WO1995006735A2 - Nucleotide sequences for novel protein tyrosine phosphatases - Google Patents

Nucleotide sequences for novel protein tyrosine phosphatases Download PDF

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WO1995006735A2
WO1995006735A2 PCT/US1994/009943 US9409943W WO9506735A2 WO 1995006735 A2 WO1995006735 A2 WO 1995006735A2 US 9409943 W US9409943 W US 9409943W WO 9506735 A2 WO9506735 A2 WO 9506735A2
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ser
leu
glu
val
ptpll
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WO1995006735A3 (en
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Leonel Jorge Gonez
Jan Saras
Lena Claesson-Welsh
Carl-Henrik Heldin
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Ludwig Cancer Research
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Ludwig Cancer Research
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Priority to EP94926675A priority patent/EP0789771A2/en
Priority to AU76443/94A priority patent/AU683299B2/en
Priority to NZ273219A priority patent/NZ273219A/en
Priority to JP7508288A priority patent/JPH09510861A/en
Priority to US08/596,291 priority patent/US5821075A/en
Publication of WO1995006735A2 publication Critical patent/WO1995006735A2/en
Publication of WO1995006735A3 publication Critical patent/WO1995006735A3/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Definitions

  • This invention relates to the isolation and cloning of nucleic acids encoding two novel protein tyrosine phosphatases (PTPs) .
  • PTPs novel protein tyrosine phosphatases
  • the present invention relates to the isolation and cloning of two PTPs from human glioblastoma cDNA which have been designated PTPLl and GL -2.
  • the present invention provides isolated PTP nucleic acid sequences; isolated PTP anti-sense sequences; vectors containing such nucleic acid sequences; cells, cell lines and animal hosts transformed by a recombinant vector so as to exhibit increased, decreased, or differently regulated expression of the PTPs; isolated probes for identifying sequences substantially similar or homologous to such sequences; substantially pure PTP proteins and variants or fragments thereof; antibodies or other agents which bind to these PTPs and variants or fragments thereof; methods of assaying for activity of these PTPs; methods of assessing the regulation of PTPLl or GLM-2; and methods of identifying and/or testing drugs which may affect the expression or activity of these PTPs.
  • Protein tyrosine phosphorylation plays an essential role in the regulation of cell growth, proliferation and differentiation (reviewed in Hunter, T. (1987) Cell 50:823-8291). This dynamic process is modulated by the counterbalancing activities of protein tyrosine kinases
  • PTPs can be classified according to their secondary structure into two broad groups, i.e. cytoplasmic and transmembrane molecules (reviewed in Charbonneau, H. , and Tonks, N.K. (1992) Annu. Rev. Cell Biol. 8:463-493; Pot, D.A., and Dixon, J.E. (1992) Biochim. Biophys. Acta 1136:35-43).
  • Transmembrane PTPs have the structural organization of receptors and thus the potential to initiate cellular signaling in response to external stimuli.
  • transmembrane PTPs that have single PTP domains are known, HPTP-P (Krueger, N.X. , et al. , (1990) EMBO J ⁇ 9:3241-3252) and DPTP10D (Tian, S.-S., et al. , (1991) Cell 67:675-685) .
  • Nonreceptor PTPs display a single catalytic domain and contain, in addition, non-catalytic amino acid sequences which appear to control intracellular localization of the molecules and which may be involved in the determination of substrate specificity (Mauro, L.J., and Dixon, J.E. (1994) TIBS 19:151-155) and have also been suggested to be regulators of PTP activity (Charbonneau, H., and Tonks, N.K. (1992) Annu. Rev. Cell Biol. 8:463-493). PTP1B (Tonks, N.K., et al ⁇ , (1988) J. Biol. Chem.
  • T-cell PTP has also been shown to be responsible for enzyme localization and functional regulation (Cool, D.E., et al. , (1990) Proc. Natl. Acad. Sci. (USA) 87:7280-7284; Cool, D.E. , et al. , (1992) Proc. Natl. Acad. Sci. (USA) 89:5422-5426).
  • PTPs containing SH2 domains have been described including PTPIC (Shen, S.-H., et al ⁇ , (1991) Nature 352:736-739), also named HCP (Yi, T., et al . , (1992) Mol. Cell. Biol. 12:836-846), SHP (Matthews, R.J., et al. , (1992) Mol. Cell. Biol 12:2396-2405) or SH-PTP1 (Plutzky, J., et al. , (1992) Proc. Natl. Acad. Sci.
  • the Drosophila csk gene product (Perkins, L.A., et al ⁇ , (1992) Cell 70:225-236) also belongs to this subfamily. PTPIC has been shown to associate via its SH2 domains with ligand-activated c-Kit and CSF-1 receptor PTKs (Yi, T., and Ihle, J.N. (1993) Mol. Cell. Biol. 13:3350-3358; Young, Y.-G. , et al., (1992) J. Biol. Chem.
  • Syp interacts with and is phosphorylated by the ligand activated receptors for epidermal growth factor and platelet-derived growth factor (Feng, G.-S., et al. , (1993) Science 259:1607-1611). Syp has also been reported to associate with tyrosine phosphorylated insulin receptor substrate 1 (Kuhne, M.R. , et al ⁇ , (1993) J. Biol. Chem. 268:11479-11481).
  • PTPa ⁇ e MEG (Gu, M. , et al ⁇ , (1991) Proc. Natl. Acad. Sci. (USA) 88:5867-5871), which contain a region in their respective N-terminal segments with similarity to the cytoskeletal- associated proteins band 4.1 (Conboy, J., et al. , (1986) Proc. Natl. Acad. Sci. (USA) 83:9512-9516), ezrin (Gould, K.L., et al.
  • PTPH1 and PTPase MEG would, like members of this family, localize at the interface between the plasma membrane and the cytoskeleton and thereby be involved in the modulation of cytoskeletal function (Tonks, N.K. , et al. , (1991) Cold Spring Harbor Symposia on Quantitative Biology LVI:265-273) .
  • the present invention is based upon the molecular cloning of previously uncloned and previously undisclosed nucleic acids encoding two novel PTPs.
  • the disclosed sequences encode PTPs which we have designated PTPLl and GLM-2.
  • PTPLl was previously designated GLM-1 in U.S. Patent Application Serial No. 08/115,573 filed September 1, 1993.
  • the present invention is based upon the molecular cloning of PTPLl and GLM-2 PTP sequences from human glioblastoma cells.
  • the invention provides isolated cDNA and RNA sequences corresponding to PTPLl and GLM-2 transcripts and encoding the novel PTPs.
  • the present invention provides vectors containing PTPLl or GLM-2 cDNA sequences, vectors capable of expressing PTPLl or GLM-2 sequences with endogenous or exogenous promoters, and hosts transformed with one or more of the above-mentioned vectors.
  • the invention further provides for the isolation of allelic variants of the disclosed sequences, endogenous PTPLl or GLM-2 regulatory sequences, and substantially similar or homologous PTPLl or GLM-2 DNA and RNA sequences from other species including mouse, rat, rabbit and non-human primates.
  • the present invention also provides fragments and variants of isolated PTPLl and GLM-2 sequences, fragments and variants of isolated PTPLl or GLM-2 RNA, vectors containing variants or fragments of PTPLl or GLM-2 sequences, vectors capable of expressing variants or fragments of PTPLl or GLM-2 sequences with endogenous or exogenous regulatory sequences, and hosts transformed with one or more of the above-mentioned vectors.
  • the invention further provides variants or fragments of substantially similar or homologous PTPLl and GLM-2 DNA and RNA sequences from species including mouse, rat, rabbit and non-human primates.
  • the present invention provides isolated PTPLl and GLM-2 anti-sense DNA, isolated PTPLl and GLM-2 anti-sense RNA, vectors containing PTPLl or GLM-2 anti-sense DNA, vectors capable of expressing PTPLl or GLM-2 anti-sense DNA with endogenous or exogenous promoters, and hosts transformed with one or more of the above-mentioned vectors.
  • the invention further provides the related PTPLl or GLM-2 anti-sense DNA and anti-sense RNA sequences from other species including mouse, rat, rabbit and non-human primates.
  • the present invention also provides fragments and variants of isolated PTPLl and GLM-2 anti-sense DNA, fragments and variants of isolated PTPLl and GLM-2 anti-sense RNA, vectors containing fragments or variants of PTPLl and GLM-2 anti-sense DNA, vectors capable of expressing fragments or variants of PTPLl and GLM-2 anti-sense DNA with endogenous or exogenous promoters, and hosts transformed with one or more of the above-mentioned vectors.
  • the invention further provides fragments or variants of the related PTPLl and GLM-2 anti-sense DNA and PTPLl and GLM-2 anti-sense RNA sequences from other species including mouse, rat, rabbit and non-human primates.
  • the invention also provides isolated probes useful for detecting the presence or level of expression of a sequence identical, substantially similar or homologous to the disclosed PTPLl and GLM-2 sequences.
  • the probes may consist of the PTPLl and GLM-2 DNA, RNA or anti-sense sequences disclosed herein.
  • the probe may be labeled with, for example, a radioactive isotope; immobilized as, for example, on a filter for Northern or Southern blotting; or may be tagged with any other sort of marker which enhances or facilitates the detection of binding.
  • the probes may be oligonucleotides or synthetic oligonucleotide analogs.
  • the invention also provides substantially pure PTPLl and GLM-2 proteins.
  • the proteins may be obtained from natural sources using the methods disclosed herein or, in particular, the invention provides substantially pure PTPLl and GLM-2 proteins produced by a host cell or transgenic animal transformed by one of the vectors disclosed herein.
  • the invention also provides substantially pure variants and fragments of PTPLl and GLM-2 proteins.
  • the present invention provides methods of obtaining and identifying agents capable of binding to either PTPLl or GLM-2.
  • agents include antibodies, peptides, carbohydrates and pharmaceutical agents.
  • the agents may include natural ligand ⁇ , co-factors, accessory proteins or associated peptides, modulators, regulators, or inhibitors.
  • the entire PTPLl or GLM-2 protein may be used to test or develop such agents or variants or fragments thereof may be employed. In particular, only certain domains of the PTPLl or GLM-2 protein may be employed.
  • the invention further provides detectably labeled, immobilized and toxin-conjugated forms of these agents.
  • the present invention also provides methods for assaying for PTPLl or GLM-2 PTP activity.
  • the presence and level of either PTPLl or GLM-2 expression may be determined by hybridizing the probes to total or selected mRNA from the cell or tissue to be studied.
  • the antibodies or other binding agents disclosed herein the presence and level of PTPLl or GLM-2 protein may be assessed. Such methods may, for example, be employed to determine the tissue-specificity of PTPLl or GLM-2 expression.
  • the present invention also provides methods for assessing the regulation of PTPLl or GLM-2 function.
  • Such methods include fusion of the regulatory regions of the PTPLl or GLM-2 nucleic acid sequences to a marker locus, introduction of this fusion product into a host cell using a vector, and testing for inducers or inhibitors of PTPLl or GLM-2 by measuring expression of the marker locus.
  • the level of expression of PTPLl or GLM-2 mRNA may be ascertained and the effect of various endogenous and exogenous compounds or treatments on PTPLl or GLM-2 expression may be determined.
  • the effect of various endogenous and exogenous compounds and treatments on PTPLl or GLM-2 expression may be assessed by measuring the level of either PTPLl or GLM-2 protein with labeled antibodies as disclosed herein.
  • the present invention provides methods for efficiently testing the activity or potency of drugs intended to enhance or inhibit PTPLl or GLM-2 expression or activity.
  • the nucleic acid sequences and vectors disclosed herein enable the development of cell lines and transgenic organisms with increased, decreased, or differently regulated expression of PTPLl or GLM-2. Such cell lines and animals are useful subjects for testing pharmaceutical compositions.
  • the present invention further provides methods of modulating the activity of PTPLl and GLM-2 PTPs in cells.
  • agents and, in particular, antibodies which are capable of binding to either PTPLl or GLM-2 PTP are provided to a cell expressing PTPLl or GLM-2.
  • the binding of such an agent to the PTP can be used either to activate or inhibit the activity of the protein.
  • PTPLl and GLM-2 anti-sense transcripts may be administered such that they enter the cell and inhibit translation of the PTPLl or GLM-2 mRNA and/or the transcription of PTPLl or GLM-2 nucleic acid sequences.
  • PTPLl or GLM-2 RNA may be administered such that it enters the cell, serves as a template for translation and thereby augments production of PTPLl or GLM-2 protein.
  • a vector capable of expressing PTPLl or GLM-2 mRNA transcripts or PTPLl or GLM-2 anti-sense RNA transcripts is administered such that it enters the cell and the transcripts are expressed.
  • FIG. 1 Comparison of PTPLl with proteins of the band 4.1 superfamily. The alignment was done using the Clustal V alignment program (Fazioli, F., et al ⁇ , (1993) Oncoqene 8:1335-1345). Identical amino acid residues conserved in two or more sequences, are boxed. A conserved tyrosine residue, which in ezrin has been shown to be phosphorylated by the epidermal growth factor receptor, is indicated by an asterisk,
  • Figure 2 Comparison of amino acid sequences of GLGF-repeats. The alignment was done manually. Numbers of the GLGF-repeats are given starting from the N-terminus of the protein. Residues conserved in at least eight (42%) repeats are showed in bold letters. Five repeats are found In PTPLl, three are found in the guanylate kinases, dlg-A gene product, PSD-95 and the 220-kDa protein. One GLGF-repeat is found in the guanylate kinase p55, in the PTPs PTPH1 and PTPase MEG, and in nitric oxide synthase (NOS) . One repeat is also found in an altered rosl transcript from the glioma cell line U-118MG,
  • Figure 3 Schematic diagram illustrating the domain strucure of PTPLl and other GLGF-repeat containing proteins. Domains and motifs indicated in the figure are L, leucine zipper motif; Band 4.1, band 4.1-like domain; G, GLGF-repeat; PTPase, catalytic PTPase domain; 3, SH3 domain; GK, guanylate kinase domain, Bind. Reg., co-enzyme binding region.
  • FIG. 4 PTP activity of PTPLl. Immunoprecipitates from COS-1 cells using an antiserum ( ⁇ LIB) against PTPLl, unblocked (open circles) or blockeod with peptide (open squares), were incubated for 2, 4, 6 or 12 minutes with myelin basic protein, 32P-labeled on tyrosine residues.
  • ⁇ LIB antiserum
  • the amount of radioactivity released as inorganic phosphate is expressed as the percentage of the total input of radioactivity.
  • a gene is a nucleic acid sequence including a promoter region operably joined to a coding sequence which may serve as a template from which an RNA molecule may be transcribed by a nucleic acid polymerase.
  • a gene contains a promoter sequence to which the polymerase binds, an initiation sequence which signals the point at which transcription should begin, and a termination sequence which signals the point at which transcription should end.
  • the gene also may contain an operator site at which a repressor may bind to block the polymerase and to prevent transcription and/or may contain ribosome binding sites, capping signals, transcription enhancers and polyadenylation signals.
  • RNA messenger RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • anti-sense RNAs of the present invention include ribosomal RNA (rRNA), transfer RNA (tRNA), and the anti-sense RNAs of the present invention.
  • coding sequences between the translation start codon (ATG) and the translation stop codon (TAA, TGA, or TAG) may be of two types: exons and introns.
  • the exons are included in processed mRNA transcripts and are generally translated into a peptide or protein.
  • Introns are excised from the RNA as it is processed into mature mRNA and are not translated into peptide or protein.
  • the word gene embraces both the gene including its introns, as may be obtained from genomic DNA, and the gene with the introns excised from the DNA, as may be obtained from cDNA.
  • Anti-sense DNA is defined as DNA that encodes anti-sense RNA and anti-sense RNA is RNA that is complementary to or capable of selectively hybridizing to some specified RNA transcript. Thus, anti-sense RNA for a particular gene would be capable of hybridizing with that gene's RNA transcript in a selective manner.
  • an anti-sense gene is defined as a segment of anti-sense DNA operably joined to regulatory sequences such that the sequences encoding the anti-sense RNA may be expressed.
  • cDNA Complementary DNA or cDNA is DNA which has been produced by reverse transcription from mature mRNA. In eukaryotes, sequences in RNA corresponding to introns in a gene are excised during mRNA processing.
  • cDNA sequences therefore, lack the intron sequences present in the genomic DNA to which they correspond.
  • cDNA sequences will lack the regulatory sequences which are not transcribed into RNA. To create a functional cDNA gene, therefore, the cDNA sequence must be operably joined to a promoter region such that transcription may occur.
  • a coding sequence and a promoter region are said to be operably joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the promoter region. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of promoter function results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • coding sequence be eventually expressed as a protein or polypeptide, as in the case of anti-sense RNA expression, there is no need to ensure that the coding sequences and promoter region are joined without a frame-shift.
  • a coding sequence which need not be eventually expressed as a protein or polypeptide is said to be operably joined to a promoter region if induction of promoter function results in the transcription of the RNA sequence of the coding sequences.
  • regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating sequences involved with initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribing regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • Such transcriptional control sequences may also include enhancer sequences or upstream activator sequences, as desired.
  • a vector may be any of a number of nucleic acid sequences into which a desired sequence may be inserted by restriction and ligation. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include plasmids, phage, phasmids and cosmids.
  • a cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to a promoter region and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., ⁇ -galactosidase or alkaline phosphatase) , and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques.
  • fragment means both unique fragments and substantially characteristic fragments. As used herein, the term “fragment” is not to be construed according to standard dictionary definitions.
  • a "substantially characteristic fragment" of a molecule such as a protein or nucleic acid sequence, is meant to refer to any portion of the molecule sufficiently rare or sufficiently characteristic of that molecule so as to identify it as derived from that molecule or to distinguish it from a class of unrelated molecules.
  • a single amino acid or nucleotide, or a sequence of only two or three, cannot be a substantially characteristic fragment because all such short sequences occur frequently in nature.
  • a substantially characteristic fragment of a nucleic acid sequence is one which would have utility as a probe in identifying the entire nucleic acid sequence from which it is derived from within a sample of total genomic or cDNA. Under stringent hybridization conditions, a substantially characteristic fragment will hybridize only to the sequence from which it was derived or to a small class of substantially similar related sequences such as allelic variants, heterospecific homologous loci, and variants with small insertions, deletions or substitutions of nucleotides or nucleotide analogues.
  • a substantially characteristic fragment may, under lower stringency hybridization conditions, hybridize with non-allelic and non-homologous loci and be used as a probe to find such loci but will not do so at higher stringency.
  • a substantially characteristic fragment of a protein would have utility in generating antibodies which would distinguish the entire protein from which it is derived, an allelomorphic protein or a heterospecific homologous protein from a mixture of many unrelated proteins. It is within the knowledge and ability of one ordinarily skilled in the art to recognize, produce and use substantially characteristic fragments of nucleic acid sequences and proteins as, for example, probes for screening DNA libraries or epitopes for generating antibodies.
  • a unique fragment of a protein or nucleic acid sequence is a substantially characteristic fragment not currently known to occur elsewhere in nature (except in allelic or heterospecific homologous variants, i.e. it is present only in the PTPLl or GLM-2 PTP or a PTPLl or GLM-2 PTP "ho ologue") .
  • a unique fragment will generally exceed 15 nucleotides or 5 amino acid residues.
  • Genbank Land Alamos National Laboratories, USA
  • SwissProt or the National Bio edical Research Foundation database.
  • a unique fragment is particularly useful, for example, in generating monoclonal antibodies or in screening DNA or cDNA libraries.
  • Stringent Hybridization conditions is a term of art understood by those of ordinary skill in the art.
  • stringent hybridization conditions are those conditions of temperature and buffer solution which will permit hybridization of that nucleic acid sequence to its complementary sequence and not to substantially different sequences.
  • the exact conditions which constitute “stringent” conditions depend upon the length of the nucleic acid sequence and the frequency of occurrence of subsets of that sequence within other non-identical sequences.
  • Stringent hybridization conditions may include hybridization conditions of 30°C-65°C and from 5X to 0. IX SSPC. Less than stringent hybridization conditions are employed to isolate nucleic acid sequences which are substantially similar, allelic or homologous to any given sequence.
  • primers that are derived from nucleic acid encoding a PTPLl or GLM-2 PTP
  • high stringency conditions e.g. annealing at 50-60°C
  • lower stringency conditions e.g. annealing at 35-37°C
  • sequences which are greater than about 40-50% homologous to the primer will be amplified.
  • DNA probes derived from a PTPLl or GLM-2 PTP for colony/plaque hybridization
  • high stringency conditions e.g. hybridization at 50-65°C, 5X SSPC, 50% formamide, wash at 50-65°C, 0.5X SSPC
  • lower stringency conditions e.g. hybridization at 35-37°C, 5X SSPC, 40-45% formamide, wash at 42°C SSPC
  • Two nucleic acid sequences are substantially similar if one of them or its anti-sense complement can bind to the other under strict hybridization conditions so as to distinguish that strand from all or substantially all other sequences in a cDNA or genomic library.
  • one sequence is substantially similar to another if it or its anti-sense complement is useful as a probe in screening for the presence of its similar DNA or RNA sequence under strict hybridization conditions.
  • Two proteins are substantially similar if they are encoded by substantially similar DNA or RNA sequences. In addition, even if they are not encoded by substantially similar nucleic acids, two proteins are substantially similar if they share sufficient primary, secondary and tertiary structure to perform the same biological role (structural or functional) with substantially the same efficacy or utility.
  • variants of a protein or nucleic acid or fragment thereof is meant to include a molecule substantially similar in structure to the protein or nucleic acid, or to a fragment thereof.
  • variants of nucleic acid sequences include sequences with conservative nucleotide substitutions, small insertions or deletions, or additions.
  • variants of proteins include proteins with conservative amino acid substitutions, small insertions or deletions, or additions.
  • nucleotide substitutions which do not effect the amino acid sequence of the subsequent translation product are particularly contemplated.
  • substitutions of structurally similar amino .acids in proteins, such as leucine for isoleucine, or insertions, deletions, and terminal additions which do not destroy the functional utility of the protein are contemplated.
  • allelic variants of nucleic acid sequences and allelomorphic variants or protein or polypeptide sequences are particularly contemplated.
  • an allelic variant is simply a naturally occurring variant of a polymorphic gene and that term is used herein as it is commonly used in the field of population genetics. The production of such variants is well known in the art and, therefore, such variants are intended to fall within the spirit and scope of the claims.
  • homologous and homologues are intended to embrace either and/or both homologous nucleic acid sequences and homologous protein sequences as the context may indicate.
  • Homologues are a class of variants, as defined above, which share a sufficient degree of structural and functional similarity so as to indicate to one of ordinary skill in the art that they share a common evolutionary origin and that the structural and functional similarity is the result of evolutionary conservation.
  • nucleic acid sequences and the proteins they encode must meet two criteria: (1)
  • the polypeptides encoded by homologous nucleic acids are at least approximately 50-60% identical and preferably at least 70% identical for at least one stretch of at least 20 amino acids.
  • both the identity and the approximate positions of the amino acid residues relative to each other must be conserved and not just the overall amino acid composition. Thus, one must be able to "line up" the conserved regions of the homologues and conclude that there is 50-60% identity; and
  • the polypeptides must retain a functional similarity to the PTPLl or GLM-2 PTP in that it is a protein tyrosine phosphatase.
  • substantially pure when applied to the proteins, variants or fragments thereof of the present invention means that the proteins are essentially free of other substances to an extent practical and appropriate for their intended use.
  • the proteins are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, protein sequencing, or producing pharmaceutical preparations.
  • substantially pure proteins, variants or fragments thereof may be produced in light of the nucleic acids of the present invention.
  • Isolated refers to a nucleic acid sequence which has been: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis.
  • An isolated nucleic acid sequence is one which is readily manipulable by recombinant DNA techniques well known in the art.
  • PCR polymerase chain reaction
  • An isolated nucleic acid may be substantially purified, but need not be.
  • a nucleic acid sequence that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides.
  • Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.
  • Im ⁇ unogenetically Effective Amount is that amount of an antigen (e.g. a protein, variant or a fragment thereof) necessary to induce the production of antibodies which will bind to the epitopes of the antigen.
  • the actual quantity comprising an "immunogenetically effective amount” will vary depending upon factors such as the nature of the antigen, the organism to be immunized, and the mode of immunization. The determination of such a quantity is well within the ability of one ordinarily skilled in the art without undue experimentation.
  • Antigen and Antibody The term "antigen" as used in this invention is meant to denote a substance that can induce a detectable immune response to it when introduced to an animal . Such substances include proteins and fragments thereof.
  • epitope is meant to refer to that portion of an antigen which can be recognized and bound by an antibody.
  • An antigen may have one, or more than one epitope.
  • An "antigen” is capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • An "immunogen” is an antigen introduced into an animal specifically for the purpose of generating an immune response to the antigen.
  • An antibody is said to be “capable of selectively binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • the selective binding of an antigen and antibody is meant to indicate that the antigen will react, in a highly specific manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
  • antibody or “monoclonal antibody” (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab') 2 fragments) which are capable of binding an antigen.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody.
  • the present invention relates to the identification, isolation and cloning of two novel protein tyrosine phosphatases designated PTPLl and GLM-2. Specifically, the present invention discloses the isolation and cloning of cDNA and the amino acid sequences of PTPLl and GLM-2 from human glioblastoma and brain cell cDNA libraries. These phosphatases are, initially, discussed separately below. As they are related in function and utility as well as structurally with respect to their catalytic domains, they are subsequently discussed in the alternative.
  • PCR-based approach was used. PCR was performed using cDNA from the human glioma cell line U-343 MGa 31L as a template and degenerate primers that were based on conserved regions of PTPs. One primer was derived from the catalytic site (HCSAG) of the PTP domain and two primers were derived from conserved regions in the N-terminal part of the domain. Several PCR-products were obtained, including some corresponding to the cytoplasmic PTPs PTPH1 (Yang, Q. , and Tonks, N.K. (1991) Proc. Natl. Acad. Sci. (USA) 88:5949-5953), PTPase MEG (Gu, M. , et al)
  • PCR-products is almost identical to a PCR-product derived from a human leukemic cell line (Honda, H., et al. , (1993) Leukemia 7:742-746) and was chosen for further characterization and was used to screen an oligo-(dT)-primed U-343 MGa 31L cDNA library which resulted in the isolation of the clone X6.15.
  • a transcript of 9.5 kb could be seen. Therefore AG1518 cDNA libraries were constructed and screened with ⁇ 6.15 in order to obtain a full-length clone.
  • the 77 bp 5' untranslated region is GC-rich and contains an inframe stop codon at positions 45-47.
  • a 3' untranslated region of 565 bp begins after a TGA stop codon at positions 7476-7478, and does not contain a poly-A tail.
  • PTPLl In the deduced amino acid sequence of PTPLl no transmembrane domain or signal sequence for secretion are found, indicating that PTPLl is a cytoplasmic PTP.
  • the sequence of the first 470 amino acid residues shows no homology to known proteins.
  • the region 470-505 contains a leucine zipper motif, with a methionine in the position where the fourth leucine usually is found (LX g L -LX g g L) ; similar replacements of leucine residues with methionine residues are also found in the leucine zippers of the transcription factors CYS-3 (Fu, Y.-H., et al. , (1989) Mol. Cell. Biol.
  • cytoplasmic PTPs PTPH1 and PTPase MEG
  • the band 4.1-like domain of PTPLl is 20% to 24% similar to most known proteins of this superfamily, including ezrin (Gould, K.L., et al. , (1989) EMBO J. 8:4133-4142), moesin (Lankes, W.T., and Furthmayr, H. (1991) Proc. Natl. Acad. Sci. (USA) 88:8297-8301), radixin (Funayama, N. , et al. , (1991) J. Cell Biol.
  • GLGF-repeats Between amino acid residues 1080 and 1940 there are five 80 amino acid repeats denoted GLGF-repeats. This repeat was first found in PSD-95 (Cho, K.-O., et al. , (1992) Neuron 9:929-942), also called SAP (Kistner, U. , et al. , (1993) J. Biol. Chem. 268:4580-4583), a protein in post-synaptic densities, i.e. structures of the submembranous cytoskeleton in synaptic junctions. Rat PSD-95 is homologous to the discs-large tumor suppressor gene in Dro ⁇ ophila (Woods, D.F., and Bryant, P.J.
  • dlg-A which encodes a protein located in septate junctions. These two proteins each contain three GLGF-repeats, one SH-3 domain and a guanylate kinase domain. Through computer searches in protein data bases complemented by manual searches, 19 GLGF-repeats in 9 different proteins, all of them enzymes, were, found (see Figure 2 and Figure 3).
  • dlg-A and PSD-95 there are two other members of the guanylate kinase family, a 220-kDa protein (Itoh, M. , et al. , (1993) J. Cell Biol.
  • GLGF-repeat is also found in nitric oxide synthase from rat brain (Bredt, D.S., et al . , (1991) Nature 351:714-718), and a glioma cell line, U-118MG, expresses an altered rosl transcript (Sharma, S., et al ⁇ , (1989) Oncoqene Res. 5:91-100), containing a GLGF-repeat probably as a result of a gene fusion.
  • the PTP domain of PTPLl is localized in the C-terminus (amino acid residues 2195-2449). It contains most of the conserved motifs of PTP domains and shows about 30% similarity to known PTPs.
  • Use of a 9.5 kb probe including SEQ ID NO.:l for Northern blot analysis for tissue-specific expression showed high expression of PTPLl in human kidney, placenta, ovaries, and testes; medium expression in human lung, pancreas, prostrate and brain; low expression in human heart, skeletal muscle, spleen, liver, small intestine and colon; and virtually no detectable expression in human leukocytes.
  • PTPLl was found to be expressed in adult rats but not in rat embryos. This latter finding suggests that PTPLl may have a role, like many PTPs, in the signal transduction process that leads to cellular growth or differentiation.
  • the rabbit antiserum ⁇ LlA (see Example 5), made against a synthetic peptide derived from amino acid residues
  • a component of about 250 kDa could also be detected in immunoprecipitations using AG1518 cells, PC-3 cells, CCL-64 cells, A549 cells and PAE cells. This component was not seen upon precipitation with the preimmune serum, or when precipitation was made with ⁇ LlA antiserum preblocked with peptide. The slight variations in sizes observed between the different cell lines could be due to species differences. A smaller component of 78 kDa was also specifically precipitated by the ⁇ LlA antiserum. The relationship between this molecule and PTPLl remains to be determined.
  • the present invention also provides an isolated nucleic acid sequence encoding a novel PTP designated GLM-2, variants and fragments thereof, and uses relating thereto.
  • GLM-2 novel PTP
  • variants and fragments thereof uses relating thereto.
  • One sequence encoding a GLM-2 PTP and surrounding nucleotides is disclosed as SEQ ID NO.:3.
  • This sequence includes the coding sequences for GLM-2 PTP as well as both 5' and 3' untranslated regions including regulatory sequences.
  • the full disclosed sequence, designated SEQ ID NO. :3 is 3090 bp in length.
  • the nucleic acid sequence of SEQ ID NO.:3 includes 1310 base pairs of 5' untranslated region and 673 bp of 3 ' untranslated region which do not appear to encode a sequence for a poly-A (polyadenylation) tail. Transcription of SEQ ID NO. :3 begins at approximately position 1146. A translation start codon (ATG) is present at positions 1311 to 1313 of SEQ ID NO.:3. The nucleotides surrounding the start codon (AGCATGG) show substantial similarity to the Kozak consensus sequence (RCCATGG) (Kozak, M. (1987) Nucl. Acids Res. 15:8125-8148).
  • a translation stop codon (TGA) is present at positions 2418 to 2420 of SEQ ID NO.:3.
  • the open reading frame of 1107 bp encodes a protein of 369 amino acid residues with a predicted molecular mass of 41 kD.
  • the deduced amino acid sequence of this protein is disclosed as SEQ ID NO.:4.
  • sequence disclosed in SEQ ID NO. :3 encodes a single domain PTP similar to the rat PTP STEP (53% identity; Lombroso, et al. , 1991) and the human PTP LC-PTP (51% identity; Adachi, M. , et al. , (1992) FEBS Letters 314:335-339). None of the sequenced regions encodes a polypeptide sequence with any substantial similarity to known signal or transmembrane domains. Further indicating that GLM-2 is a cytoplasmic PTP.
  • an isolated DNA, cDNA or RNA sequence encoding a PTPLl or GLM-2 PTP, or a variant or fragment thereof is provided.
  • the procedures described above, which were employed to isolate the first PTPLl and GLM-2 sequences no longer need be employed. Rather, using the sequences disclosed herein, a genomic DNA or cDNA library may be readily screened to isolate a clone containing at least a fragment of a PTPLl or GLM-2 sequence and, if desired, a full sequence. Alternatively, one may synthesize PTPLl and GLM-2 encoding nucleic acids using the sequences disclosed herein.
  • the present invention further provides vectors containing nucleic acid sequences encoding PTPLl and GLM-2.
  • vectors include, but are not limited to, plasmids, phage, plasmids and cosmid vectors.
  • plasmids include, but are not limited to, plasmids, phage, plasmids and cosmid vectors.
  • one of ordinary skill in the art can readily place the nucleic acid sequences of the present invention into any of a great number of known suitable vectors using routine procedures.
  • the source nucleic acids for a DNA library may be genomic DNA or cDNA. Which of these is employed depends upon the nature of the sequences sought to be cloned and the intended use of those sequences.
  • Genomic DNA may be obtained by methods well known to those or ordinary skill in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et al. , eds. , Academic Press (1987)). Genomic DNA is preferred when it is desired to clone the entire gene including its endogenous regulatory sequences. Similarly, genomic DNA is used when it is only the regulatory sequences which are of interest.
  • Complementary or cDNA may be produced by reverse transcription methods which are well known to those of ordinary skill in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et al. , eds. , Academic Press (1987)).
  • the mRNA preparation for reverse transcription should be enriched in the mRNA of the desired sequence. This may be accomplished by selecting cells in which the mRNA is produced at high levels or by inducing high levels of production. Alternatively, in vitro techniques may be used such as sucrose gradient centrifugation to isolate mRNA transcripts of a particular size.
  • cDNA is preferred when the regulatory sequences of a gene are not needed or when the genome is very large in comparison with the expressed transcripts. In particular, cDNA is preferred when a eukaryotic gene containing introns is to be expressed in a prokaryotic host.
  • DNA or cDNA preparations are randomly sheared or enzymatically cleaved by restriction endonucleases to create fragments appropriate in size for the chosen library vector.
  • the DNA or cDNA fragments may be inserted into the vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation. Typically, this is accomplished by restriction enzyme digestion to provide appropriate termini, the filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligase ⁇ . Techniques for such manipulations are well known in the art and may be found, for example, in Sambrook, et al .
  • the library will consist of a great many clones, each containing a fragment of the total DNA or cDNA.
  • a great variety of cloning vectors, restriction endonucleases and ligases are commercially available and their use in creating DNA libraries is well known to those of ordinary skill in the art. See, for example, Sambrook, et al ⁇ , Molecular Cloning, A Laboratory Manual, 2d ed. , Cold Spring Harbor Laboratory Pres ⁇ , Plainview, NY (1989).
  • DNA or cDNA libraries containing sequences coding for PTPLl or GLM-2 nucleic acid sequences may be screened and a sequence coding for either PTPLl or GLM-2 identified by any means which specifically selects for that sequence.
  • Such means include (a) hybridization with an appropriate nucleic acid probe(s) containing a unique or ⁇ ub ⁇ tantially characteristic fragment of the desired DNA or cDNA (b) hybridization-selected tran ⁇ lational analysis in which native mRNA which hybridizes to the clone in question is tran ⁇ lated in vitro and the translation products are further characterized (c) if the cloned genetic sequences are themselves capable of expressing mRNA, immunoprecipitation of a translated PTPLl or GLM-2 recombinant product produced by the host containing the clone, or preferarably (d) by u ⁇ ing a unique or ⁇ ubstantially characteristic fragment of the desired sequence as a PCR primer to amplify those
  • the probe or primer is a ⁇ ubstantially characteristic fragment of one of the disclosed ⁇ equence ⁇ . More preferably, the probe i ⁇ a unique fragment of one of the di ⁇ clo ⁇ ed sequences. In choosing a fragment, unique and ⁇ ubstantially characteristic fragment ⁇ can be identified by comparing the sequence of a proposed probe to the known sequences found in ⁇ equence databa ⁇ es. Alternatively, the entire PTPLl or GLM-2 sequence may be used as a probe. In a preferred embodiment, the probe is a 32P random-labeled unique fragment of the PTPLl or GLM-2 nucleic acid sequences disclosed herein. In a most preferred embodiment, the probe serves a ⁇ a PCR primer containing a unique or ⁇ ubstantially characteristic fragment of the PTPLl or GLM-2 sequences disclosed herein.
  • the library to be screened may be DNA or cDNA.
  • a U-343 MGa 31L human gliobla ⁇ toma Ni ⁇ ter, M. , et al. , (1988) Cancer Re ⁇ . 48:3910-3918
  • AG1518 human fibrobla ⁇ t Human Genetic Mutant Cell Repo ⁇ itory, In ⁇ titute for Medical Re ⁇ earch, Camden, NJ
  • cDNA libraries from many tis ⁇ ue ⁇ may be employedN n another preferred embodiment, a ⁇ gtlO human brain cDNA library (Clontech, Calif.) i ⁇ ⁇ creened with a probe to a unique or ⁇ ubstantially characteristic fragment of the GLM-2 sequence. Because expression of GLM-2 appears to be high in brain ti ⁇ ues but low or absent in other tis ⁇ ue ⁇ te ⁇ ted, a brain cDNA library i ⁇ recommended for the cloning of GLM-2.
  • the ⁇ elected fragment ⁇ may be cloned into any of a great number of vectors known to those of ordinary ⁇ kill in the art.
  • the cloning vector is a plasmid ⁇ uch a ⁇ pUCl ⁇ or Blue ⁇ cript (Stratagene) .
  • the cloned ⁇ equence ⁇ ⁇ hould be examined to determine whether or not they contain the entire PTPLl or GLM-2 ⁇ equence ⁇ or de ⁇ ired portion ⁇ thereof.
  • a ⁇ erie ⁇ of overlapping clone ⁇ of partial ⁇ equence ⁇ may be ⁇ elected and combined to produce a complete sequence by methods well known in the art.
  • a library is prepared using an expre ⁇ ion vector.
  • the library i ⁇ then ⁇ creened for clone ⁇ which expre ⁇ the PTPLl or GLM-2 protein, for example, by ⁇ creening the library with antibodie ⁇ to the protein or with labeled probes for the desired RNA ⁇ equences or by as ⁇ aying for PTPLl or GLM-2 PTP activity on a pho ⁇ phorylated ⁇ ub ⁇ trate such as para-nitrylphenyl phosphate.
  • the above discussed methods are, therefore, capable of identifying cloned genetic sequences which are capable of expressing PTPLl or GLM-2 PTPs, or variants or fragments thereof.
  • RNA ⁇ criptional and translational signal ⁇ recognizable by an appropriate ho ⁇ t are nece ⁇ ary.
  • the cloned PTPLl or GLM-2 encoding sequences obtained through the methods described above, and preferably in a double- ⁇ tranded form, may be operably joined to regulatory ⁇ equence ⁇ in an expre ⁇ ion vector, and introduced into a ho ⁇ t cell, either prokaryote or eukaryote, to produce recombinant PTPLl or GLM-2 PTP, a variant or fragment thereof, PTPLl or GLM-2 anti-sense RNA, or a variant or fragment thereof.
  • the host may be eukaryotic or prokaryotic.
  • the host i ⁇ preferably eukaryotic.
  • the eukaryotic ho ⁇ t cell ⁇ are COS cells derived from monkey kidney.
  • the host cells are human fibroblasts. Many other eukaryotic ho ⁇ t cells may be employed a ⁇ is well known in the art.
  • Xenopus oocytes comprise a cell system useful for the functional expre ⁇ ion of eukaryotic messenger RNA or DNA.
  • This sy ⁇ tem has, for example, been used to clone the ⁇ odium:gluco ⁇ e cotran ⁇ porter in rabbit ⁇ (Hediger, M.A. , et. al. , Proc. Natl. Acad. Sci. (USA) 84:2634-2637 (1987)).
  • a prokaryotic expre ⁇ ion ⁇ y ⁇ tem i ⁇ preferred.
  • the choice of an appropriate expre ⁇ ion ⁇ y ⁇ tem i ⁇ within the ability and discretion of one of ordinary skill in the art.
  • the expres ⁇ ion vectors will produce either PTPLl or GLM-2 PTP ⁇ , variant ⁇ or fragment ⁇ thereof, or will expre ⁇ PTPLl and GLM-2 anti-sense RNA, variants or fragment ⁇ thereof.
  • Such PTPLl and GLM-2 anti- ⁇ en ⁇ e RNA may be used to inhibit expres ⁇ ion of the PTPLl or GLM-2 PTP and/or the replication of tho ⁇ e sequences.
  • Expres ⁇ ion of a protein in different ho ⁇ t ⁇ may result in different post-translational modification ⁇ which may alter the propertie ⁇ of the protein. This is particularly true when eukaryotic genes are expressed in prokaryotic host ⁇ . In the pre ⁇ ent invention, however, thi ⁇ is of less concern as PTPLl and GLM-2 are cytoplasmic PTPs and are unlikely to be post-translationally glycosylated.
  • Transcriptional initiation regulatory sequences can be selected which allow for repre ⁇ ion or activation, ⁇ o that expre ⁇ ion of the operably joined ⁇ equence ⁇ can be modulated.
  • Such regulatory sequences include regulatory sequence ⁇ which are temperature- ⁇ en ⁇ itive ⁇ o that by varying the temperature, expre ⁇ ion can be repressed or initiated, or which are ⁇ ubject to chemical regulation by inhibitor ⁇ or inducers.
  • intere ⁇ t are con ⁇ tructs wherein both PTPLl or GLM-2 mRNA and PTPLl or GLM-2 anti-sense RNA are provided in a transcribable form but with different promoter ⁇ or other tran ⁇ criptional regulatory elements such that induction of PTPLl or GLM-2 mRNA expres ⁇ ion is accompanied by repres ⁇ ion of the expre ⁇ ion of the corre ⁇ ponding anti- ⁇ en ⁇ e RNA, or alternatively, repre ⁇ sion of PTPLl or GLM-2 mRNA expression is accompanied by induction of expres ⁇ ion of the corresponding anti-sen ⁇ e RNA. Tran ⁇ lational ⁇ equence ⁇ are not necessary when it is de ⁇ ired to expre ⁇ PTPLl and GLM-2 anti- ⁇ en ⁇ e RNA sequences.
  • a non-transcribed and/or non-translated sequence 5' or 3 ' to the ⁇ equence coding for PTPLl or GLM-2 PTP can be obtained by the above-described cloning methods using one of the probes disclo ⁇ ed herein to ⁇ elect a clone from a genomic DNA library.
  • a 5' region may be u ⁇ ed for the endogenou ⁇ regulatory sequences of the PTPLl or GLM-2 PTP.
  • a 3'-non-transcribed region may be utilized for a transcriptional termination regulatory sequence or for a translational termination regulatory ⁇ equence. Where the native regulatory sequences do not function sati ⁇ factorily in the host cell, then exogenous sequence ⁇ functional in the ho ⁇ t cell may be utilized.
  • the vector ⁇ of the invention further comprise other operably joined regulatory elements ⁇ uch a ⁇ DNA element ⁇ which confer ti ⁇ ue or cell-type ⁇ pecific expression of an operably joined coding sequence.
  • Oligonucleotide probes derived from the nucleotide ⁇ equence of PTPLl or GLM-2 can be u ⁇ ed to identify genomic or cDNA library clone ⁇ po ⁇ essing a related nucleic acid ⁇ equence such as an allelic variant or homologous ⁇ equence.
  • a suitable oligonucleotide or set of oligonucleotides which i ⁇ capable of encoding a fragment of the PTPLl or GLM-2 coding ⁇ equence ⁇ , or a PTPLl or GLM-2 anti- ⁇ en ⁇ e complement of ⁇ uch an oligonucleotide or set of oligonucleotides, may be ⁇ ynthesized by means well known in the art (see, for example, Synthesis and Application of DNA and RNA, S.A. Narang, ed., 1987, Academic Press, San Diego, CA) and employed as a probe to identify and isolate a cloned PTPLl or GLM-2 ⁇ equence, variant or fragment thereof by technique ⁇ known in the art.
  • a unique or ⁇ ub ⁇ tantially characteri ⁇ tic fragment of a PTPLl or GLM-2 ⁇ equence disclosed herein is preferred.
  • Techniques of nucleic acid hybridization and clone identification are di ⁇ clo ⁇ ed by Sambrook, et al . , Molecular Cloning, A Laboratory Manual, 2d ed. , Cold Spring Harbor Laboratory Pre ⁇ , Plainview, NY (1989), and by Hame ⁇ , B.D., et al. , in Nucleic Acid Hybridization, A Practical Approach, IRL Pre ⁇ s, Washington, DC (1985).
  • the above-described probe ⁇ may be labeled with a detectable group.
  • a detectable group may be any material having a detectable phy ⁇ ical or chemical property.
  • Such material ⁇ have been we11-developed in the field of nucleic acid hybridization and in general mo ⁇ t any label useful in such methods can be applied to the present invention.
  • Particularly useful are radioactive labels. Any radioactive label may be employed which provide ⁇ for an adequate ⁇ ignal and ha ⁇ a ⁇ ufficient half-life.
  • the oligonucleotide may be radioactively labeled using kinase reaction ⁇ .
  • oligonucleotide ⁇ are al ⁇ o u ⁇ eful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group. See, for example, Leary, J.J., et al ⁇ , Proc. Natl. Acad. Sci. (USA) 80:4045 (1983); Renz, M. et al ⁇ , Nucl. Acids Res. 12:3435 (1984); and Renz, M. , EMBO J. 6:817 (1983).
  • a ⁇ probe ⁇ or a ⁇ primer ⁇ By using the sequences disclo ⁇ ed herein a ⁇ probe ⁇ or a ⁇ primer ⁇ , and technique ⁇ such as PCR cloning and colony/plaque hybridization, it is within the abilities of one ⁇ killed in the art to obtain human allelic variant ⁇ and sequences sub ⁇ tantially similar or homologous to PTPLl or GLM-2 nucleic acid ⁇ equence ⁇ from species including mouse, rat, rabbit and non-human primates. Thus, the present invention is further directed to mou ⁇ e, rat, rabbit and primate PTPLl and GLM-2.
  • the protein sequence ⁇ disclosed herein for PTPLl and GLM-2 may be used to generate sets of degenerate probes or PCR primers useful in isolating ⁇ imilar and potentially evolutionarily ⁇ imilar ⁇ equences encoding protein ⁇ related to the PTPLl or GLM-2 PTP ⁇ .
  • Such degenerate probe ⁇ may not be ⁇ ub ⁇ tantially ⁇ imilar to any fragments of the PTPLl or GLM-2 nucleic acid sequences but, as derived from the protein ⁇ equence ⁇ di ⁇ clo ⁇ ed herein, are intended to fall within the ⁇ pirit and ⁇ cope of the claim ⁇ .
  • the antibodies of the present invention are prepared by any of a variety of method ⁇ .
  • purified PTPLl or GLM-2 PTP, a variant or a fragment thereof i ⁇ admini ⁇ tered to an animal in order to induce the production of ⁇ era containing polyclonal antibodie ⁇ that are capable of binding the PTP, variant or fragment thereof.
  • Cell ⁇ expressing PTPLl or GLM-2 PTP, a variant or a fragment thereof, or, a mixture of such proteins, variants or fragments, can be administered to an animal in order to induce the production of sera containing polyclonal antibodies, some of which will be capable of binding the PTPLl or GLM-2 PTP.
  • ⁇ uch PTPLl or GLM-2 antibody may be purified from other polyclonal antibodie ⁇ by ⁇ tandard protein purification techniques and especially by affinity chromatography with purified PTPLl or GLM-2 protein or variants or fragments thereof.
  • a PTPLl or GLM-2 protein fragment may also be chemically synthesized and purified by HPLC to render it sub ⁇ tantially pure. Such a preparation i ⁇ then introduced into an animal in order to produce polyclonal anti ⁇ era of high ⁇ pecific activity.
  • the protein may be coupled to a carrier protein ⁇ uch as bovine serum albumin or keyhole limpet hemocyanin (KLH), and and u ⁇ ed to immunogenize a rabbit utilizing techniques well-known and commonly used in the art.
  • the PTPLl or GLM-2 protein can be admixed with an immunologically inert or active carrier. Carriers which promote or induce immune responses, such a ⁇ Freund' ⁇ complete adjuvant, can be utilized.
  • Monoclonal antibodie ⁇ can be prepared u ⁇ ing hybridoma technology (Kohler et al. , Nature 256:495 (1975); Kohler, et al. , Eur. J. Immuno1. 6:511 (1976); Kohler, et al . , Eur. J. Immunol. 6:292 (1976); Hammerling, et al . , in Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y. , pp. 563-681 (1981)).
  • such procedures involve immunizing an animal with PTPLl or GLM-2 PTP, or a variant or a fragment thereof.
  • the splenocytes of ⁇ uch animal ⁇ are extracted and fu ⁇ ed with a ⁇ uitable myeloma cell line. After fu ⁇ ion, the re ⁇ ulting hybridoma cell ⁇ are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wand ⁇ , J.R., et al. , Ga ⁇ tro-enterology 80:225-232 (1981), which reference i ⁇ herein incorporated by reference.
  • the hybridoma cells obtained through such a ⁇ election are then a ⁇ ayed to identify clone ⁇ which ⁇ ecrete antibodies capable of binding the PTP and/or the PTP antigen.
  • the proliferation of transfected cell lines is potentially more promising than clas ⁇ ical myeloma technology, using methods available in the art.
  • antibodies can be used clinically as markers (both quantitative and qualitative) of the PTPLl and GLM-2 PTPs in brain, blastoma or other ti ⁇ ue. Additionally, the antibodie ⁇ are u ⁇ eful in a method to a ⁇ sess PTP function in cancer or other patients.
  • the protein is purified from tis ⁇ ues or cells which naturally produce the protein.
  • an expres ⁇ ion vector may be introduced into cell ⁇ to cau ⁇ e production of the protein.
  • human fibrobla ⁇ t or monkey kidney COS cell ⁇ may be employed.
  • mRNA tran ⁇ cript ⁇ may be microinjected into cells, such as Xenopus oocytes or rabbit reticulocyte ⁇ .
  • mRNA is used with an in vitro tran ⁇ lation ⁇ y ⁇ tem.
  • bacterial cell ⁇ are used to make large quantities of the protein.
  • fusion protein ⁇ uch a ⁇ a bacterial GST fusion (Pharmacia) may be employed, the fusion product purified by affinity chromatography, and the PTPLl or GLM-2 protein may be relea ⁇ ed from the hybrid by cleaving the amino acid ⁇ equence joining them.
  • Determinations of purity may be performed by physical characterization ⁇ ( ⁇ uch a ⁇ molecular ma ⁇ in size fractionation) , immunological techniques or enzymatic as ⁇ ay ⁇ .
  • PTPLl or GLM-2 PTP, variants or fragments thereof, purified in the above manner, or in a manner wherein equivalents of the above sequence of steps are utilized, are useful in the preparation of polyclonal and monoclonal antibodies, for pharmaceutical preparation ⁇ to inhibit or enhance PTP activity and for in vitro depho ⁇ phorylation ⁇ .
  • Variants of PTPLl or GLM-2 having an altered nucleic acid sequence can be prepared by mutagene ⁇ i ⁇ of the DNA. Thi ⁇ can be accompli ⁇ hed using one of the mutagenesis procedure ⁇ known in the art.
  • Preparation of variant ⁇ of PTPLl or GLM-2 are preferably achieved by ⁇ ite-directed mutagene ⁇ i ⁇ .
  • Site-directed mutagenesis allows the production of variants of these PTPs through the use of a specific oligonucleotide which contain ⁇ the de ⁇ ired mutated DNA ⁇ equence.
  • Site-directed mutagenesis typically employs a phage vector that exists in both a single- ⁇ tranded and double-stranded form.
  • Typical vectors u ⁇ eful in ⁇ ite-directed mutagenesis include vectors ⁇ uch as the M13 phage, as disclosed by Messing, et al ⁇ , Third Cleveland Sympo ⁇ ium on Macromolecule ⁇ and Recombinant DNA, A. Walton, ed. , Elsevier, Amsterdam (1981), the disclo ⁇ ure of which is incorporated herein by reference.
  • These phage are commercially available and their use is generally well known to those skilled in the art.
  • plasmid vector ⁇ containing a ⁇ ingle- ⁇ tranded phage origin of replication may be employed to obtain single-stranded DNA.
  • site-directed mutagenesis in accordance herewith is performed by fir ⁇ t obtaining a ⁇ ingle-stranded vector that includes within it ⁇ sequence the DNA sequence which is to be altered.
  • An oligonucleotide primer bearing the desired mutated ⁇ equence i ⁇ prepared, generally synthetically, for example by the method of Crea, et al. , Proc. Natl. Acad. Sci. (USA) 75:5765 (1978).
  • the primer i ⁇ then annealed with the ⁇ ingle- ⁇ tranded vector containing the ⁇ equence which is to be altered, and the created vector is incubated with a DNA-polymerizing enzyme such as E. coli polymerase I Klenow fragment in an appropriate reaction buffer.
  • the polymerase will complete the ⁇ ynthe ⁇ i ⁇ of a mutation-bearing ⁇ trand. Thu ⁇ , the ⁇ econd ⁇ trand will contain the de ⁇ ired mutation. Thi ⁇ heteroduplex vector is then used to tran ⁇ form appropriate cell ⁇ and clone ⁇ are ⁇ elected that contain recombinant vector ⁇ bearing the mutated sequence.
  • the mutation per se need not be predetermined.
  • random mutagenesis may be conducted at a target region and the newly generated sequence ⁇ can be screened for the optimal combination of desired activity.
  • One skilled in the art can evaluate the functionality of the variant by routine ⁇ creening a ⁇ ays.
  • the present invention further comprises fusion product ⁇ of the PTPLl or GLM-2 PTPs.
  • translation of eukaryotic mRNA is initiated at the codon which encodes the fir ⁇ t methionine.
  • the pre ⁇ ence of ⁇ uch codon ⁇ between a eukaryotic promoter and a PTPLl or GLM-2 ⁇ equence re ⁇ ult ⁇ either in the formation of a fu ⁇ ion protein (if the ATG codon i ⁇ in the ⁇ ame reading frame a ⁇ the PTP encoding DNA ⁇ equence) or a frame- ⁇ hift mutation (if the ATG codon i ⁇ not in the ⁇ ame reading frame a ⁇ the PTP encoding ⁇ equence) .
  • Fu ⁇ ion protein ⁇ may be con ⁇ tructed with enhanced immuno ⁇ pecificity for the detection of the ⁇ e PTP ⁇ .
  • the ⁇ equence coding for the PTPLl or GLM-2 PTP may al ⁇ o be joined to a signal sequence which will allow secretion of the protein from, or the compartmentalization of the protein in, a particular ho ⁇ t.
  • signal sequences may be designed with or without specific protea ⁇ e ⁇ ite ⁇ ⁇ uch that the ⁇ ignal peptide ⁇ equence i ⁇ amenable to ⁇ ubsequent removal.
  • the invention further provides detectably labeled, immobilized and toxin conjugated forms of PTPLl and GLM-2 PTP ⁇ , and variant ⁇ or fragments thereof.
  • the production of such labeled, immobilized or toxin conjugated form ⁇ of a protein are well known to tho ⁇ e of ordinary ⁇ kill in the art.
  • radiolabeling represents one embodiment
  • the PTP ⁇ or variants or fragments thereof may also be labeled using fluorescent labels, enzyme labels, free radical labels, avidin-biotin labels, or bacteriophage labels, u ⁇ ing technique ⁇ known to the art (Chard, Laboratory Technique ⁇ in Biology, "An Introduction to Radioimmunoa ⁇ ay and Related Techniques," North Holland Publishing Company (1978)).
  • Typical fluorescent labels include fluore ⁇ cein i ⁇ othiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluore ⁇ camine.
  • Typical chemilumine ⁇ cent compounds include luminol, isoluminol, aromatic acridinium e ⁇ ters, imidazoles, and the oxalate ester ⁇ .
  • Typical biolumine ⁇ cent compounds include luciferin, and luciferase.
  • Typical enzymes include alkaline phosphatase, ⁇ -galactosida ⁇ e, glucose-6-phosphate dehydrogena ⁇ e, maleate dehydrogena ⁇ e, gluco ⁇ e oxidase, and peroxidase. Transformed cells, cell lines and hosts.
  • vector sy ⁇ tems are available depending upon whether it is de ⁇ ired to in ⁇ ert the recombinant DNA con ⁇ truct into the ho ⁇ t cell's chromosomal DNA, or to allow it to exist in an extrachromo ⁇ omal form.
  • a non-replicating DNA (or RNA) molecule may be a linear molecule or, more preferably, a closed covalent circular molecule which is incapable of autonomous replication.
  • genetically ⁇ table tran ⁇ formant ⁇ may be con ⁇ tructed with vector system ⁇ , or tran ⁇ formation ⁇ y ⁇ tem ⁇ , whereby recombinant PTPLl or GLM-2 PTP DNA i ⁇ integrated into the ho ⁇ t chromo ⁇ ome.
  • Such integration may occur de novo within the cell or, in a mo ⁇ t preferred embodiment, be a ⁇ i ⁇ ted by tran ⁇ formation with a vector which functionally in ⁇ ert ⁇ it ⁇ elf into the host chromosome with, for example, retro vectors, transpo ⁇ ons or other DNA elements which promote integration of DNA sequence ⁇ in chromosomes.
  • a vector is employed which is capable of integrating the desired ⁇ equence ⁇ into a mammalian ho ⁇ t cell chromo ⁇ ome.
  • the tran ⁇ formed cell ⁇ are human fibrobla ⁇ t ⁇ .
  • the tran ⁇ formed cell ⁇ are monkey kidney COS cell ⁇ .
  • Cells which have stably integrated the introduced DNA into their chromo ⁇ ome ⁇ may be ⁇ elected by al ⁇ o introducing one or more markers which allow for selection of ho ⁇ t cells which contain the expres ⁇ ion vector in the chromo ⁇ ome, for example the marker may provide biocide re ⁇ i ⁇ tance, e.g., re ⁇ i ⁇ tance to antibiotics, or heavy metals, such a ⁇ copper, or the like.
  • the selectable marker can either be directly 1inked to the DNA sequences to be expressed, or introduced into the same cell by co-transfection.
  • the introduced sequence is incorporated into a vector capable of autonomous replication in the recipient ho ⁇ t.
  • a vector capable of autonomous replication in the recipient ho ⁇ t Any of a wide variety of vector ⁇ may be employed for this purpose, as outlined below.
  • Factors of importance in selecting a particular plasmid or vector include: the ea ⁇ e with which recipient cell ⁇ that contain the vector may be recognized and ⁇ elected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is de ⁇ irable to be able to " ⁇ huttle" the vector between ho ⁇ t cell ⁇ of different species.
  • Preferred eukaryotic plasmid ⁇ include tho ⁇ e derived from the bovine papilloma viru ⁇ , SV40, and, in yea ⁇ t, pla ⁇ mid ⁇ containing the 2-micron circle, etc., or their derivative ⁇ .
  • pla ⁇ mid ⁇ are well known in the art (Bot ⁇ tein, D., et al . , Miami Wntr. Symp. 19:265-274 (1982); Broach, J.R., in The Molecular Biology of the Yea ⁇ t Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p.
  • mammalian expre ⁇ ion vector ⁇ ystems which utilize the MSV-LTR promoter to drive expres ⁇ ion of the cloned gene and with which it is possible to co-transfect with a helper viru ⁇ to amplify pla ⁇ mid copy number and to integrate the plasmid into the chromosomes of host cells have been de ⁇ cribed (Perkin ⁇ , A.S., et al. , Mol. Cell Biol. 3:1123 (1983); Clontech, Palo Alto, California).
  • the vector or DNA sequence is prepared for expres ⁇ ion, it is introduced into an appropriate host cell by any of a variety of suitable mean ⁇ , including tran ⁇ fection.
  • recipient cells may be grown in a selective medium, which select ⁇ for the growth of vector-containing cell ⁇ .
  • Expre ⁇ ion of the cloned nucleic acid sequence(s) re ⁇ ults in the production of PTPLl or GLM-2 PTP, or the production of a variant or fragment of the PTP, or the expression of a PTPLl or GLM-2 anti-sense RNA, or a variant or fragment thereof.
  • This expre ⁇ ion can take place in a transient manner, in a continuous manner, or in a controlled manner a ⁇ , for example, expression which follows induction of differentiation of the tran ⁇ formed cell ⁇ (for example, by admini ⁇ tration of bromodeoxyuracil to neurobla ⁇ toma cell ⁇ or the like) .
  • the ho ⁇ t i ⁇ a human ho ⁇ t. Thu ⁇ a vector may be employed which will introduce into a human with deficient PTPLl or GLM-2 PTP activity, operable PTPLl or GLM-2 ⁇ eguence ⁇ which can ⁇ upplement the patient' ⁇ endogenous production.
  • the patient suffers from a cancer caused by an oncogene which is a protein tyro ⁇ ine kina ⁇ e (PTK) .
  • PTK protein tyro ⁇ ine kina ⁇ e
  • a vector capable of expre ⁇ sing the PTPLl or GLM-2 protein is introduced within the patient to counteract the PTK activity.
  • the recombinant PTPLl or GLM-2 PTP cDNA coding sequences may be used to obtain PTPLl or GLM-2 anti- ⁇ en ⁇ e RNA ⁇ equence ⁇ .
  • An expre ⁇ sion vector may be constructed which contain ⁇ a DNA ⁇ equence operably joined to regulatory ⁇ equence ⁇ ⁇ uch that the DNA ⁇ equence expre ⁇ e ⁇ the PTPLl or GLM-2 anti- ⁇ ense RNA sequence. Tran ⁇ formation with thi ⁇ vector results in a host capable of expre ⁇ sion of a PTPLl or GLM-2 anti-sense RNA in the transformed cell.
  • expre ⁇ ion occur ⁇ in a regulated manner wherein it may be induced and/or repre ⁇ ed a ⁇ desired.
  • anti- ⁇ ense PTPLl or GLM-2 RNA interacts with an endogenous PTPLl or GLM-2 DNA or RNA in a manner which inhibit ⁇ or repre ⁇ e ⁇ tran ⁇ cription and/or tran ⁇ lation of the PTPLl or GLM-2 PTP DNA ⁇ equence ⁇ and/or mRNA tran ⁇ cripts in a highly specific manner.
  • the cloning of PTPLl and GLM-2 now makes pos ⁇ ible the production and u ⁇ e of high through-put a ⁇ ay ⁇ for the identification and evaluation of new agoni ⁇ t ⁇ (inducer ⁇ /enhancers) and antagonist ⁇ (repre ⁇ or ⁇ /inhibitor ⁇ ) of PTPLl or GLM-2 PTP ⁇ for therapeutic strategies using ⁇ ingle or combination ⁇ of drug ⁇ .
  • the a ⁇ ay may, for example, test for PTPLl or GLM-2 PTP activity in transfected cells (e.g. fibroblasts) to identify drugs that interfere with, enhance, or otherwise alter the expression or regulation of these PTPs.
  • probes developed for the disclo ⁇ ed PTPLl and GLM-2 nucleic acid sequences or proteins may be used as qualitative and/or quantitative indicators for the PTP ⁇ in cell ly ⁇ ates, whole cells or whole tis ⁇ ue.
  • human fibroblast cells are transformed with the PTPLl or GLM-2 PTP ⁇ equence ⁇ and vector ⁇ di ⁇ clo ⁇ ed herein.
  • the cell ⁇ may then be treated with a variety of compound ⁇ to identify tho ⁇ e which enhance or inhibit PTPLl or GLM-2 tran ⁇ cription, tran ⁇ lation, or PTP activity.
  • assay ⁇ for PDGF (platelet derived growth factor) signalling, cell growth, chemotaxis, and actin reorganization are preferred to as ⁇ e ⁇ a compound' ⁇ affect on PTPLl or GLM-2 PTP tran ⁇ cription, tran ⁇ lation or activity.
  • the ability of a compound to enhance or inhibit PTPLl or GLM-2 PTP activity is assayed in vitro.
  • the ability of various compounds to enhance or inhibit the phosphata ⁇ e activity of PTPLl or GLM-2 may be assayed.
  • the phosphorylated ⁇ ubstrate is para-nitrylphenyl phosphate (which turn ⁇ yellow upon dephosphorylation) .
  • the ability of a compound to enhance or inhibit PTPLl or GLM-2 transcription i ⁇ a ⁇ ayed.
  • U ⁇ ing the PTPLl or GLM-2 cDNA ⁇ equence ⁇ di ⁇ clo ⁇ ed herein, one of ordinary ⁇ kill in the art can clone the 5' regulatory ⁇ equence ⁇ of the PTPLl or GLM-2 gene ⁇ .
  • the ⁇ e regulatory ⁇ equences may then be operably joined to a sequence encoding a marker.
  • the marker may be an enzyme with an easily as ⁇ ayable activity or may cau ⁇ e the ho ⁇ t cell ⁇ to change phenotypically or in their ⁇ en ⁇ itivity or re ⁇ i ⁇ tance to certain molecules.
  • markers are known to those of ordinary skill in the art and appropriate markers may be cho ⁇ en depending upon the ho ⁇ t u ⁇ ed.
  • Compound ⁇ which may alter the tran ⁇ cription of PTPLl or GLM-2 PTP may be tested by exposing cells transformed with the PTPLl or GLM-2 regulatory sequences operably joined to the marker and as ⁇ aying for increa ⁇ ed or decrea ⁇ ed expression of the marker.
  • DMEM Gibco Dulbeco Modified Eagles Medium
  • FCS Fetal Calf Serum
  • the human glioma cell line u ⁇ ed was U-343 MGa 31L (Nister, M., et al. , (1988) Cancer Re ⁇ . 48:3910-3918).
  • the AG1518 human foreskin fibrobla ⁇ t ⁇ were from the Human Genetic Mutant Cell Repo ⁇ itory, In ⁇ titute for Medical Research, Camden, NJ.
  • RNA was prepared from U-343 MGa 31L cell ⁇ or AG1518 human fibrobla ⁇ t ⁇ by guanidine thiocyanate (Merck, Darm ⁇ tadt) extraction (Chirgwin et al. , 1979). Briefly, cell ⁇ were harvested, washed in pho ⁇ phate buffered ⁇ aline (PBS), and ly ⁇ ed in 4 M guanidine thiocyanate containing 25 mM sodium citrate (pH 7.0) and 0.1 M 2-mercaptoethanol.
  • PBS pho ⁇ phate buffered ⁇ aline
  • RNA was sedimented through 5.7 M cesium chloride, the RNA pellet wa ⁇ then dissolved in 10 mM Tris hydrochloride (pH 7.5), 5 mM EDTA (TE buffer), extracted with phenol and chloroform, precipitated with ethanol, and the final pellet stored at -70°C or resu ⁇ pended in TE buffer for subsequent manipulations.
  • Polyadenylated [poly(A)+] RNA was prepared by chromatography on oligo (dT)-cellulose a ⁇ de ⁇ cribed in Maniatis et al ⁇ , 1982.
  • Poly(A)+ RNA (5 ⁇ g) from U-343 MGa 31 L cells wa ⁇ u ⁇ ed to make a cDNA library by oligo (dT)-primed cDNA ⁇ ynthe ⁇ i ⁇ u ⁇ ing an Amer ⁇ ham ⁇ gtlO cDNA cloning ⁇ y ⁇ tem.
  • a random and oligo (dT) primed cDNA library wa ⁇ prepared from AG1518 fibrobla ⁇ t ⁇ u ⁇ ing 5 ⁇ g of poly(A)+ RNA, a RiboClone cDNA ⁇ ynthesis ⁇ y ⁇ tem (Promega Corporation, Madison, Wl .
  • Sense oligonucleotides corresponded to the ⁇ equence ⁇ FWRM I/V WEQ (5'- TTCTGG A/C GNATGATNTGGGAACA-3 ' , 23mer with 32-fold degeneracy) and KC A/D Q/E YWP (5'-AA A/G TG C/T GANCAGTA C/T TGGCC-3 ' , 20mer with 32-fold degeneracy), and the anti- ⁇ ense oligonucleotide was based on the ⁇ equence HCSAG V/I G (5'-CCNACNCC A/C GC A/G CTGCAGTG-3 ' , 20mer with 64-fold degeneracy).
  • Unpackaged template cDNA from the U-343 MGa 31L library (100 ng) wa ⁇ amplified u ⁇ ing Tag polymera ⁇ e (Perkin Elmer-Cetu ⁇ ) and 100 ng of either sense primer in combination with 100 ng of the anti-sense primer as described (Saiki et al. , 1985) .
  • PCR was carried out for 25 cycles each con ⁇ isting of denaturation at 94°C for 30 sec, annealing at 40°C for 2 min followed by 55°C for 1 min, and extension at 72°C for 2 min.
  • PCR products were ⁇ eparated on a 2.0% low gelling temperature agaro ⁇ e gel (FMC BioProduct ⁇ , Rockland, USA) and DNA fragments of approximately 368 base pair ⁇ (with FWRM sense primer) and approximately 300 bp (with KC A/D Q sense primer) were excised, eluted from the gel, subcloned into a T-tailed vector (TA Cloning Kit, Invitrogen Corporation, San Diego, CA, USA), and ⁇ equenced.
  • FMC BioProduct ⁇ 2.0% low gelling temperature agaro ⁇ e gel
  • DNA fragments of approximately 368 base pair ⁇ with FWRM sense primer
  • approximately 300 bp with KC A/D Q sense primer
  • Nucleotide sequences from several of the PCR cDNA clone ⁇ analy ⁇ ed were representative of both cytoplasmic and receptor types of PTPs. Thirteen clone ⁇ encoded cytopla ⁇ mic enzy e ⁇ including MEG (Gu et al. , 1991; 8 clone ⁇ ), PTPHl (Yang and Tonk ⁇ , 1991; 2 clone ⁇ ), P19PTP (den Hertog et al ⁇ , 1992), and TC-PTP (Cool et al.
  • Double-stranded pla ⁇ mid DNA wa ⁇ prepared by a ⁇ ingle-tube mini preparation method (Del Sal et al. , 1988) or u ⁇ ing Magic mini or maxiprep kit ⁇ (Promega) according to the manufacturer' ⁇ specifications. Double-stranded DNA was denatured and used a ⁇ template for ⁇ equencing by the dideoxynucleotide chain-termination procedure with T7 DNA polymera ⁇ e (Pharmacia-LKB) , and M13-univer ⁇ al and rever ⁇ e primer ⁇ or ⁇ ynthetic oligonucleotide ⁇ derived from the cDNA ⁇ equence ⁇ being determined.
  • the 8040 bp ⁇ equence is disclosed as SEQ ID NO.: 1.
  • the human glioma cell line U-343 MGa 31L (Nister, M. , et al. , (1988) Cancer Re ⁇ . 48:3910-3918) wa ⁇ cultured in Dulbecco' Modified Eagles Medium (DMEM Gibco) supplemented with 10% Fetal Calf Serum (FCS, Flow Laboratories), 100 unit ⁇ of penicillin, 50 ⁇ g/ml ⁇ treptomycin and 2mM glutamine.
  • DMEM Gibco Dulbecco' Modified Eagles Medium
  • FCS Fetal Calf Serum
  • RNA wa ⁇ prepared from U-343 MGa 31L cell ⁇ by guanidine thiocyanate (Merck, Darm ⁇ tadt) extraction (Chirgwin, et al ⁇ , 1979). Briefly, cell ⁇ were harve ⁇ ted, wa ⁇ hed in pho ⁇ phate buffered ⁇ aline (PBS), and ly ⁇ ed in 4 M guanidine thiocyanate containing 25mM ⁇ odium citrate (pH 7.0) and 0.1 M 2-mercaptoethanol .
  • PBS pho ⁇ phate buffered ⁇ aline
  • RNA wa ⁇ sedimented through 5.7 M cesium chloride the RNA pellet was then di ⁇ olved in 10 mM Tri ⁇ hydrochloride (pH 7.5), 5 mM EDTA (TE buffer), extracted with phenol and chloroform, precipitated with ethanol, and the final pellet ⁇ tored at -70°C or re ⁇ u ⁇ pended in TE buffer for ⁇ ub ⁇ equent manipulations.
  • Polyadenylated [poly(A)+] RNA was prepared by chromatography on oligo (dT)-cellulose a ⁇ de ⁇ cribed in Maniati ⁇ et al . (1982).
  • RNA Poly(A)+ RNA (5 ⁇ g) i ⁇ olated from U-343 MGa 31L cell ⁇ wa ⁇ u ⁇ ed to make a cDNA library by oligo (dT)-primed cDNA ⁇ ynthe ⁇ i ⁇ u ⁇ ing an Amer ⁇ ham ⁇ gtlO cDNA cloning ⁇ y ⁇ tem.
  • Degenerate primer ⁇ were designed based on conserved amino acid regions of known PTP sequence ⁇ , and ⁇ ynthesized u ⁇ ing a Gene A ⁇ embler Plu ⁇ (Pharmacia-LKB) .
  • Unpackaged template cDNA from the U-343 MGa 31L library (100 ng) was amplified using Tag polymera ⁇ e (Perkin Elmer-Cetu ⁇ ) and 100 ng of either ⁇ en ⁇ e primer in combination with 100 ng of the anti- ⁇ en ⁇ e primer a ⁇ de ⁇ cribed (Saiki, et al. , 1985).
  • PCR wa ⁇ carried out for 25 cycle ⁇ each con ⁇ isting of denaturation at 94°C for 30 sec, annealing at 40°C for 2 min followed by 55°C for 1 min, and extension at 72°C for 2 min.
  • PCR products were separated on a 2.0% low gelling temperature agarose gel (FMC BioProduct ⁇ , Rockland, USA) and DNA fragment ⁇ of approximately 368 ba ⁇ e pair ⁇ (with FWRM sense primer) and approximately 300 bp (with KC A/D Q sen ⁇ e primer) were exci ⁇ ed, eluted from the gel, ⁇ ubcloned into a T-tailed vector (TA Cloning Kit, Invitrogen Corporation, San Diego, CA, USA), and ⁇ equenced.
  • FMC BioProduct ⁇ , Rockland, USA 2.0% low gelling temperature agarose gel
  • DNA fragment ⁇ of approximately 368 ba ⁇ e pair ⁇ with FWRM sense primer
  • approximately 300 bp with KC A/D Q sen ⁇ e primer
  • Double-stranded plasmid DNA was prepared by a ⁇ ingle-tube mini preparation method (Del Sal, et al ⁇ , 1988) or by u ⁇ ing Magic mini or maxiprep kit ⁇ (Pro ega) according to the manufacturer ' ⁇ ⁇ pecification ⁇ .
  • RNA (20 ⁇ g) or poly(A)+ RNA (2 ⁇ g) denatured in formaldehyde and formamide was separated by electrophoresis on a formaldehyde/1% agarose gel and transferred to nitrocellulose.
  • the filters were hybridized for 16 hr ⁇ at 42°C with 32P-labeled probe ⁇ in a ⁇ olution containing 5x ⁇ tandard ⁇ aline citrate (SSC; lx SSC i ⁇ 50 M ⁇ odium citrate, pH 7.0, 150 mM ⁇ odium chloride), 50% formamide, 0.1% sodium dodecyl sulfate (SDS), 50 mM sodium phosphate and 0.1 mg/ml salmon sperm DNA. All probe ⁇ were labeled by random priming (Feinberg and Vogel ⁇ tein, 1983) and unincorporated 32P wa ⁇ removed by Sephadex G-25
  • RNAs from various human tissues showed that the 9.5 kb PTPLl transcript i ⁇ expre ⁇ ed at different level ⁇ with kidney, placenta, ovaries and testes showing high expres ⁇ ion, compared to medium expre ⁇ ion in lung, pancrea ⁇ , pro ⁇ tate and brain ti ⁇ ues, low in heart, skeletal mu ⁇ cle, spleen, liver, small intestine and colon and virtually no detectable expression in leukocytes.
  • EXAMPLE 4 Tis ⁇ ue-Specific Expre ⁇ ion of GLM-2
  • the filter was hybridized according to manufacturer's ⁇ pecification ⁇ with 32P-labeled GLM-2 PCR product a ⁇ probe, wa ⁇ hed twice for 30 min at 60°C in 2x ⁇ tandard ⁇ aline citrate (SSC; lx SSC i ⁇ 50 mM ⁇ odium citrate, pH 7.0, 150 mM ⁇ odium chloride), containing 0.1% ⁇ odium dodecyl ⁇ ulfate (SDS), once for 30 min at 60°C in 0.5x SSC/0.1% SDS, and expo ⁇ ed to X-ray film (Fuji, RX) with inten ⁇ ifying ⁇ creen (Cronex Lighting Plu ⁇ , Dupont) at -70°C.
  • SSC 2x ⁇ tandard ⁇ aline citrate
  • SDS ⁇ odium dodecyl ⁇ ulfate
  • Rabbit antisera denoted ⁇ LlA and ⁇ LIB were prepared against peptides corresponding to amino acid residues 1802 to 1823 (PAKSDGRLKPGDRLIKVNDTDV) and 450 to 470 (DETLSQGQSQRPSRQYETPFE) , respectively, of PTPLl.
  • the peptides were synthe ⁇ ized in an Applied Biosy ⁇ tems 430A Peptide Synthe ⁇ izer u ⁇ ing t-butoxycarbonyl chemi ⁇ try and purified by rever ⁇ e pha ⁇ e high performance liquid chromatography.
  • the peptide ⁇ were coupled to keyhole limpet hemocyanin (Calbiochem-Behring) u ⁇ ing glutaraldehyde, a ⁇ de ⁇ cribed (Gullick, W.J., et al. , (1985) EMBO J. 4:2869-2877), and then mixed with Freund's adjuvant and used to Immunize a rabbit.
  • the ⁇ LlA anti ⁇ erum wa ⁇ purified by affinity chomatography on protein A-Sepharo ⁇ e CL4B (Pharmacia-LKB) a ⁇ de ⁇ cribed by the manufacturer.
  • EXAMPLE 6 Tran ⁇ fection of the PTPLl cDNA Into COS-1 Cell ⁇ .
  • the full length PTPLl cDNA was con ⁇ tructed using overlapping clones and cloned into the SV40-based expression vector pSV7d (Truett, M.A. , et al. , (1985) DNA 4:333-349), and transfected into COS-1 cells by the calcium phosphate precipitation method (Wigler, M. , et al ⁇ , (1979) Cell
  • KC1 0.7 mM CaCl 2 , 0.5 mM MgCl 2 and 0.6 mM Na 2 HP0 4 , and then incubated with Dulbecco's modified Eagle' ⁇ medium containing 10% fetal calf ⁇ erum and antibiotic ⁇ . Two day ⁇ after tran ⁇ fection, the cell ⁇ were u ⁇ ed for metabolic labeling followed by immunoprecipitation and SDS-gel electrophore ⁇ i ⁇ , or immunoprecipitation followed by depho ⁇ phorylation experiment ⁇ .
  • Immune complexe ⁇ were then mixed with 50 ⁇ l of a protein A-Sepharo ⁇ e (Pharmacia-LKB) ⁇ lurry (50% packed bead ⁇ in 150 mM NaCl, 20 mM Tri ⁇ -HCl, pH 7.4, 0.2% Triton X-100) and incubated for 45 min at 4°C.
  • the bead ⁇ were pelleted and wa ⁇ hed four time ⁇ with wa ⁇ hing buffer (20 mM Tri ⁇ -HCl, pH 7.4, 500 mM NaCl, 1% Triton X-100, 1% deoxycholate and 0.2% SDS), followed by one wash in distilled water.
  • the immune complexes were eluted by boiling for 5 min in the SDS-sample buffer (100 mM Tris-HCl, pH 8.8, 0.01% bromophenol blue, 36% glycerol, 4% SDS) in the pre ⁇ ence of 10 mM dithiothreitol (DTT) , and analyzed by SDS-gel electrophore ⁇ i ⁇ u ⁇ ing 4-7% polyacrylamide gels (Blobel, G., and Dobberstein, B. (1975) J. Cell Biol. 67:835-851). The gel was fixed, incubated with Amplify (Amersham) for 20 min, dried and ⁇ ubjected to fluorography.
  • SDS-sample buffer 100 mM Tris-HCl, pH 8.8, 0.01% bromophenol blue, 36% glycerol, 4% SDS
  • DTT dithiothreitol
  • COS-1 cell ⁇ were ly ⁇ ed in 20 mM Tri ⁇ -HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholate, 1.5% Trasylol, 1 mM PMSF and 1 M DTT, for 15 min. Lysates were cleared by centrifugation, 3 ⁇ l of the anti ⁇ erum ⁇ LIB, with or without preblocking with 10 ⁇ g peptide, were added and samples were incubated for 2 h at 4°C. Protein A-Sepharose ⁇ lurry (25 ⁇ l) was then added and incubation was prolonged another 30 min at 4°C.
  • the beads were pelleted and washed four time ⁇ with lysis buffer and one time with dephosphorylation assay buffer (25 mM imidazole-HCl, pH 7.2, 1 mg/ml bovine serum albumin and 1 mM DTT), and finally resuspended in depho ⁇ phorylation a ⁇ ay buffer containing 2 ⁇ M myelin ba ⁇ ic protein 32P-labeled on tyrosine residue ⁇ by Baculo-viru ⁇ expressed intracellular part of the insulin receptor, kindly provided by A.J. Flint (Cold Spring Harbor Laboratory) and M.M. Cobb (University of Texas). After incubation for indicated times at 30°C, the reactions were stopped with a charcoal mixture (Streull, M.
  • NAME WOLF, GREENFIELD & SACKS, P.C.
  • AAA AGT GAT GGA AGG CTA
  • AAA CCT GGG GAC CGG CTC ATA AAG GTT AAT 5534 Lys Ser Asp Gly Arg Leu Lys Pro Gly Asp Arg Leu He Lys Val Asn 1805 1810 1815
  • ATTTTAACAC TAACCAAACA ATGCAGATCT TAGGGATGAT TAAAGGCAGC ATTGATGATA 7745
  • AGA CCA AAA AAT GTA ACC GAT TCA TTG AGC ACC TAC ATT AAT GCT AAT 1796 Arg Pro Lys Asn Val Thr Asp Ser Leu Ser Thr Tyr He Asn Ala Asn 150 155 160
  • GGC CCC ATG ATC AAC ACC GTG GAT GAT TTC TGG CAG ATG GTT TGG CAG 1892 Gly Pro Met He Asn Thr Val Asp Asp Phe Trp Gin Met Val Trp Gin 180 185 190
  • AGA GGT GGA ATG GTG CAA ACC
  • CTGTTAAACT GTTCCTGTTT TGAAGTGCTA TTACCTTTCT CAGTTACCAG AATCTTGCTG 2907
  • AAAAATCAAA AACAGTAACT ATTTTATATG GAAATGTGTC TTGATAATAT TACCTATTAA 3027

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Abstract

The invention relates to the cloning of two novel protein tyrosine phosphatases. Nucleic acid sequences encoding these phosphatases (PTPL1 and GLM-2) as well as anti-sense sequences also are provided. The recombinantly produced PTPL1 and GLM-2 proteins also are provided, as well as antibodies to these proteins. Methods relating to isolating the phosphatases, using the nucleic acid sequences, and using the phosphatases also are provided.

Description

PRIMARY STRUCTURE AND FUNCTIONAL EXPRESSION
OF NUCLEOTIDE SEQUENCES FOR NOVEL PROTEIN
TYROSINE PHOSPHATASES i
Field of the Invention
This invention relates to the isolation and cloning of nucleic acids encoding two novel protein tyrosine phosphatases (PTPs) . Specifically, the present invention relates to the isolation and cloning of two PTPs from human glioblastoma cDNA which have been designated PTPLl and GL -2. The present invention provides isolated PTP nucleic acid sequences; isolated PTP anti-sense sequences; vectors containing such nucleic acid sequences; cells, cell lines and animal hosts transformed by a recombinant vector so as to exhibit increased, decreased, or differently regulated expression of the PTPs; isolated probes for identifying sequences substantially similar or homologous to such sequences; substantially pure PTP proteins and variants or fragments thereof; antibodies or other agents which bind to these PTPs and variants or fragments thereof; methods of assaying for activity of these PTPs; methods of assessing the regulation of PTPLl or GLM-2; and methods of identifying and/or testing drugs which may affect the expression or activity of these PTPs.
Brief Description of the Background Art
Protein tyrosine phosphorylation plays an essential role in the regulation of cell growth, proliferation and differentiation (reviewed in Hunter, T. (1987) Cell 50:823-8291). This dynamic process is modulated by the counterbalancing activities of protein tyrosine kinases
M (PTKs and protein tyrosine phophatases (PTPs). The recent
{ elucidation of int? acellular signaling pathways has revealed important roles fot PTKS. Conserved domains like the Src homology 2 (SH2) (Suh, P.-G., et al. , (1988) Proc. Na l. Acad. Sci. (USA) 85:5419-5423) and the Src homology 3 (SH3) (Mayer, B.J., et al■ , (1988) Nature 352:272-275) domains have been found to determine the interaction between activated PTKs and signal transducing molecules (reviewed in Pawson, T., and Schiessinger, J. (1993) Current Biol. 3:434-442; Koch, C.A., et al■ , (1991) Science 252:668-674). The overall effect of such protein interactions is the formation of signaling cascades in which phosphorylation and dephosphorylation of proteins on tyrosine residues are major events. The involvement of PTPs in such signaling cascades is beginning to emerge from studies on the regulation and mechanisms of action of several representatives of this broad family of proteins.
Similarly to PTKS, PTPs can be classified according to their secondary structure into two broad groups, i.e. cytoplasmic and transmembrane molecules (reviewed in Charbonneau, H. , and Tonks, N.K. (1992) Annu. Rev. Cell Biol. 8:463-493; Pot, D.A., and Dixon, J.E. (1992) Biochim. Biophys. Acta 1136:35-43). Transmembrane PTPs have the structural organization of receptors and thus the potential to initiate cellular signaling in response to external stimuli. These molecules are characterized by the presence of a single transmembrane segment and two tandem PTP domains; only two examples of transmembrane PTPs that have single PTP domains are known, HPTP-P (Krueger, N.X. , et al. , (1990) EMBO J^ 9:3241-3252) and DPTP10D (Tian, S.-S., et al. , (1991) Cell 67:675-685) .
Nonreceptor PTPs display a single catalytic domain and contain, in addition, non-catalytic amino acid sequences which appear to control intracellular localization of the molecules and which may be involved in the determination of substrate specificity (Mauro, L.J., and Dixon, J.E. (1994) TIBS 19:151-155) and have also been suggested to be regulators of PTP activity (Charbonneau, H., and Tonks, N.K. (1992) Annu. Rev. Cell Biol. 8:463-493). PTP1B (Tonks, N.K., et al■ , (1988) J. Biol. Chem. 263:6731-6737) is localized to the cytosolic face of the endoplasmic reticulu via its C-terminal 35 amino acids (Frangioni, J.V. , et al. , (1992) Cell 68:545-560). The proteolytic cleavage of PTP1B by the calcium dependent neutral protease calpain occurs upstream from this targeting sequence, and results in the relocation of the enzyme from the endoplasmic reticulum to the cytosol; such relocation is concomitant with a two-fold stimulation of PTP1B enzymatic activity (Frangioni, J.V. , et al. , (1993) EMBO J. 12:4843-4856). Similarly, the 11 kDa C-terminal domain of T-cell PTP (Cool, D.E., et al. , (1989) Proc. Natl. Acad. Sci. (USA) 86:5257-5261) has also been shown to be responsible for enzyme localization and functional regulation (Cool, D.E., et al. , (1990) Proc. Natl. Acad. Sci. (USA) 87:7280-7284; Cool, D.E. , et al. , (1992) Proc. Natl. Acad. Sci. (USA) 89:5422-5426).
PTPs containing SH2 domains have been described including PTPIC (Shen, S.-H., et al ■ , (1991) Nature 352:736-739), also named HCP (Yi, T., et al . , (1992) Mol. Cell. Biol. 12:836-846), SHP (Matthews, R.J., et al. , (1992) Mol. Cell. Biol 12:2396-2405) or SH-PTP1 (Plutzky, J., et al. , (1992) Proc. Natl. Acad. Sci. (USA) 89:1123-1127), and the related phosphatase PTP2C (Ahmad, S., et al■ , (1993) Proc. Natl. Acad. Sci. (USA) 90:2197-2201), also termed SH-PTP2 (Freeman Jr., R.M. , et al . , (1992) Proc. Natl. Acad. Sci. (USA) 89:11239-11243), SH-PTP3 (Adachi, M. , et al. ,
(1992) FEBS Letters 314:335-339), PTP1D (Vogel, W. , et al . ,
(1993) Science 259:1611-1614) or Syp (Feng, G.-S., et al . , (1993) Science 259:1607-1611). The Drosophila csk gene product (Perkins, L.A., et al ■ , (1992) Cell 70:225-236) also belongs to this subfamily. PTPIC has been shown to associate via its SH2 domains with ligand-activated c-Kit and CSF-1 receptor PTKs (Yi, T., and Ihle, J.N. (1993) Mol. Cell. Biol. 13:3350-3358; Young, Y.-G. , et al., (1992) J. Biol. Chem. 267:23447-23450) but only association with activated CSF-1 receptor is followed by tyrosine phosphorylation of PTPIC. Syp interacts with and is phosphorylated by the ligand activated receptors for epidermal growth factor and platelet-derived growth factor (Feng, G.-S., et al. , (1993) Science 259:1607-1611). Syp has also been reported to associate with tyrosine phosphorylated insulin receptor substrate 1 (Kuhne, M.R. , et al■ , (1993) J. Biol. Chem. 268:11479-11481).
Two PTPs have been identified, PTPH1 (Yang, Q., and Tonks, N.K. (1991) Proc. Natl. Acad. Sci. (USA) 88:5949-5953) and PTPaεe MEG (Gu, M. , et al ■ , (1991) Proc. Natl. Acad. Sci. (USA) 88:5867-5871), which contain a region in their respective N-terminal segments with similarity to the cytoskeletal- associated proteins band 4.1 (Conboy, J., et al. , (1986) Proc. Natl. Acad. Sci. (USA) 83:9512-9516), ezrin (Gould, K.L., et al. , (1989) EMBO J. 8:4133-4142), talin (Rees, D.J.G., et al■ , (1990) Nature 347:685-689) and radixin (Funayama, N., et al. , (1991) J. Cell Biol. 115:1039-1048). The function of proteins of the band 4.1 family appears to be the provision of anchors for cytoskeletal proteins at the inner surface of the plasma membrane (Conboy, J., et al. , (1986) Proc. Natl. Acad. Sci. (USA) 83:9512-9516; Gould, K.L., et al. , (1989) EMBO J. 8:4133-4142). It has been postulated that PTPH1 and PTPase MEG would, like members of this family, localize at the interface between the plasma membrane and the cytoskeleton and thereby be involved in the modulation of cytoskeletal function (Tonks, N.K. , et al. , (1991) Cold Spring Harbor Symposia on Quantitative Biology LVI:265-273) .
The interest in studying PTKs and PTPs is particularly great in cancer research. For example, approximately one third of the known oncogenes include PTKs (Hunter, T. (1989) In Oncogenes and Molecular Origins of Cancer, R. Weinberg, Ed., Coldspring Harbor Laboratory Press, New York). In addition, the extent of tyrosine phosphorylation closely correlates with the manifestation of the transformed phenotype in cells infected by temperature-sensitive mutants of rous sarcoma virus. (Sefton, B. , et al . , (1980) Cell 20:807-816) Similarly, Brown-Shirner and colleagues demonstrated that over-expression of PTP1B in 3T3 cells suppressed the transforming potential of oncogenic neu, as measured by focus formation, anchorage-independent growth and tumorigenicity (Brown-Shirner, S., et al. , (1992) Cancer Res. 52:478-482). Because they are direct antagonists of PTK activity, the PTPs also may provide an avenue of treatment for cancers caused by excessive PTK activity. Therefore, the isolation, characterization and cloning of various PTPs is an important step in developing, for example, gene therapy to treat PTK oncogene cancers.
Summary of the Invention
The present invention is based upon the molecular cloning of previously uncloned and previously undisclosed nucleic acids encoding two novel PTPs. The disclosed sequences encode PTPs which we have designated PTPLl and GLM-2. (PTPLl was previously designated GLM-1 in U.S. Patent Application Serial No. 08/115,573 filed September 1, 1993.) In particular, the present invention is based upon the molecular cloning of PTPLl and GLM-2 PTP sequences from human glioblastoma cells. The invention provides isolated cDNA and RNA sequences corresponding to PTPLl and GLM-2 transcripts and encoding the novel PTPs. In addition, the present invention provides vectors containing PTPLl or GLM-2 cDNA sequences, vectors capable of expressing PTPLl or GLM-2 sequences with endogenous or exogenous promoters, and hosts transformed with one or more of the above-mentioned vectors. Using the sequences disclosed herein as probes or primers in conjunction with such techniques as PCR cloning, targeted gene walking, and colony/plaque hybridization with genomic or cDNA libraries, the invention further provides for the isolation of allelic variants of the disclosed sequences, endogenous PTPLl or GLM-2 regulatory sequences, and substantially similar or homologous PTPLl or GLM-2 DNA and RNA sequences from other species including mouse, rat, rabbit and non-human primates. The present invention also provides fragments and variants of isolated PTPLl and GLM-2 sequences, fragments and variants of isolated PTPLl or GLM-2 RNA, vectors containing variants or fragments of PTPLl or GLM-2 sequences, vectors capable of expressing variants or fragments of PTPLl or GLM-2 sequences with endogenous or exogenous regulatory sequences, and hosts transformed with one or more of the above-mentioned vectors. The invention further provides variants or fragments of substantially similar or homologous PTPLl and GLM-2 DNA and RNA sequences from species including mouse, rat, rabbit and non-human primates.
The present invention provides isolated PTPLl and GLM-2 anti-sense DNA, isolated PTPLl and GLM-2 anti-sense RNA, vectors containing PTPLl or GLM-2 anti-sense DNA, vectors capable of expressing PTPLl or GLM-2 anti-sense DNA with endogenous or exogenous promoters, and hosts transformed with one or more of the above-mentioned vectors. The invention further provides the related PTPLl or GLM-2 anti-sense DNA and anti-sense RNA sequences from other species including mouse, rat, rabbit and non-human primates.
The present invention also provides fragments and variants of isolated PTPLl and GLM-2 anti-sense DNA, fragments and variants of isolated PTPLl and GLM-2 anti-sense RNA, vectors containing fragments or variants of PTPLl and GLM-2 anti-sense DNA, vectors capable of expressing fragments or variants of PTPLl and GLM-2 anti-sense DNA with endogenous or exogenous promoters, and hosts transformed with one or more of the above-mentioned vectors. The invention further provides fragments or variants of the related PTPLl and GLM-2 anti-sense DNA and PTPLl and GLM-2 anti-sense RNA sequences from other species including mouse, rat, rabbit and non-human primates.
Based upon the sequences disclosed herein and techniques well known in the art, the invention also provides isolated probes useful for detecting the presence or level of expression of a sequence identical, substantially similar or homologous to the disclosed PTPLl and GLM-2 sequences. The probes may consist of the PTPLl and GLM-2 DNA, RNA or anti-sense sequences disclosed herein. The probe may be labeled with, for example, a radioactive isotope; immobilized as, for example, on a filter for Northern or Southern blotting; or may be tagged with any other sort of marker which enhances or facilitates the detection of binding. The probes may be oligonucleotides or synthetic oligonucleotide analogs.
The invention also provides substantially pure PTPLl and GLM-2 proteins. The proteins may be obtained from natural sources using the methods disclosed herein or, in particular, the invention provides substantially pure PTPLl and GLM-2 proteins produced by a host cell or transgenic animal transformed by one of the vectors disclosed herein.
The invention also provides substantially pure variants and fragments of PTPLl and GLM-2 proteins.
Using the substantially pure PTPLl or GLM-2 protein or variants or fragments of the PTPLl or GLM-2 protein which are disclosed herein, the present invention provides methods of obtaining and identifying agents capable of binding to either PTPLl or GLM-2. Specifically, such agents include antibodies, peptides, carbohydrates and pharmaceutical agents. The agents may include natural ligandε, co-factors, accessory proteins or associated peptides, modulators, regulators, or inhibitors. The entire PTPLl or GLM-2 protein may be used to test or develop such agents or variants or fragments thereof may be employed. In particular, only certain domains of the PTPLl or GLM-2 protein may be employed. The invention further provides detectably labeled, immobilized and toxin-conjugated forms of these agents.
The present invention also provides methods for assaying for PTPLl or GLM-2 PTP activity. For example, using the PTPLl and GLM-2 anti-sense probes disclosed herein, the presence and level of either PTPLl or GLM-2 expression may be determined by hybridizing the probes to total or selected mRNA from the cell or tissue to be studied. Alternatively, using the antibodies or other binding agents disclosed herein, the presence and level of PTPLl or GLM-2 protein may be assessed. Such methods may, for example, be employed to determine the tissue-specificity of PTPLl or GLM-2 expression.
The present invention also provides methods for assessing the regulation of PTPLl or GLM-2 function. Such methods include fusion of the regulatory regions of the PTPLl or GLM-2 nucleic acid sequences to a marker locus, introduction of this fusion product into a host cell using a vector, and testing for inducers or inhibitors of PTPLl or GLM-2 by measuring expression of the marker locus. In addition, by using labeled PTPLl and GLM-2 anti-sense transcripts, the level of expression of PTPLl or GLM-2 mRNA may be ascertained and the effect of various endogenous and exogenous compounds or treatments on PTPLl or GLM-2 expression may be determined. Similarly, the effect of various endogenous and exogenous compounds and treatments on PTPLl or GLM-2 expression may be assessed by measuring the level of either PTPLl or GLM-2 protein with labeled antibodies as disclosed herein.
The present invention provides methods for efficiently testing the activity or potency of drugs intended to enhance or inhibit PTPLl or GLM-2 expression or activity. In particular, the nucleic acid sequences and vectors disclosed herein enable the development of cell lines and transgenic organisms with increased, decreased, or differently regulated expression of PTPLl or GLM-2. Such cell lines and animals are useful subjects for testing pharmaceutical compositions.
The present invention further provides methods of modulating the activity of PTPLl and GLM-2 PTPs in cells. Specifically, agents and, in particular, antibodies which are capable of binding to either PTPLl or GLM-2 PTP are provided to a cell expressing PTPLl or GLM-2. The binding of such an agent to the PTP can be used either to activate or inhibit the activity of the protein. In addition, PTPLl and GLM-2 anti-sense transcripts may be administered such that they enter the cell and inhibit translation of the PTPLl or GLM-2 mRNA and/or the transcription of PTPLl or GLM-2 nucleic acid sequences. Alternatively, PTPLl or GLM-2 RNA may be administered such that it enters the cell, serves as a template for translation and thereby augments production of PTPLl or GLM-2 protein. In another embodiment, a vector capable of expressing PTPLl or GLM-2 mRNA transcripts or PTPLl or GLM-2 anti-sense RNA transcripts is administered such that it enters the cell and the transcripts are expressed.
Brief Description of the Drawings
Figure 1. Comparison of PTPLl with proteins of the band 4.1 superfamily. The alignment was done using the Clustal V alignment program (Fazioli, F., et al■ , (1993) Oncoqene 8:1335-1345). Identical amino acid residues conserved in two or more sequences, are boxed. A conserved tyrosine residue, which in ezrin has been shown to be phosphorylated by the epidermal growth factor receptor, is indicated by an asterisk,
Figure 2. Comparison of amino acid sequences of GLGF-repeats. The alignment was done manually. Numbers of the GLGF-repeats are given starting from the N-terminus of the protein. Residues conserved in at least eight (42%) repeats are showed in bold letters. Five repeats are found In PTPLl, three are found in the guanylate kinases, dlg-A gene product, PSD-95 and the 220-kDa protein. One GLGF-repeat is found in the guanylate kinase p55, in the PTPs PTPH1 and PTPase MEG, and in nitric oxide synthase (NOS) . One repeat is also found in an altered rosl transcript from the glioma cell line U-118MG,
Figure 3. Schematic diagram illustrating the domain strucure of PTPLl and other GLGF-repeat containing proteins. Domains and motifs indicated in the figure are L, leucine zipper motif; Band 4.1, band 4.1-like domain; G, GLGF-repeat; PTPase, catalytic PTPase domain; 3, SH3 domain; GK, guanylate kinase domain, Bind. Reg., co-enzyme binding region.
Figure 4. PTP activity of PTPLl. Immunoprecipitates from COS-1 cells using an antiserum (αLIB) against PTPLl, unblocked (open circles) or blockeod with peptide (open squares), were incubated for 2, 4, 6 or 12 minutes with myelin basic protein, 32P-labeled on tyrosine residues.
The amount of radioactivity released as inorganic phosphate is expressed as the percentage of the total input of radioactivity.
Detailed Description of the Invention Definitions.
In the description that follows, a number of terms used in biochemistry, molecular biology, recombinant DNA (rDNA) technology and immunology are extensively utilized. In addition, certain new terms are introduced for greater ease of exposition and to more clearly and distinctly point out the subject matter of the invention. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
Gene. A gene is a nucleic acid sequence including a promoter region operably joined to a coding sequence which may serve as a template from which an RNA molecule may be transcribed by a nucleic acid polymerase. A gene contains a promoter sequence to which the polymerase binds, an initiation sequence which signals the point at which transcription should begin, and a termination sequence which signals the point at which transcription should end. The gene also may contain an operator site at which a repressor may bind to block the polymerase and to prevent transcription and/or may contain ribosome binding sites, capping signals, transcription enhancers and polyadenylation signals. The promoter, initiation, termination and, when present, operator sequences, ribosome binding sites, capping signals, transcription enhancers and polyadenylation signals are collectively referred to as regulatory sequences. Regulatory sequences 5' of the transcription initiation codon are collectively referred to as the promoter region. The sequences which are transcribed into RNA are the coding sequences. The RNA may or may not code for a protein. RNA that codes for a protein is processed into messenger RNA (mRNA) . Other RNA molecules may serve functions or uses without ever being translated into protein. These include ribosomal RNA (rRNA), transfer RNA (tRNA), and the anti-sense RNAs of the present invention. In eukaryotes, coding sequences between the translation start codon (ATG) and the translation stop codon (TAA, TGA, or TAG) may be of two types: exons and introns. The exons are included in processed mRNA transcripts and are generally translated into a peptide or protein. Introns are excised from the RNA as it is processed into mature mRNA and are not translated into peptide or protein. As used herein, the word gene embraces both the gene including its introns, as may be obtained from genomic DNA, and the gene with the introns excised from the DNA, as may be obtained from cDNA.
Anti-sense DNA is defined as DNA that encodes anti-sense RNA and anti-sense RNA is RNA that is complementary to or capable of selectively hybridizing to some specified RNA transcript. Thus, anti-sense RNA for a particular gene would be capable of hybridizing with that gene's RNA transcript in a selective manner. Finally, an anti-sense gene is defined as a segment of anti-sense DNA operably joined to regulatory sequences such that the sequences encoding the anti-sense RNA may be expressed. cDNA. Complementary DNA or cDNA is DNA which has been produced by reverse transcription from mature mRNA. In eukaryotes, sequences in RNA corresponding to introns in a gene are excised during mRNA processing. cDNA sequences, therefore, lack the intron sequences present in the genomic DNA to which they correspond. In addition, cDNA sequences will lack the regulatory sequences which are not transcribed into RNA. To create a functional cDNA gene, therefore, the cDNA sequence must be operably joined to a promoter region such that transcription may occur.
Operably Joined. A coding sequence and a promoter region are said to be operably joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the promoter region. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of promoter function results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
If it is not desired that the coding sequence be eventually expressed as a protein or polypeptide, as in the case of anti-sense RNA expression, there is no need to ensure that the coding sequences and promoter region are joined without a frame-shift. Thus, a coding sequence which need not be eventually expressed as a protein or polypeptide is said to be operably joined to a promoter region if induction of promoter function results in the transcription of the RNA sequence of the coding sequences.
The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating sequences involved with initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' non-transcribing regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Such transcriptional control sequences may also include enhancer sequences or upstream activator sequences, as desired.
Vector. A vector may be any of a number of nucleic acid sequences into which a desired sequence may be inserted by restriction and ligation. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include plasmids, phage, phasmids and cosmids. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to a promoter region and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase or alkaline phosphatase) , and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques.
Fragment. As used herein, the term "fragment" means both unique fragments and substantially characteristic fragments. As used herein, the term "fragment" is not to be construed according to standard dictionary definitions.
Substantially Characteristic Fragment. A "substantially characteristic fragment" of a molecule, such as a protein or nucleic acid sequence, is meant to refer to any portion of the molecule sufficiently rare or sufficiently characteristic of that molecule so as to identify it as derived from that molecule or to distinguish it from a class of unrelated molecules. A single amino acid or nucleotide, or a sequence of only two or three, cannot be a substantially characteristic fragment because all such short sequences occur frequently in nature.
A substantially characteristic fragment of a nucleic acid sequence is one which would have utility as a probe in identifying the entire nucleic acid sequence from which it is derived from within a sample of total genomic or cDNA. Under stringent hybridization conditions, a substantially characteristic fragment will hybridize only to the sequence from which it was derived or to a small class of substantially similar related sequences such as allelic variants, heterospecific homologous loci, and variants with small insertions, deletions or substitutions of nucleotides or nucleotide analogues. A substantially characteristic fragment may, under lower stringency hybridization conditions, hybridize with non-allelic and non-homologous loci and be used as a probe to find such loci but will not do so at higher stringency.
A substantially characteristic fragment of a protein would have utility in generating antibodies which would distinguish the entire protein from which it is derived, an allelomorphic protein or a heterospecific homologous protein from a mixture of many unrelated proteins. It is within the knowledge and ability of one ordinarily skilled in the art to recognize, produce and use substantially characteristic fragments of nucleic acid sequences and proteins as, for example, probes for screening DNA libraries or epitopes for generating antibodies.
Unique Fragment. As used herein, a unique fragment of a protein or nucleic acid sequence is a substantially characteristic fragment not currently known to occur elsewhere in nature (except in allelic or heterospecific homologous variants, i.e. it is present only in the PTPLl or GLM-2 PTP or a PTPLl or GLM-2 PTP "ho ologue") . A unique fragment will generally exceed 15 nucleotides or 5 amino acid residues. One of ordinary skill in the art can identify unique fragments by searching available computer databases of nucleic acid and protein sequences such as Genbank (Los Alamos National Laboratories, USA), SwissProt or the National Bio edical Research Foundation database. A unique fragment is particularly useful, for example, in generating monoclonal antibodies or in screening DNA or cDNA libraries.
Stringent Hybridization Conditions. "Stringent hybridization conditions" is a term of art understood by those of ordinary skill in the art. For any given nucleic acid sequence, stringent hybridization conditions are those conditions of temperature and buffer solution which will permit hybridization of that nucleic acid sequence to its complementary sequence and not to substantially different sequences. The exact conditions which constitute "stringent" conditions, depend upon the length of the nucleic acid sequence and the frequency of occurrence of subsets of that sequence within other non-identical sequences. By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, one of ordinary skill in the art can, without undue experimentation, determine conditions which will allow a given sequence to hybridize only with identical sequences. Suitable ranges of such stringency conditions are described in Krause, M.H.. and S.A. Aaronson, Methods in Enzymoloqy, 200:546-556 (1991). Stringent hybridization conditions, depending upon the length and commonality of a sequence, may include hybridization conditions of 30°C-65°C and from 5X to 0. IX SSPC. Less than stringent hybridization conditions are employed to isolate nucleic acid sequences which are substantially similar, allelic or homologous to any given sequence.
When using primers that are derived from nucleic acid encoding a PTPLl or GLM-2 PTP, one skilled in the art will recognize that by employing high stringency conditions (e.g. annealing at 50-60°C), sequences which are greater than about 75% homologous to the primer will be amplified. By employing lower stringency conditions (e.g. annealing at 35-37°C), sequences which are greater than about 40-50% homologous to the primer will be amplified.
When using DNA probes derived from a PTPLl or GLM-2 PTP for colony/plaque hybridization, one skilled in the art will recognize that by employing high stringency conditions (e.g. hybridization at 50-65°C, 5X SSPC, 50% formamide, wash at 50-65°C, 0.5X SSPC), sequences having regions which are greater than about 90% homologous to the probe can be obtained, and by employing lower stringency conditions (e.g. hybridization at 35-37°C, 5X SSPC, 40-45% formamide, wash at 42°C SSPC), sequences having regions which are greater than 35-45% homologous to the probe will be obtained.
Substantially similar. Two nucleic acid sequences are substantially similar if one of them or its anti-sense complement can bind to the other under strict hybridization conditions so as to distinguish that strand from all or substantially all other sequences in a cDNA or genomic library. Alternatively, one sequence is substantially similar to another if it or its anti-sense complement is useful as a probe in screening for the presence of its similar DNA or RNA sequence under strict hybridization conditions. Two proteins are substantially similar if they are encoded by substantially similar DNA or RNA sequences. In addition, even if they are not encoded by substantially similar nucleic acids, two proteins are substantially similar if they share sufficient primary, secondary and tertiary structure to perform the same biological role (structural or functional) with substantially the same efficacy or utility.
Variant■ A "variant" of a protein or nucleic acid or fragment thereof is meant to include a molecule substantially similar in structure to the protein or nucleic acid, or to a fragment thereof. Variants of nucleic acid sequences include sequences with conservative nucleotide substitutions, small insertions or deletions, or additions. Variants of proteins include proteins with conservative amino acid substitutions, small insertions or deletions, or additions. Thus, nucleotide substitutions which do not effect the amino acid sequence of the subsequent translation product are particularly contemplated. Similarly, substitutions of structurally similar amino .acids in proteins, such as leucine for isoleucine, or insertions, deletions, and terminal additions which do not destroy the functional utility of the protein are contemplated. Allelic variants of nucleic acid sequences and allelomorphic variants or protein or polypeptide sequences are particularly contemplated. As is well known in the art, an allelic variant is simply a naturally occurring variant of a polymorphic gene and that term is used herein as it is commonly used in the field of population genetics. The production of such variants is well known in the art and, therefore, such variants are intended to fall within the spirit and scope of the claims.
Homologous and homologues. As used herein, the term "homologues" is intended to embrace either and/or both homologous nucleic acid sequences and homologous protein sequences as the context may indicate. Homologues are a class of variants, as defined above, which share a sufficient degree of structural and functional similarity so as to indicate to one of ordinary skill in the art that they share a common evolutionary origin and that the structural and functional similarity is the result of evolutionary conservation. To be considered homologues of the PTPLl or GLM-2 PTP, nucleic acid sequences and the proteins they encode must meet two criteria: (1) The polypeptides encoded by homologous nucleic acids are at least approximately 50-60% identical and preferably at least 70% identical for at least one stretch of at least 20 amino acids. As is well known in the art, both the identity and the approximate positions of the amino acid residues relative to each other must be conserved and not just the overall amino acid composition. Thus, one must be able to "line up" the conserved regions of the homologues and conclude that there is 50-60% identity; and (2) The polypeptides must retain a functional similarity to the PTPLl or GLM-2 PTP in that it is a protein tyrosine phosphatase.
Substantially Pure. The term "substantially pure" when applied to the proteins, variants or fragments thereof of the present invention means that the proteins are essentially free of other substances to an extent practical and appropriate for their intended use. In particular, the proteins are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, protein sequencing, or producing pharmaceutical preparations. By techniques well known in the art, substantially pure proteins, variants or fragments thereof may be produced in light of the nucleic acids of the present invention.
Isolated. Isolated refers to a nucleic acid sequence which has been: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid sequence is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleic acid sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid sequence that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.
Imπunogenetically Effective Amount. An "immunogenetically effective amount" is that amount of an antigen (e.g. a protein, variant or a fragment thereof) necessary to induce the production of antibodies which will bind to the epitopes of the antigen. The actual quantity comprising an "immunogenetically effective amount" will vary depending upon factors such as the nature of the antigen, the organism to be immunized, and the mode of immunization. The determination of such a quantity is well within the ability of one ordinarily skilled in the art without undue experimentation.
Antigen and Antibody. The term "antigen" as used in this invention is meant to denote a substance that can induce a detectable immune response to it when introduced to an animal . Such substances include proteins and fragments thereof.
The term "epitope" is meant to refer to that portion of an antigen which can be recognized and bound by an antibody. An antigen may have one, or more than one epitope. An "antigen" is capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An "immunogen" is an antigen introduced into an animal specifically for the purpose of generating an immune response to the antigen. An antibody is said to be "capable of selectively binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The selective binding of an antigen and antibody is meant to indicate that the antigen will react, in a highly specific manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
The term "antibody" (Ab) or "monoclonal antibody" (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab')2 fragments) which are capable of binding an antigen. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. Single chain antibodies, humanized antibodies, and fragments thereof, also are included.
Description of the Preferred Embodiments
The present invention relates to the identification, isolation and cloning of two novel protein tyrosine phosphatases designated PTPLl and GLM-2. Specifically, the present invention discloses the isolation and cloning of cDNA and the amino acid sequences of PTPLl and GLM-2 from human glioblastoma and brain cell cDNA libraries. These phosphatases are, initially, discussed separately below. As they are related in function and utility as well as structurally with respect to their catalytic domains, they are subsequently discussed in the alternative.
In order to identify novel PTPs, a PCR-based approach was used. PCR was performed using cDNA from the human glioma cell line U-343 MGa 31L as a template and degenerate primers that were based on conserved regions of PTPs. One primer was derived from the catalytic site (HCSAG) of the PTP domain and two primers were derived from conserved regions in the N-terminal part of the domain. Several PCR-products were obtained, including some corresponding to the cytoplasmic PTPs PTPH1 (Yang, Q. , and Tonks, N.K. (1991) Proc. Natl. Acad. Sci. (USA) 88:5949-5953), PTPase MEG (Gu, M. , et al
(1991) Proc. Natl. Acad. Sci. (USA) 88:5867-5871), P19PTP (den Hertog, J. , et al . , (1992) Biochem. Biophys. Res■ Commun■ 184:1241-1249), and TC-PTP (Cool, D.E., et al. , (1989) Proc. Natl. Acad. Sci. (USA) 86:5257-5261), as well as to the receptor-like PTPs HPTP- , HPTP-γ, and HPTP-δ (Krueger, N.X. , et al. , (1990) EMBO J. 9:3241-3252). In addition to these known sequences, three PCR-products encoding novel PTP-like sequences were found.
One of these PCR-products is almost identical to a PCR-product derived from a human leukemic cell line (Honda, H., et al. , (1993) Leukemia 7:742-746) and was chosen for further characterization and was used to screen an oligo-(dT)-primed U-343 MGa 31L cDNA library which resulted in the isolation of the clone X6.15. Upon Northern blot analysis of mRNA from human foreskin fibroblasts AG1518, probed with the X6.15 insert, a transcript of 9.5 kb could be seen. Therefore AG1518 cDNA libraries were constructed and screened with \6.15 in order to obtain a full-length clone. Screening of these libraries with X6.15, and thereafter with subsequently isolated clones, resulted in several overlapping clones which together covered 8040 bp including the whole coding sequence of a novel phosphatase, denoted PTPLl. The total length of the open reading frame was 7398 bp coding for 2466 amino acids with a predicted molecular mass of 275 kDa. The nucleotide and deduced amino acid sequence of PTPLl are disclosed as SEQ ID NO.:l and SEQ ID NO.:2, respectively. Although the sequence surrounding the putative initiator codon at positions 78-80 does not conform well to the Kozak consensus sequence (Kozak, M. (1987) Nucl. Acids Res. 15:8125-8148) there is a purine at position -3 which is an important requirement for an initiation site. The 77 bp 5' untranslated region is GC-rich and contains an inframe stop codon at positions 45-47. A 3' untranslated region of 565 bp begins after a TGA stop codon at positions 7476-7478, and does not contain a poly-A tail.
In the deduced amino acid sequence of PTPLl no transmembrane domain or signal sequence for secretion are found, indicating that PTPLl is a cytoplasmic PTP. Starting from the N-terminus, the sequence of the first 470 amino acid residues shows no homology to known proteins. The region 470-505 contains a leucine zipper motif, with a methionine in the position where the fourth leucine usually is found (LXgL -LXg gL) ; similar replacements of leucine residues with methionine residues are also found in the leucine zippers of the transcription factors CYS-3 (Fu, Y.-H., et al. , (1989) Mol. Cell. Biol. 9:1120-1127) and dFRA (Perkins, K.K. , et al. , (1990) Genes Dev. 4:822-834). Furthermore, consistent with the notion that this is a functional leucine zipper, no helix breaking residues (glycine and proline) are present in this region. The leucine zipper motif is followed by a 300 amino acid region (570-885) with homology to the band 4.1 superfamily (see Figure 1). The members of this superfamily are cytoskeleton-associated proteins with a homologous domain in the N-terminus (Tsukita, S., et al■ , (1992) Curr. Opin. Cell Biol. 4:834-839). Interestingly, two cytoplasmic PTPs, PTPH1 and PTPase MEG, contain a band 4.1-1ike domain. The band 4.1-like domain of PTPLl is 20% to 24% similar to most known proteins of this superfamily, including ezrin (Gould, K.L., et al. , (1989) EMBO J. 8:4133-4142), moesin (Lankes, W.T., and Furthmayr, H. (1991) Proc. Natl. Acad. Sci. (USA) 88:8297-8301), radixin (Funayama, N. , et al. , (1991) J. Cell Biol. 115:1039-1048), merlin (Trofatter, J.A. , et al. , (1993) Cell 72:791-800), band 4.1 protein (Conboy, J. , et al. , (1986) Proc. Natl. Acad. Sci. (USA) 83:9512-9516), PTPH1 (Yang, Q. , and Tonks, N.K. (1991) Proc. Natl. Acad. Sci. (USA) 88:5949-5953) and PTPase MEG (Gu, M. , et al. , (1991) Proc. Natl. Acad. Sci. (USA) 88:5867-5871). Between amino acid residues 1080 and 1940 there are five 80 amino acid repeats denoted GLGF-repeats. This repeat was first found in PSD-95 (Cho, K.-O., et al. , (1992) Neuron 9:929-942), also called SAP (Kistner, U. , et al. , (1993) J. Biol. Chem. 268:4580-4583), a protein in post-synaptic densities, i.e. structures of the submembranous cytoskeleton in synaptic junctions. Rat PSD-95 is homologous to the discs-large tumor suppressor gene in Droεophila (Woods, D.F., and Bryant, P.J. (1991) Cell 66:451-464), dlg-A, which encodes a protein located in septate junctions. These two proteins each contain three GLGF-repeats, one SH-3 domain and a guanylate kinase domain. Through computer searches in protein data bases complemented by manual searches, 19 GLGF-repeats in 9 different proteins, all of them enzymes, were, found (see Figure 2 and Figure 3). Besides dlg-A and PSD-95, there are two other members of the guanylate kinase family, a 220-kDa protein (Itoh, M. , et al. , (1993) J. Cell Biol. 121:491-502) which is a constitutive protein of the plasma membrane undercoat with three GLGF-repeats, and p55 (Ruff, P., et al. , (1991) Proc. Natl. Acad. Sci. (USA) 88:6595-6599) which is a palmitoylated protein from erythrocyte membranes with one GLGF-repeat. A close look into the sequence of PTPHl and PTPase MEG revealed that each of them has one GLGF-repeat between the band 4.1 homology domain and the PTP domain. One GLGF-repeat is also found in nitric oxide synthase from rat brain (Bredt, D.S., et al . , (1991) Nature 351:714-718), and a glioma cell line, U-118MG, expresses an altered rosl transcript (Sharma, S., et al■ , (1989) Oncoqene Res. 5:91-100), containing a GLGF-repeat probably as a result of a gene fusion.
The PTP domain of PTPLl is localized in the C-terminus (amino acid residues 2195-2449). It contains most of the conserved motifs of PTP domains and shows about 30% similarity to known PTPs. Use of a 9.5 kb probe including SEQ ID NO.:l for Northern blot analysis for tissue-specific expression showed high expression of PTPLl in human kidney, placenta, ovaries, and testes; medium expression in human lung, pancreas, prostrate and brain; low expression in human heart, skeletal muscle, spleen, liver, small intestine and colon; and virtually no detectable expression in human leukocytes. Furthermore, using a rat PCR product for PTPLl as a probe, PTPLl was found to be expressed in adult rats but not in rat embryos. This latter finding suggests that PTPLl may have a role, like many PTPs, in the signal transduction process that leads to cellular growth or differentiation.
The rabbit antiserum αLlA (see Example 5), made against a synthetic peptide derived from amino acid residues
1802-1823 in the PTPLl sequence, specifically precipitated a component of 250 kDa from [ 35S]methionine and t 35S]cysteme labeled COS-1 cells transfected with the
PTPLl cDNA. This component could not be detected in untransfected cells, or in transfected cells using either pre-immune serum or antiserum pre-blocked with the immunogenic peptide. Identical results were obtained using the antiserum αLIB (see Example 5) made against residues
450-470 of PTPLl. A component of about 250 kDa could also be detected in immunoprecipitations using AG1518 cells, PC-3 cells, CCL-64 cells, A549 cells and PAE cells. This component was not seen upon precipitation with the preimmune serum, or when precipitation was made with αLlA antiserum preblocked with peptide. The slight variations in sizes observed between the different cell lines could be due to species differences. A smaller component of 78 kDa was also specifically precipitated by the αLlA antiserum. The relationship between this molecule and PTPLl remains to be determined.
In order to demonstrate that PTPLl has PTP activity, immunoprecipitates from COS-1 cells transfected with PTPLl cDNA were incubated with myelm basic protein, 32P-labeled on tyrosine residues, as a substrate. The amount of radioactivity released as inorganic phosphate was measured. Immunoprecipitates with αLIB (open circles) gave a time-dependent increase in dephosphorylation with over 30% dephosphorylation after 12 minutes compared to 2% dephosphorylation when the antiserum was pre-blocked with peptide (open squares) (see Figure 4).
The present invention also provides an isolated nucleic acid sequence encoding a novel PTP designated GLM-2, variants and fragments thereof, and uses relating thereto. One sequence encoding a GLM-2 PTP and surrounding nucleotides is disclosed as SEQ ID NO.:3. This sequence includes the coding sequences for GLM-2 PTP as well as both 5' and 3' untranslated regions including regulatory sequences. The full disclosed sequence, designated SEQ ID NO. :3 is 3090 bp in length.
The nucleic acid sequence of SEQ ID NO.:3 includes 1310 base pairs of 5' untranslated region and 673 bp of 3 ' untranslated region which do not appear to encode a sequence for a poly-A (polyadenylation) tail. Transcription of SEQ ID NO. :3 begins at approximately position 1146. A translation start codon (ATG) is present at positions 1311 to 1313 of SEQ ID NO.:3. The nucleotides surrounding the start codon (AGCATGG) show substantial similarity to the Kozak consensus sequence (RCCATGG) (Kozak, M. (1987) Nucl. Acids Res. 15:8125-8148). A translation stop codon (TGA) is present at positions 2418 to 2420 of SEQ ID NO.:3. The open reading frame of 1107 bp encodes a protein of 369 amino acid residues with a predicted molecular mass of 41 kD. The deduced amino acid sequence of this protein is disclosed as SEQ ID NO.:4.
The sequence disclosed in SEQ ID NO. :3 encodes a single domain PTP similar to the rat PTP STEP (53% identity; Lombroso, et al. , 1991) and the human PTP LC-PTP (51% identity; Adachi, M. , et al. , (1992) FEBS Letters 314:335-339). None of the sequenced regions encodes a polypeptide sequence with any substantial similarity to known signal or transmembrane domains. Further indicating that GLM-2 is a cytoplasmic PTP.
Use of a 3.6 kb probe including SEQ ID NO.:3 for Northern blot analysis for tissue-specific expression showed a strong association with human brain tissue and little or no expression in human heart, placenta, lung, liver, skeletal muscle, kidney or pancreas. This is similar to to the pattern of tissue-specific expression shown by STEP.
Cloning and expression of PTPLl and GLM-2.
In one series of embodiments of the present invention, an isolated DNA, cDNA or RNA sequence encoding a PTPLl or GLM-2 PTP, or a variant or fragment thereof, is provided. The procedures described above, which were employed to isolate the first PTPLl and GLM-2 sequences no longer need be employed. Rather, using the sequences disclosed herein, a genomic DNA or cDNA library may be readily screened to isolate a clone containing at least a fragment of a PTPLl or GLM-2 sequence and, if desired, a full sequence. Alternatively, one may synthesize PTPLl and GLM-2 encoding nucleic acids using the sequences disclosed herein.
The present invention further provides vectors containing nucleic acid sequences encoding PTPLl and GLM-2. Such vectors include, but are not limited to, plasmids, phage, plasmids and cosmid vectors. In light of the present disclosure, one of ordinary skill in the art can readily place the nucleic acid sequences of the present invention into any of a great number of known suitable vectors using routine procedures.
The source nucleic acids for a DNA library may be genomic DNA or cDNA. Which of these is employed depends upon the nature of the sequences sought to be cloned and the intended use of those sequences.
Genomic DNA may be obtained by methods well known to those or ordinary skill in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et al. , eds. , Academic Press (1987)). Genomic DNA is preferred when it is desired to clone the entire gene including its endogenous regulatory sequences. Similarly, genomic DNA is used when it is only the regulatory sequences which are of interest.
Complementary or cDNA may be produced by reverse transcription methods which are well known to those of ordinary skill in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et al. , eds. , Academic Press (1987)). Preferably, the mRNA preparation for reverse transcription should be enriched in the mRNA of the desired sequence. This may be accomplished by selecting cells in which the mRNA is produced at high levels or by inducing high levels of production. Alternatively, in vitro techniques may be used such as sucrose gradient centrifugation to isolate mRNA transcripts of a particular size. cDNA is preferred when the regulatory sequences of a gene are not needed or when the genome is very large in comparison with the expressed transcripts. In particular, cDNA is preferred when a eukaryotic gene containing introns is to be expressed in a prokaryotic host.
To create a DNA or cDNA library, suitable DNA or cDNA preparations are randomly sheared or enzymatically cleaved by restriction endonucleases to create fragments appropriate in size for the chosen library vector. The DNA or cDNA fragments may be inserted into the vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation. Typically, this is accomplished by restriction enzyme digestion to provide appropriate termini, the filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligaseε. Techniques for such manipulations are well known in the art and may be found, for example, in Sambrook, et al . , Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY (1989). The library will consist of a great many clones, each containing a fragment of the total DNA or cDNA. A great variety of cloning vectors, restriction endonucleases and ligases are commercially available and their use in creating DNA libraries is well known to those of ordinary skill in the art. See, for example, Sambrook, et al■ , Molecular Cloning, A Laboratory Manual, 2d ed. , Cold Spring Harbor Laboratory Presε, Plainview, NY (1989).
DNA or cDNA libraries containing sequences coding for PTPLl or GLM-2 nucleic acid sequences may be screened and a sequence coding for either PTPLl or GLM-2 identified by any means which specifically selects for that sequence. Such means include (a) hybridization with an appropriate nucleic acid probe(s) containing a unique or εubεtantially characteristic fragment of the desired DNA or cDNA (b) hybridization-selected tranεlational analysis in which native mRNA which hybridizes to the clone in question is tranεlated in vitro and the translation products are further characterized (c) if the cloned genetic sequences are themselves capable of expressing mRNA, immunoprecipitation of a translated PTPLl or GLM-2 recombinant product produced by the host containing the clone, or preferarably (d) by uεing a unique or εubstantially characteristic fragment of the desired sequence as a PCR primer to amplify those clones with which it hybridizes.
Preferably, the probe or primer is a εubstantially characteristic fragment of one of the disclosed εequenceε. More preferably, the probe iε a unique fragment of one of the diεcloεed sequences. In choosing a fragment, unique and εubstantially characteristic fragmentε can be identified by comparing the sequence of a proposed probe to the known sequences found in εequence databaεes. Alternatively, the entire PTPLl or GLM-2 sequence may be used as a probe. In a preferred embodiment, the probe is a 32P random-labeled unique fragment of the PTPLl or GLM-2 nucleic acid sequences disclosed herein. In a most preferred embodiment, the probe serves aε a PCR primer containing a unique or εubstantially characteristic fragment of the PTPLl or GLM-2 sequences disclosed herein.
The library to be screened may be DNA or cDNA. Preferably, a cDNA library iε εcreened. In a preferred embodiment, a U-343 MGa 31L human glioblaεtoma (Niεter, M. , et al. , (1988) Cancer Reε. 48:3910-3918) or AG1518 human fibroblaεt (Human Genetic Mutant Cell Repoεitory, Inεtitute for Medical Reεearch, Camden, NJ) cDNA library iε screened with a probe to a unique or substantially characteristic fragment of the PTPLl sequence. Because PTPLl is expressed in a wide variety of tissues, cDNA libraries from many tisεueε may be employedN n another preferred embodiment, a λgtlO human brain cDNA library (Clontech, Calif.) iε εcreened with a probe to a unique or εubstantially characteristic fragment of the GLM-2 sequence. Because expression of GLM-2 appears to be high in brain tiεεues but low or absent in other tisεueε teεted, a brain cDNA library iε recommended for the cloning of GLM-2.
The εelected fragmentε may be cloned into any of a great number of vectors known to those of ordinary εkill in the art. In one preferred embodiment, the cloning vector is a plasmid εuch aε pUClδ or Blueεcript (Stratagene) . The cloned εequenceε εhould be examined to determine whether or not they contain the entire PTPLl or GLM-2 εequenceε or deεired portionε thereof. A εerieε of overlapping cloneε of partial εequenceε may be εelected and combined to produce a complete sequence by methods well known in the art.
In an alternative embodiment of cloning a PTPLl or GLM-2 nucleotide sequence, a library is prepared using an expreεεion vector. The library iε then εcreened for cloneε which expreεε the PTPLl or GLM-2 protein, for example, by εcreening the library with antibodieε to the protein or with labeled probes for the desired RNA εequences or by asεaying for PTPLl or GLM-2 PTP activity on a phoεphorylated εubεtrate such as para-nitrylphenyl phosphate. The above discussed methods are, therefore, capable of identifying cloned genetic sequences which are capable of expressing PTPLl or GLM-2 PTPs, or variants or fragments thereof.
To expresε a PTPLl or GLM-2 PTP, variantε or fragments thereof, or PTPLl or GLM-2 anti-senεe RNA, and variants or fragments thereof, tranεcriptional and translational signalε recognizable by an appropriate hoεt are neceεεary. The cloned PTPLl or GLM-2 encoding sequences, obtained through the methods described above, and preferably in a double-εtranded form, may be operably joined to regulatory εequenceε in an expreεεion vector, and introduced into a hoεt cell, either prokaryote or eukaryote, to produce recombinant PTPLl or GLM-2 PTP, a variant or fragment thereof, PTPLl or GLM-2 anti-sense RNA, or a variant or fragment thereof.
Depending upon the purpose for which expresεion is desired, the host may be eukaryotic or prokaryotic. For example, if the intention is to εtudy the regulation of PTPLl or GLM-2 PTP in a εearch for inducerε or inhibitorε of its activity, the host iε preferably eukaryotic. In one preferred embodiment, the eukaryotic hoεt cellε are COS cells derived from monkey kidney. In a particularly preferred embodiment, the host cells are human fibroblasts. Many other eukaryotic hoεt cells may be employed aε is well known in the art. For example, it is known in the art that Xenopus oocytes comprise a cell system useful for the functional expreεεion of eukaryotic messenger RNA or DNA. This syεtem has, for example, been used to clone the εodium:glucoεe cotranεporter in rabbitε (Hediger, M.A. , et. al. , Proc. Natl. Acad. Sci. (USA) 84:2634-2637 (1987)). Alternatively, if the intention iε to produce large quantitieε of the PTPLl or GLM-2 PTPε, a prokaryotic expreεεion εyεtem iε preferred. The choice of an appropriate expreεεion εyεtem iε within the ability and discretion of one of ordinary skill in the art.
Depending upon which strand of the PTPLl or GLM-2 PTP encoding sequence is operably joined to the regulatory sequences, the expresεion vectors will produce either PTPLl or GLM-2 PTPε, variantε or fragmentε thereof, or will expreεε PTPLl and GLM-2 anti-sense RNA, variants or fragmentε thereof. Such PTPLl and GLM-2 anti-εenεe RNA may be used to inhibit expresεion of the PTPLl or GLM-2 PTP and/or the replication of thoεe sequences.
Expresεion of a protein in different hoεtε may result in different post-translational modificationε which may alter the propertieε of the protein. This is particularly true when eukaryotic genes are expressed in prokaryotic hostε. In the preεent invention, however, thiε is of less concern as PTPLl and GLM-2 are cytoplasmic PTPs and are unlikely to be post-translationally glycosylated.
Transcriptional initiation regulatory sequences can be selected which allow for repreεεion or activation, εo that expreεεion of the operably joined εequenceε can be modulated. Such regulatory sequences include regulatory sequenceε which are temperature-εenεitive εo that by varying the temperature, expreεεion can be repressed or initiated, or which are εubject to chemical regulation by inhibitorε or inducers. Also of intereεt are conεtructs wherein both PTPLl or GLM-2 mRNA and PTPLl or GLM-2 anti-sense RNA are provided in a transcribable form but with different promoterε or other tranεcriptional regulatory elements such that induction of PTPLl or GLM-2 mRNA expresεion is accompanied by represεion of the expreεεion of the correεponding anti-εenεe RNA, or alternatively, repreεsion of PTPLl or GLM-2 mRNA expression is accompanied by induction of expresεion of the corresponding anti-senεe RNA. Tranεlational εequenceε are not necessary when it is deεired to expreεε PTPLl and GLM-2 anti-εenεe RNA sequences.
A non-transcribed and/or non-translated sequence 5' or 3 ' to the εequence coding for PTPLl or GLM-2 PTP can be obtained by the above-described cloning methods using one of the probes discloεed herein to εelect a clone from a genomic DNA library. A 5' region may be uεed for the endogenouε regulatory sequences of the PTPLl or GLM-2 PTP. A 3'-non-transcribed region may be utilized for a transcriptional termination regulatory sequence or for a translational termination regulatory εequence. Where the native regulatory sequences do not function satiεfactorily in the host cell, then exogenous sequenceε functional in the hoεt cell may be utilized.
The vectorε of the invention further comprise other operably joined regulatory elements εuch aε DNA elementε which confer tiεεue or cell-type εpecific expression of an operably joined coding sequence.
Oligonucleotide probes derived from the nucleotide εequence of PTPLl or GLM-2 can be uεed to identify genomic or cDNA library cloneε poεεessing a related nucleic acid εequence such as an allelic variant or homologous εequence. A suitable oligonucleotide or set of oligonucleotides, which iε capable of encoding a fragment of the PTPLl or GLM-2 coding εequenceε, or a PTPLl or GLM-2 anti-εenεe complement of εuch an oligonucleotide or set of oligonucleotides, may be εynthesized by means well known in the art (see, for example, Synthesis and Application of DNA and RNA, S.A. Narang, ed., 1987, Academic Press, San Diego, CA) and employed as a probe to identify and isolate a cloned PTPLl or GLM-2 εequence, variant or fragment thereof by techniqueε known in the art. Aε noted above, a unique or εubεtantially characteriεtic fragment of a PTPLl or GLM-2 εequence disclosed herein is preferred. Techniques of nucleic acid hybridization and clone identification are diεcloεed by Sambrook, et al . , Molecular Cloning, A Laboratory Manual, 2d ed. , Cold Spring Harbor Laboratory Preεε, Plainview, NY (1989), and by Hameε, B.D., et al. , in Nucleic Acid Hybridization, A Practical Approach, IRL Preεs, Washington, DC (1985). To facilitate the detection of a desired PTPLl or GLM-2 nucleic acid sequence, whether for cloning purpoεeε or for the mere detection of the preεence of PTPLl or GLM-2 εequences, the above-described probeε may be labeled with a detectable group. Such a detectable group may be any material having a detectable phyεical or chemical property. Such materialε have been we11-developed in the field of nucleic acid hybridization and in general moεt any label useful in such methods can be applied to the present invention. Particularly useful are radioactive labels. Any radioactive label may be employed which provideε for an adequate εignal and haε a εufficient half-life. If εingle εtranded, the oligonucleotide may be radioactively labeled using kinase reactionε. Alternatively, oligonucleotideε are alεo uεeful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group. See, for example, Leary, J.J., et al■ , Proc. Natl. Acad. Sci. (USA) 80:4045 (1983); Renz, M. et al ■ , Nucl. Acids Res. 12:3435 (1984); and Renz, M. , EMBO J. 6:817 (1983).
By using the sequences discloεed herein aε probeε or aε primerε, and techniqueε such as PCR cloning and colony/plaque hybridization, it is within the abilities of one εkilled in the art to obtain human allelic variantε and sequences subεtantially similar or homologous to PTPLl or GLM-2 nucleic acid εequenceε from species including mouse, rat, rabbit and non-human primates. Thus, the present invention is further directed to mouεe, rat, rabbit and primate PTPLl and GLM-2.
In particular the protein sequenceε disclosed herein for PTPLl and GLM-2 may be used to generate sets of degenerate probes or PCR primers useful in isolating εimilar and potentially evolutionarily εimilar εequences encoding proteinε related to the PTPLl or GLM-2 PTPε. Such degenerate probeε may not be εubεtantially εimilar to any fragments of the PTPLl or GLM-2 nucleic acid sequences but, as derived from the protein εequenceε diεcloεed herein, are intended to fall within the εpirit and εcope of the claimε.
Antibodies to PTPLl and GLM-2.
In the following deεcription, reference will be made to variouε methodologieε well-known to those skilled in the art of immunology. Standard reference works setting forth the general principles of immunology include Catty, D. Antibodies, A Practical Approach, Vols. I and II, IRL Press, Washington, DC (1988); Klein, J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley & Sons, New York (1982); Kennett, R. , et al. in Monoclonal Antibodies, Hybridoma: A New Dimenεion in Biological Analyεeε, Plenum Press, New York (1980); Campbell, A., "Monoclonal Antibody Technology," in Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon, R. , et al ■ , eds. ) , Elεevier, Amsterdam (1984); and Eisen, H.N., in Microbiology, 3rd Ed. (Davis, B.D., et al■ , eds.) Harper & Row, Philadelphia (1980) .
The antibodies of the present invention are prepared by any of a variety of methodε. In one embodiment, purified PTPLl or GLM-2 PTP, a variant or a fragment thereof, iε adminiεtered to an animal in order to induce the production of εera containing polyclonal antibodieε that are capable of binding the PTP, variant or fragment thereof.
The preparation of antiεera in animalε iε a well known technique (εee, for example, Chard, Laboratory Techniques in Biology, "An Introduction to Radioimmunoasεay and Related Techniques," North Holland Publishing Company (1978), pp. 385-396; and Antibodies, A Practical Handbook, Vols. I and II, D. Catty, ed., IRL Presε, Waεhington, D.C. (1988)). The choice of animal iε uεually determined by a balance between the facilitieε available and the likely requirements in terms of volume of the resultant antiserum. A large species such as goat, donkey and horse may be preferred, because of the larger volumes of serum readily obtained. However, it iε alεo possible to use smaller species such aε rabbit or guinea pig which often yield higher titer antiεera. Uεually, a εubcutaneous injection of the antigenic material (the protein or fragment thereof or a hapten-carrier protein conjugate) is used. The detection of appropriate antibodies may be carried out by testing the antiεera with appropriately labeled tracer-containing molecules. Fractions that bind tracer-containing moleculeε are then isolated and further purified if necesεary.
Cellε expressing PTPLl or GLM-2 PTP, a variant or a fragment thereof, or, a mixture of such proteins, variants or fragments, can be administered to an animal in order to induce the production of sera containing polyclonal antibodies, some of which will be capable of binding the PTPLl or GLM-2 PTP. If desired, εuch PTPLl or GLM-2 antibody may be purified from other polyclonal antibodieε by εtandard protein purification techniques and especially by affinity chromatography with purified PTPLl or GLM-2 protein or variants or fragments thereof.
A PTPLl or GLM-2 protein fragment may also be chemically synthesized and purified by HPLC to render it subεtantially pure. Such a preparation iε then introduced into an animal in order to produce polyclonal antiεera of high εpecific activity. In a preferred embodiment, the protein may be coupled to a carrier protein εuch as bovine serum albumin or keyhole limpet hemocyanin (KLH), and and uεed to immunogenize a rabbit utilizing techniques well-known and commonly used in the art. Additionally, the PTPLl or GLM-2 protein can be admixed with an immunologically inert or active carrier. Carriers which promote or induce immune responses, such aε Freund'ε complete adjuvant, can be utilized.
Monoclonal antibodieε can be prepared uεing hybridoma technology (Kohler et al. , Nature 256:495 (1975); Kohler, et al. , Eur. J. Immuno1. 6:511 (1976); Kohler, et al . , Eur. J. Immunol. 6:292 (1976); Hammerling, et al . , in Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y. , pp. 563-681 (1981)). In general, such procedures involve immunizing an animal with PTPLl or GLM-2 PTP, or a variant or a fragment thereof. The splenocytes of εuch animalε are extracted and fuεed with a εuitable myeloma cell line. After fuεion, the reεulting hybridoma cellε are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wandε, J.R., et al. , Gaεtro-enterology 80:225-232 (1981), which reference iε herein incorporated by reference. The hybridoma cells obtained through such a εelection are then aεεayed to identify cloneε which εecrete antibodies capable of binding the PTP and/or the PTP antigen. The proliferation of transfected cell lines is potentially more promising than clasεical myeloma technology, using methods available in the art.
Through application of the above-deεcribed methodε, additional cell lines capable of producing antibodies which recognize epitopes of the PTPLl and GLM-2 PTPs can be obtained.
These antibodies can be used clinically as markers (both quantitative and qualitative) of the PTPLl and GLM-2 PTPs in brain, blastoma or other tiεεue. Additionally, the antibodieε are uεeful in a method to aεsess PTP function in cancer or other patients.
The method whereby two antibodies to PTPLl were produced iε outlined in Example 5.
Substantially pure PTPLl and GLM-2 proteinε.
A variety of methodologies known in the art can be utilized to obtain a purified PTPLl or GLM-2 PTP. In one method, the protein is purified from tisεues or cells which naturally produce the protein. Alternatively, an expresεion vector may be introduced into cellε to cauεe production of the protein. For example, human fibroblaεt or monkey kidney COS cellε may be employed. In another embodiment, mRNA tranεcriptε may be microinjected into cells, such as Xenopus oocytes or rabbit reticulocyteε. In another embodiment, mRNA is used with an in vitro tranεlation εyεtem. In preferred embodiment, bacterial cellε are used to make large quantities of the protein. In a particularly preferred embodiment, a fusion protein, εuch aε a bacterial GST fusion (Pharmacia) may be employed, the fusion product purified by affinity chromatography, and the PTPLl or GLM-2 protein may be releaεed from the hybrid by cleaving the amino acid εequence joining them.
In light of the preεent diεclosure, one skilled in the art can readily follow known methods for isolating proteins in order to obtain subεtantially pure PTPLl or GLM-2 PTP, free of natural contaminants. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chro atography, ion-exchange chromatography, and immuno-affinity chromatography.
Determinations of purity may be performed by physical characterizationε (εuch aε molecular maεε in size fractionation) , immunological techniques or enzymatic asεayε.
PTPLl or GLM-2 PTP, variants or fragments thereof, purified in the above manner, or in a manner wherein equivalents of the above sequence of steps are utilized, are useful in the preparation of polyclonal and monoclonal antibodies, for pharmaceutical preparationε to inhibit or enhance PTP activity and for in vitro dephoεphorylationε.
Variants of PTPLl and GLM-2 nucleic acids and proteins.
Variants of PTPLl or GLM-2 having an altered nucleic acid sequence can be prepared by mutageneεiε of the DNA. Thiε can be accompliεhed using one of the mutagenesis procedureε known in the art.
Preparation of variantε of PTPLl or GLM-2 are preferably achieved by εite-directed mutageneεiε. Site-directed mutagenesis allows the production of variants of these PTPs through the use of a specific oligonucleotide which containε the deεired mutated DNA εequence.
Site-directed mutagenesis typically employs a phage vector that exists in both a single-εtranded and double-stranded form. Typical vectors uεeful in εite-directed mutagenesis include vectors εuch as the M13 phage, as disclosed by Messing, et al■ , Third Cleveland Sympoεium on Macromoleculeε and Recombinant DNA, A. Walton, ed. , Elsevier, Amsterdam (1981), the discloεure of which is incorporated herein by reference. These phage are commercially available and their use is generally well known to those skilled in the art. Alternatively, plasmid vectorε containing a εingle-εtranded phage origin of replication (Veira, et al■ , Meth. Enzymol . 153:3 (1987)) may be employed to obtain single-stranded DNA.
In general, site-directed mutagenesis in accordance herewith is performed by firεt obtaining a εingle-stranded vector that includes within itε sequence the DNA sequence which is to be altered. An oligonucleotide primer bearing the desired mutated εequence iε prepared, generally synthetically, for example by the method of Crea, et al. , Proc. Natl. Acad. Sci. (USA) 75:5765 (1978). The primer iε then annealed with the εingle-εtranded vector containing the εequence which is to be altered, and the created vector is incubated with a DNA-polymerizing enzyme such as E. coli polymerase I Klenow fragment in an appropriate reaction buffer. The polymerase will complete the εyntheεiε of a mutation-bearing εtrand. Thuε, the εecond εtrand will contain the deεired mutation. Thiε heteroduplex vector is then used to tranεform appropriate cellε and cloneε are εelected that contain recombinant vectorε bearing the mutated sequence.
While the site for introducing a sequence variation is predetermined, the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at a target region and the newly generated sequenceε can be screened for the optimal combination of desired activity. One skilled in the art can evaluate the functionality of the variant by routine εcreening aεεays.
The present invention further comprises fusion productε of the PTPLl or GLM-2 PTPs. As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the firεt methionine. The preεence of εuch codonε between a eukaryotic promoter and a PTPLl or GLM-2 εequence reεultε either in the formation of a fuεion protein (if the ATG codon iε in the εame reading frame aε the PTP encoding DNA εequence) or a frame-εhift mutation (if the ATG codon iε not in the εame reading frame aε the PTP encoding εequence) . Fuεion proteinε may be conεtructed with enhanced immunoεpecificity for the detection of theεe PTPε. The εequence coding for the PTPLl or GLM-2 PTP may alεo be joined to a signal sequence which will allow secretion of the protein from, or the compartmentalization of the protein in, a particular hoεt. Such signal sequences may be designed with or without specific proteaεe εiteε εuch that the εignal peptide εequence iε amenable to εubsequent removal.
The invention further provides detectably labeled, immobilized and toxin conjugated forms of PTPLl and GLM-2 PTPε, and variantε or fragments thereof. The production of such labeled, immobilized or toxin conjugated formε of a protein are well known to thoεe of ordinary εkill in the art. While radiolabeling represents one embodiment, the PTPε or variants or fragments thereof may also be labeled using fluorescent labels, enzyme labels, free radical labels, avidin-biotin labels, or bacteriophage labels, uεing techniqueε known to the art (Chard, Laboratory Techniqueε in Biology, "An Introduction to Radioimmunoaεεay and Related Techniques," North Holland Publishing Company (1978)).
Typical fluorescent labels include fluoreεcein iεothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluoreεcamine.
Typical chemilumineεcent compounds include luminol, isoluminol, aromatic acridinium eεters, imidazoles, and the oxalate esterε.
Typical biolumineεcent compounds include luciferin, and luciferase. Typical enzymes include alkaline phosphatase, β-galactosidaεe, glucose-6-phosphate dehydrogenaεe, maleate dehydrogenaεe, glucoεe oxidase, and peroxidase. Transformed cells, cell lines and hosts.
To transform a mammalian cell with the nucleic acid εequences of the invention many vector syεtems are available depending upon whether it is deεired to inεert the recombinant DNA conεtruct into the hoεt cell's chromosomal DNA, or to allow it to exist in an extrachromoεomal form. If the PTPLl or GLM-2 PTP coding εequence, along with an operably joined regulatory εequence iε introduced into a recipient eukaryotic cell aε a non-replicating DNA (or RNA) molecule, the expreεεion of PTPLl or GLM-2 PTP may occur through the tranεient expreεεion of the introduced εequence. Such a non-replicating DNA (or RNA) molecule may be a linear molecule or, more preferably, a closed covalent circular molecule which is incapable of autonomous replication.
In a preferred embodiment, genetically εtable tranεformantε may be conεtructed with vector systemε, or tranεformation εyεtemε, whereby recombinant PTPLl or GLM-2 PTP DNA iε integrated into the hoεt chromoεome. Such integration may occur de novo within the cell or, in a moεt preferred embodiment, be aεεiεted by tranεformation with a vector which functionally inεertε itεelf into the host chromosome with, for example, retro vectors, transpoεons or other DNA elements which promote integration of DNA sequenceε in chromosomes. A vector is employed which is capable of integrating the desired εequenceε into a mammalian hoεt cell chromoεome. In a preferred embodiment, the tranεformed cellε are human fibroblaεtε. In another preferred embodiment, the tranεformed cellε are monkey kidney COS cellε.
Cells which have stably integrated the introduced DNA into their chromoεomeε may be εelected by alεo introducing one or more markers which allow for selection of hoεt cells which contain the expresεion vector in the chromoεome, for example the marker may provide biocide reεiεtance, e.g., reεiεtance to antibiotics, or heavy metals, such aε copper, or the like. The selectable marker can either be directly 1inked to the DNA sequences to be expressed, or introduced into the same cell by co-transfection.
In another embodiment, the introduced sequence is incorporated into a vector capable of autonomous replication in the recipient hoεt. Any of a wide variety of vectorε may be employed for this purpose, as outlined below.
Factors of importance in selecting a particular plasmid or vector include: the eaεe with which recipient cellε that contain the vector may be recognized and εelected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is deεirable to be able to "εhuttle" the vector between hoεt cellε of different species.
Preferred eukaryotic plasmidε include thoεe derived from the bovine papilloma viruε, SV40, and, in yeaεt, plaεmidε containing the 2-micron circle, etc., or their derivativeε. Such plaεmidε are well known in the art (Botεtein, D., et al . , Miami Wntr. Symp. 19:265-274 (1982); Broach, J.R., in The Molecular Biology of the Yeaεt Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, J.R., Cell 28:203-204 (1982); Bolion, D.P., et al■ , J. Clin. Hematol . Oncol. 10:39-48 (1980); Maniatis, T., in Cell Biology: A Comprehenεive Treatise, Vol. 3, Gene Expression, Academic Press, NY, pp. 563-608 (1980)), and are commercially available. For example, mammalian expreεεion vector εystems which utilize the MSV-LTR promoter to drive expresεion of the cloned gene and with which it is possible to co-transfect with a helper viruε to amplify plaεmid copy number and to integrate the plasmid into the chromosomes of host cells have been deεcribed (Perkinε, A.S., et al. , Mol. Cell Biol. 3:1123 (1983); Clontech, Palo Alto, California).
Once the vector or DNA sequence is prepared for expresεion, it is introduced into an appropriate host cell by any of a variety of suitable meanε, including tranεfection. After the introduction of the vector, recipient cells may be grown in a selective medium, which selectε for the growth of vector-containing cellε. Expreεεion of the cloned nucleic acid sequence(s) reεults in the production of PTPLl or GLM-2 PTP, or the production of a variant or fragment of the PTP, or the expression of a PTPLl or GLM-2 anti-sense RNA, or a variant or fragment thereof. This expreεεion can take place in a transient manner, in a continuous manner, or in a controlled manner aε, for example, expression which follows induction of differentiation of the tranεformed cellε (for example, by adminiεtration of bromodeoxyuracil to neuroblaεtoma cellε or the like) .
In another embodiment of the invention the hoεt iε a human hoεt. Thuε, a vector may be employed which will introduce into a human with deficient PTPLl or GLM-2 PTP activity, operable PTPLl or GLM-2 εeguenceε which can εupplement the patient'ε endogenous production. In another embodiment, the patient suffers from a cancer caused by an oncogene which is a protein tyroεine kinaεe (PTK) . A vector capable of expreεsing the PTPLl or GLM-2 protein is introduced within the patient to counteract the PTK activity.
The recombinant PTPLl or GLM-2 PTP cDNA coding sequences, obtained through the methods above, may be used to obtain PTPLl or GLM-2 anti-εenεe RNA εequenceε. An expreεsion vector may be constructed which containε a DNA εequence operably joined to regulatory εequenceε εuch that the DNA εequence expreεεeε the PTPLl or GLM-2 anti-εense RNA sequence. Tranεformation with thiε vector results in a host capable of expreεsion of a PTPLl or GLM-2 anti-sense RNA in the transformed cell. Preferably such expreεεion occurε in a regulated manner wherein it may be induced and/or repreεεed aε desired. Most preferably, when expreεεed, anti-εense PTPLl or GLM-2 RNA interacts with an endogenous PTPLl or GLM-2 DNA or RNA in a manner which inhibitε or repreεεeε tranεcription and/or tranεlation of the PTPLl or GLM-2 PTP DNA εequenceε and/or mRNA tranεcripts in a highly specific manner. Uεe of anti-εenεe RNA probeε to block gene expreεεion iε discusεed in Lichtenεtein, C. , Nature 333:801-802 (1988) .
Assays for agonists and antagonists.
The cloning of PTPLl and GLM-2 now makes posεible the production and uεe of high through-put aεεayε for the identification and evaluation of new agoniεtε (inducerε/enhancers) and antagonistε (repreεεorε/inhibitorε) of PTPLl or GLM-2 PTPε for therapeutic strategies using εingle or combinationε of drugε. The aεεay may, for example, test for PTPLl or GLM-2 PTP activity in transfected cells (e.g. fibroblasts) to identify drugs that interfere with, enhance, or otherwise alter the expression or regulation of these PTPs. In addition, probes developed for the discloεed PTPLl and GLM-2 nucleic acid sequences or proteins (e.g. DNA or RNA probeε or or primerε or antibodieε to the proteins) may be used as qualitative and/or quantitative indicators for the PTPε in cell lyεates, whole cells or whole tisεue.
In a preferred embodiment, human fibroblast cells are transformed with the PTPLl or GLM-2 PTP εequenceε and vectorε diεcloεed herein. The cellε may then be treated with a variety of compoundε to identify thoεe which enhance or inhibit PTPLl or GLM-2 tranεcription, tranεlation, or PTP activity. In addition, assayε for PDGF (platelet derived growth factor) signalling, cell growth, chemotaxis, and actin reorganization are preferred to asεeεε a compound'ε affect on PTPLl or GLM-2 PTP tranεcription, tranεlation or activity.
In another embodiment, the ability of a compound to enhance or inhibit PTPLl or GLM-2 PTP activity is assayed in vitro. Using the εubεtantially pure PTPLl or GLM-2 PTPε diεcloεed herein, and a detectable phoεphorylated substrate, the ability of various compounds to enhance or inhibit the phosphataεe activity of PTPLl or GLM-2 may be assayed. In a particularly preferred embodiment the phosphorylated εubstrate is para-nitrylphenyl phosphate (which turnε yellow upon dephosphorylation) .
In another embodiment, the ability of a compound to enhance or inhibit PTPLl or GLM-2 transcription iε aεεayed. Uεing the PTPLl or GLM-2 cDNA εequenceε diεcloεed herein, one of ordinary εkill in the art can clone the 5' regulatory εequenceε of the PTPLl or GLM-2 geneε. Theεe regulatory εequences may then be operably joined to a sequence encoding a marker. The marker may be an enzyme with an easily asεayable activity or may cauεe the hoεt cellε to change phenotypically or in their εenεitivity or reεiεtance to certain molecules. A wide variety of markers are known to those of ordinary skill in the art and appropriate markers may be choεen depending upon the hoεt uεed. Compoundε which may alter the tranεcription of PTPLl or GLM-2 PTP may be tested by exposing cells transformed with the PTPLl or GLM-2 regulatory sequences operably joined to the marker and asεaying for increaεed or decreaεed expression of the marker.
The following examples further describe the particular materials and methods used in developing and carrying out some of the embodimentε of the present invention. These exampleε are merely illustrative of techniques employed to date and are not intended to limit the scope of the invention in any manner.
EXAMPLE 1 Original Cloning of PTPLl
All cells, unless stated otherwise, were cultured in Dulbeco Modified Eagles Medium (DMEM Gibco) supplemented with 10% Fetal Calf Serum (FCS, Flow Laboratories), 100 unitε of penicillin, 50 μg/ml εtrepto ycin and glutamine. The human glioma cell line uεed was U-343 MGa 31L (Nister, M., et al. , (1988) Cancer Reε. 48:3910-3918). The AG1518 human foreskin fibroblaεtε were from the Human Genetic Mutant Cell Repoεitory, Inεtitute for Medical Research, Camden, NJ.
RNA was prepared from U-343 MGa 31L cellε or AG1518 human fibroblaεtε by guanidine thiocyanate (Merck, Darmεtadt) extraction (Chirgwin et al. , 1979). Briefly, cellε were harvested, washed in phoεphate buffered εaline (PBS), and lyεed in 4 M guanidine thiocyanate containing 25 mM sodium citrate (pH 7.0) and 0.1 M 2-mercaptoethanol. RNA was sedimented through 5.7 M cesium chloride, the RNA pellet waε then dissolved in 10 mM Tris hydrochloride (pH 7.5), 5 mM EDTA (TE buffer), extracted with phenol and chloroform, precipitated with ethanol, and the final pellet stored at -70°C or resuεpended in TE buffer for subsequent manipulations. Polyadenylated [poly(A)+] RNA was prepared by chromatography on oligo (dT)-cellulose aε deεcribed in Maniatis et al■ , 1982.
Poly(A)+ RNA (5 μg) from U-343 MGa 31 L cells waε uεed to make a cDNA library by oligo (dT)-primed cDNA εyntheεiε uεing an Amerεham λgtlO cDNA cloning εyεtem. Similarly, a random and oligo (dT) primed cDNA library waε prepared from AG1518 fibroblaεtε uεing 5 μg of poly(A)+ RNA, a RiboClone cDNA εynthesis εyεtem (Promega Corporation, Madison, Wl . , USA), a Lambda ZAPII synthesiε kit (Stratagene), and Gigapack Gold II packaging extract (Stratagene). Degenerate primers were designed based on conserved amino acid-regions of known PTP sequenceε and were εynthesized using a Gene Assembler Plus (Pharmacia-LKB) . Sense oligonucleotides corresponded to the εequenceε FWRM I/V WEQ (5'- TTCTGG A/C GNATGATNTGGGAACA-3 ' , 23mer with 32-fold degeneracy) and KC A/D Q/E YWP (5'-AA A/G TG C/T GANCAGTA C/T TGGCC-3 ' , 20mer with 32-fold degeneracy), and the anti-εense oligonucleotide was based on the εequence HCSAG V/I G (5'-CCNACNCC A/C GC A/G CTGCAGTG-3 ' , 20mer with 64-fold degeneracy). Unpackaged template cDNA from the U-343 MGa 31L library (100 ng) waε amplified uεing Tag polymeraεe (Perkin Elmer-Cetuε) and 100 ng of either sense primer in combination with 100 ng of the anti-sense primer as described (Saiki et al. , 1985) . PCR was carried out for 25 cycles each conεisting of denaturation at 94°C for 30 sec, annealing at 40°C for 2 min followed by 55°C for 1 min, and extension at 72°C for 2 min. The PCR products were εeparated on a 2.0% low gelling temperature agaroεe gel (FMC BioProductε, Rockland, USA) and DNA fragments of approximately 368 base pairε (with FWRM sense primer) and approximately 300 bp (with KC A/D Q sense primer) were excised, eluted from the gel, subcloned into a T-tailed vector (TA Cloning Kit, Invitrogen Corporation, San Diego, CA, USA), and εequenced.
Nucleotide sequences from several of the PCR cDNA cloneε analyεed were representative of both cytoplasmic and receptor types of PTPs. Thirteen cloneε encoded cytoplaεmic enzy eε including MEG (Gu et al. , 1991; 8 cloneε), PTPHl (Yang and Tonkε, 1991; 2 cloneε), P19PTP (den Hertog et al■ , 1992), and TC-PTP (Cool et al. , 1989, one clone); 11 clones encoded receptor-type enzymes such as HPTP-α (Kruger et al■ , 1990, 7 clones), HPTP-γ (Kruger et al. , 1990, 3 clones) and HPTP-δ (Kruger et al■ , 1990, 1 clone), and three clones defined novel PTP εequences. Two of these were named PTPLl and GLM-2.
The U-343 MGa 31L cDNA library waε εcreened with
32 P-random prime-labeled (Megapπme Kit, Amerεham) approximately 368 bp inεertε correεponding to PTPLl as described elsewhere (Huynh et al. , 1986); clone X6.15 was iεolated, exciεed from purified phage DNA by Eco RI (Biolabε) digeεtion and εubcloned into pUC18 for εequencing. All other cDNA cloneε were iεolated from the AG1518 human fibroblaεt cDNA library which waε εcreened with 32P-labeled X6.15 inεert and with εubεequently iεolated partial cDNA clones.
Double-stranded plaεmid DNA waε prepared by a εingle-tube mini preparation method (Del Sal et al. , 1988) or uεing Magic mini or maxiprep kitε (Promega) according to the manufacturer'ε specifications. Double-stranded DNA was denatured and used aε template for εequencing by the dideoxynucleotide chain-termination procedure with T7 DNA polymeraεe (Pharmacia-LKB) , and M13-univerεal and reverεe primerε or εynthetic oligonucleotideε derived from the cDNA εequenceε being determined. The complete 7395 bp open reading frame of PTPLl, waε derived from εix overlapping cDNA cloneε totalling 8040 bp and predictε a protein of 2465 amino acids with an approximate molecular maεε of 275 kDa. The 8040 bp εequence is disclosed as SEQ ID NO.: 1.
EXAMPLE 2 Original Cloning of GLM-2
The human glioma cell line U-343 MGa 31L (Nister, M. , et al. , (1988) Cancer Reε. 48:3910-3918) waε cultured in Dulbecco' Modified Eagles Medium (DMEM Gibco) supplemented with 10% Fetal Calf Serum (FCS, Flow Laboratories), 100 unitε of penicillin, 50 μg/ml εtreptomycin and 2mM glutamine.
Total RNA waε prepared from U-343 MGa 31L cellε by guanidine thiocyanate (Merck, Darmεtadt) extraction (Chirgwin, et al■ , 1979). Briefly, cellε were harveεted, waεhed in phoεphate buffered εaline (PBS), and lyεed in 4 M guanidine thiocyanate containing 25mM εodium citrate (pH 7.0) and 0.1 M 2-mercaptoethanol . RNA waε sedimented through 5.7 M cesium chloride, the RNA pellet was then diεεolved in 10 mM Triε hydrochloride (pH 7.5), 5 mM EDTA (TE buffer), extracted with phenol and chloroform, precipitated with ethanol, and the final pellet εtored at -70°C or reεuεpended in TE buffer for εubεequent manipulations. Polyadenylated [poly(A)+] RNA was prepared by chromatography on oligo (dT)-cellulose aε deεcribed in Maniatiε et al . (1982).
Poly(A)+ RNA (5 μg) iεolated from U-343 MGa 31L cellε waε uεed to make a cDNA library by oligo (dT)-primed cDNA εyntheεiε uεing an Amerεham λgtlO cDNA cloning εyεtem. Degenerate primerε were designed based on conserved amino acid regions of known PTP sequenceε, and εynthesized uεing a Gene Aεεembler Pluε (Pharmacia-LKB) . Senεe oligonucleotides correεponded to the εequences FWRM I/V WEQ (5'-TTCTGG A/C GNATGATNTGGGAACA-3' , 23mer with 32-fold degeneracy=primer PI) and KC A/D Q/E YWP (5'-AA A/G TG C/T GANCAGTA C/T TGGCC-3' , 20mer with 32-fold degeneracy=primer P2), and the anti-sense oligonucleotide was baεed on the εequence HCSAG V/I G (5'-CCNACNCC A/C GC A/G CTGCAGTG-3' , 20mer with 64-fold degeneracy=primer P3). Unpackaged template cDNA from the U-343 MGa 31L library (100 ng) was amplified using Tag polymeraεe (Perkin Elmer-Cetuε) and 100 ng of either εenεe primer in combination with 100 ng of the anti-εenεe primer aε deεcribed (Saiki, et al. , 1985). PCR waε carried out for 25 cycleε each conεisting of denaturation at 94°C for 30 sec, annealing at 40°C for 2 min followed by 55°C for 1 min, and extension at 72°C for 2 min. The PCR products were separated on a 2.0% low gelling temperature agarose gel (FMC BioProductε, Rockland, USA) and DNA fragmentε of approximately 368 baεe pairε (with FWRM sense primer) and approximately 300 bp (with KC A/D Q senεe primer) were exciεed, eluted from the gel, εubcloned into a T-tailed vector (TA Cloning Kit, Invitrogen Corporation, San Diego, CA, USA), and εequenced. Double-stranded plasmid DNA was prepared by a εingle-tube mini preparation method (Del Sal, et al ■ , 1988) or by uεing Magic mini or maxiprep kitε (Pro ega) according to the manufacturer ' ε εpecificationε. Double-εtranded DNA waε denatured and uεed aε template for εequencing by the dideoxynucleotide chain-termination procedure (Sanger, et al ■ , 1977) with T7 DNA polymerase (Pharmacia-LKB), and M13-universal and reverεe primerε or, in the caεe of cDNA cloneε iεolated from the brain cDNA library, uεing alεo εynthetic oligonucleotides derived from the cDNA sequenceε being determined.
A human brain cDNA library conεtructed in λgtlO
(Clontech, Calif.) waε screened as described elεewhere
(Huynh, et al . , 1986) with 32P-random prime-labeled
(Megaprime Kit, Amerεham) approximately 360 bp inserts correεponding to GLM-2. Clone HBM1 was isolated, exciεed from purified phage DNA by Eco RI (Biolabs) digestion and subcloned into the plaεmid vectorε pUC18 or Blueεcript (Stratagene) for εequencing. The resulting sequence iε diεcloεed aε SEQ ID NO. : 3.
EXAMPLE 3 Tissue-Specific Expression of PTPLl
Total RNA (20 μg) or poly(A)+ RNA (2 μg) denatured in formaldehyde and formamide was separated by electrophoresis on a formaldehyde/1% agarose gel and transferred to nitrocellulose. The filters were hybridized for 16 hrε at 42°C with 32P-labeled probeε in a εolution containing 5x εtandard εaline citrate (SSC; lx SSC iε 50 M εodium citrate, pH 7.0, 150 mM εodium chloride), 50% formamide, 0.1% sodium dodecyl sulfate (SDS), 50 mM sodium phosphate and 0.1 mg/ml salmon sperm DNA. All probeε were labeled by random priming (Feinberg and Vogelεtein, 1983) and unincorporated 32P waε removed by Sephadex G-25
(Pharmacia-LKB) chromatography. Human tissue blots (Clontech, Calif.) were hybridized with PTPLl specific probes according to manufacturer's specifications. Filters were washed twice for 30 min at 60°C in 2x SSC/0.1% SDS, once for 30 min at 60°C in 0.5x SSC/0.1% SDS, and exposed to X-ray film (Fuji, XR) with intensifying screen (Cronex Lighting Plus, Dupont) at -70°C.
Northern blot analysis of RNAs from various human tissues showed that the 9.5 kb PTPLl transcript iε expreεεed at different levelε with kidney, placenta, ovaries and testes showing high expresεion, compared to medium expreεεion in lung, pancreaε, proεtate and brain tiεεues, low in heart, skeletal muεcle, spleen, liver, small intestine and colon and virtually no detectable expression in leukocytes. EXAMPLE 4 Tisεue-Specific Expreεεion of GLM-2
To inveεtigate the expreεεion of GLM-2 mRNA in human tiεεueε, Northern blot analysis was performed on a commercially available filter (Clontech, California) containing mRNAs from human heart, brain, placenta, lung, liver, εkeletal muεcle, kidney and pancreaε tiεsue. The filter was hybridized according to manufacturer's εpecificationε with 32P-labeled GLM-2 PCR product aε probe, waεhed twice for 30 min at 60°C in 2x εtandard εaline citrate (SSC; lx SSC iε 50 mM εodium citrate, pH 7.0, 150 mM εodium chloride), containing 0.1% εodium dodecyl εulfate (SDS), once for 30 min at 60°C in 0.5x SSC/0.1% SDS, and expoεed to X-ray film (Fuji, RX) with intenεifying εcreen (Cronex Lighting Pluε, Dupont) at -70°C.
EXAMPLE 5 Production of PTPLl specific antisera
Rabbit antisera denoted αLlA and αLIB were prepared against peptides corresponding to amino acid residues 1802 to 1823 (PAKSDGRLKPGDRLIKVNDTDV) and 450 to 470 (DETLSQGQSQRPSRQYETPFE) , respectively, of PTPLl. The peptides were syntheεized in an Applied Biosyεtems 430A Peptide Syntheεizer uεing t-butoxycarbonyl chemiεtry and purified by reverεe phaεe high performance liquid chromatography. The peptideε were coupled to keyhole limpet hemocyanin (Calbiochem-Behring) uεing glutaraldehyde, aε deεcribed (Gullick, W.J., et al. , (1985) EMBO J. 4:2869-2877), and then mixed with Freund's adjuvant and used to Immunize a rabbit. The αLlA antiεerum waε purified by affinity chomatography on protein A-Sepharoεe CL4B (Pharmacia-LKB) aε deεcribed by the manufacturer. EXAMPLE 6 Tranεfection of the PTPLl cDNA Into COS-1 Cellε.
The full length PTPLl cDNA was conεtructed using overlapping clones and cloned into the SV40-based expression vector pSV7d (Truett, M.A. , et al. , (1985) DNA 4:333-349), and transfected into COS-1 cells by the calcium phosphate precipitation method (Wigler, M. , et al■ , (1979) Cell
16:777-785). Briefly, cellε were εeeded into 6-well cell
5 culture plates at a density of 5x10 cells/well, and transfected the following day with 10 μg of plasmid. After overnight incubation, cells were washed three times with a buffer containing 25 mM Tris-HCl, pH 7.4, 138 mM NaCl, 5 mM
KC1, 0.7 mM CaCl2, 0.5 mM MgCl2 and 0.6 mM Na2HP04, and then incubated with Dulbecco's modified Eagle'ε medium containing 10% fetal calf εerum and antibioticε. Two dayε after tranεfection, the cellε were uεed for metabolic labeling followed by immunoprecipitation and SDS-gel electrophoreεiε, or immunoprecipitation followed by dephoεphorylation experimentε.
EXAMPLE 7
Metabolic Labeling, Immunoprecipitation and
Electrophoreεis of PTPLl
Metabolic labeling of COS-1 cellε, AG1518 cellε, PC-3 cells, CCL-64 cellε, A549 cellε and PAE cellε waε performed for 4 h in methionine- and cysteine-free MCDB 104 medium (Gibco) with 150 μCi/ml of [ 35Slmethionine and [ 35Sjcysteine (in vivo labeling mix; Amersham) . After labeling, the cellε were εolubilized in a buffer containing
20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 0.5% Triton
X-100, 0.5% deoxycholate, 1.5% Trasylol (Bayer) and 1 mM phenylmethylεulfonyl fluoride (PMSF; Sigma) . After 15 min on ice, cell debriε waε removed by centrifugation. Sampleε (1 ml) were then incubated for 1.5 h at 4°C with either αLlA antibodieε or αLlA antibodieε preblocked with 10 μg of peptide. Immune complexeε were then mixed with 50 μl of a protein A-Sepharoεe (Pharmacia-LKB) εlurry (50% packed beadε in 150 mM NaCl, 20 mM Triε-HCl, pH 7.4, 0.2% Triton X-100) and incubated for 45 min at 4°C. The beadε were pelleted and waεhed four timeε with waεhing buffer (20 mM Triε-HCl, pH 7.4, 500 mM NaCl, 1% Triton X-100, 1% deoxycholate and 0.2% SDS), followed by one wash in distilled water. The immune complexes were eluted by boiling for 5 min in the SDS-sample buffer (100 mM Tris-HCl, pH 8.8, 0.01% bromophenol blue, 36% glycerol, 4% SDS) in the preεence of 10 mM dithiothreitol (DTT) , and analyzed by SDS-gel electrophoreεiε uεing 4-7% polyacrylamide gels (Blobel, G., and Dobberstein, B. (1975) J. Cell Biol. 67:835-851). The gel was fixed, incubated with Amplify (Amersham) for 20 min, dried and εubjected to fluorography.
EXAMPLE 8 Dephoεphorylation Aεεay for PTPLl
COS-1 cellε were lyεed in 20 mM Triε-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholate, 1.5% Trasylol, 1 mM PMSF and 1 M DTT, for 15 min. Lysates were cleared by centrifugation, 3 μl of the antiεerum αLIB, with or without preblocking with 10 μg peptide, were added and samples were incubated for 2 h at 4°C. Protein A-Sepharose εlurry (25 μl) was then added and incubation was prolonged another 30 min at 4°C. The beads were pelleted and washed four timeε with lysis buffer and one time with dephosphorylation assay buffer (25 mM imidazole-HCl, pH 7.2, 1 mg/ml bovine serum albumin and 1 mM DTT), and finally resuspended in dephoεphorylation aεεay buffer containing 2 μM myelin baεic protein 32P-labeled on tyrosine residueε by Baculo-viruε expressed intracellular part of the insulin receptor, kindly provided by A.J. Flint (Cold Spring Harbor Laboratory) and M.M. Cobb (University of Texas). After incubation for indicated times at 30°C, the reactions were stopped with a charcoal mixture (Streull, M. , et al■ , (1988) J. Exp. Med. 168:1523-1530) and the radioactivity in the supernatants waε determined by Cerenkov counting. For each ssaammppllee,, ]lysate correεpondmg to 5 cm 2 of confluent cellε was used,
It εhould be underεtood that the preceding iε merely a detailed deεcription of certain preferred embodimentε and exampleε of particular laboratory embodimentε. It therefore should be apparent to those skilled in the art that various modifications and equivalents can be made without departing from the spirit or scope of the invention aε defined in the appended claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: LUDWIG INSTITUTE FOR CANCER RESEARCH
(B) STREET: 1345 AVENUE OF THE AMERICAS
(C) CITY: NEW YORK
(D) STATE: NEW YORK
(E) COUNTRY: USA
(F) POSTAL CODE: 10105
(C) TELEPHONE: 212-765-3000
(i) APPLICANT/INVENTOR:
(A) NAME: GONEZ, LEONEL JORGE
(B) STREET: OVRE SLOTTSGATAN 11
(C) CITY: UPPSALA
(E) COUNTRY: SWEDEN
(F) POSTAL CODE: S-753 40
(G) TELEPHONE: 46-18- 17-41-46
(i) APPLICANT/INVENTOR:
(A) NAME: SARAS, JAN
(B) STREET: LINGSBERGSGATAN 15B
(C) CITY: UPPSALA
(E) COUNTRY: SWEDEN
(F) POSTAL CODE: S-752 40
(G) TELEPHONE: 46-18-17-41-46
(i) APPLICANT/INVF,NTOR:
(A) NAME: CLAESSON-WELSH, LENA
(B) STREET: GRANITVAGEN 16A
(C) CITY: UPPSALA
(E) COUNTRY: SWEDEN
(F) POSTAL CODE: S-752 43
(G) TELEPHONE: 46-18-17-41-46
( i ) APPLICANT/ INVENTO :
(A) NAME: HELD1N, CARL-HENRIK
(B) STREET: HESSELMAUS VAG 35
(C) CITY: UPPSALA
(E) COUNTRY: SWEDEN
(F) POSTA! CODE: S-7 2 6λ
(G) TELEPHONE: 46-18-17 ■ 41-4b
(ii) TITLE OF INVENTION: PRIMARY STRUCTURE AND FUNCTIONAL FXPRfcSSl'JN OF NUCLEOTIDE SFQUENCES FOR NOVEL PROTEIN TYR 1STN PHOSPHATASES liii.i NI'MBF OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:
(A) NAME: WOLF, GREENFIELD & SACKS, P.C.
(B) STREET: 600 ATLANTIC AVENUE
(C) CITY: BOSTON
(D) STATE: MASSACHUSETTS
(E) COUNTRY: USA
(F) POSTAL CODE: 02210
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 01-SEP-1994
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/115,573
(B) FILING DATE: 01-SEP-1993
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: TWOMEY, MICHAEL J.
(B) REGISTRATION NUMBER: P-38,349
(C) REFERENCE/DOCKET NUMBER: LO461/7000WO
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617/720-3500
(B) TELEFAX: 617/720-2441
(C) TELEX: 92-1742 EZEKIEL
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8043 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 78..7478 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
CCCGCCCCGA CGCCGCGTCC CTGCAGCCCT GCCCGGCGCT CCAGTAGCAG GACCCGGTCT 60
CGGGACCAGC CGGTAAT ATG CAC GTG TCA CTA GCT GAG GCC CTG GAG GTT 110
Met His Val Ser Leu Ala Glu Ala Leu Glu Val
1 5 10
CGG GGT GGA CCA CTT CAG GAG GAA GAA ATA TGG GCT GTA TTA AAT CAA 158
Arg Gly Gly Pro Leu Gin Glu Glu Glu lie Trp Ala Val Leu Asn Gin 15 20 25
AGT GCT GAA AGT CTC CAA GAA TTA TTC AGA AAA GTA AGC CTA GCT GAT 206
Ser Ala Glu Ser Leu Gin Glu Leu Phe Arg Lys Val Ser Leu Ala Asp
30 35 40
CCT GCT GCC CTT GGC TTC ATC ATT TCT CCA TGG TCT CTG CTG TTG CTG 254
Pro Ala Ala Leu Gly Phe lie lie Ser Pro Trp Ser Leu Leu Leu Leu 45 50 55
CCA TCT GGT AGT GTG TCA TTT ACA GAT GAA AAT ATT TCC AAT CAG GAT 302
Pro Ser Gly Ser Val Ser Phe Thr Asp Glu Asn lie Ser Asn Gin Asp
60 65 70 75
CTT CGA GCA TTC ACT GCA CCA GAG GTT CTT CAA AAT CAG TCA CTA ACT 350
Leu Arg Ala Phe Thr Ala Pro Glu Val Leu Gin Asn Gin Ser Leu Thr
80 85 90
TCT CTC TCA GAT GTT GAA AAG ATC CAC ATT TAT TCT CTT GGA ATG ACA 398
Ser Leu Ser Asp Val Glu Lys lie His lie Tyr Ser Leu Gly Met Thr 95 100 105
CTG TAT TGG GGG GCT GAT TAT GAA GTG CCT CAG AGC CAA CCT ATT AAG 446
Leu Tyr Trp Gly Ala Asp Tyr Glu Val Pro Gin Ser Gin Pro lie Lys
110 115 120
CTT GGA GAT CAT CTC AAC AGC ATA CTG CTT GGA ATG TGT GAG GAT GTT 494
Leu Gly Asp His Leu Asn Ser lie Leu Leu Gly Met Cys Glu Asp Val 125 130 135
ATT TAC GCT CGA GTT TCT GTT CGG ACT GTG CTG GAT GCT TGC AGT GCC 542
He Tyr Ala Arg Val Ser Val Arg Thr Val Leu Asp Ala Cys Ser Ala
140 145 150 155
CAC ATT AGG AAT AGC AAT TGT GCA CCC TCA TTT TCC TAC GTG AAA CAC 590
His He Arg Asn Ser Asn Cys Ala Pro Ser Phe Ser Tyr Val Lys His
160 165 170
TTG GTA AAA CTG GTT CTG GGA AAT CTT TCT GGG ACA GAT CAG CTT TCC 638
Leu Val Lys Leu Val Leu Gly Asn Leu Ser Gly Thr Asp Gin Leu Ser 175 180 185 686
734
782
830
878
926
974
1022
1070
1118
1166
1214
1262
Figure imgf000059_0001
1310
1358
1406
1454
1502
1550
1598
1646
1694
1742
1790
1838
1886
Figure imgf000060_0001
1934
1982
2030
2078
2126
2174
2222
2270
2318
2366
2414
2462
2510
Figure imgf000061_0001
CGC ACA TTG GTC CTT CGC TTT CCA TGG AGG GAA ACC AAG AAA ATA TCT 2558
Arg Thr Leu Val Leu Arg Phe Pro Trp Arg Glu Thr Lys Lys He Ser 815 820 825
TTT TCT AAA AAG AAA ATC ACA TTG CAA AAT ACA TCA GAT GGA ATA AAA 2606
Phe Ser Lys Lys Lys He Thr Leu Gin Asn Thr Ser Asp Gly He Lys 830 835 840
CAT GGC TTC CAG ACA GAC AAC AGT AAG ATA TGC CAG TAC CTG CTG CAC 2654
His Gly Phe Gin Thr Asp Asn Ser Lys He Cys Gin Tyr Leu Leu His 845 850 855
CTC TGC TCT TAC CAG CAT AAG TTC CAG CTA CAG ATG AGA GCA AGA CAG 2702
Leu Cys Ser Tyr Gin His Lys Phe Gin Leu Gin Met Arg Ala Arg Gin
860 865 870 875
AGC AAC CAA GAT GCC CAA GAT ATT GAG AGA GCT TCG TTT AGG AGC CTG 2750
Ser Asn Gin Asp Ala Gin Asp He Glu Arg Ala Ser Phe Arg Ser Leu 880 885 890
AAT CTC CAA GCA GAG TCT GTT AGA GGA TTT AAT ATG GGA CGA GCA ATC 2798
Asn Leu Gin Ala Glu Ser Val Arg Gly Phe Asn Met Gly Arg Ala He 895 900 905
AGC ACT GGC AGT CTG GCC AGC AGC ACC CTC AAC AAA CTT GCT GTT CGA 2846
Ser Thr Gly Ser Leu Ala Ser Ser Thr Leu Asn Lys Leu Ala Val Arg 910 915 920
CCT TTA TCA GTT CAA GCT GAG ATT CTG AAG AGG CTA TCC TGC TCA GAG 2894
Pro Leu Ser Val Gin Ala Glu He Leu Lys Arg Leu Ser Cys Ser Glu 925 930 935
CTG TCG CTT TAC CAG CCA TTG CAA AAC AGT TCA AAA GAG AAG AAT GAC 2942
Leu Ser Leu Tyr Gin Pro Leu Gin Asn Ser Ser Lys Glu Lys Asn Asp
940 945 950 955
AAA GCT TCA TGG GAG GAA AAG CCT AGA GAG ATG AGT AAA TCA TAC CAT 2990
Lys Ala Ser Trp Glu Glu Lys Pro Arg Glu Met Ser Lys Ser Tyr His 960 965 970
GAT CTC AGT CAG GCC TCT CTC TAT CCA CAT CGG AAA AAT GTC ATT GTT 3038
Asp Leu Ser Gin Ala Ser Leu Tyr Pro His Arg Lys Asn Val He Val 975 980 985
AAC ATG GAA CCC CCA CCA CAA ACC GTT GCA GAG TTG GTG GGA AAA CCT 3086
Asn Met Glu Pro Pro Pro Gin Thr Val Ala Glu Leu Val Gly Lys Pro 990 995 1000
TCT CAC CAG ATG TCA AGA TCT GAT GCA GAA TCT TTG GCA GGA GTG ACA 3134
Ser His Gin Met Ser Arg Ser Asp Ala Glu Ser Leu Ala Gly Val Thr 1005 1010 1015 AAA CTT AAT AAT TCA AAG TCT GTT GCG AGT TTA AAT AGA AGT CCT GAA 3182 Lys Leu Asn Asn Ser Lys Ser Val Ala Ser Leu Asn Arg Ser Pro Glu 1020 1025 1030 1035
AGG AGG AAA CAT GAA TCA GAC TCC TCA TCC ATT GAA GAC CCT GGG CAA 3230 Arg Arg Lys His Glu Ser Asp Ser Ser Ser He Glu Asp Pro Gly Gin 1040 1045 1050
GCA TAT GTT CTA GAT GTG CTA CAC AAA AGA TGG AGC ATA GTA TCT TCA 3278 Ala Tyr Val Leu Asp Val Leu His Lys Arg Trp Ser He Val Ser Ser 1055 1060 1065
CCA GAA AGG GAG ATC ACC TTA GTG AAC CTG AAA AAA GAT GCA AAG TAT 3326 Pro Glu Arg Glu He Thr Leu Val Asn Leu Lys Lys Asp Ala Lys Tyr 1070 1075 1080
GGC TTG GGA TTT CAA ATT ATT GGT GGG GAG AAG ATG GGA AGA CTG GAC 3374 Gly Leu Gly Phe Gin He He Gly Gly Glu Lys Met Gly Arg Leu Asp 1085 1090 1095
CTA GGC ATA TTT ATC AGC TCA GTT GCC CCT GGA GGA CCA GCT GAC TTC 3422 Leu Gly He Phe He Ser Ser Val Ala Pro Gly Gly Pro Ala Asp Phe 1100 1105 1110 1115
CAT GGA TGC TTG AAG CCA GGA GAC CGT TTG ATA TCT GTG AAT AGT GTG 3470 His Gly Cys Leu Lys Pro Gly Asp Arg Leu He Ser Val Asn Ser Val 1120 1125 1130
AGT CTG GAG GGA GTC AGC CAC CAT GCT GCA ATT GAA ATT TTG CAA AAT 3518 Ser Leu Glu Gly Val Ser His His Ala Ala He Glu He Leu Gin Asn 1135 1140 1145
GCA CCT GAA GAT GTG ACA CTT GTT ATC TCT CAG CCA AAA GAA AAG ATA 3566 Ala Pro Glu Asp Val Thr Leu Val He Ser Gin Pro Lys Glu Lys He 1150 1155 1160
TCC AAA GTG CCT TCT ACT CCT GTG CAT CTC ACC AAT GAG ATG AAA AAC 3614 Ser Lys Val Pro Ser Thr Pro Val His Leu Thr Asn Glu Met Lys Asn 1165 1170 1175
TAC ATG AAG AAA TCT TCC TAC ATG CAA GAC AGT GCT ATA GAT TCT TCT 3662 Tyr Met Lys Lys Ser Ser Tyr Met Gin Asp Ser Ala He Asp Ser Ser 1180 1185 1190 1195
TCC AAG GAT CAC CAC TGG TCA CGT GGT ACC CTG AGG CAC ATC TCG GAG 3710 Ser Lys Asp His His Trp Ser Arg Gly Thr Leu Arg His He Ser Glu 1200 1205 1210
AAC TCC TTT GGG CCG TCT GGG GGC CTG CGG GAA GGA AGC CTG AGT TCT 3758 Asn Ser Phe Gly Pro Ser Gly Gly Leu Arg Glu Gly Ser Leu Ser Ser 1215 1220 1225 CAA GAT TCC AGG ACT GAG AGT GCC AGC TTG TCT CAA AGC CAG GTC AAT 3806
Gin Asp Ser Arg Thr Glu Ser Ala Ser Leu Ser Gin Ser Gin Val Asn
1230 1235 1240
GGT TTC TTT GCC AGC CAT TTA GGT GAC CAA ACC TGG CAG GAA TCA CAG 3854
Gly Phe Phe Ala Ser His Leu Gly Asp Gin Thr Trp Gin Glu Ser Gin 1245 1250 1255
CAT GGC AGC CCT TCC CCA TCT GTA ATA TCC AAA GCC ACC GAG AAA GAG 3902
His Gly Ser Pro Ser Pro Ser Val He Ser Lys Ala Thr Glu Lys Glu
1260 1265 1270 1275
ACT TTC ACT GAT AGT AAC CAA AGC AAA ACT AAA AAG CCA GGC ATT TCT 3950
Thr Phe Thr Asp Ser Asn Gin Ser Lys Thr Lys Lys Pro Gly He Ser 1280 1285 1290
GAT GTA ACT GAT TAC TCA GAC CGT GGA GAT TCA GAC ATG GAT GAA GCC 3998
Asp Val Thr Asp Tyr Ser Asp Arg Gly Asp Ser Asp Met Asp Glu Ala
1295 1300 1305
ACT TAC TCC AGC AGT CAG GAT CAT CAA ACA CCA AAA CAG GAA TCT TCC 4046
Thr Tyr Ser Ser Ser Gin Asp His Gin Thr Pro Lys Gin Glu Ser Ser
1310 1315 1320
TCT TCA GTG AAT ACA TCC AAC AAG ATG AAT TTT AAA ACT TTT TCT TCA 4094
Ser Ser Val Asn Thr Ser Asn Lys Met Asn Phe Lys Thr Phe Ser Ser 1325 1330 1335
TCA CCT CCT AAG CCT GGA GAT ATC TTT GAG GTT GAA CTG GCT AAA AAT 4142
Ser Pro Pro Lys Pro Gly Asp He Phe Glu Val Glu Leu Ala Lys Asn
1340 1345 1350 1355
GAT AAC AGC TTG GGG ATA AGT GTC ACG GGA GGT GTG AAT ACG AGT GTC 4190
Asp Asn Ser Leu Gly He Ser Val Thr Gly Gly Val Asn Thr Ser Val 1360 1365 1370
AGA CAT GGT GGC ATT TAT GTG AAA GCT GTT ATT CCC CAG GGA GCA GCA 4238
Arg His Gly Gly He Tyr Val Lys Ala Val He Pro Gin Gly Ala Ala
1375 1380 1385
GAG TCT GAT GGT AGA ATT CAC AAA GGT GAT CGC GTC CTA GCT GTC AAT 4286
Glu Ser Asp Gly Arg He His Lys Gly Asp Arg Val Leu Ala Val Asn
1390 1395 1400
GGA GTT AGT CTA GAA GGA GCC ACC CAT AAG CAA GCT GTG GAA ACA CTG 4334
Gly Val Ser Leu Glu Gly Ala Thr His Lys Gin Ala Val Glu Thr Leu 1405 1410 1415
AGA AAT ACA GGA CAG GTG GTT CAT CTG TTA TTA GAA AAG GGA CAA TCT 4382
Arg Asn Thr Gly Gin Val Val His Leu Leu Leu Glu Lys Gly Gin Ser
1420 1425 1430 1435 CCA ACA TCT AAA GAA CAT GTC CCG GTA ACC CCA CAG TGT ACC CTT TCA 4430 Pro Thr Ser Lys Glu His Val Pro Val Thr Pro Gin Cys Thr Leu Ser 1440 1445 1450
GAT CAG AAT GCC CAA GGT CAA GGC CCA GAA AAA GTG AAG AAA ACA ACT 4478 Asp Gin Asn Ala Gin Gly Gin Gly Pro Glu Lys Val Lys Lys Thr Thr 1455 1460 1465
CAG GTC AAA GAC TAC AGC TTT GTC ACT GAA GAA AAT ACA TTT GAG GTA 4526 Gin Val Lys Asp Tyr Ser Phe Val Thr Glu Glu Asn Thr Phe Glu Val 1470 1475 1480
AAA TTA TTT AAA AAT AGC TCA GGT CTA GGA TTC AGT TTT TCT CGA GAA 4574 Lys Leu Phe Lys Asn Ser Ser Gly Leu Gly Phe Ser Phe Ser Arg Glu 1485 1490 1495
GAT AAT CTT ATA CCG GAG CAA ATT AAT GCC AGC ATA GTA AGG GTT AAA 4622 Asp Asn Leu He Pro Glu Gin He Asn Ala Ser He Val Arg Val Lys 1500 1505 1510 1515
AAG CTC TTT GCT GGA CAG CCA GCA GCA GAA AGT GGA AAA ATT GAT GTA 4670 Lys Leu Phe Ala Gly Gin Pro Ala Ala Glu Ser Gly Lys He Asp Val 1520 1525 1530
GGA GAT GTT ATC TTG AAA GTG AAT GGA GCC TCT TTG AAA GGA CTA TCT 4718 Gly Asp Val He Leu Lys Val Asn Gly Ala Ser Leu Lys Gly Leu Ser 1535 1540 1545
CAG CAG GAA GTC ATA TCT GCT CTC AGG GGA ACT GCT CCA GAA GTA TTC 4766 Gin Gin Glu Val He Ser Ala Leu Arg Gly Thr Ala Pro Glu Val Phe 1550 1555 1560
TTG CTT CTC TGC AGA CCT CCA CCT GGT GTG CTA CCG GAA ATT GAT ACT 4814 Leu Leu Leu Cys Arg Pro Pro Pro Gly Val Leu Pro Glu He Asp Thr 1565 1570 1575
GCG CTT TTG ACC CCA CTT CAG TCT CCA GCA CAA GTA CTT CCA AAC AGC 4862 Ala Leu Leu Thr Pro Leu Gin Ser Pro Ala Gin Val Leu Pro Asn Ser 1580 1585 1590 1595
AGT AAA GAC TCT TCT CAG CCA TCA TGT GTG GAG CAA AGC ACC AGC TCA 4910 Ser Lys Asp Ser Ser Gin Pro Ser Cys Val Glu Gin Ser Thr Ser Ser 1600 1605 1610
GAT GAA AAT GAA ATG TCA GAC AAA AGC AAA AAA CAG TGC AAG TCC CCA 4958 Asp Glu Asn Glu Met Ser Asp Lys Ser Lys Lys Gin Cys Lys Ser Pro 1615 1620 1625
TCC AGA AGA GAC AGT TAC AGT GAC AGC AGT GGG AGT GGA GAA GAT GAC 5006 Ser Arg Arg Asp Ser Tyr Ser Asp Ser Ser Gly Ser Gly Glu Asp Asp 1630 1635 1640 TTA GTC ACA GCT CCA GCA AAC ATA TCA AAT TCG ACC TGG AGT TCA GCT 5054 Leu Val Thr Ala Pro Ala Asn He Ser Asn Ser Thr Trp Ser Ser Ala 1645 1650 1655
TTG CAT CAG ACT CTA AGC AAC ATG GTA TCA CAG GCA CAG AGT CAT CAT 5102 Leu His Gin Thr Leu Ser Asn Met Val Ser Gin Ala Gin Ser His His 1660 1665 1670 1675
GAA GCA CCC AAG AGT CAA GAA GAT ACC ATT TGT ACC ATG TTT TAC TAT 5150 Glu Ala Pro Lys Ser Gin Glu Asp Thr He Cys Thr Met Phe Tyr Tyr 1680 1685 1690
CCT CAG AAA ATT CCC AAT AAA CCA GAG TTT GAG GAC AGT AAT CCT TCC 5198 Pro Gin Lys He Pro Asn Lys Pro Glu Phe Glu Asp Ser Asn Pro Ser 1695 1700 1705
CCT CTA CCA CCG GAT ATG GCT CCT GGG CAG AGT TAT CAA CCC CAA TCA 5246 Pro Leu Pro Pro Asp Met Ala Pro Gly Gin Ser Tyr Gin Pro Gin Ser 1710 1715 1720
GAA TCT GCT TCC TCT AGT TCG ATG GAT AAG TAT CAT ATA CAT CAC ATT 5294 Glu Ser Ala Ser Ser Ser Ser Met Asp Lys Tyr His He His His He 1725 1730 1735
TCT GAA CCA ACT AGA CAA GAA AAC TGG ACA CCT TTG AAA AAT GAC TTG 5342 Ser Glu Pro Thr Arg Gin Glu Asn Trp Thr Pro Leu Lys Asn Asp Leu 1740 1745 1750 1755
GAA AAT CAC CTT GAA GAC TTT GAA CTG GAA GTA GAA CTC CTC ATT ACC 5390 Glu Asn His Leu Glu Asp Phe Glu Leu Glu Val Glu Leu Leu He Thr 1760 1765 1770
CTA ATT AAA TCA GAA AAA GCA AGC CTG GGT TTT ACA GTA ACC AAA GGC 5438 Leu He Lys Ser Glu Lys Ala Ser Leu Gly Phe Thr Val Thr Lys Gly 1775 1780 1785
AAT CAG AGA ATT GGT TGT TAT GTT CAT GAT GTC ATA CAG GAT CCA GCC 5486 Asn Gin Arg He Gly Cys Tyr Val His Asp Val He Gin Asp Pro Ala 1790 1795 1800
AAA AGT GAT GGA AGG CTA AAA CCT GGG GAC CGG CTC ATA AAG GTT AAT 5534 Lys Ser Asp Gly Arg Leu Lys Pro Gly Asp Arg Leu He Lys Val Asn 1805 1810 1815
GAT ACA GAT GTT ACT AAT ATG ACT CAT ACA GAT GCA GTT AAT CTG CTC 5582 Asp Thr Asp Val Thr Asn Met Thr His Thr Asp Ala Val Asn Leu Leu 1820 1825 1830 1835
CGG GCT GCA TCC AAA ACA GTC AGA TTA GTT ATT GGA CGA GTT CTA GAA 5630 Arg Ala Ala Ser Lys Thr Val Arg Leu Val He Gly Arg Val Leu Glu 1840 1845 1850 TTA CCC AGA ATA CCA ATG TTG CCT CAT TTG CTA CCG GAC ATA ACA CTA 5678 Leu Pro Arg He Pro Met Leu Pro His Leu Leu Pro Asp He Thr Leu 1855 1860 1865
ACG TGC AAC AAA GAG GAG TTG GGT TTT TCC TTA TGT GGA GGT CAT GAC 5726 Thr Cys Asn Lys Glu Glu Leu Gly Phe Ser Leu Cys Gly Gly His Asp 1870 1875 1880
AGC CTT TAT CAA GTG GTA TAT ATT AGT GAT ATT AAT CCA AGG TCC GTC 5774 Ser Leu Tyr Gin Val Val Tyr He Ser Asp He Asn Pro Arg Ser Val 1885 1890 1895
GCA GCC ATT GAG GGT AAT CTC CAG CTA TTA GAT GTC ATC CAT TAT GTG 5822 Ala Ala He Glu Gly Asn Leu Gin Leu Leu Asp Val He His Tyr Val 1900 1905 1910 1915
AAC GGA GTC AGC ACA CAA GGA ATG ACC TTG GAG GAA GTT AAC AGA GCA 5870 Asn Gly Val Ser Thr Gin Gly Met Thr Leu Glu Glu Val Asn Arg Ala 1920 1925 1930
TTA GAC ATG TCA CTT CCT TCA TTG GTA TTG AAA GCA ACA AGA AAT GAT 5918 Leu Asp Met Ser Leu Pro Ser Leu Val Leu Lys Ala Thr Arg Asn Asp 1935 1940 1945
CTT CCA GTG GTT CCC AGC TCA AAG AGG TCT GCT GTT TCA GCT CCA AAG 5966 Leu Pro Val Val Pro Ser Ser Lys Arg Ser Ala Val Ser Ala Pro Lys 1950 1955 1960
TCA ACC AAA GGC AAT GGT TCC TAC AGT GTG GGG TCT TGC AGC CAG CCT 6014 Ser Thr Lys Gly Asn Gly Ser Tyr Ser Val Gly Ser Cys Ser Gin Pro 1965 1970 1975
GCC CTC ACT CCT AAT GAT TCA TTC TCC ACG GTT GCT GGG GAA GAA ATA 6062 Ala Leu Thr Pro Asn Asp Ser Phe Ser Thr Val Ala Gly Glu Glu He 1980 1985 1990 1995
AAT GAA ATA TCG TAC CCC AAA GGA AAA TGT TCT ACT TAT CAG ATA AAG 6110 Asn Glu He Ser Tyr Pro Lys Gly Lys Cys Ser Thr Tyr Gin He Lys 2000 2005 2010
GGA TCA CCA AAC TTG ACT CTG CCC AAA GAA TCT TAT ATA CAA GAA GAT 6158 Gly Ser Pro Asn Leu Thr Leu Pro Lys Glu Ser Tyr He Gin Glu Asp 2015 2020 2025
GAC ATT TAT GAT GAT TCC CAA GAA GCT GAA GTT ATC CAG TCT CTG CTG 6206 Asp He Tyr Asp Asp Ser Gin Glu Ala Glu Val He Gin Ser Leu Leu 2030 2035 2040
GAT GTT GTT GAT GAG GAA GCC CAG AAT CTT TTA AAC GAA AAT AAT GCA 6254 Asp Val Val Asp Glu Glu Ala Gin Asn Leu Leu Asn Glu Asn Asn Ala 2045 2050 2055 GCA GGA TAC TCC TGT GGT CCA GGT ACA TTA AAG ATG AAT GGG AAG TTA 6302 Ala Gly Tyr Ser Cys Gly Pro Gly Thr Leu Lys Met Asn Gly Lys Leu 2060 2065 2070 2075
TCA GAA GAG AGA ACA GAA GAT ACA GAC TGC GAT GGT TCA CCT TTA CCT 6350 Ser Glu Glu Arg Thr Glu Asp Thr Asp Cys Asp Gly Ser Pro Leu Pro 2080 2085 2090
GAG TAT TTT ACT GAG GCC ACC AAA ATG AAT GGC TGT GAA GAA TAT TGT 6398 Glu Tyr Phe Thr Glu Ala Thr Lys Met Asn Gly Cys Glu Glu Tyr Cys 2095 2100 2105
GAA GAA AAA GTA AAA AGT GAA AGC TTA ATT CAG AAG CCA CAA GAA AAG 6446 Glu Glu Lys Val Lys Ser Glu Ser Leu He Gin Lys Pro Gin Glu Lys 2110 2115 2120
AAG ACT GAT GAT GAT GAA ATA ACA TGG GGA AAT GAT GAG TTG CCA ATA 6494 Lys Thr Asp Asp Asp Glu He Thr Trp Gly Asn Asp Glu Leu Pro He 2125 2130 2135
GAG AGA ACA AAC CAT GAA GAT TCT GAT AAA GAT CAT TCC TTT CTG ACA 6542 Glu Arg Thr Asn His Glu Asp Ser Asp Lys Asp His Ser Phe Leu Thr 2140 2145 2150 2155
AAC GAT GAG CTC GCT GTA CTC CCT GTC GTC AAA GTG CTT CCC TCT GGT 6590 Asn Asp Glu Leu Ala Val Leu Pro Val Val Lys Val Leu Pro Ser Gly 2160 2165 2170
AAA TAC ACG GGT GCC AAC TTA AAA TCA GTC ATT CGA GTC CTG CGG GGT 6638 Lys Tyr Thr Gly Ala Asn Leu Lys Ser Val He Arg Val Leu Arg Gly 2175 2180 2185
TTG CTA GAT CAA GGA ATT CCT TCT AAG GAG CTG GAG AAT CTT CAA GAA 6686 Leu Leu Asp Gin Gly He Pro Ser Lys Glu Leu Glu Asn Leu Gin Glu 2190 2195 2200
TTA AAA CCT TTG GAT CAG TGT CTA ATT GGG CAA ACT AAG GAA AAC AGA 6734 Leu Lys Pro Leu Asp Gin Cys Leu He Gly Gin Thr Lys Glu Asn Arg 2205 2210 2215
AGG AAG AAC AGA TAT AAA AAT ATA CTT CCC TAT GAT GCT ACA AGA GTG 6782 Arg Lys Asn Arg Tyr Lys Asn He Leu Pro Tyr Asp Ala Thr Arg Val 2220 2225 2230 2235
CCT CTT GGA GAT GAA GGT GGC TAT ATC AAT GCC AGC TTC ATT AAG ATA 6830 Pro Leu Gly Asp Glu Gly Gly Tyr He Asn Ala Ser Phe He Lys He 2240 2245 2250
CCA GTT GGG AAA GAA GAG TTC GTT TAC ATT GCC TGC CAA GGA CCA CTG 6878 Pro Val Gly Lys Glu Glu Phe Val Tyr He Ala Cys Gin Gly Pro Leu 2255 2260 2265 CCT ACA ACT GTT GGA GAC TTC TGG CAG ATG ATT TGG GAG CAA AAA TCC 6926 Pro Thr Thr Val Gly Asp Phe Trp Gin Met He Trp Glu Gin Lys Ser 2270 2275 2280
ACA GTG ATA GCC ATG ATG ACT CAA GAA GTA GAA GGA GAA AAA ATC AAA 6974 Thr Val He Ala Met Met Thr Gin Glu Val Glu Gly Glu Lys He Lys 2285 2290 2295
TGC CAG CGC TAT TGG CCC AAC ATC CTA GGC AAA ACA ACA ATG GTC AGC 7022 Cys Gin Arg Tyr Trp Pro Asn He Leu Gly Lys Thr Thr Met Val Ser 2300 2305 2310 2315
AAC AGA CTT CGA CTG GCT CTT GTG AGA ATG CAG CAG CTG AAG GGC TTT 7070 Asn Arg Leu Arg Leu Ala Leu Val Arg Met Gin Gin Leu Lys Gly Phe 2320 2325 2330
GTG GTG AGG GCA ATG ACC CTT GAA GAT ATT CAG ACC AGA GAG GTG CGC 7118 Val Val Arg Ala Met Thr Leu Glu Asp He Gin Thr Arg Glu Val Arg 2335 2340 2345
CAT ATT TCT CAT CTG AAT TTC ACT GCC TGG CCA GAC CAT GAT ACA CCT 7166 His He Ser His Leu Asn Phe Thr Ala Trp Pro Asp His Asp Thr Pro 2350 2355 2360
TCT CAA CCA GAT GAT CTG CTT ACT TTT ATC TCC TAC ATG AGA CAC ATC 7214 Ser Gin Pro Asp Asp Leu Leu Thr Phe He Ser Tyr Met Arg His He 2365 2370 2375
CAC AGA TCA GGC CCA ATC ATT ACG CAC TGC AGT GCT GGC ATT GGA CGT 7262 His Arg Ser Gly Pro He He Thr His Cys Ser Ala Gly He Gly Arg 2380 2385 2390 2395
TCA GGG ACC CTG ATT TGC ATA GAT GTG GTT CTG GGA TTA ATC AGT CAG 7310 Ser Gly Thr Leu He Cys He Asp Val Val Leu Gly Leu He Ser Gin 2400 2405 2410
GAT CTT GAT TTT GAC ATC TCT GAT TTG GTG CGC TGC ATG AGA CTA CAA 7358 Asp Leu Asp Phe Asp He Ser Asp Leu Val Arg Cys Met Arg Leu Gin 2415 2420 2425
AGA CAC GGA ATG GTT CAG ACA GAG GAT CAA TAT ATT TTC TGC TAT CAA 7406 Arg His Gly Met Val Gin Thr Glu Asp Gin Tyr He Phe Cys Tyr Gin 2430 2435 2440
GTC ATC CTT TAT GTC CTG ACA CGT CTT CAA GCA GAA GAA GAG CAA AAA 7454 Val He Leu Tyr Val Leu Thr Arg Leu Gin Ala Glu Glu Glu Gin Lys 2445 2450 2455
CAG CAG CCT CAG CTT CTG AAG TGACATGAAA AGAGCCTCTG GATGCATTTC 7505
Gin Gin Pro Gin Leu Leu Lys 2460 2465 CATTTCTCTC CTTAACCTCC AGCAGACTCC TGCTCTCTAT CCAAATAAAG ATCACAGAGC 7565
AGCAAGTTCA TACAACATGC ATGTTCTCCT CTATCTTAGA GGGGTATTCT TCTTGAAAAT 7625
AAAAAATATT GAAATGCTGT ATTTTTACAG CTACTTTAAC CTATGATAAT TATTTACAAA 7685
ATTTTAACAC TAACCAAACA ATGCAGATCT TAGGGATGAT TAAAGGCAGC ATTGATGATA 7745
GCAAGACATT GTTACAAGGA CATGGTGAGT CTATTTTTAA TGCACCAATC TTGTTTATAG 7805
CAAAAATGTT TTCCAATATT TTAATAAAGT AGTTATTTTA TAGGGCATAC TTGAAACCAG 7865
TATTTAAGCT TTAAATGACA GTAATATTGG CATAGAAAAA AGTAGCAAAT GTTTACTGTA 7925
TCAATTTCTA ATGTTTACTA TATAGAATTT CCTGTAATAT ATTTATATAC TTTTTCATGA 7985
AAATGGAGTT ATCAGTTATC TGTTTGTTAC TGCATCATCT GTTTGTAATC ATTATCTC 8043
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2466 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met His Val Ser Leu Ala Glu Ala Leu Glu Val Arg Gly Gly Pro Leu 1 5 10 15
Gin Glu Glu Glu He Trp Ala Val Leu Asn Gin Ser Ala Glu Ser Leu 20 25 30
Gin Glu Leu Phe Arg Lys Val Ser Leu Ala Asp Pro Ala Ala Leu Gly 35 40 45
Phe He He Ser Pro Trp Ser Leu Leu Leu Leu Pro Ser Gly Ser Val 50 55 60
Ser Phe Thr Asp Glu Asn He Ser Asn Gin Asp Leu Arg Ala Phe Thr 65 70 75 80
Ala Pro Glu Val Leu Gin Asn Gin Ser Leu Thr Ser Leu Ser Asp Val 85 90 95
Glu Lys He His He Tyr Ser Leu Gly Met Thr Leu Tyr Trp Gly Ala 100 105 110
Asp Tyr Glu Val Pro Gin Ser Gin Pro He Lys Leu Gly Asp His Leu 115 120 125 Asn Ser He Leu Leu Gly Met Cys Glu Asp Val He Tyr Ala Arg Val 130 135 140
Ser Val Arg Thr Val Leu Asp Ala Cys Ser Ala His He Arg Asn Ser 145 150 155 160
Asn Cys Ala Pro Ser Phe Ser Tyr Val Lys His Leu Val Lys Leu Val 165 170 175
Leu Gly Asn Leu Ser Gly Thr Asp Gin Leu Ser Cys Asn Ser Glu Gin 180 185 190
Lys Pro Asp Arg Ser Gin Ala He Arg Asp Arg Leu Arg Gly Lys Gly 195 200 205
Leu Pro Thr Gly Arg Ser Ser Thr Ser Asp Val Leu Asp He Gin Lys 210 215 220
Pro Pro Leu Ser His Gin Thr Phe Leu Asn Lys Gly Leu Ser Lys Ser 225 230 235 240
Met Gly Phe Leu Ser He Lys Asp Thr Gin Asp Glu Asn Tyr Phe Lys 245 250 255
Asp He Leu Ser Asp Asn Ser Gly Arg Glu Asp Ser Glu Asn Thr Phe 260 265 270
Ser Pro Tyr Gin Phe Lys Thr Ser Gly Pro Glu Lys Lys Pro He Pro 275 280 285
Gly He Asp Val Leu Ser Lys Lys Lys He Trp Ala Ser Ser Met Asp 290 295 300
Leu Leu Cys Thr Ala Asp Arg Asp Phe Ser Ser Gly Glu Thr Ala Thr 305 310 315 320
Tyr Arg Arg Cys His Pro Glu Ala Val Thr Val Arg Thr Ser Thr Thr 325 330 335
Pro Arg Lys Lys Glu Ala Arg Tyr Ser Asp Gly Ser He Ala Leu Asp 340 345 350
He Phe Gly Pro Gin Lys Met Asp Pro He Tyr His Thr Arg Glu Leu 355 360 365
Pro Thr Ser Ser Ala He Ser Ser Ala Leu Asp Arg He Arg Glu Arg 370 375 380
Gin Lys Lys Leu Gin Val Leu Arg Glu Ala Met Asn Val Glu Glu Pro 385 390 395 400
Val Arg Arg Tyr Lys Thr Tyr His Gly Asp Val Phe Ser Thr Ser Ser 405 410 415 Glu Ser Pro Ser He He Ser Ser Glu Ser Asp Phe Arg Gin Val Arg 420 425 430
Arg Ser Glu Ala Ser Lys Arg Phe Glu Ser Ser Ser Gly Leu Pro Gly 435 440 445
Val Asp Glu Thr Leu Ser Gin Gly Gin Ser Gin Arg Pro Ser Arg Gin 450 455 460
Tyr Glu Thr Pro Phe Glu Gly Asn Leu He Asn Gin Glu He Met Leu 465 470 475 480
Lys Arg Gin Glu Glu Glu Leu Met Gin Leu Gin Ala Lys Met Ala Leu 485 490 495
Arg Gin Ser Arg Leu Ser Leu Tyr Pro Gly Asp Thr He Lys Ala Ser 500 505 510
Met Leu Asp He Thr Arg Asp Pro Leu Arg Glu He Ala Leu Glu Thr 515 520 525
Ala Met Thr Gin Arg Lys Leu Arg Asn Phe Phe Gly Pro Glu Phe Val 530 535 540
Lys Met Thr He Glu Pro Phe He Ser Leu Asp Leu Pro Arg Ser He 545 550 555 560
Leu Thr Lys Lys Gly Lys Asn Glu Asp Asn Arg Arg Lys Val Asn He 565 570 575
Met Leu Leu Asn Gly Gin Arg Leu Glu Leu Thr Cys Asp Thr Lys Thr 580 585 590
He Cys Lys Asp Val Phe Asp Met Val Val Ala His He Gly Leu Val 595 600 605
Glu His His Leu Phe Ala Leu Ala Thr Leu Lys Asp Asn Glu Tyr Phe 610 615 620
Phe Val Asp Pro Asp Leu Lys Leu Thr Lys Val Ala Pro Glu Gly Trp 625 630 635 640
Lys Glu Glu Pro Lys Lys Lys Thr Lys Ala Thr Val Asn Phe Thr Leu 645 650 655
Phe Phe Arg He Lys Phe Phe Met Asp Asp Val Ser Leu He Gin His 660 665 670
Thr Leu Thr Cys His Gin Tyr Tyr Leu Gin Leu Arg Lys Asp He Leu 675 680 685
Glu Glu Arg Met His Cys Asp Asp Glu Thr Ser Leu Leu Leu Ala Ser 690 695 700 Leu Ala Leu Gin Ala Glu Tyr Gly Asp Tyr Gin Pro Glu Val His Gly 705 710 715 720
Val Ser Tyr Phe Arg Met Glu His Tyr Leu Pro Ala Arg Val Met Glu 725 730 735
Lys Leu Asp Leu Ser Tyr He Lys Glu Glu Leu Pro Lys Leu His Asn 740 745 750
Thr Tyr Val Gly Ala Ser Glu Lys Glu Thr Glu Leu Glu Phe Leu Lys 755 760 765
Val Cys Gin Arg Leu Thr Glu Tyr Gly Val His Phe His Arg Val His 770 775 780
Pro Glu Lys Lys Ser Gin Thr Gly He Leu Leu Gly Val Cys Ser Lys 785 790 795 800
Gly Val Leu Val Phe Glu Val His Asn Gly Val Arg Thr Leu Val Leu 805 810 815
Arg Phe Pro Trp Arg Glu Thr Lys Lys He Ser Phe Ser Lys Lys Lys 820 825 830
He Thr Leu Gin Asn Thr Ser Asp Gly He Lys His Gly Phe Gin Thr 835 840 845
Asp Asn Ser Lys He Cys Gin Tyr Leu Leu His Leu Cys Ser Tyr Gin 850 855 860
His Lys Phe Gin Leu Gin Met Arg Ala Arg Gin Ser Asn Gin Asp Ala 865 870 875 880
Gin Asp He Glu Arg Ala Ser Phe Arg Ser Leu Asn Leu Gin Ala Glu 885 890 895
Ser Val Arg Gly Phe Asn Met Gly Arg Ala He Ser Thr Gly Ser Leu 900 905 910
Ala Ser Ser Thr Leu Asn Lys Leu Ala Val Arg Pro Leu Ser Val Gin 915 920 925
Ala Glu He Leu Lys Arg Leu Ser Cys Ser Glu Leu Ser Leu Tyr Gin 930 935 940
Pro Leu Gin Asn Ser Ser Lys Glu Lys Asn Asp Lys Ala Ser Trp Glu 945 950 955 960
Glu Lys Pro Arg Glu Met Ser Lys Ser Tyr His Asp Leu Ser Gin Ala 965 970 975
Ser Leu Tyr Pro His Arg Lys Asn Val He Val Asn Met Glu Pro Pro 980 985 990 Pro Gin Thr Val Ala Glu Leu Val Gly Lys Pro Ser His Gin Met Ser 995 1000 1005
Arg Ser Asp Ala Glu Ser Leu Ala Gly Val Thr Lys Leu Asn Asn Ser 1010 1015 1020
Lys Ser Val Ala Ser Leu Asn Arg Ser Pro Glu Arg Arg Lys His Glu 1025 1030 1035 1040
Ser Asp Ser Ser Ser He Glu Asp Pro Gly Gin Ala Tyr Val Leu Asp 1045 1050 1055
Val Leu His Lys Arg Trp Ser He Val Ser Ser Pro Glu Arg Glu He 1060 1065 1070
Thr Leu Val Asn Leu Lys Lys Asp Ala Lys Tyr Gly Leu Gly Phe Gin 1075 1080 1085
He He Gly Gly Glu Lys Met Gly Arg Leu Asp Leu Gly He Phe He 1090 1095 1100
Ser Ser Val Ala Pro Gly Gly Pro Ala Asp Phe His Gly Cys Leu Lys 1105 1110 1115 1120
Pro Gly Asp Arg Leu He Ser Val Asn Ser Val Ser Leu Glu Gly Val 1125 1130 1135
Ser His His Ala Ala He Glu He Leu Gin Asn Ala Pro Glu Asp Val 1140 1145 1150
Thr Leu Val He Ser Gin Pro Lys Glu Lys He Ser Lys Val Pro Ser 1155 1160 1165
Thr Pro Val His Leu Thr Asn Glu Met Lys Asn Tyr Met Lys Lys Ser 1170 1175 1180
Ser Tyr Met Gin Asp Ser Ala He Asp Ser Ser Ser Lys Asp His His 1185 1190 1195 1200
Trp Ser Arg Gly Thr Leu Arg His He Ser Glu Asn Ser Phe Gly Pro 1205 1210 1215
Ser Gly Gly Leu Arg Glu Gly Ser Leu Ser Ser Gin Asp Ser Arg Thr 1220 1225 1230
Glu Ser Ala Ser Leu Ser Gin Ser Gin Val Asn Gly Phe Phe Ala Ser 1235 1240 1245
His Leu Gly Asp Gin Thr Trp Gin Glu Ser Gin His Gly Ser Pro Ser 1250 1255 1260
Pro Ser Val He Ser Lys Ala Thr Glu Lys Glu Thr Phe Thr Asp Ser 1265 1270 1275 1280 Asn Gin Ser Lys Thr Lys Lys Pro Gly He Ser Asp Val Thr Asp Tyr 1285 1290 1295
Ser Asp Arg Gly Asp Ser Asp Met Asp Glu Ala Thr Tyr Ser Ser Ser 1300 1305 1310
Gin Asp His Gin Thr Pro Lys Gin Glu Ser Ser Ser Ser Val Asn Thr 1315 1320 1325
Ser Asn Lys Met Asn Phe Lys Thr Phe Ser Ser Ser Pro Pro Lys Pro 1330 1335 1340
Gly Asp He Phe Glu Val Glu Leu Ala Lys Asn Asp Asn Ser Leu Gly 1345 1350 1355 1360
He Ser Val Thr Gly Gly Val Asn Thr Ser Val Arg His Gly Gly He 1365 1370 1375
Tyr Val Lys Ala Val He Pro Gin Gly Ala Ala Glu Ser Asp Gly Arg 1380 1385 1390
He His Lys Gly Asp Arg Val Leu Ala Val Asn Gly Val Ser Leu Glu 1395 1400 1405
Gly Ala Thr His Lys Gin Ala Val Glu Thr Leu Arg Asn Thr Gly Gin 1410 1415 1420
Val Val His Leu Leu Leu Glu Lys Gly Gin Ser Pro Thr Ser Lys Glu 1425 1430 1435 1440
His Val Pro Val Thr Pro Gin Cys Thr Leu Ser Asp Gin Asn Ala Gin 1445 1450 1455
Gly Gin Gly Pro Glu Lys Val Lys Lys Thr Thr Gin Val Lys Asp Tyr 1460 1465 1470
Ser Phe Val Thr Glu Glu Asn Thr Phe Glu Val Lys Leu Phe Lys Asn 1475 1480 1485
Ser Ser Gly Leu Gly Phe Ser Phe Ser Arg Glu Asp Asn Leu He Pro 1490 1495 1500
Glu Gin He Asn Ala Ser He Val Arg Val Lys Lys Leu Phe Ala Gly 1505 1510 1515 1520
Gin Pro Ala Ala Glu Ser Gly Lys He Asp Val Gly Asp Val He Leu 1525 1530 1535
Lys Val Asn Gly Ala Ser Leu Lys Gly Leu Ser Gin Gin Glu Val He 1540 1545 1550
Ser Ala Leu Arg Gly Thr Ala Pro Glu Val Phe Leu Leu Leu Cys Arg 1555 1560 1565 Pro Pro Pro Gly Val Leu Pro Glu He Asp Thr Ala Leu Leu Thr Pro 1570 1575 1580
Leu Gin Ser Pro Ala Gin Val Leu Pro Asn Ser Ser Lys Asp Ser Ser 1585 1590 1595 1600
Gin Pro Ser Cys Val Glu Gin Ser Thr Ser Ser Asp Glu Asn Glu Met 1605 1610 1615
Ser Asp Lys Ser Lys Lys Gin Cys Lys Ser Pro Ser Arg Arg Asp Ser 1620 1625 1630
Tyr Ser Asp Ser Ser Gly Ser Gly Glu Asp Asp Leu Val Thr Ala Pro 1635 1640 1645
Ala Asn He Ser Asn Ser Thr Trp Ser Ser Ala Leu His Gin Thr Leu 1650 1655 1660
Ser Asn Met Val Ser Gin Ala Gin Ser His His Glu Ala Pro Lys Ser 1665 1670 1675 1680
Gin Glu Asp Thr He Cys Thr Met Phe Tyr Tyr Pro Gin Lys He Pro 1685 1690 1695
Asn Lys Pro Glu Phe Glu Asp Ser Asn Pro Ser Pro Leu Pro Pro Asp 1700 1705 1710
Met Ala Pro Gly Gin Ser Tyr Gin Pro Gin Ser Glu Ser Ala Ser Ser 1715 1720 1725
Ser Ser Met Asp Lys Tyr His He His His He Ser Glu Pro Thr Arg 1730 1735 1740
Gin Glu Asn Trp Thr Pro Leu Lys Asn Asp Leu Glu Asn His Leu Glu 1745 1750 1755 1760
Asp Phe Glu Leu Glu Val Glu Leu Leu He Thr Leu He Lys Ser Glu 1765 1770 1775
Lys Ala Ser Leu Gly Phe Thr Val Thr Lys Gly Asn Gin Arg He Gly 1780 1785 1790
Cys Tyr Val His Asp Val He Gin Asp Pro Ala Lys Ser Asp Gly Arg 1795 1800 1805
Leu Lys Pro Gly Asp Arg Leu He Lys Val Asn Asp Thr Asp Val Thr 1810 1815 1820
Asn Met Thr His Thr Asp Ala Val Asn Leu Leu Arg Ala Ala Ser Lys 1825 1830 1835 1840
Thr Val Arg Leu Val He Gly Arg Val Leu Glu Leu Pro Arg He Pro 1845 1850 1855 Met Leu Pro His Leu Leu Pro Asp He Thr Leu Thr Cys Asn Lys Glu 1860 .1865 1870
Glu Leu Gly Phe Ser Leu Cys Gly Gly His Asp Ser Leu Tyr Gin Val 1875 1880 1885
Val Tyr He Ser Asp He Asn Pro Arg Ser Val Ala Ala He Glu Gly 1890 1895 1900
Asn Leu Gin Leu Leu Asp Val He His Tyr Val Asn Gly Val Ser Thr 1905 1910 1915 1920
Gin Gly Met Thr Leu Glu Glu Val Asn Arg Ala Leu Asp Met Ser Leu 1925 1930 1935
Pro Ser Leu Val Leu Lys Ala Thr Arg Asn Asp Leu Pro Val Val Pro 1940 1945 1950
Ser Ser Lys Arg Ser Ala Val Ser Ala Pro Lys Ser Thr Lys Gly Asn 1955 1960 1965
Gly Ser Tyr Ser Val Gly Ser Cys Ser Gin Pro Ala Leu Thr Pro Asn 1970 1975 1980
Asp Ser Phe Ser Thr Val Ala Gly Glu Glu He Asn Glu He Ser Tyr 1985 1990 1995 2000
Pro Lys Gly Lys Cys Ser Thr Tyr Gin He Lys Gly Ser Pro Asn Leu 2005 2010 2015
Thr Leu Pro Lys Glu Ser Tyr He Gin Glu Asp Asp He Tyr Asp Asp 2020 2025 2030
Ser Gin Glu Ala Glu Val He Gin Ser Leu Leu Asp Val Val Asp Glu 2035 2040 2045
Glu Ala Gin Asn Leu Leu Asn Glu Asn Asn Ala Ala Gly Tyr Ser Cys 2050 2055 2060
Gly Pro Gly Thr Leu Lys Met Asn Gly Lys Leu Ser Glu Glu Arg Thr 2065 2070 2075 2080
Glu Asp Thr Asp Cys Asp Gly Ser Pro Leu Pro Glu Tyr Phe Thr Glu 2085 2090 2095
Ala Thr Lys Met Asn Gly Cys Glu Glu Tyr Cys Glu Glu Lys Val Lys 2100 2105 2110
Ser Glu Ser Leu He Gin Lys Pro Gin Glu Lys Lys Thr Asp Asp Asp 2115 2120 2125
Glu He Thr Trp Gly Asn Asp Glu Leu Pro He Glu Arg Thr Asn His 2130 2135 2140 Glu Asp Ser Asp Lys Asp His Ser Phe Leu Thr Asn Asp Glu Leu Ala 2145 2150 2155 2160
Val Leu Pro Val Val Lys Val Leu Pro Ser Gly Lys Tyr Thr Gly Ala 2165 2170 2175
Asn Leu Lys Ser Val He Arg Val Leu Arg Gly Leu Leu Asp Gin Gly 2180 2185 2190
He Pro Ser Lys Glu Leu Glu Asn Leu Gin Glu Leu Lys Pro Leu Asp
2195 2200 2205
Gin Cys Leu He Gly Gin Thr Lys Glu Asn Arg Arg Lys Asn Arg Tyr 2210 2215 2220
Lys Asn He Leu Pro Tyr Asp Ala Thr Arg Val Pro Leu Gly Asp Glu 2225 2230 2235 2240
Gly Gly Tyr He Asn Ala Ser Phe He Lys He Pro Val Gly Lys Glu 2245 2250 2255
Glu Phe Val Tyr He Ala Cys Gin Gly Pro Leu Pro Thr Thr Val Gly 2260 2265 2270
Asp Phe Trp Gin Met He Trp Glu Gin Lys Ser Thr Val He Ala Met
2275 2280 2285
Met Thr Gin Glu Val Glu Gly Glu Lys He Lys Cys Gin Arg Tyr Trp 2290 2295 2300
Pro Asn He Leu Gly Lys Thr Thr Met Val Ser Asn Arg Leu Arg Leu 2305 2310 2315 2320
Ala Leu Val Arg Met Gin Gin Leu Lys Gly Phe Val Val Arg Ala Met 2325 2330 2335
Thr Leu Glu Asp He Gin Thr Arg Glu Val Arg His He Ser His Leu 2340 2345 2350
Asn Phe Thr Ala Trp Pro Asp His Asp Thr Pro Ser Gin Pro Asp Asp
2355 2360 2365
Leu Leu Thr Phe He Ser Tyr Met Arg His He His Arg Ser Gly Pro 2370 2375 2380
He He Thr His Cys Ser Ala Gly He Gly Arg Ser Gly Thr Leu He 2385 2390 2395 2400
Cys He Asp Val Val Leu Gly Leu He Ser Gin Asp Leu Asp Phe Asp 2405 2410 2415
He Ser Asp Leu Val Arg Cys Met Arg Leu Gin Arg His Gly Met Val 2420 2425 2430 Gln Thr Glu Asp Gin Tyr He Phe Cys Tyr Gin Val He Leu Tyr Val 2435 2440 2445
Leu Thr Arg Leu Gin Ala Glu Glu Glu Gin Lys Gin Gin Pro Gin Leu
2450 2455 2460
Leu Lys 2465
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3090 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1311..2420
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAATTCCGGA TTTACCTCAG TCTGTATCCC TTGAATAGCT CACAATAATC GACACATGCA 60
GCTGGGGACT GTGGGTGGGA TACTTAGGTG TGGGACACCA TATCTTCCAG CAGTAATAAA 120
GAAGTCAGGT GGGAATATGT AACATCTTGA GTGCTCATCC AGGTAGGTAC TAAGGTATGA 180
TCAACTCTAT GGAAGATCGA TTAGGAAACT CCCTGAAAGA GAGTTCAGCC TGAAGAGAGA 240
ACCAAAGGCC AACATCTTGG AGCTGGCTAC AGGACAGTAG GATGTAAGCT CGAGGGGAGG 300
AGAGGGTTAG GCGCAGTGGC TCACGCCTGT AGTCCCAACC ATTTGGGAGG CTGAGGCAGG 360
CAGATCGCTT GAGCCCGGGG GTTCAAGACC AGCCTGGGCA ACATGGCGAA ACCCCATCTC 420
TACAAAAAAA TACAAAAAAA ATGTAGCTGC GTGTGGTGGC ATGCACCTGT AGTCACAGCC 480
ACCACAGAGG TTGAGGTGGG AGGACTGCTT GAGCCTGGGA GGTGGAGGCT GCAGCGAACC 540 GAGATTGTGC CACTGCACTC CAGGATGGGC GACAGAGTGA GACCCGGACA GAGTGAGACC 600
CTGTCTCATT CATTCATTCA TAAATAAGAA GAGGGGGAAA ACGGGTGCCC AGATTGCTCT 660
CAGGCTCCTC CTCCCTTTCA GCTGGTACTT AACCACTCTT AACTTCAGCC TGCTCATGAA 720
TGAAATGGGA ATGACAATTC CTAACTCAGG CAGTTTTTGC AAAGACCAGA GAAAATCATG 780
TATTAATACT AGTACCCAGC ACCATTCCAA ACATACAATA CAAATGCCCC ATAAATGACA 840
GCCAAGGTAA CTGTTCTTTG CTTCCTCTCT TAGGAGACGT GTGAGGTTCT CTGTTGCTCC 900
TTTTGACTCC CAACTCCTGC TACAATGACT GATTTGACAC TGATTACCTC ACAGTACACA 960
CTGGGTGCTG GCCAACTGCA GCATGCTACG TATCCCACAC CCCCTCCCTG AGTGGTGGGA 1020
CATTAATGGT GGGATGGTAG AATGTGCAGT CCGGTCTTGT ACATTGAGTG TTAAACCTAC 1080
AATGTTTTGG ATGATAGAAG GGACATTCCA TCTTCTTACA AGCAGGGAAG TAACGGCAGA 1140
GCTGACTACT GGAAGGTGGT GCTGGTGGTG CAACAGGTTC TGGAGTTAAA ACCAATGGAA 1200
AAGAAAGATT TCAGCTTTCC TTAAGACAAG ACAAAGAGAA AAACCAGGAG ATCCACCTAT 1260
CGCCCATCAC ATTACAGCCA GCACTGTCCG AGGCAAAGAC AGTCCACAGC ATG GTC 1316
Met Val 1
CAA CCT GAG CAG GCC CCA AAG GTA CTG AAT GTT GTC GTG GAC CCT CAA 1364 Gin Pro Glu Gin Ala Pro Lys Val Leu Asn Val Val Val Asp Pro Gin 5 10 15
GGC CGA GGT GCT CCT GAG ATC AAA GCT ACC ACC GCT ACC TCT GTT TGC 1412 Gly Arg Gly Ala Pro Glu He Lys Ala Thr Thr Ala Thr Ser Val Cys 20 25 30
CCT TCT CCT TTC AAA ATG AAG CCC ATA GGA CTT CAA GAG AGA AGA GGG 1460 Pro Ser Pro Phe Lys Met Lys Pro He Gly Leu Gin Glu Arg Arg Gly 35 40 45 50
TCC AAC GTA TCT CTT ACA TTG GAC ATG AGT AGC TTG GGG AAC ATT GAA 1508 Ser Asn Val Ser Leu Thr Leu Asp Met Ser Ser Leu Gly Asn He Glu 55 60 65
CCC TTT GTG TCT ATA CCA ACA CCA CGG GAG AAG GTA GCA ATG GAG TAT 1556 Pro Phe Val Ser He Pro Thr Pro Arg Glu Lys Val Ala Met Glu Tyr 70 75 80
CTG CAG TCA GCC AGC CGA ATT CTC GAC AAG GTT CAG CTG AGG GAC GTC 1604 Leu Gin Ser Ala Ser Arg He Leu Asp Lys Val Gin Leu Arg Asp Val 85 90 95 GTG GCA AGT TCA CAT TTA CTC CAA AGT GAA TTC ATG GAA ATA CCA ATG 1652 Val Ala Ser Ser His Leu Leu Gin Ser Glu Phe Met Glu He Pro Met 100 105 110
AAC TTT GTG GAT CCC AAA GAA ATT GAT ATT CCG CGT CAT GGA ACT AAA 1700 Asn Phe Val Asp Pro Lys Glu He Asp He Pro Arg His Gly Thr Lys 115 120 125 130
AAT CGC TAT AAG ACC ATT TTA CCA AAT CCC CTC AGC AGA GTG TGT TTA 1748 Asn Arg Tyr Lys Thr He Leu Pro Asn Pro Leu Ser Arg Val Cys Leu 135 140 145
AGA CCA AAA AAT GTA ACC GAT TCA TTG AGC ACC TAC ATT AAT GCT AAT 1796 Arg Pro Lys Asn Val Thr Asp Ser Leu Ser Thr Tyr He Asn Ala Asn 150 155 160
TAT ATT AGG GGC TAC AGT GGC AAG GAG AAA GCC TTC ATT GCC ACG CAG 1844 Tyr He Arg Gly Tyr Ser Gly Lys Glu Lys Ala Phe He Ala Thr Gin 165 170 175
GGC CCC ATG ATC AAC ACC GTG GAT GAT TTC TGG CAG ATG GTT TGG CAG 1892 Gly Pro Met He Asn Thr Val Asp Asp Phe Trp Gin Met Val Trp Gin 180 185 190
GAA GAC AGC CCT GTG ATT GTT ATG ATC ACA AAA CTC AAA GAA AAA AAT 1940 Glu Asp Ser Pro Val He Val Met He Thr Lys Leu Lys Glu Lys Asn 195 200 205 210
GAG AAA TGT GTG CTA TAC TGG CCG GAA AAG AGA GGG ATA TAT GGA AAA 1988 Glu Lys Cys Val Leu Tyr Trp Pro Glu Lys Arg Gly He Tyr Gly Lys 215 220 225
GTT GAG GTT CTG GTT ATC AGT GTA AAT GAA TGT GAT AAC TAC ACC ATT 2036 Val Glu Val Leu Val He Ser Val Asn Glu Cys Asp Asn Tyr Thr He 230 235 240
CGA AAC CTT GTC TTA AAG CAA GGA AGC CAC ACC CAA CAT GTG AGC AAT 2084 Arg Asn Leu Val Leu Lys Gin Gly Ser His Thr Gin His Val Ser Asn 245 250 255
TAC TGG TAC ACC TCA TGG CCT GAT CAC AAG ACT CCA GAC AGT GCC CAG 2132 Tyr Trp Tyr Thr Ser Trp Pro Asp His Lys Thr Pro Asp Ser Ala Gin 260 265 270
CCC CTC CTA CAG CTC ATG CTG GAT GTA GAA GAA GAC AGA CTT GCT TCC 2180 Pro Leu Leu Gin Leu Met Leu Asp Val Glu Glu Asp Arg Leu Ala Ser 275 280 285 290
CAG GGG CCG AGG GCT GTG GTT GTC CAC TGC AGT GCA GGA ATA GGT AGA 2228 Gin Gly Pro Arg Ala Val Val Val His Cys Ser Ala Gly He Gly Arg 295 300 305 ACA GGG TGT TTT ATT GCT ACA TCC ATT GGC TGT CAA CAG CTG AAA GAA 2276 Thr Gly Cys Phe He Ala Thr Ser He Gly Cys Gin Gin Leu Lys Glu 310 315 320
GAA GGA GTT GTG GAT GCA CTA AGC ATT GTC TGC CAG CTT CGT ATG GAT 2324 Glu Gly Val Val Asp Ala Leu Ser He Val Cys Gin Leu Arg Met Asp 325 330 335
AGA GGT GGA ATG GTG CAA ACC AGT GAG CAG TAT GAA TTT GTG CAC CAT 2372 Arg Gly Gly Met Val Gin Thr Ser Glu Gin Tyr Glu Phe Val His His 340 345 350
GCT CTG TGC CTG TAT GAG AGC AGA CTT TCA GCA GAG ACT GTC CAG TGAGTCATTG
2427
Ala Leu Cys Leu Tyr Glu Ser Arg Leu Ser Ala Glu Thr Val Gin
355 360 365 370
AAGACTTGTC AGACCATCAA TCTCTTGGGG TGATTAACAA ATTACCCACC CAAGGCTTCA 2487
TGAAGGAGCT TCCTGCAATG GAAGGAAGGA GAAGCTCTGA AGCCCATGTA TGGCATGGAT 2547
TGTGGAAGAC TGGGCAACAT ATTTAAGATT TCCAGCTCCT TGTGTATATG AATGCATTTG 2607
TAAGCATCCC CCAAATTATT CTGAAGGTTT TTTGATGATG GAGGTATGAT AGGTTTATCA 2667
CACAGCCTAA GGCAGATTTT GTTTTGTCTG TACTGACTCT ATCTGCCACA CAGAATGTAT 2727
GTATGTAATA TTCAGTAATA AATGTCATCA GGTGATGACT GGATGAGCTG CTGAAGACAT 2787
TCGTATTATG TGTTAGATGC TTTAATGTTT GCAAAATCTG TCTTGTGAAT GGACTGTCAG 2847
CTGTTAAACT GTTCCTGTTT TGAAGTGCTA TTACCTTTCT CAGTTACCAG AATCTTGCTG 2907
CTAAAGTTGC AAGTGATTGA TAATGGATTT TTAACAGAGA AGTCTTTGTT TTTGAAAAAC 2967
AAAAATCAAA AACAGTAACT ATTTTATATG GAAATGTGTC TTGATAATAT TACCTATTAA 3027
ATGTGTATTT ATAGTCCCTC CTATCAAACA ATTACAGAGC ACAATGATTG TCATCCGGAA 3087
TTC 3090
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 369 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Val Gin Pro Glu Gin Ala Pro Lys Val Leu Asn Val Val Val Asp 1 5 10 15
Pro Gin Gly Arg Gly Ala Pro Glu He Lys Ala Thr Thr Ala Thr Ser 20 25 30
Val Cys Pro Ser Pro Phe Lys Met Lys Pro He Gly Leu Gin Glu Arg 35 40 45
Arg Gly Ser Asn Val Ser Leu Thr Leu Asp Met Ser Ser Leu Gly Asn 50 55 60
He Glu Pro Phe Val Ser He Pro Thr Pro Arg Glu Lys Val Ala Met 65 70 75 80
Glu Tyr Leu Gin Ser Ala Ser Arg He Leu Asp Lys Val Gin Leu Arg 85 90 95
Asp Val Val Ala Ser Ser His Leu Leu Gin Ser Glu Phe Met Glu He 100 105 110
Pro Met Asn Phe Val Asp Pro Lys Glu He Asp He Pro Arg His Gly 115 120 125
Thr Lys Asn Arg Tyr Lys Thr He Leu Pro Asn Pro Leu Ser Arg Val 130 135 140
Cys Leu Arg Pro Lys Asn Val Thr Asp Ser Leu Ser Thr Tyr He Asn 145 150 155 160
Ala Asn Tyr He Arg Gly Tyr Ser Gly Lys Glu Lys Ala Phe He Ala 165 170 175
Thr Gin Gly Pro Met He Asn Thr Val Asp Asp Phe Trp Gin Met Val 180 185 190
Trp Gin Glu Asp Ser Pro Val He Val Met He Thr Lys Leu Lys Glu 195 200 205
Lys Asn Glu Lys Cys Val Leu Tyr Trp Pro Glu Lys Arg Gly He Tyr 210 215 220
Gly Lys Val Glu Val Leu Val He Ser Val Asn Glu Cys Asp Asn Tyr 225 230 235 240
Thr He Arg Asn Leu Val Leu Lys Gin Gly Ser His Thr Gin His Val 245 250 255
Ser Asn Tyr Trp Tyr Thr Ser Trp Pro Asp His Lys Thr Pro Asp Ser 260 265 270
Ala Gin Pro Leu Leu Gin Leu Met Leu Asp Val Glu Glu Asp Arg Leu 275 280 285 Ala Ser Gin Gly Pro Arg Ala Val Val Val His Cys Ser Ala Gly He 290 295 300
Gly Arg Thr Gly Cys Phe He Ala Thr Ser He Gly Cys Gin Gin Leu 305 310 315 320
Lys Glu Glu Gly Val Val Asp Ala Leu Ser He Val Cys Gin Leu Arg 325 330 335
Met Asp Arg Gly Gly Met Val Gin Thr Ser Glu Gin Tyr Glu Phe Val 340 345 350
His His Ala Leu Cys Leu Tyr Glu Ser Arg Leu Ser Ala Glu Thr Val 355 360 365
Gin

Claims

1. An iεolated nucleic acid compriεing a nucleotide εequence encoding at least a fragment of a PTPLl protein tyrosine phoεphataεe.
2. An iεolated nucleic acid aε in claim 1 wherein εaid PTPLl comprises at least a fragment of SEQ ID NO.:2.
3. An isolated nucleic acid as in claim 1 wherein said nucleotide sequence comprises at least a fragment of SEQ ID NO. :1.
4. An iεolated nucleic acid aε in any one of claimε 1-3 wherein εaid nucleotide εequence iε operably joined to regulatory sequences such that mRNA encoding at least a fragment of a PTPLl protein tyrosine phosphataεe may be expreεεed.
5. An iεolated nucleic acid aε in any one of claimε 1-3 wherein εaid nucleotide is operably joined to regulatory sequences such that RNA which is anti-εenεe to mRNA encoding at leaεt a fragment of a PTPLl protein tyroεine phoεphataεe iε expreεεed.
6. A transgenic host into which haε been introduced the iεolated nucleic acid of any of of claimε 1-5.
7. A tranεgenic hoεt aε in claim 6 wherein εaid host is choεen from the group conεisting of E. coli, yeast, COS cells, fibroblasts, oocytes, and embryonic εtem cellε.
8. A substantially pui e protein comprising at least a fragment of a PTPLl protein tyrosine phoεphataεe.
9. A εubstantially pure protein as in claim 8 wherein said PTPLl is at least a fragment of SEQ ID NO.:2.
10. A substantially pure antibody capable of selectively binding at least a fragment of a PTPLl protein tyrosine phosphataεe.
11. An antibody aε in claim 10 wherein εaid PTPLl iε at leaεt a fragment of SEQ ID NO.:2.
12. A method of detecting compoundε capable of altering expreεsion or activity of a PTPLl comprising the steps of
(a) introducing within a cell a nucleic acid encoding a PTPLl protein tyrosine phosphataεe;
(b) growing said cell or a descendant of said cell for a period of time and under conditions which allow for expreεεion of said receptor;
(c) contacting said cell or said deεcendant of εaid cell with a test compound;
(d) performing an assay on said cell or said deεcendant of said cell for an indication of activity of said PTPLl.
13. A method aε in claim 12 further compriεing the εtep of performing an aεεay on εaid cell or εaid deεcendant of εaid cell for an indication of activity of εaid PTPLl prior to contacting εaid cell or said descendant of εaid cell with said test compound.
14. An iεolated nucleic acid comprising a nucleotide sequence encoding at least a fragment of a GLM-2 protein tyrosine phosphatase.
15. An iεolated nucleic acid aε in claim 14 wherein εaid GLM-2 compriεeε at leaεt a fragment of SEQ ID NO.:2.
16. An isolated nucleic acid as in claim 14 wherein said nucleotide sequence comprises at least a fragment of SEQ ID NO. :1.
17. An isolated nucleic acid aε in any one of claimε 14-16 wherein εaid nucleotide εequence iε operably joined to regulatory εequences εuch that mRNA encoding at leaεt a fragment of a GLM-2 protein tyrosine phoεphataεe may be expreεsed.
18. An isolated nucleic acid as in any one of claims 14-16 wherein said nucleotide is operably joined to regulatory sequences such that RNA which is anti-sense to mRNA encoding at least a fragment of a GLM-2 protein tyrosine phosphataεe is expresεed.
19. A transgenic host into which has been introduced the isolated nucleic acid of any of of claims 14-18.
20. A transgenic host as in claim 19 wherein εaid host is chosen from the group consisting of E. coli, yeast, COS cellε, fibroblaεtε, oocyteε, and embryonic εtem cellε.
21. A εubstantially pure protein comprising at least a fragment of a GLM-2 protein tyrosine phosphatase.
22. A εubεtantially pure protein aε in claim 21 wherein εaid GLM-2 iε at leaεt a fragment of SEQ ID NO.:2.
23. A εubεtantially pure antibody capable of selectively binding at least a fragment of a GLM-2 protein tyrosine phosphataεe.
24. An antibody aε in claim 23 wherein εaid GLM-2 is at luast a fragment of SEQ ID NO.:2.
25. A method of detecting compounds capable of altering expression or activity of a GLM-2 comprising the steps of
(a) introducing within a cell a nucleic acid encoding a GLM-2 protein tyrosine phoεphataεe;
(b) growing εaid cell or a deεcendant of said cell for a period of time and under conditions which allow for expreεsion of said receptor;
(c) contacting said cell or said descendant of said cell with a test compound;
(d) performing an aεεay on εaid cell or εaid deεcendant of εaid cell for an indication of activity of εaid GLM-2.
26. A method aε in claim 25 further compriεing the step of performing an asεay on εaid cell or said descendant of εaid cell for an indication of activity of said GLM-2 prior to contacting said cell or said descendant of εaid cell with εaid teεt compound.
PCT/US1994/009943 1993-09-01 1994-09-01 Nucleotide sequences for novel protein tyrosine phosphatases Ceased WO1995006735A2 (en)

Priority Applications (6)

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CA002170515A CA2170515C (en) 1993-09-01 1994-09-01 Nucleotide sequences for novel protein tyrosine phosphatases
EP94926675A EP0789771A2 (en) 1993-09-01 1994-09-01 Primary structure and functional expression of nucleotide sequences for novel protein tyrosine phosphatases
AU76443/94A AU683299B2 (en) 1993-09-01 1994-09-01 Nucleotide sequences for novel protein tyrosine phosphatases
NZ273219A NZ273219A (en) 1993-09-01 1994-09-01 Nucleotide sequences for protein tyrosine phosphatases
JP7508288A JPH09510861A (en) 1993-09-01 1994-09-01 Primary structure and functional expression of nucleotide sequences for a novel protein tyrosine phosphatase
US08/596,291 US5821075A (en) 1993-09-01 1994-09-01 Nucleotide sequences for novel protein tyrosine phosphatases

Applications Claiming Priority (2)

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US11557393A 1993-09-01 1993-09-01
US08/115,573 1993-09-01

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EP (1) EP0789771A2 (en)
JP (1) JPH09510861A (en)
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CA (1) CA2170515C (en)
NZ (1) NZ273219A (en)
WO (1) WO1995006735A2 (en)

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WO1997006245A1 (en) * 1995-08-04 1997-02-20 Medical Research Council Map kinase phosphatase gene and uses thereof
WO1997006262A1 (en) * 1995-08-10 1997-02-20 Incyte Pharmaceuticals, Inc. Non-receptor type human protein tyrosine phosphatase
WO1998001551A1 (en) * 1995-10-13 1998-01-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Tyrosine kinase associated protein-1 (tka-1), nucleotide and amino acid sequences and their use in diagnosis and treatment of tka-1 related disorders
WO1998037196A1 (en) * 1997-02-25 1998-08-27 Ludwig Institute For Cancer Research PARG, A GTPase ACTIVATING PROTEIN WHICH INTERACTS WITH PTPL1
WO1998049317A3 (en) * 1997-04-28 1999-02-25 Sugen Inc Diagnosis and treatment of phosphatase- 0r kinase-related disorders
US5945523A (en) * 1995-10-13 1999-08-31 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Diagnosis and treatment of TKA-1 related disorders
US5951979A (en) * 1996-07-25 1999-09-14 Cold Spring Harbor Laboratory Substrate trapping protein tyrosine phosphatases
WO2000053801A1 (en) * 1999-03-08 2000-09-14 Merck Frosst Canada & Co. Intact cell assay for protein tyrosine phosphatases
US6337192B1 (en) 1998-01-20 2002-01-08 Myraid Genetics, Inc. MMSC1-an MMAC1 interacting protein
US6388063B1 (en) 1997-06-18 2002-05-14 Sugen, Inc. Diagnosis and treatment of SAD related disorders
US6818440B2 (en) 1997-04-28 2004-11-16 Sugen, Inc. Diagnosis and treatment of alk-7 related disorders
US6844177B2 (en) 1997-05-20 2005-01-18 Sugen, Inc. Diagnosis and treatment of PTP04 related disorders

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US6521414B2 (en) * 2000-02-01 2003-02-18 Agy Therapeutics, Inc. Methods for identifying a modulator of the interaction of NMDA receptor with protein tyrosine phosphatase L1
US20030092071A1 (en) * 2000-02-01 2003-05-15 Jasna Jerecic Interaction of NMDA receptor with protein tyrosine phosphatase
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US20040115635A1 (en) * 2002-12-11 2004-06-17 Isis Pharmaceuticals Inc. Modulation of PTPN13 expression
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WO1997006245A1 (en) * 1995-08-04 1997-02-20 Medical Research Council Map kinase phosphatase gene and uses thereof
WO1997006262A1 (en) * 1995-08-10 1997-02-20 Incyte Pharmaceuticals, Inc. Non-receptor type human protein tyrosine phosphatase
WO1998001551A1 (en) * 1995-10-13 1998-01-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Tyrosine kinase associated protein-1 (tka-1), nucleotide and amino acid sequences and their use in diagnosis and treatment of tka-1 related disorders
US5945523A (en) * 1995-10-13 1999-08-31 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Diagnosis and treatment of TKA-1 related disorders
US5951979A (en) * 1996-07-25 1999-09-14 Cold Spring Harbor Laboratory Substrate trapping protein tyrosine phosphatases
WO1998037196A1 (en) * 1997-02-25 1998-08-27 Ludwig Institute For Cancer Research PARG, A GTPase ACTIVATING PROTEIN WHICH INTERACTS WITH PTPL1
US6083721A (en) * 1997-02-25 2000-07-04 Ludwig Institute For Cancer Research Isolated nucleic acid molecules encoding PARG, a GTPase activating protein which interacts with PTPL1
US6475775B1 (en) 1997-02-25 2002-11-05 Ludwig Institute For Cancer Research PARG, a GTPase activating protein which interacts with PTPL1
AU746135B2 (en) * 1997-02-25 2002-04-18 Ludwig Institute For Cancer Research PARG, a GTPase activating protein which interacts with PTPL1
WO1998049317A3 (en) * 1997-04-28 1999-02-25 Sugen Inc Diagnosis and treatment of phosphatase- 0r kinase-related disorders
US6818440B2 (en) 1997-04-28 2004-11-16 Sugen, Inc. Diagnosis and treatment of alk-7 related disorders
US6844177B2 (en) 1997-05-20 2005-01-18 Sugen, Inc. Diagnosis and treatment of PTP04 related disorders
US6388063B1 (en) 1997-06-18 2002-05-14 Sugen, Inc. Diagnosis and treatment of SAD related disorders
US6337192B1 (en) 1998-01-20 2002-01-08 Myraid Genetics, Inc. MMSC1-an MMAC1 interacting protein
US6329137B1 (en) 1999-03-08 2001-12-11 Merck Frosst Canada & Co. Intact cell assay for protein tyrosine phosphatases using recombinant baculoviruses
WO2000053801A1 (en) * 1999-03-08 2000-09-14 Merck Frosst Canada & Co. Intact cell assay for protein tyrosine phosphatases

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CA2170515A1 (en) 1995-03-09
NZ273219A (en) 1998-06-26
EP0789771A2 (en) 1997-08-20
JPH09510861A (en) 1997-11-04
US5821075A (en) 1998-10-13
CA2170515C (en) 2002-11-12
WO1995006735A3 (en) 1995-06-22
US6066472A (en) 2000-05-23
AU7644394A (en) 1995-03-22
AU683299B2 (en) 1997-11-06

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