FIG. 4A was analyzed by phosphorimager scanning (Molecular Dynamics) and the results were normalized to the level of cyclophilin in each lane. OST-PTP expression was low during proliferation, increased 10 fold during differentiation and started to decline during mineralization. The 4.8 kb OST transcript was expressed at highest levels during proliferation, declined during differentiation and was absent during the mineralization phase.
DESCRIPTION OF SEQUENCE LISTING
The Sequence Listing (SEQ ID NO.: 1) and SEQ ID NO:2) at the end of the Specification is the nucleotide sequence of cDNA together with the deduced amino acid sequence encoding OST-PTP. DNA sequencing was performed using an Applied Biosystems automated DNA sequencer. Numbers on the right of the columns indicate base positions. The first base position corresponds to the first nucleotide in the 5' extension clone #20. The ten fibronectin type III-like domains (FNI-FNX) are located as follows (for the purposes of these listings, the first amino acid listed begins the segment and the final amino acid listed terminates it): FNI=amino acid (aa) 32 (Arg) to aa 124 (Ala); FNII=aa 125 (Arg) to aa 215 (Trp); FNIII=aa 216 (Thr) to aa 303 (Trp); FNIV=aa 304 (Thr) to aa 392 (Ala); FNV=aa 393 (Ala) to aa 470 (Tyr); FNVI=aa 471 (Thr) to aa 562 (Cys); FNVII=aa 563 (Thr) to aa 652 (Gly); FNVIII=aa 653 (Trp) to aa 741 (Trp); FNIX=aa 742 (Thr) to aa 830 (Ser); and FNX=aa 831 (Val) to aa 921 (Ser). The two predicted hydrophobic domains are boxed from aa 3 (Pro) to aa 18 (Ala) and from aa 1082 (Leu) to aa 1104 (Leu). The 5' hydrophobic region demonstrates homology to a signal sequence. A predicted cleavage site is located between aa 18 (Ala) and aa 19 (Glu) as indicated by an arrow. The 3' hydrophobic end is the putative transmembrane domain. The catalytic includes aa 1348 (Val) to aa 1359 (Thr) and is 3' to the transmembrane domain, in the cytoplasmic region. A second catalytic domain which is missing key amino acids required for activity is located from aa 1668 (Val) to aa 1678 (Leu). The predicted N-linked glycosylation sites include aa 117 to aa 119 (Asn-Val-Thr), aa 239 to aa 241 (Asn-Ser-Ser), aa 259 to aa 261 (Asn-Thr-Thr), aa 431 to aa 433 (Asn-Ile-Ser), aa 551 to aa 553 (Asn-Leu-Ser), aa 570 to aa 572 (Asn-Leu-Ser), aa 620 to aa 622 (Asn-Phe-Ser), aa 649 to aa 651 (Asn-Ala-Thr), aa 663 to aa 665 (Asn-Val-Thr), aa 737 to aa 739 (Asn-Val-Ser), aa 851 to aa 853 (Asn-Trp-Thr), aa 882 to aa 884 (Asn-Thr-Ser), aa 970 to aa 972 (Asn-Met-Thr), aa 982 to aa 984 (Asn-Tyr-Thr), aa 1266 to aa 1268 (Asn-Ser-Thr), and aa 1569 to aa 1571 (Asn-Thr-Thr). The termination codon is at 5340 and the 3' noncoding sequence are included with the poly (A) tract beginning at nucleotide 5449.
MODES OF CARRYING OUT THE INVENTION
Utilizing recombinant DNA technology, the inventors identified a unique mammalian (rat) bone-testis tyrosine phosphatase protein termed osteotesticular protein tyrosine phosphatase (OST-PTP) and a truncated derivative (OST). The reverse transcriptase-polymerase chain reaction methodology was employed using degenerate primers based on the conserved regions of previously characterized tyrosine phosphatase catalytic domains. Osteogenic tissue was screened to resolve the novel OST-PTP clones from among other tyrosine phosphatase proteins. Sequence analysis including Genebank comparisons established that the nearest homologue to the OST-PTP sequence was PTP-.epsilon., which exhibited only 45% identity in the catalytic region. Further unusual characteristics of OST-PTP are its low abundance RNA and the presence of a single active carboxy-terminus catalytic domain, in contrast to the bipartite active catalytic domains found in 14 of the 16 transmembrane PTP molecules isolated thus far. The protein also contains an extracellular domain of 10 FN-III repeats, a motif known to be important in homophilic binding of cells as well as in the attachment of cells to an extracellular matrix. These structural features and the restricted expression pattern of OST-PTP distinguish this protein as a previously undiscovered member of the protein tyrosine phosphatase family.
OST-PTP expression increases markedly (5-8 fold) in association with osteoblast differentiation and the bone mineralization, in contrast to the low levels expressed during the osteoblast proliferation. Moreover, OST-PTP expression in vitro is exquisitely sensitive to parathyroid hormone (PTH), a critical modulator of bone metabolism in vivo which inhibits osteoblast proliferation and stimulates bone resorption. Stimulation of the protein kinase A (PKA) pathway using the cyclic AMP analogue, chlorophenylthio-cAMP resulted in an increase in OST-PTP mRNA levels comparable to PTH treatment and consistent with the idea that OST-PTP is modulated by PTH through the PKA pathway. These findings indicate that the OST-PTP program is critical to proper bone growth and metabolism and, therefore, an important potential target for manipulation of bone growth, mineralization and treatment of bone pathology. The discrete timing and spatial expression pattern of OST-PTP provides a basis for targeting pharmacological agents with the capacity to alter major pathways involved in osteogenesis and maintenance of bone integrity. The transmembrane receptor structure of the OST-PTP protein suggests that ligands in the extracellular environment control the activity of this phosphatase and supports its potential role as a tissue specific target for pharmacological agents. A variety of proven methods are presently available for the purpose of screening for natural or synthetic ligands. These methods are outlined below.
(a) By attaching intact OST-PTP or its ligand-binding domain to a solid phase matrix, an affinity probe is created for screening of biological products or chemical agents to determine their capacity to interact with (i.e., bind to) the receptor of the invention. Bound material can then be eluted from the affinity probe in purified form. Methods for coupling of peptides and proteins to a solid phase matrix and means for elution are well known to those of skill in the art. Note that this technique may be applied separately to the truncated OST receptor molecule which contains the extracellular domain of OST-PTP. Furthermore, secondary screening analysis may be used to test the effect of systematic deletions or mutations in regions of the OST-PTP protein perceived to be critical to biological function. Standard recombinant technology will be used to identify, alter and characterize such mutations in previously identified ligand binding sequences deemed critical.
(b) OST-PTP or active derivatives can be used for testing of compounds to determine their ability to enhance or inhibit phosphatase activity. Modification of phosphatase activity can be assayed in vitro by addition of test compounds to purified OST-PTP or an enzymatically active derivative followed by standard enzymological analysis well known to those of skill in the art. This technology applies as well to testing of antibodies for binding and correlative antagonist or agonist properties. This assay may be performed in the presence of a known activator of OST-PTP or in the presence of an activator tyrosine kinase.
(c) The action of a compound on OST-PTP can be measured in whole cell preparations using live or fixed cells, e.g., isolated primary cells or cell lines including primary rat osteoblasts or UMR106 osteosarcoma cells which express high levels of OST-PTP under regulated conditions. A test compound is incubated with the cells and the amount of cellular phosphotyrosine is then measured according to established methods (Margolis et al., 1989, Cell 57:1101-1107) for comparison with cultures treated identically but lacking the test compound. In addition, this method is useful for determining whether test compounds act via the extracellular receptor portion of the OST-PTP protein. This assay may be performed in the presence of a known activator of OST-PTP or in the presence of an activator tyrosine kinase.
(d) Alternatively, method (c) can be employed with a cellular membrane fraction containing high levels of OST-PTP, e. g., primary cells or osteogenic cell lines or from transfection of cells with constructs designed to yield high levels of membrane integrated OST-PTP such as transfected COS or NIH-3T3 cells. The use of a membrane fraction allows one skilled in the art to screen compounds for action directed at the extracellular receptor portion versus the cytoplasmic enzymatic subunit. This assay may be performed in the presence of an activator of OST-PTP or the presence of an activator tyrosine kinase.
(e) The methods described above, i. e., whole cell or membrane preparations, may be applied to examine the role of the FN-III domain in cellular aggregation and differentiation. The role of the FN-III domain in intercellular aggregation and communication may be tested directly by systematic and sequential deletion of regions of the FN-III domains and analyzing their effects in primary culture or cell lines during proliferation and differentiation phases. In conjunction with these studies, drugs or antibodies previously screened as targeting the extracellular FN-III domain may be tested for effects on either inhibition or stimulation of cell adhesion and subsequent effects on cellular differentiation both in the process of osteogenesis and spermatogenesis.
(f) The OST-PTP invention may be utilized to determine the relationship between the FN-III extracellular domain and the enzymatic capabilities of the OST-PTP protein. Deletion of the extracellular ligand binding region or the use of drugs or antibodies which block the signal sequence allow analysis of phosphatase activation via the remaining extracellular region particularly the FN-III repeat domain. DNA constructs incorporating modifications of the extensive FN-III domains may be generated, transfected and expressed in transient or stable primary cells or cell lines to assay for effects on catalytic activity in combination with or separate from a role in cellular aggregation.
In summary, the cloning of OST-PTP including the OST receptor form of the invention provides a novel tool with which to screen naturally occurring ligands as well as natural or synthesized pharmacological agents with the potential for therapeutic application to a variety of bone disorders. The unique tissue specificity of the invention defines a distinct drug target while eliminating potential side effects in other tissues. Furthermore, useful chimeric molecules can be constructed which contain an OST binding region, e.g., either an anti-OST antibody or a ligand for OST, as well as a region which can modulate the process of bone remodeling or spermatogenesis. This technology has recently been utilized to design tissue specific pharmaceutical agents where a tissue specific receptor--other than bone or testis--provides the target (Trail et al., 1993, Science 261:212-215) in a manner analogous to that described for the present invention. Consequently, the present invention will allow this technology to extend to the process of osteogenesis as well as bone disorders such as osteoporosis, osteopetrosis and Paget's disease.
Recent evidence indicates that protein tyrosine phosphatase activation provides a counterregulatory mechanism to inhibit mitogenic influence on cells mediated through tyrosine kinase growth factor receptors, e.g., epidermal growth factor (EGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF). Consequently, reduced or blocked phosphatase function is implicated in the process of oncogenic transformation of cells and susceptibility to cancer. Specifically, mutation of the OST-PTP genomic DNA or mutation or dysregulation of the OST-PTP receptor/enzyme system at the RNA or protein level may play a role in development of bone-related cancers. The present invention provides methods for analysis of the genomic sequence encoding the OST-PTP invention and for evaluating or level of normal or aberrant OST-PTP RNA or protein expression which may predict or diagnose bone pathologies including cancer.
Analysis of the organization and structure of the genomic DNA encoding the OST-PTP invention can be performed by standardized procedures including but not restricted to Southern blotting (Southern, E., 1975, J. Mol. Biol. 98:503-517). High molecular weight genomic DNA is purified, cleaved with a variety of restriction enzymes, fractionated via agarose gel electrophoresis and transferred to a filter or membrane for hybridization with a series of OST-PTP specific probes of either purified RNA, cloned cDNA or short synthetic oligonucleotides. A preferred oligonucleotide probe would be one directed to the nucleic acid sequence of at least 5 amino acid residues of the invention. The resulting pattern of bands obtained after hybridization and stringent washing delineate the number and size of the DNA fragments containing OST-PTP sequences. These data are used to generate a restriction map of the OST-PTP gene and for comparative analysis of DNA samples from a variety of normal subjects and those with identified bone pathologies. Aberrant restriction patterns on Southern blots allow detection of deletions or rearrangements which may be associated with particular bone pathologies or serve as a predictor of bone disorders or susceptibility to bone cancer.
Analysis of RNA size and expression pattern can be performed by standardized procedures, including but not restricted to Northern blotting. Analogous to Southern blotting, total or polyadenylated RNA is purified, size-separated via agarose electrophoresis, transferred to a solid matrix and sequentially hybridized to a panel of OST-PTP cDNA or oligonucleotide probes as well as a standard probe for a constitutively expressed RNA as a loading control, e.g., alkaline phosphatase, glyceraldehyde 3-phosphate dehydrogenase or .beta.-cytoplasmic actin. OST-PTP mRNA expression levels in a particular cell or tissue preparations from multiple subjects can then be quantified by comparison to the expression of the constitutively expressed loading control. This technology then provides a means for comparison of RNA expression levels among multiple samples to establish normal levels as well as define altered OST-PTP levels or patterns of expression. Moreover, this analysis provides a screening mechanism to detect modified or aberrant RNA products associated with bone pathologies.
These screening methods can be used even with very small amounts of DNA or RNA obtained from an individual, following use of selective amplification techniques known to those of skill in the art. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized including 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 (Cohen et al. (U.S. Pat. No. 4,237,224); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 2d ed., 1989)). Moreover, the polymerase chain reaction (PCR) provides a standardized method for selectively increasing the concentration of a particular nucleic acid even when that sequence has not been previously purified and is present only in a single copy in a particular sample (Mullis et al., 1986, Cold Spring Harbor Symp. Quart. Biol. 51:263-273; Erlich et al., EP 50,424; EP 84,796; EP 258,017; EP 237,362; Mullis, K., EP 201,184; Mullis et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki et al., U.S. Pat. No. 4,683,194).
The PCR method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired DNA sequence. Where RNA is the initial starting substrate, the enzyme reverse transcriptase is used to produce cDNA copies of each mRNA for PCR amplification and analysis. The precise nature of the two oligonucleotide primers of the PCR method is critical to the success of the method as it is the primers which define the sequence to be amplified. The oligonucleotide sequences of the probes of the PCR are selected such that they contain sequences identical to, or complementary to, sequences which flank the particular nucleic acid sequence whose amplification is desired. Therefore, the present invention is critical as it provides novel OST-PTP sequences to serve as PCR primers to exclusively amplify regions of the OST-PTP genomic DNA or RNA. Thus, by permitting cycles of polymerization, and denaturation, a geometric increase in the concentration of the desired OST-PTP sequence can be achieved (Saiki et al., 1985, Biotechnology 3:1008-1012; and Mullis et al., 1987, Meth. Enzymol. 155:335-350). As described for Southern and Northern analysis, the OST-PTP PCR products can be analyzed to detect rearrangements or deletions associated with bone pathology. Multiplex PCR involving simultaneous amplification of a constitutively expressed transcript as well as the OST-PTP sequence allows quantitative analysis to be performed by normalizing OST-PTP expression level on the basis of the constitutive product level. Comparisons of normalized expression levels then can be made among subjects for correlation with bone pathology.
The present invention provides methods for evaluating the level of normal or mutant OST-PTP both as a functional enzyme and based on molecular weight. The presence of normally functioning OST-PTP in a subject can be tested using direct enzymatic assay for the tyrosine phosphatase activity. Such biochemical measurements can be performed in vitro using purified enzymes which allow precise measurements of enzyme activity, or with membrane preparations, or whole cells, where the net phosphotyrosine level is determined. In addition, a polyclonal or monoclonal antibody specific to the OST-PTP invention may be used to characterize the molecular weight of the OST-PTP protein using a standardized technique of Western blotting. Protein is separated by electrophoresis using a sodium dodecyl sulfate polyacrylamide gel and then transferred to a filter for hybridization with an OST-PTP specific antibody. Variations in the molecular weight of the OST-PTP protein associated with mutations or post-translational modification can be detected by this technique for correlation with bone pathology.
The antibodies useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of OST-PTP expression. In situ detection may be accomplished by removal of a histological specimen from a subject for incubation with the OST-PTP specific antibody which may be detected by any of a number of techniques well-known in the art. Through the use of such a procedure, it is possible to determine not only the presence of OST-PTP but also its distribution throughout the biological sample. Thus OST-PTP may be an important diagnostic tool in analysis of bone development and a hallmark of certain bone disorders.
It will be appreciated that delineation of the nucleotide and amino acid sequences and general structural domains of the OST-PTP molecule allows one skilled in the art to generate a biological equivalent of this protein using readily available RNA in vitro translation systems or through biosynthetic peptide assembly systems. Moreover, truncated molecules expressing one or a portion of the functional attributes of the parent OST-PTP can be generated by utilizing partial or modified nucleotide and/or amino acid sequence information. Such truncated OST-PTP versions borrowing on the features of the present invention can, in principle, eventually recover the biological properties of native OST-PTP without necessarily utilizing the exact sequence as disclosed. For example, in one embodiment, the inventors constructed an enzymatically active fusion protein from the catalytic portion of OST-PTP and glutathione-S-transferase using a bacterial expression vector. In another embodiment of the invention, the minimal ligand binding sequence can be defined and similarly incorporated into truncated OST-PTP mini-gene constructs adding ligand specificity to catalytic activity of expressed proteins. Likewise, identification of tissue specific regulatory elements associated with OST-PTP expression will provide another important incremental step in assembly of the OST-PTP molecule. Thus, the present invention provides the necessary framework for the parsimonious dissection of OST-PTP nucleotide sequence to derive the minimal functional mini-gene construct for therapeutic and diagnostic applications. The product of such dissection is considered to be a derivative of OST-PTP inasmuch as it yields a protein with the biological characteristics described for OST-PTP and utilizes regions of sequence homologous to OST-PTP in order to obtain those characteristics. These definitions are specifically clarified as follows:
The invention provides the naturally occurring molecule "substantially free of other proteins or glycoproteins." It will be understood that the mammalian OST-PTP protein of the present invention can be biochemically purified from a variety of cell or tissue sources including primary cells and cell lines by standard protein purification techniques well known to those of skill in the art. "Substantially pure" indicates that the protein has been purified away from at least 70 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.
In another embodiment, the invention is directed to a recombinant mammalian OST-PTP protein. 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 well known to those of skill in the art (Wu et al., 1978, Prog. Nucl. Acid. Res. Molec. Biol. 21:101-141). Because the gene for the OST-PTP protein can be isolated or synthesized, the polypeptide can be synthesized substantially free of other proteins or glycoproteins of an origin in a prokaryotic organism or in a non-mammalian eukaryotic organism, if desired. As intended by the present invention, a recombinant OST-PTP molecule produced in mammalian cells, such as transfected COS, NIH-3T3, or CHO cells, for example, is either a naturally occurring protein sequence or 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.
In a further embodiment, the invention provides "functional derivatives" of OST-PTP. By "functional derivative" is meant a "fragment," "variant," "analog," or "chemical derivative" of the OST-PTP molecule, which terms are defined below. A functional derivative retains at least a portion of the function of the OST-PTP, such as binding to a specific antibody, phosphatase enzymatic activity or binding of the extracellular domain to a ligand which permits its utility in accordance with the present invention.
A "fragment" of OST-PTP refers to any subset of the molecule, that is, a shorter peptide. The term "fragment" is used to indicate a polypeptide which is derived from the OST-PTP protein or glycoprotein having a naturally occurring protein sequence by appropriately modifying the DNA sequence encoding the OST-PTP protein or glycoprotein, resulting in deletion of one or more amino acids at one or more sites of the C-terminal, N-terminal, and within the native sequence. Fragments of the OST-PTP protein or glycoprotein are useful for screening for compounds that are antagonists or agonists. It is understood that such fragments of the OST-PTP protein or glycoprotein may retain characterizing portion(s) of the native OST-PTP or glycoprotein. In particular, such fragments should retain one or more biological activities or functions which are characteristic for the intact OST-PTP proteins or glycoproteins. Examples, which are not intended to be in any way limiting to the scope of the invention claimed of OST-PTP fragments are: a) the catalytic domain; b) the signal sequence c) regions of the OST-PTP proteins or glycoproteins which interact with other molecules in the intact cell; d) the FN-III repeat extracellular domain; and e) regulatory parts of OST-PTP.
A "variant" of OST-PTP 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 of OST-PTP protein or glycoprotein, provided that the final construct possesses the desired activity or function present in the intact OST-PTP proteins or glycoproteins. Examples of such activities and functions are: a) the catalytic activity; b) substrate specificity; c) interaction with other molecules in vitro and in vivo; and d) regulatory functions. Only one of such activities or functions needs to be retained after any combination of deletion, insertion, and substitution. These examples are not intended to be in any way limiting to the scope of the invention claimed. Obviously, the modifications or mutations that will be made in the DNA encoding the OST-PTP protein or glycoprotein must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see EP 75,444).
In a further aspect, the invention provides for an OST-PTP protein or glycoprotein having additional amino acids that are derived from a naturally occurring OST-PTP protein or glycoprotein. This OST-PTP protein or glycoprotein is derived by appropriately modifying the DNA sequence encoding the OST-PTP protein or glycoprotein, resulting in addition of one or more amino acids at one or more sites of the C-terminal, N-terminal, and within the native sequence. It is understood that such an OST-PTP protein or glycoprotein having additional amino acids may retain characterizing portion(s) of the native OST-PTP protein or glycoprotein. In particular, such OST-PTP proteins or glycoproteins with additional amino acids should retain one or more biological activities or functions which are characteristic for the intact OST-PTP proteins or glycoproteins. Examples of such characteristics, of which at least one should be retained: a) the catalytic activity; b) the substrate specificity; c) interaction with other molecules in the intact cell; d) intercellular homophilic binding; and e) regulatory functions of OST-PTP. These examples are not intended to be in any way limiting to the scope of the invention claimed.
At the genetic level, these variants ordinarily are prepared by site-directed mutagenesis (Adelman et al., 1983, DNA 2:183) 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 OST-PTP refers to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
A "chemical derivative" of the OST-PTP 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. 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. Moieties capable of mediating such effects are exemplified in Remington's Pharmaceutical Sciences, 16th ed., Hack Publishing Co., Easton, Pa. (1980).
In a further aspect, the invention encompasses so-called chimeric molecules which are made up of OST-PTP in which one or more specific amino acid sequences are replaced with homologous sequence(s) from another protein or glycoprotein. Chimeric molecules include, for example, an OST-PTP protein or glycoprotein having a ligand-binding extracellular domain that is grafted onto a portion of another protein or glycoprotein.
"Homologous sequences" are defined as sequences in two or more PTP proteins which are similarly positioned in the primary sequence and which may exhibit sequence homology. It should be emphasized that "homologous sequences" should not be limited to cases with high degree of homology. Chimeric molecules are important tools for elucidating structure-function relationships and for identifying specific compounds (drugs). Therefore, the most useful chimeras are often, but not always, molecules in which a certain portion of one molecule has been replaced with the similarly positioned, but divergent, sequence from another, otherwise homologous, molecule. Thus, the exchanged portions will quite often represent the parts of the molecules where they differ the most.
This invention is also directed to an antibody specific for an epitope of OST-PTP, and the use of such antibody to detect the presence of, or measure the quantity or concentration of, OST-PTP in a cell, a cell or tissue extract, or a biological fluid. The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAhs), 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 (Kohler and Milstein, 1975, Nature 256:495-497) and U.S. Pat. No. 4,376,110. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof.
Chimeric antibodies are molecules, different portions of which are derived from different animal species, such as those having variable region derived from a murine mAh and a human immunoglobulin constant region. Chimeric antibodies and methods for their production are known in the art (Cabilly et al., 1984, Proc. Natl. Acad. Sci. USA 81:3273-3277; Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855; Boulianne et al., 1984, Nature 312:643-646; Cabilly et al., European Patent Application 125023 (published Nov. 14, 1984); 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., 1986, J. Immunol. 137:1066-1074; Robinson et al., PCT/US86/02269 (published 7 May 1987); Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Better et al., 1988, Science 240:1041-1043). 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 isotypic 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 clones expressing antibodies of identical specificity.
Accordingly, mAbs generated against the OST-PTP of the present invention 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 final mAb specific for a R-PTPase epitope. The anti-Id mAbs thus have their idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as OST-PTP.
The term "antibody" is also meant to include both intact molecules as well as fragments such as Fab which are capable of binding antigen. Fab 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., 1983, J. Nucl. Med. 24:316-325). It will be appreciated that Fab and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of OST-PTP according to the methods for intact antibody molecules.
In additional embodiments of the present invention, a DNA sequence encoding a OST-PTP 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 PTP molecules, of human or other mammalian species, which have sequence homology to the OST-PTP 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 OST-PTP resulting in chimeric molecules. Non-limiting examples of such chimeric molecules include the OST-PTP 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 (Riedel et al., 1986, Nature 324:628-670).
Genetic constructs encoding OST-PTP, functional derivative thereof, and chimeric molecules such as those described above, can be used in gene therapy. An abnormal or dysfunctional OST-PTP, which results in disease, may be replaced by infusion of cells of the osteogenic lineage transfected with a normal OST-PTP. Alternatively, or additionally, cells carrying a chimeric OST-PTP having a receptor to a ligand of choice can be used for such gene therapy.
EXAMPLE I
Isolation of Rat OST-PTP cDNA Clones
The bone-testis specific phosphatase cDNA sequence was first identified in fetal rat calvaria and the rat osteosarcoma cell line, UMR106, messenger RNA by a reverse transcription polymerase chain reaction (RT-PCR). These two sources of bone starting material, fetal rat calvaria, and rat osteosarcoma cell line (UMR106), were chosen based on both information in the literature and availability. PCR derivation of a hybridization screening probe was accomplished utilizing three sets of degenerate oligonucleotide primers designed to amplify conserved regions within the tyrosine phosphatase catalytic regions. Three of the 5' primers corresponding to the conserved amino acids, DYINA (SEQ ID NO:6) ((5'-CAGTGGATCC(A/C/T)GA(C/T)TA(C/T)AT(A/C/T)AA(T/C)GC-3' (SEQ ID NO:7)); (72-fold degeneracy), YIATQGP (SEQ ID NO:8) ((5'-CAGTGGATCCTACAT(C/T)G(C/T)(A/C/T)(A/G)C(C/A)CA(A/G)GG-3') (SEQ ID NO:9); (96-fold degeneracy), and KCDQYW (SEQ ID NO:10) ((5'CAGTGGATCCAA(A/G)TG(C/T)(C/G)(A/C)(A/C/G/T)(C/G)A(A/G)TA(C/T)TGGCC-3') (SEQ ID NO:11); (512-fold degeneracy) were paired with a common 3' primer corresponding to the active site, HCSAGVGR (SEQ ID NO:12) ((5'-CTAGTCTAGACCNA(T/A)(T/A/G)CCNGC(A/G)CA(A/G)TG-3') (SEQ ID NO:13); (768-fold degeneracy). The 5' and 3' primers included sites for restriction enzymes BamHI and XbaI, respectively. The template for PCR reactions was first-strand cDNA synthesized using poly A+ RNA isolated from rat osteosarcoma line UMR106 and fetal rat calvaria. Total RNA was isolated using RNAzol B (Tel-Test Inc.) and the poly A+ RNA was isolated following the recommended conditions in the Poly ATtract mRNA Isolation System IV (Promega). The cDNA synthesis reactions were performed with the Invitrogen cDNA cycle kit using instructions recommended by the manufacturer. The PCR reactions included 1/5 and 1/10 volume of the original cDNA synthesis reaction and 500 ng of each primer along with the recommended reagent concentrations in the GeneAmp kit (Perkin-Elmer). The PCR conditions were: 94.degree. C., 1 minute; 55.degree. C., 1 minute; 72.degree. C., 1 minute; for 35 cycles. For PCR from calvaria, reamplification of original PCR reactions was necessary to see products. Products of the expected size using the DYINA (SEQ ID NO:6) pair (495 bp) and the YIATQGP (SEQ ID NO:8) pair (432 bp) were isolated after fractionation, digested with BamHI and XbaI and cloned into the pBluescript II vector (Stratagene). Approximately 200 independent clones were sequenced using the T3 and T7 primer sites within the vector. Sequencing was done following the recommended conditions in the Sequenase sequencing kit (GibcoBRL Life Technologies).
Two rat UMR106 cDNA libraries were screened to isolate the full length clone of OST-PTP. The rat osteosarcoma cell line (UMR106) was obtained from American Type Culture Collection (ATCC) and were grown in Dulbecco's Minimal Eagle Media (DMEM), 10% fetal bovine serum (Hyclone Laboratories, Inc.), 1% glutamine (GibcoBRL Life Technologies), and 1% penicillin-streptomycin (GibcoBRL Life Technologies). The cells were discarded after the twentieth passage. One of these libraries was made from poly A+ RNA of normal confluent UMR106 cells and primed with oligo dT and random primers using the TimeSaver cDNA Synthesis Kit from Pharmacia. This kit allowed for synthesis of cDNA with NotI restriction sites flanking the cohesive EcoRI end, and facilitated cloning into the predigested Lambda ZapII vector (Stratagene) which also has cohesive EcoRI ends. The second library was a Lambda ZapII cDNA library custom made by Stratagene using poly A+ RNA from UMR106 cells stimulated with 100 nM PTH for 18 hours. The PCR fragment of OST-PTP was .sup.32 P-labeled either by random priming following the recommended conditions in the Random Primers DNA labelling kit (GibcoBRL Life Technologies) or by synthesizing a riboprobe using the procedure recommended by the manufacturer (Promega). All probes had the unincorporated nucleotides removed by using a NICK column as recommended by the manufacturer (Pharmacia). Approximately 106 plaques were plated and duplicate filters were screened by hybridization. The hybridization conditions for the filters identical to the conditions described in detail under the section on RNA analysis. Initial screens yielded incomplete, poly A-tailed clones which were 2.6 kb in length.
To obtain the 5' end, 5' Rapid Amplification of cDNA Ends (RACE) was performed using the 5'RACE system (GibcoBRL Life Technologies) as recommended by the manufacturers. Several independent clones (5 total) were synthesized and sequenced using an automated sequencer (Applied Biosystems, Inc.) and procedures recommended by the manufacturer to define the full-length clone. To verify the authenticity of the sequence, an 870 base pair probe (random-prime labeled) corresponding to one of the RACE products, was used to rescreen the custom Stratagene library.
Analysis of RNA for Expression of OST-PTP
RNA was analyzed by Northern blot hybridization to determine the tissue specificity of the OST-PTP clone. Poly A.sup.+ RNA was extracted from an array of adult rat tissues: uterus, ovary, testis, kidney, heart, liver, lung, spleen, brain, skeletal muscle, smooth muscle, pancreas and retina. Poly A.sup.+ RNA derived from UMR106 was used as a standard to control for expression in bone. The RNA preparations were quantitated by absorbance at 260 nm and, depending on the message abundance, either total cellular RNA or poly A+ RNA was electrophoretically separated in a 6.6% formaldehyde-1% agarose gel. RNA fractionated in such gels was transferred to Magnagraph nylon membrane (MSI) in 20.times. SSC by capillary action method. Two different types of probe; riboprobe (RNA) or random primed cDNA probe, were used depending on the experiment. The riboprobe used for hybridization corresponded to the extracellular domain of OST-PTP, and was labeled by in vitro transcription using .sup.32 P-a (3000 Ci/mM) as described by manufacturers (Promega). The DNA probes used for hybridization were rat H4 histone (Grimes et al., 1987 Exp. Cell Res. 173:534-545) rat type I collagen (Genovese et al., 1984, Biochemistry 23:6210-6216) rat type II collagen (Kohno et al., 1984, J. Biol. Chem. 259(22):13668-13673), rat alkaline phosphatase (Noda et al., 1987, J. Bone and Mineral Res. 2(2):161-164), rat osteopontin (Oldberg et al., 1986, Proc. Natl. Acad. Sci. USA 83:8819-8823), rat osteocalcin (Lian et al., 1989, Proc. Natl. Acad. Sci. USA 86:1143-1147) and cyclophilin. All DNA probes were labeled with .sup.32 P-a (3000 Ci/mM) by the Random Primers DNA labeling kit using manufacturer's recommended conditions (GibcoBRL Life Technologies). Prehybridizations and hybridizations for the OST-PTP riboprobe were performed in 50% formamide; 5.times. SSC; 5.times. Denhardts, 50 mM NaPO.sub.4 buffer, pH 6.5; 1% SDS; and 250 ug/ml salmon sperm DNA at 68.degree. C. for 16 hours. For hybridizations, 10.sup.6 cpms/ml probe was added. Following hybridization, the blots were washed twice at room temperature in 1.times. SSC+0.1% SDS (30 minutes per wash), and then washed twice in 0.1.times. SSC+0.1% SDS (30 minutes per wash) once at room temperature and once at 68.degree. C. The prehybridization/hybridization conditions for the DNA probes were the same as described above except the temperature was lowered to 43.degree. C. For hybridization, 10.sup.6 cpms/ml probe and 10% dextran sulfate were added (Maniatis et al., 1982, Molecular Cloning: a Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)). The washes were done as described above. The results were visualized either by autoradiography or by scanning with a phosphorimager (Molecular Dynamics). All quantifications were normalized against the amount of cyclophilin message which was considered to be standard for any given conditions. It can be seen in FIG. 3 that the OST-PTP clone appeared to have a message size of 5.8 kb and was expressed in rat tissue only in RNA derived from bone and testis.
OST-PTP Expression During Osteogenesis
The expression of OST-PTP was further characterized by measuring its expression during rat primary osteoblast development (osteogenesis). Primary rat osteoblasts were derived from calvaria of fetal rats of 21 days gestation. The calvaria were surgically isolated, and the periosteum and suture lines removed. The calvaria were then subjected to sequential digestions of 20, 60, 90 minutes at 37.degree. C. in 2 mg/ml collagenase B (Boehringer Mannheim, Lot# EHA131)/0.25% trypsin (GibcoBRL Life Technologies). (This procedure is a modification of Aronow et al., 1990, J. Cell Physiol. 143:213-221). The cells in the first digest were discarded, while those released from the other two digests were pooled for plating in minimal essential media (GibcoBRL Life Technologies) supplemented with 10% defined fetal bovine serum (Hyclone Laboratories, Inc., Lot# 11112275), and 1% penicillin-streptomycin-amphotericin B (GibcoBRL Life Technologies), in 75 cm.sup.2 flasks at a density of 1.times.10.sup.6 cells. The total yield of cells ranged from 3 to 5.times.10.sup.6 per pregnant rat. After 24 hours the media was changed and cell viability ranged from 90-95%. At confluence (day 5), the cells were cultured in BGJb media (GibcoBRL Life Technologies) supplemented with 10% defined fetal bovine serum, 1% penicillin-streptomycin-amphotericin B, 50 ug/ml ascorbic acid, and 10 mM .alpha.-glycerol phosphate.
Cellular RNA was isolated at various times during the differentiation time course (5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and 33 days) and assayed for the steady state levels of various transcripts by Northern blot analysis. The cells were initially characterized using a series of markers of is osteoblast differentiation, including probes for the extracellular matrix-associated gene, type I collagen, and the osteoblast phenotype marker genes, osteopontin, osteocalcin, and alkaline phosphatase. The time of maximal expression of phenotypic markers of the 33 day developmental sequence did not vary by more than 48 hours in independent experiments using the standardized lots of culture reagents as described above. The results were quantitated by Phosphorimager scanning and normalized to cyclophilin expression to derive the maximal expression level for each transcript.
The results indicated that the cells were moving through differentiation in a manner similar to that reported in the literature (Aronow et al., 1990, J. Cell. Phys. 143:213-221). The cultures formed mineralizing nodules with very little chondrocyte contamination and developed along a pathway which could be divided into three stages, proliferation (day 1-7), differentiation (day 5-29), and mineralization (day 27-33). Once the developmental pathway of the primary cells was established, Northern blot analysis was carried out to determine OST-PTP expression. Results showed that OST-PTP expression was very low during proliferation, increased 10 fold during the differentiation stage and started to decline during mineralization (2 fold). A second transcript (OST) was expressed at the highest level during proliferation, declined during differentiation and was absent during mineralization.
Tissue Distribution of OST-PTP
Based on the results of the Northern analysis, in situ hybridizations were performed with testis from adult rats to further characterize expression in that tissue. In these hybridizations unperfused testis from Sprague-Dawley rats were collected and frozen in Tissue Tek embedding medium (Miles) on dry ice. Tissue sections (10-15 Mm) were cut at -15.degree. C. and thaw mounted on poly-L-lysine slides. Double-labeled .sup.35 S-UTP and CTP riboprobes were prepared using the Maxiscript (T3/T7/SP6) in vitro transcription kit (Ambion) with modifications to the manufacturer's protocol. The template for antisense and sense riboprobes encoded the original 470 bp PCR fragment. Hybridizations were performed as previously described with minor modifications for testis tissue (S. Watson et al., pp. 4-29, Society For Neuroscience Short Course I Syllabus (A. Sunderman, ed. , Society for Neuroscience, Washington, D.C., 1988). Sequential sections with sense probe and RNase treatment were run for each antisense section. Completed slides were dipped in Kodak photographic emulsion (NBT-2) and exposed for 14-21 days. Sections were developed and counterstained with hematoxylin-eosin (Richard Allan, Inc.). The slides were viewed and photographed on a Zeiss Axioskop with a (Micro Video Instruments, Inc.) darkfield illumination step.
These steps revealed that OST-PTP transcripts are spatially restricted to the basal portion of the seminiferous tubule, suggesting localization to the Sertoli cell and/or primary spermatogonia. It is interesting to note that this expression is stage-specific. Abundance of the OST-PTP transcripts appears greatest between stages I-VII when maturing spermatids remain buried within the Sertoli epithelium. Those tubules with low or non-detectable signals possess mature spermatids at the luminal surface of the Sertoli epithelium (stages VIII-IX) or immature spermatids with heads lacking the densely staining chromatin and the strong "bent rod" appearance (stages X-XIII). Analysis of the OST-PTP expression in neonates (day 10-18) revealed both temporal and spatial regulation of its expression within the developing seminiferous tubules.
The analysis of RNA expression by the combination of Northern blots and in situ hybridization demonstrated a restricted tissue distribution for OST and OST-PTP to bone and testis. Both tissues possess highly regulated temporal and spatial organization which is necessary for the continuous differentiation and function of specific cell populations. Within the seminiferous tubule, coupled paracrine and cell surface interactions between the Sertoli cell, germ cell and the extracellular matrix (basement membrane) are essential to spermatogenesis (see Skinner, M. K., 1991, Endocrine Rev. 12:45, and A. L. Kerszenbaum, 1994, Endocrine Rev. 15:116) in a manner analogous to the coupling of the osteoblast-osteoclast function in bone remodeling. Consequently, in addition to its role in bone, OST-PTP also appears to function in the regulation of germ cell differentiation in the testis.
Parathyroid Hormone Treatment of UMR106 Cells
Parathyroid hormone is an important bone hormone. It has been reported to inhibit cell proliferation, and DNA synthesis, in the UMR106 cells and increase expression of proteins, such as collagenase, that potentiate the onset of resorption by osteoclasts (Scott et al., 1992, Mol. Endo. 6(12):2153-2159 and Clohisy et al., 1992, Mol. Endo. 6(11):1834-1842). The UMR106 cells were cultured as described above until confluence. The cells were then washed twice in phosphate buffered saline followed by incubation for 5 hours at 37.degree. C. in serum free DMEM. After serum deprivation, 100 nM parathyroid hormone (PTH) was added to the cultures and incubation continued for 4, 8, 12, and 16 hours at 37.degree. C. RNA was then isolated from the cells as described above. Northern blot analysis revealed that the PTP-OST message was increased as much as 5 fold upon treatment of the UMR106 cells with 100 nM parathyroid hormone (PTH) for 4 hours with further increases at later time points. Neither of the other two novel PTPase clones were altered by PTH. In addition, serum deprivation and refeeding also did not significantly affect OST-PTP expression. PTH concentrations as low as 1 nM for 18 hours were effective in increasing OST-PTP transcripts but exposure for less than 4 hours regardless of concentration (10 pM-100 nM) had no significant effect. These results demonstrated that OST-PTP was important not only for its exclusionary tissue distribution but also that it functioned in a PTH sensitive manner that indicated a critical role in proper bone metabolism.
Parathyroid hormone modulation of OST-PTP may be mediated through a C-protein coupled pathway utilizing the cyclic AMP-dependent protein kinase (PKA) signalling pathway. Stimulation of osteosarcoma cells using the cyclic AMP analogue, chlorophenylthio-cAMP for 18 hours (10 .mu.M) resulted in an increase in OST-PTP mRNA levels comparable (5 fold) to that seen with PTH. These results are consistent with the idea that OST-PTP expression is regulated by PTH through stimulation of the PKA signalling pathway which has been demonstrated to modulate expression of other genes involved in osteoblast differentiation (Scott et al., 1992, Mol. Endo. 6(12):1834-1842).
EXAMPLE II
Expression and Characterization of Recombinant OST-PTP
The catalytic portion of OST-PTP was expressed as a glutathione-S-transferase fusion protein using the pGEX-KG E. coli expression vector. A blunted NotI-XbaI fragment corresponding to the entire cytoplasmic domain of OST-PTP was subcloned into SmaI-XbaI digested pGEX-KG vector. For protein expression, this construct, pKG-OST, was freshly transformed into BL21 strain of E. coli and the GST-OST-PTP fusion protein was produced and purified as described previously (Guan et al., 1990, Proc. Natl. Acad. Sci. USA 87:1501-1505). The recombinant fusion protein was approximately 70% pure as determined by SDS-PAGE electrophoresis.
Basic kinetic analysis of the recombinant protein was performed using the hydrolysis of the artificial substrate, para-nitrophenylphosphate (pNPP), as the measure of phosphatase activity. Enzyme activity was measured in a 200 .mu.l reaction comprised of 1) a buffer consisting of 0.1M imidazole, 0.1 mM EDTA, 0.1% .beta.-mercaptoethanol, 2) 20 mM pNPP (saturating conditions), and enzyme. Incubation was carried out at 30.degree. C. for 10 minutes over a range of pH. The reaction was terminated by addition of 800 .mu.l of 0.25N NaOH. The amount of hydrolyzed product was determined by measurement of absorbance at 410 nm. The extinction coefficient of 1.8.times.10.sup.4 M.sup.-1 cm.sup.-1 was used to determine the molar concentration of hydrolyzed pNPP. The maximum activity of OST-PTP incubated with paranitrophenylphosphate (pNPP) at 30.degree. C. for 10 minutes occurred at pH 5.6.
Substrate specificity was determined using tyrosine-phosphorylated Raytide Peptide Substrates (Oncogene Sciences) and serine phosphorylated Kemptide phosphate acceptor peptide (Oncogene Sciences). Raytide Peptide Substrate was phosphorylated on tyrosine by incubating 10 .mu.g of Raytide Peptide Substrate in a 30 .mu.l reaction including assay buffer (50 mM Hepes, pH 7.5, 0.1 mM EDTA, 0.1 mg/ml BSA, 10 mM MgCl.sub.2, 0.1 mM ATP, 0.2% b-mercaptoethanol), 1.2 .mu.l .sup.32 P-ATP (6000 Ci/mMol) and 0.5 mg GST-v-src at 30.degree. C. for 30 minutes. Kemptide phosphate acceptor peptide was phosphorylated on serine by incubating 10 .mu.g of the peptide in a 50 ml reaction including assay buffer (40 mM Tris:HCl, pH 7.5, 10 mM MgCl.sub.2, 10 mM dithiothreitol, 0.1 mM ATP), 3 ml .sup.32 P-ATP (6000 Ci/mMol) and 5 .mu.g of the catalytic subunit of bovine protein kinase A (Fluka) at 30.degree. C. for 30 minutes. The reactions were terminated by addition of 120 .mu.l of 10% phosphoric acid and applied to P81 filter. Free ATP was removed by 3.times.100 ml washes with 0.5% phosphoric acid and the peptide eluted with 2.times.1 ml aliquots of 0.5M ammonium bicarbonate. Aliquots were lyophilized, resuspended in distilled water and radioactivity determined. Phosphatase activity as determined in a 50 .mu.l reaction containing assay buffer (0.1M imidazole, pH 5.6 or 7.0, 5 mM EDTA, 0.2% b-mercaptoethanol), 100,000 cpms of phosphorylated substrate, and 100 ng-4 .mu.g of enzyme, incubated at 30.degree. C. for 10 minutes. The reaction was terminated by adding 750 .mu.l of charcoal mix (0.9M HCl, 90 mM sodium pyrophosphate, 2 mM sodium pyrophosphate dibasic and 4% (vol/vol) Norit. The supernatant was counted to determine the amount of .sup.32 P released. The enzyme rapidly dephosphorylated Raytide Peptide Substrates but not Kemptide phosphate acceptor peptide, demonstrating its specificity to tyrosine residues. These data established that the OST-PTP enzyme dephosphorylated tyrosine residues in proteins with specificity and activity levels typical of previously identified PTPases which control the level of phosphorylation in various cell types.
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: 13 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5455 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 205..5337 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AGAACAGCCTACAACAGCTGCCTTCCGGGAGGGACCAGGCTAGTTCACACTTGGAAGTTG60 GGATGCCAGGAGCAGCCTTCTGTCTTCCGAGGCCTTCCTGGGTCTCCTGGTCAGCTCATT120 CCACACTGAGATGATTCTAAAGAAAGATCCTCACACAGACTCTGCTGGAAGAAACAAAGT180 GAAGTGTCCCCAGACTTTATCAGGATGAGGCCCCTGATTCTGTTAGCTGCC231 MetArgProLeuIleLeuLeuAlaAla 15 CTCCTCTGGCTCCAGGGCTTTTTGGCCGAGGACGACGCATGCTCATCC279 LeuLeuTrpLeuGlnGlyPheLeuAlaGluAspAspAlaCysSerSer 10152025 TTGGAAGGGAGCCCAGACAGGCAGGGTGGAGGTCCACTTCTGAGTGTG327 LeuGluGlySerProAspArgGlnGlyGlyGlyProLeuLeuSerVal 303540 AACGTCAGTAGCCATGGAAAGTCTACCAGCCTGTTTCTGAGCTGGGTA375 AsnValSerSerHisGlyLysSerThrSerLeuPheLeuSerTrpVal 455055 GCTGCAGAGCTGGGCGGATTTGACTATGCCCTCAGCCTCAGGAGTGTG423 AlaAlaGluLeuGlyGlyPheAspTyrAlaLeuSerLeuArgSerVal 606570 AACTCCTCAGGTTCTCCAGAAGGGCAACAGCTCCAGGCTCACACAAAT471 AsnSerSerGlySerProGluGlyGlnGlnLeuGlnAlaHisThrAsn 758085 GAGTCCGGCTTTGAGTTCCATGGCCTGGTGCCAGGGAGTCGCTACCAG519 GluSerGlyPheGluPheHisGlyLeuValProGlySerArgTyrGln 9095100105 CTAAAACTGACTGTCCTAAGACCCTGTTGGCAGAATGTCACAATTACC567 LeuLysLeuThrValLeuArgProCysTrpGlnAsnValThrIleThr 110115120 CTCACTGCCCGAACTGCCCCGACAGTGGTCCGTGGACTGCAGCTGCAT615 LeuThrAlaArgThrAlaProThrValValArgGlyLeuGlnLeuHis 125130135 AGCGCTGGGAGCCCAGCCAGGCTGGAAGCCTCGTGGAGTGATGCCCCT663 SerAlaGlySerProAlaArgLeuGluAlaSerTrpSerAspAlaPro 140145150 GGAGATCAAGACAGCTACCAACTTCTCCTCTACCACCTGGAATCCCAA711 GlyAspGlnAspSerTyrGlnLeuLeuLeuTyrHisLeuGluSerGln 155160165 ACTCTGGCATGCAATGTCTCTGTGTCCCCTGACACCCTGTCTTACAGT759 ThrLeuAlaCysAsnValSerValSerProAspThrLeuSerTyrSer 170175180185 TTTGGCGACCTTTTGCCAGGTACTCAGTATGTCTTGGAGGTTATCACC807 PheGlyAspLeuLeuProGlyThrGlnTyrValLeuGluValIleThr 190195200 TGGGCTGGCAGTCTCCATGCGAAGACTAGTATCCTCCAGTGGACAGAG855 TrpAlaGlySerLeuHisAlaLysThrSerIleLeuGlnTrpThrGlu 205210215 CCTGTCCCTCCTGATCACCTAGCACTACGTGCCTTGGGTACCAGTAGC903 ProValProProAspHisLeuAlaLeuArgAlaLeuGlyThrSerSer 220225230 CTGCAAGCCTTCTGGAACAGCTCTGAAGGGGCCACCTCGTTTCACCTG951 LeuGlnAlaPheTrpAsnSerSerGluGlyAlaThrSerPheHisLeu 235240245 ATGCTCACAGACCTCCTCGGGGGCACCAACACGACTGCGGTGATCAGA999 MetLeuThrAspLeuLeuGlyGlyThrAsnThrThrAlaValIleArg 250255260265 CAAGGGGTCTCGACCCACACCTTTCTTCACCTATCTCCGGGTACACCT1047 GlnGlyValSerThrHisThrPheLeuHisLeuSerProGlyThrPro 270275280 CATGAGCTGAAGATTTGTGCTTCTGCTGGGCCCCACCAGATCTGGGGA1095 HisGluLeuLysIleCysAlaSerAlaGlyProHisGlnIleTrpGly 285290295 CCCAGTGCCACCGAGTGGACCTATCCCTCTTACCCATCTGACCTGGTG1143 ProSerAlaThrGluTrpThrTyrProSerTyrProSerAspLeuVal 300305310 CTGACTCCCTTACGGAATGAGCTCTGGGCCAGCTGGAAGGCAGGGCTG1191 LeuThrProLeuArgAsnGluLeuTrpAlaSerTrpLysAlaGlyLeu 315320325 GGAGCCCGGGACGGCTATGTACTGAAGTTAAGTGGGCCAATGGAGAGT1239 GlyAlaArgAspGlyTyrValLeuLysLeuSerGlyProMetGluSer 330335340345 ACGTCTACCCTGGGCCCGGAAGAGTGCAATGCAGTCTTCCCAGGGCCC1287 ThrSerThrLeuGlyProGluGluCysAsnAlaValPheProGlyPro 350355360 CTGCCTCCGGGACACTACACTTTGCAGCTGAAGGTTCTAGCTGGACCT1335 LeuProProGlyHisTyrThrLeuGlnLeuLysValLeuAlaGlyPro 365370375 TATGATGCCTGGGTGGAGGGCAGTACCTGGCTGGCTGAATCTGCTGCC1383 TyrAspAlaTrpValGluGlySerThrTrpLeuAlaGluSerAlaAla 380385390 CTTCCCAGGGAGGTCCCTGGTGCCAGACTGTGGCTAGATGGACTGGAA1431 LeuProArgGluValProGlyAlaArgLeuTrpLeuAspGlyLeuGlu 395400405 GCTTCCAAGCAGCCTGGGAGACGGGCGCTACTCTATTCTGACGATGCC1479 AlaSerLysGlnProGlyArgArgAlaLeuLeuTyrSerAspAspAla 410415420425 CCAGGCTCCCTAGGGAACATCTCTGTGCCCTCTGGTGCCACTCACGTC1527 ProGlySerLeuGlyAsnIleSerValProSerGlyAlaThrHisVal 430435440 ATTTTCTGTGGCCTGGTACCTGGAGCCCACTATAGGGTGGACATTGCC1575 IlePheCysGlyLeuValProGlyAlaHisTyrArgValAspIleAla 445450455 TCATCCACGGGGGACATCTCTCAGAGCATCTCAGGCTATACAAGTCCC1623 SerSerThrGlyAspIleSerGlnSerIleSerGlyTyrThrSerPro 460465470 CTGCCACCGCAGTCACTGGAGGTCATCAGCAGGAGCAGCCCATCTGAC1671 LeuProProGlnSerLeuGluValIleSerArgSerSerProSerAsp 475480485 CTGACTATTGCTTGGGGTCCAGCACCAGGGCAGCTGGAAGGTTATAAG1719 LeuThrIleAlaTrpGlyProAlaProGlyGlnLeuGluGlyTyrLys 490495500505 GTTACCTGGCATCAGGATGGCAGCCAGAGGTCTCCTGGCGACCTTGTT1767 ValThrTrpHisGlnAspGlySerGlnArgSerProGlyAspLeuVal 510515520 GACTTGGGCCCTGACACTTTGAGCCTGACTCTGAAATCTCTGGTACCC1815 AspLeuGlyProAspThrLeuSerLeuThrLeuLysSerLeuValPro 525530535 GGCTCCTGCTACACCGTGTCAGCATGGGCCTGGGCCGGGAACCTCAGC1863 GlySerCysTyrThrValSerAlaTrpAlaTrpAlaGlyAsnLeuSer 540545550 TCTGACTCTCAGAAGATTCACAGCTGCACCCGCCCCGCTCCTCCCACC1911 SerAspSerGlnLysIleHisSerCysThrArgProAlaProProThr 555560565 AACCTGAGTCTGGGCTTTGCCCACCAGCCTGCGGCACTGAAGGCTTCC1959 AsnLeuSerLeuGlyPheAlaHisGlnProAlaAlaLeuLysAlaSer 570575580585 TGGTATCACCCACCGGGTGGCAGGGATGCCTTTCACTTACGGCTTTAC2007 TrpTyrHisProProGlyGlyArgAspAlaPheHisLeuArgLeuTyr 590595600 AGGCTGAGGCCTCTGACACTGGAAAGTGAGAAGGTCCTACCTCGGGAG2055 ArgLeuArgProLeuThrLeuGluSerGluLysValLeuProArgGlu 605610615 GCCCAGAACTTCTCCTGGGCCCAGCTGACTGCAGGCTGTGAGTTCCAG2103 AlaGlnAsnPheSerTrpAlaGlnLeuThrAlaGlyCysGluPheGln 620625630 GTACAGCTGTCTACCTTGTGGGGGTCTGAGAGAAGCAGCAGTGCCAAC2151 ValGlnLeuSerThrLeuTrpGlySerGluArgSerSerSerAlaAsn 635640645 GCCACAGGCTGGACACCCCCTTCAGCTCCTACACTGGTAAACGTGACC2199 AlaThrGlyTrpThrProProSerAlaProThrLeuValAsnValThr 650655660665 AGCGATGCTCCTACCCAGCTCCAAGTATCCTGGGCCCACGTTCCTGGG2247 SerAspAlaProThrGlnLeuGlnValSerTrpAlaHisValProGly 670675680 GGCCGGAGCCGCTACCAAGTGACCCTATACCAGGAGAGTACCCGGACA2295 GlyArgSerArgTyrGlnValThrLeuTyrGlnGluSerThrArgThr 685690695 GCCACCAGCATCATGGGGCCCAAGGAAGATGGCACGAGCTTTTTGGGT2343 AlaThrSerIleMetGlyProLysGluAspGlyThrSerPheLeuGly 700705710 TTGACTCCTGGCACTAAGTACAAGGTGGAAGTCATCTCCTGGGCTGGG2391 LeuThrProGlyThrLysTyrLysValGluValIleSerTrpAlaGly 715720725 CCCCTCTACACTGCAGCAGCCAACGTTTCTGCCTGGACCTACCCACTC2439 ProLeuTyrThrAlaAlaAlaAsnValSerAlaTrpThrTyrProLeu 730735740745 ATACCCAATGAGCTGCTCGTGTCAATGCAGGCAGGCAGTGCTGTGGTT2487 IleProAsnGluLeuLeuValSerMetGlnAlaGlySerAlaValVal 750755760 AACCTGGCCTGGCCCAGTGGTCCCCTGGGGCAAGGGGCATGCCACGCC2535 AsnLeuAlaTrpProSerGlyProLeuGlyGlnGlyAlaCysHisAla 765770775 CAACTCTCAGATGCTGGACACCTCTCATGGGAGCAACCCCTGAAACTA2583 GlnLeuSerAspAlaGlyHisLeuSerTrpGluGlnProLeuLysLeu 780785790 GGCCAAGAGCTCTTCATGCTAAGGGATCTCACACCAGGACATACCATC2631 GlyGlnGluLeuPheMetLeuArgAspLeuThrProGlyHisThrIle 795800805 TCGATGTCAGTGAGGTGTCGGGCAGGGCCGCTCCAGGCCTCTACGCAC2679 SerMetSerValArgCysArgAlaGlyProLeuGlnAlaSerThrHis 810815820825 CTTGTGGTGCTGTCTGTGGAGCCTGGCCCTGTGGAAGATGTGCTCTGT2727 LeuValValLeuSerValGluProGlyProValGluAspValLeuCys 830835840 CATCCAGAGGCCACCTACCTGGCCCTGAACTGGACGATGCCTGCTGGA2775 HisProGluAlaThrTyrLeuAlaLeuAsnTrpThrMetProAlaGly 845850855 GACGTGGATGTCTGTCTGGTGGTGGTAGAGCGGCTGGTGCCGGGAGGG2823 AspValAspValCysLeuValValValGluArgLeuValProGlyGly 860865870 GGCACTCATTTTGTCTTCCAGGTCAACACCTCAGGGGATGCTCTTCTG2871 GlyThrHisPheValPheGlnValAsnThrSerGlyAspAlaLeuLeu 875880885 TTGCCCAACTTGATGCCCACCACTTCTTACCGCCTTAGCCTCACCGTT2919 LeuProAsnLeuMetProThrThrSerTyrArgLeuSerLeuThrVal 890895900905 CTGGGCAGGAATAGTCGGTGGAGCCGGGCGGTTTCCCTGGTGTGCAGT2967 LeuGlyArgAsnSerArgTrpSerArgAlaValSerLeuValCysSer 910915920 ACTTCTGCTGAGGCTTGGCACCCCCCAGAGCTAGCTGAGCCCCCCCAG3015 ThrSerAlaGluAlaTrpHisProProGluLeuAlaGluProProGln 925930935 GTGGAGCTGGGGACAGGGATGGGTGTGACAGTCATGCGTGGCATGTTT3063 ValGluLeuGlyThrGlyMetGlyValThrValMetArgGlyMetPhe 940945950 GGTAAAGATGACGGGCAGATCCAGTGGTATGGCATAATTGCCACCATC3111 GlyLysAspAspGlyGlnIleGlnTrpTyrGlyIleIleAlaThrIle 955960965 AACATGACGCTGGCCCAGCCTTCCCGGGAAGCCATCAATTACACATGG3159 AsnMetThrLeuAlaGlnProSerArgGluAlaIleAsnTyrThrTrp 970975980985 TATGACCACTACTATAGAGGATGTGAGTCCTTCCTGGCTCTCCTGTTC3207 TyrAspHisTyrTyrArgGlyCysGluSerPheLeuAlaLeuLeuPhe 9909951000 CCAAACCCCTTCTACCCAGAGCCTTGGGCTGGGCCAAGATCCTGGACA3255 ProAsnProPheTyrProGluProTrpAlaGlyProArgSerTrpThr 100510101015 GTACCTGTGGGTACTGAGGACTGTGACAACACCCAAGAGATATGCAAT3303 ValProValGlyThrGluAspCysAspAsnThrGlnGluIleCysAsn 102010251030 GGGCGTCTCAAGTCAGGCTTCCAGTATAGGTTCAGCGTTGTGGCCTTT3351 GlyArgLeuLysSerGlyPheGlnTyrArgPheSerValValAlaPhe 103510401045 AGTAGGCTCAACACTCCAGAGACCATCCTCGCCTTCTCGGCCTTCTCA3399 SerArgLeuAsnThrProGluThrIleLeuAlaPheSerAlaPheSer 1050105510601065 GAGCCCCGGGCCAGCATCTCTCTGGCGATCATTCCCCTGACAGTTATG3447 GluProArgAlaSerIleSerLeuAlaIleIleProLeuThrValMet 107010751080 CTGGGGGCTGTGGTGGGCAGCATTGTCATTGTGTGTGCAGTGCTATGC3495 LeuGlyAlaValValGlySerIleValIleValCysAlaValLeuCys 108510901095 TTGCTCCGCTGGCGGTGCCTGAAGGGACCAAGATCAGAGAAGGATGGC3543 LeuLeuArgTrpArgCysLeuLysGlyProArgSerGluLysAspGly 110011051110 TTTTCCAAGGAGCTGATGCCTTACAACCTGTGGCGGACCCATCGGCCT3591 PheSerLysGluLeuMetProTyrAsnLeuTrpArgThrHisArgPro 111511201125 ATCCCCATCCATAGCTTCCGGCAGAGCTATGAGGCCAAGAGCGCACAT3639 IleProIleHisSerPheArgGlnSerTyrGluAlaLysSerAlaHis 1130113511401145 GCACACCAGACCTTCTTCCAGGAATTTGAGGAGTTGAAGGAGGTAGGC3687 AlaHisGlnThrPhePheGlnGluPheGluGluLeuLysGluValGly 115011551160 AAGGACCAGCCCCGACTAGAGGCTGAGCATCCGGACAACATCATCAAG3735 LysAspGlnProArgLeuGluAlaGluHisProAspAsnIleIleLys 116511701175 AACCGGTACCCACACGTGCTGCCCTATGACCACTCCAGGGTCAGGCTG3783 AsnArgTyrProHisValLeuProTyrAspHisSerArgValArgLeu 118011851190 ACCCAGCTACCAGGAGAGCCTCATTCTGACTACATCAATGCCAACTTC3831 ThrGlnLeuProGlyGluProHisSerAspTyrIleAsnAlaAsnPhe 119512001205 ATCCCAGGCTATAGCCACACACAGGAGATCATTGCCACCCAGGGGCCT3879 IleProGlyTyrSerHisThrGlnGluIleIleAlaThrGlnGlyPro 1210121512201225 CTCAAAAAGACGCTAGAGGACTTCTGGCGGTTGGTATGGGAGCAGCAA3927 LeuLysLysThrLeuGluAspPheTrpArgLeuValTrpGluGlnGln 123012351240
GTCCACGTGATCATCATGCTGACTGTGGGCATGGAGAACGGGCGGGTA3975 ValHisValIleIleMetLeuThrValGlyMetGluAsnGlyArgVal 124512501255 CTGTGTGAGCACTACTGGCCAGCCAACTCCACGCCTGTTACTCACGGT4023 LeuCysGluHisTyrTrpProAlaAsnSerThrProValThrHisGly 126012651270 CACATCACCATCCACCTCCTGGCAGAGGAGCCTGAGGATGAGTGGACC4071 HisIleThrIleHisLeuLeuAlaGluGluProGluAspGluTrpThr 127512801285 AGGAGGGAATTCCAGCTGCAGCACGGTACCGAGCAAAAACAGAGGCGA4119 ArgArgGluPheGlnLeuGlnHisGlyThrGluGlnLysGlnArgArg 1290129513001305 GTGAAGCAGCTGCAGTTCACTACCTGGCCAGACCACAGTGTCCCGGAG4167 ValLysGlnLeuGlnPheThrThrTrpProAspHisSerValProGlu 131013151320 GCTCCCAGCTCTCTGCTCGCTTTTGTAGAACTGGTACAGGAGCAGGTG4215 AlaProSerSerLeuLeuAlaPheValGluLeuValGlnGluGlnVal 132513301335 CAGGCCACTCAGGGCAAGGGACCCATCCTGGTGCATTGCAGTGCTGGC4263 GlnAlaThrGlnGlyLysGlyProIleLeuValHisCysSerAlaGly 134013451350 GTGGGGAGGACAGGCACCTTTGTGGCTCTCTTGCGGCTACTGCGACAA4311 ValGlyArgThrGlyThrPheValAlaLeuLeuArgLeuLeuArgGln 135513601365 CTAGAGGAAGAGAAGGTGGCCGATGTGTTCAACACTGTGTACATACTC4359 LeuGluGluGluLysValAlaAspValPheAsnThrValTyrIleLeu 1370137513801385 CGGTTGCACCGGCCCCTCATGATCCAGACCCTGAGTCAATACATCTTC4407 ArgLeuHisArgProLeuMetIleGlnThrLeuSerGlnTyrIlePhe 139013951400 CTGCACAGTTGCCTGCTGAACAAGATTCTGGAAGGGCCCCCTGACAGC4455 LeuHisSerCysLeuLeuAsnLysIleLeuGluGlyProProAspSer 140514101415 TCCGACTCCGGCCCCATCTCTGTGATGGATTTTGCACAGGCTTGTGCC4503 SerAspSerGlyProIleSerValMetAspPheAlaGlnAlaCysAla 142014251430 AAGAGGGCAGCCAACGCCAATGCTGGTTTCTTGAAGGAGTACAAGCTC4551 LysArgAlaAlaAsnAlaAsnAlaGlyPheLeuLysGluTyrLysLeu 143514401445 CTGAAGCAGGCCATCAAGGATGGGACTGGCTCTCTGCTGCCCCCTCCT4599 LeuLysGlnAlaIleLysAspGlyThrGlySerLeuLeuProProPro 1450145514601465 GACTACAATCAGAACAGCATTGTCTCCCGTCGTCATTCTCAGGAGCAG4647 AspTyrAsnGlnAsnSerIleValSerArgArgHisSerGlnGluGln 147014751480 TTCGCCCTGGTGGAGGAGTGCCCTGAGGATAGCATGCTGGAAGCCTCA4695 PheAlaLeuValGluGluCysProGluAspSerMetLeuGluAlaSer 148514901495 CTCTTCCCTGGTGGTCCGTCTGGTTGTGATCATGTGGTGCTGACTGGC4743 LeuPheProGlyGlyProSerGlyCysAspHisValValLeuThrGly 150015051510 TCAGCCGGACCAAAGGAACTCTGGGAAATGGTGTGGGAGCATGATGCC4791 SerAlaGlyProLysGluLeuTrpGluMetValTrpGluHisAspAla 151515201525 CATGTGCTCGTCTCCCTGGGCCTGCCTGATACCAAGGAGAAGCCACCA4839 HisValLeuValSerLeuGlyLeuProAspThrLysGluLysProPro 1530153515401545 GACATCTGGCCAGTGGAGATGCAGCCTATTGTCACAGACATGGTGACA4887 AspIleTrpProValGluMetGlnProIleValThrAspMetValThr 155015551560 GTGCACAGAGTGTCTGAGAGCAACACAACAACTGGCTGGCCCAGCACC4935 ValHisArgValSerGluSerAsnThrThrThrGlyTrpProSerThr 156515701575 CTCTTCAGAGTCATACACGGGGAGAGTGGAAAGGAAAGGCAGGTTCAA4983 LeuPheArgValIleHisGlyGluSerGlyLysGluArgGlnValGln 158015851590 TGCCTGCAATTTCCATGCTCTGAGTCTGGGTGTGAGCTCCCAGCTAAC5031 CysLeuGlnPheProCysSerGluSerGlyCysGluLeuProAlaAsn 159516001605 ACCCTACTGACCTTCCTTGATGCTGTGGGCCAGTGCTGCTTCCGGGGC5079 ThrLeuLeuThrPheLeuAspAlaValGlyGlnCysCysPheArgGly 1610161516201625 AAGAGCAAGAAGCCAGGGACCCTGCTCAGCCACTCCAGCAAAAACACA5127 LysSerLysLysProGlyThrLeuLeuSerHisSerSerLysAsnThr 163016351640 AACCAGCTGGGCACCTTCTTGGCTATGGAACAGCTGTTACAGCAAGCA5175 AsnGlnLeuGlyThrPheLeuAlaMetGluGlnLeuLeuGlnGlnAla 164516501655 GGGACAGAGCGCACAGTGGACGTCTTCAATGTGGCCCTGAAGCAGTCA5223 GlyThrGluArgThrValAspValPheAsnValAlaLeuLysGlnSer 166016651670 CAGGCCTGCGGCCTTATGACCCCAACACTGGAGCAGTATATCTACCTC5271 GlnAlaCysGlyLeuMetThrProThrLeuGluGlnTyrIleTyrLeu 167516801685 TACAACTGTCTGAACAGCGCACTGCTGAACGGGCTGCCCAGAGCTGGG5319 TyrAsnCysLeuAsnSerAlaLeuLeuAsnGlyLeuProArgAlaGly 1690169517001705 AAGTGGCCTGCGCCCTGCTAGGCGTCATGTTCCAGCAAATCCACCCAG5367 LysTrpProAlaProCys 1710 GCCTGACTTCCCTAGGAGAGTGGATCCACCGGGCCTCACACTGTCCAAGGGCAGAGTCCA5427 GGAATAAAGAGACATGGTCAAAAAAAAA5455 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1711 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: MetArgProLeuIleLeuLeuAlaAlaLeuLeuTrpLeuGlnGlyPhe 151015 LeuAlaGluAspAspAlaCysSerSerLeuGluGlySerProAspArg 202530 GlnGlyGlyGlyProLeuLeuSerValAsnValSerSerHisGlyLys 354045 SerThrSerLeuPheLeuSerTrpValAlaAlaGluLeuGlyGlyPhe 505560 AspTyrAlaLeuSerLeuArgSerValAsnSerSerGlySerProGlu 65707580 GlyGlnGlnLeuGlnAlaHisThrAsnGluSerGlyPheGluPheHis 859095 GlyLeuValProGlySerArgTyrGlnLeuLysLeuThrValLeuArg 100105110 ProCysTrpGlnAsnValThrIleThrLeuThrAlaArgThrAlaPro 115120125 ThrValValArgGlyLeuGlnLeuHisSerAlaGlySerProAlaArg 130135140 LeuGluAlaSerTrpSerAspAlaProGlyAspGlnAspSerTyrGln 145150155160 LeuLeuLeuTyrHisLeuGluSerGlnThrLeuAlaCysAsnValSer 165170175 ValSerProAspThrLeuSerTyrSerPheGlyAspLeuLeuProGly 180185190 ThrGlnTyrValLeuGluValIleThrTrpAlaGlySerLeuHisAla 195200205 LysThrSerIleLeuGlnTrpThrGluProValProProAspHisLeu 210215220 AlaLeuArgAlaLeuGlyThrSerSerLeuGlnAlaPheTrpAsnSer 225230235240 SerGluGlyAlaThrSerPheHisLeuMetLeuThrAspLeuLeuGly 245250255 GlyThrAsnThrThrAlaValIleArgGlnGlyValSerThrHisThr 260265270 PheLeuHisLeuSerProGlyThrProHisGluLeuLysIleCysAla 275280285 SerAlaGlyProHisGlnIleTrpGlyProSerAlaThrGluTrpThr 290295300 TyrProSerTyrProSerAspLeuValLeuThrProLeuArgAsnGlu 305310315320 LeuTrpAlaSerTrpLysAlaGlyLeuGlyAlaArgAspGlyTyrVal 325330335 LeuLysLeuSerGlyProMetGluSerThrSerThrLeuGlyProGlu 340345350 GluCysAsnAlaValPheProGlyProLeuProProGlyHisTyrThr 355360365 LeuGlnLeuLysValLeuAlaGlyProTyrAspAlaTrpValGluGly 370375380 SerThrTrpLeuAlaGluSerAlaAlaLeuProArgGluValProGly 385390395400 AlaArgLeuTrpLeuAspGlyLeuGluAlaSerLysGlnProGlyArg 405410415 ArgAlaLeuLeuTyrSerAspAspAlaProGlySerLeuGlyAsnIle 420425430 SerValProSerGlyAlaThrHisValIlePheCysGlyLeuValPro 435440445 GlyAlaHisTyrArgValAspIleAlaSerSerThrGlyAspIleSer 450455460 GlnSerIleSerGlyTyrThrSerProLeuProProGlnSerLeuGlu 465470475480 ValIleSerArgSerSerProSerAspLeuThrIleAlaTrpGlyPro 485490495 AlaProGlyGlnLeuGluGlyTyrLysValThrTrpHisGlnAspGly 500505510 SerGlnArgSerProGlyAspLeuValAspLeuGlyProAspThrLeu 515520525 SerLeuThrLeuLysSerLeuValProGlySerCysTyrThrValSer 530535540 AlaTrpAlaTrpAlaGlyAsnLeuSerSerAspSerGlnLysIleHis 545550555560 SerCysThrArgProAlaProProThrAsnLeuSerLeuGlyPheAla 565570575 HisGlnProAlaAlaLeuLysAlaSerTrpTyrHisProProGlyGly 580585590 ArgAspAlaPheHisLeuArgLeuTyrArgLeuArgProLeuThrLeu 595600605 GluSerGluLysValLeuProArgGluAlaGlnAsnPheSerTrpAla 610615620 GlnLeuThrAlaGlyCysGluPheGlnValGlnLeuSerThrLeuTrp 625630635640 GlySerGluArgSerSerSerAlaAsnAlaThrGlyTrpThrProPro 645650655 SerAlaProThrLeuValAsnValThrSerAspAlaProThrGlnLeu 660665670 GlnValSerTrpAlaHisValProGlyGlyArgSerArgTyrGlnVal 675680685 ThrLeuTyrGlnGluSerThrArgThrAlaThrSerIleMetGlyPro 690695700 LysGluAspGlyThrSerPheLeuGlyLeuThrProGlyThrLysTyr 705710715720 LysValGluValIleSerTrpAlaGlyProLeuTyrThrAlaAlaAla 725730735 AsnValSerAlaTrpThrTyrProLeuIleProAsnGluLeuLeuVal 740745750 SerMetGlnAlaGlySerAlaValValAsnLeuAlaTrpProSerGly 755760765 ProLeuGlyGlnGlyAlaCysHisAlaGlnLeuSerAspAlaGlyHis 770775780 LeuSerTrpGluGlnProLeuLysLeuGlyGlnGluLeuPheMetLeu 785790795800 ArgAspLeuThrProGlyHisThrIleSerMetSerValArgCysArg 805810815 AlaGlyProLeuGlnAlaSerThrHisLeuValValLeuSerValGlu 820825830 ProGlyProValGluAspValLeuCysHisProGluAlaThrTyrLeu 835840845 AlaLeuAsnTrpThrMetProAlaGlyAspValAspValCysLeuVal 850855860 ValValGluArgLeuValProGlyGlyGlyThrHisPheValPheGln 865870875880 ValAsnThrSerGlyAspAlaLeuLeuLeuProAsnLeuMetProThr 885890895 ThrSerTyrArgLeuSerLeuThrValLeuGlyArgAsnSerArgTrp 900905910 SerArgAlaValSerLeuValCysSerThrSerAlaGluAlaTrpHis 915920925 ProProGluLeuAlaGluProProGlnValGluLeuGlyThrGlyMet 930935940 GlyValThrValMetArgGlyMetPheGlyLysAspAspGlyGlnIle 945950955960 GlnTrpTyrGlyIleIleAlaThrIleAsnMetThrLeuAlaGlnPro 965970975 SerArgGluAlaIleAsnTyrThrTrpTyrAspHisTyrTyrArgGly 980985990 CysGluSerPheLeuAlaLeuLeuPheProAsnProPheTyrProGlu 99510001005 ProTrpAlaGlyProArgSerTrpThrValProValGlyThrGluAsp 101010151020 CysAspAsnThrGlnGluIleCysAsnGlyArgLeuLysSerGlyPhe 1025103010351040 GlnTyrArgPheSerValValAlaPheSerArgLeuAsnThrProGlu 104510501055 ThrIleLeuAlaPheSerAlaPheSerGluProArgAlaSerIleSer 106010651070 LeuAlaIleIleProLeuThrValMetLeuGlyAlaValValGlySer 107510801085 IleValIleValCysAlaValLeuCysLeuLeuArgTrpArgCysLeu 109010951100 LysGlyProArgSerGluLysAspGlyPheSerLysGluLeuMetPro 1105111011151120 TyrAsnLeuTrpArgThrHisArgProIleProIleHisSerPheArg 112511301135 GlnSerTyrGluAlaLysSerAlaHisAlaHisGlnThrPhePheGln 114011451150 GluPheGluGluLeuLysGluValGlyLysAspGlnProArgLeuGlu 115511601165 AlaGluHisProAspAsnIleIleLysAsnArgTyrProHisValLeu 117011751180 ProTyrAspHisSerArgValArgLeuThrGlnLeuProGlyGluPro 1185119011951200 HisSerAspTyrIleAsnAlaAsnPheIleProGlyTyrSerHisThr 120512101215
GlnGluIleIleAlaThrGlnGlyProLeuLysLysThrLeuGluAsp 122012251230 PheTrpArgLeuValTrpGluGlnGlnValHisValIleIleMetLeu 123512401245 ThrValGlyMetGluAsnGlyArgValLeuCysGluHisTyrTrpPro 125012551260 AlaAsnSerThrProValThrHisGlyHisIleThrIleHisLeuLeu 1265127012751280 AlaGluGluProGluAspGluTrpThrArgArgGluPheGlnLeuGln 128512901295 HisGlyThrGluGlnLysGlnArgArgValLysGlnLeuGlnPheThr 130013051310 ThrTrpProAspHisSerValProGluAlaProSerSerLeuLeuAla 131513201325 PheValGluLeuValGlnGluGlnValGlnAlaThrGlnGlyLysGly 133013351340 ProIleLeuValHisCysSerAlaGlyValGlyArgThrGlyThrPhe 1345135013551360 ValAlaLeuLeuArgLeuLeuArgGlnLeuGluGluGluLysValAla 136513701375 AspValPheAsnThrValTyrIleLeuArgLeuHisArgProLeuMet 138013851390 IleGlnThrLeuSerGlnTyrIlePheLeuHisSerCysLeuLeuAsn 139514001405 LysIleLeuGluGlyProProAspSerSerAspSerGlyProIleSer 141014151420 ValMetAspPheAlaGlnAlaCysAlaLysArgAlaAlaAsnAlaAsn 1425143014351440 AlaGlyPheLeuLysGluTyrLysLeuLeuLysGlnAlaIleLysAsp 144514501455 GlyThrGlySerLeuLeuProProProAspTyrAsnGlnAsnSerIle 146014651470 ValSerArgArgHisSerGlnGluGlnPheAlaLeuValGluGluCys 147514801485 ProGluAspSerMetLeuGluAlaSerLeuPheProGlyGlyProSer 149014951500 GlyCysAspHisValValLeuThrGlySerAlaGlyProLysGluLeu 1505151015151520 TrpGluMetValTrpGluHisAspAlaHisValLeuValSerLeuGly 152515301535 LeuProAspThrLysGluLysProProAspIleTrpProValGluMet 154015451550 GlnProIleValThrAspMetValThrValHisArgValSerGluSer 155515601565 AsnThrThrThrGlyTrpProSerThrLeuPheArgValIleHisGly 157015751580 GluSerGlyLysGluArgGlnValGlnCysLeuGlnPheProCysSer 1585159015951600 GluSerGlyCysGluLeuProAlaAsnThrLeuLeuThrPheLeuAsp 160516101615 AlaValGlyGlnCysCysPheArgGlyLysSerLysLysProGlyThr 162016251630 LeuLeuSerHisSerSerLysAsnThrAsnGlnLeuGlyThrPheLeu 163516401645 AlaMetGluGlnLeuLeuGlnGlnAlaGlyThrGluArgThrValAsp 165016551660 ValPheAsnValAlaLeuLysGlnSerGlnAlaCysGlyLeuMetThr 1665167016751680 ProThrLeuGluGlnTyrIleTyrLeuTyrAsnCysLeuAsnSerAla 168516901695 LeuLeuAsnGlyLeuProArgAlaGlyLysTrpProAlaProCys 170017051710 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "This position is (I/V)." (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 10 (D) OTHER INFORMATION: /note= "This position is (S/T)." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: XaaHisCysXaaAlaGlyXaaXaaArgXaaGly 1510 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ValHisCysSerAlaGlyValGlyArgThrGly 1510 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: SerHisSerSerLysAsnThrAsnGlnLeuGly 1510 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AspTyrIleAsnAla 15 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CAGTGGATCCHGAYTAYATHAAYGC25 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TyrIleAlaThrGlnGlyPro 15 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CAGTGGATCCTACATYGYHRCMCARGG27 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: LysCysAspGlnTyrTrp 15 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CAGTGGATCCAARTGYSMNSARTAYTGGCC30 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: HisCysSerAlaGlyValGlyArg 15 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CTAGTCTAGACCNAWDCCNGCRCARTG27 __________________________________________________________________________