WO1994003610A2 - Isolation and cloning of a protein with tyrosine-phosphatase activity - Google Patents
Isolation and cloning of a protein with tyrosine-phosphatase activity Download PDFInfo
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- WO1994003610A2 WO1994003610A2 PCT/EP1993/002036 EP9302036W WO9403610A2 WO 1994003610 A2 WO1994003610 A2 WO 1994003610A2 EP 9302036 W EP9302036 W EP 9302036W WO 9403610 A2 WO9403610 A2 WO 9403610A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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Definitions
- the present invention relates to transmembrane tyrosine phosphatase proteins, DNA coding therefor, methods for production and identification of these proteins, and methods for screening compounds capable of binding to and inhibiting or stimulating phosphatase enzymatic activity.
- the phosphorylation state of protein tyrosyl residues plays a key role in cellular growth control.
- Cellular phosphotyrosine levels are regulated by the relative activities of opposing protein-tyrosine kinases and tyrosine phosphatases.
- Several growth factor receptors, as well as insulin receptor, are tyrosine-specific protein kinases.
- receptor tyrosine kinases The activation of receptor tyrosine kinases is mediated by interaction with their respective growth factors or cytokines, such as FGF, EGF, PDGF, GM-CSF and IL-1, and usually induces mitogenesis and growth (Yarden, Y. and Ullrich, A.: Annual Rev. Biochem. 52, 443-478; 1988).
- cytokines such as FGF, EGF, PDGF, GM-CSF and IL-1
- Class I contains the low molecular weight non-receptor molecules possessing a single catalytic domain and includes placental PTPase IB (Charbonneau, H. et al., Proc. Natl. Acad. Sci. 86; 5252-5256, 1989; Chernoff, J. et al., Proc. Natl. Acad. Sci. 82:2735-2789,1990), T-cell PTPase (Cool, D.E. et al., Proc. Natl. Acad. Sci. j$6_:5257-5261,1989) and rat brain PTPase (Guan,K. et al., Proc. Natl, Acad. Sci.
- Class II and III PTPases are receptor-like transmembrane receptors. While the sole members of Class II described so far (HPTP3 and DPTP 10 D) have a single cytoplasmic catalytic domain (Kreuger, N.X. et al.: EMBO J., 9, 3241-3252, 1990; Shin-Shay, T. et al : Cell 67, 675-685, 1991) , Class III members possess two repeated putative catalytic domains in the cytoplasmic region of the molecule. Class III includes the leukocyte common antigen (LCA) (Ralph, S.J., EMBO J., .6:1251-1257, 1987; Charbonneau, H.
- LCA leukocyte common antigen
- PTPases have adhesion molecule-like extracellular domains consisting of Fibronectin-type (FN) repeats alone (HPTP0) or of immunoglobulin domains and FN repeats together (LAR, DLAR and DPTP) , suggesting that they may couple cell-cell recognition to tyrosine phosphorylation. Indeed, it has been shown that density-dependent inhibition of cell growth involves the regulated elevation of tyrosine phosphatase activity, thus supporting the concept that this activity may play a role in the control of cell growth, differentiation and oncogenesis.
- the invention provides mammalian receptor- type protein tyrosine phosphatases (PTPases) whose extracellular domains include an Arg-Gly-Asp sequence, and functional derivatives thereof.
- PTPases are Class II PTPases.
- a Class II PTPase may be the novel PTPase PTP35 which has a transmembrane topology. Two different forms of PTP35 have been isolated, sharing common structural characteristics and differing only in their N-terminal sequences.
- the extracellular domain of PTP35 is unrelated to any other receptor-type phosphatase described so far and contains an Arg-Gly-Asp (RGD) sequence.
- PTP35 seems to be particularly significant in brain, suggesting that it may have an important role in the control of central nervous system metabolism and differentiation. Moreover, when PTP35 is studied in cultured fibroblasts, it appears to be involved in the control of cell proliferation. Also, the levels of mRNA for phosphatase PTP35 seem to be specifically increased by stimulation of 3T3 cells in culture with basic Fibroblast Growth Factor (bFGF) or other cell mitogens.
- bFGF basic Fibroblast Growth Factor
- the present inventors have identified a novel receptor-type protein tyrosine phosphatase.
- the analysis performed at the DNA level as well as the predicted amino acid structure of the proteins indicate that the proteins, lacking duplication of the catalytic domain, belong to Class II of the Protein Tyrosine Phosphatases (PTPases) , thus representing new examples of this particular group of phosphatases.
- PTPases Protein Tyrosine Phosphatases
- PTP35 The different forms of PTP35 described herein share common features. On the basis of the predicted amino acid sequence various functional domains can be recognised. Based on the presence of a hydrophobic stretch (see Figures la and lb) , PTP35 is classified as a transmembrane protein, comprising an extracellular domain N-terminal to the hydrophobic stretch, and an intracellular domain C-terminal to the hydrophobic stretch.
- the predicted intracellular portion of the protein which is identical in the different forms of PTP35 so far isolated, consists of a unique domain displaying significant homology to the intracellular catalytic regions of the previously described transmembrane phosphatases.
- the conserved amino acid sequence IIVHCSDGAGRTG (one letter code) , which has been proposed to be part of the phosphatase catalytic domain, is also present in PTP35 (see Figs, la and lb) .
- the greatest degree of homology was found with mouse LRPA (Matthews, R.J., Cahir, E.D. and Thomas, M.L. Proc. Natl. Acad. Sci., 82, 4444-4448, 1990; Sap. J. et al.,: Proc. Natl. Acad. Sci, l_, 6112-6116, 1990) and mouse CD45 (Charbonneau, H., Tonks, N. , Walsh, K. and Fischer, E. Proc. Natl. Acad. Sci. JL5, 7182-7186, 1988) tyrosine phosphatases.
- the present invention thus includes a new mammalian receptor-type protein tyrosine phosphatase PTPase, named PTP35, whose extracellular domain includes an Arg-Gly-Asp sequence, or any functional derivative thereof. PTP35 does not share any significant homology with other known PTPases in its extracellular domain. The sequences of two forms of PTP35 are shown in Figures la and lb.
- the protein of the invention may be of natural origin or, alternatively, may be prepared by chemical or recombinant means.
- the molecule When the molecule is of natural origin it may be obtained by subjecting the cells, tissues or fluids containing the PTPase to standard protein purification techniques so as to obtain a preparation substantially free of other proteins with which the PTPase is natively associated.
- An example of purification techniques is represented by an affinity purification in which a solid-phase substrate or ligand binds the enzymatic or, respectively, the receptor domain of the PTPase.
- the purification can be achieved by a combination of standard methods, such as ammonium sulfate precipitation, molecular sieve chromatography and ion exchange chromatography.
- the PTPase of the invention may be biochemically purified from a variety of tissues and cell lines. Among these, mouse brain and NIH-3T3 cell line are preferred.
- the invention includes a fusion protein comprising a PTPase or derivative thereof according to the invention, fused to a carrier polypeptide.
- the protein produced by the above methods is substantially free from other proteins. “Substantially free of other proteins” indicates that the protein is at least 90% or even at least 95% free of other proteins. Most preferably, the protein is 98% of more pure.
- other proteins includes proteins with which the PTPase is associated naturally in a cell and proteins present in a recombinant host cell.
- functional derivative is meant any fragment, variant, analog or chemical derivative of the PTPase, for example of PTP35, retaining at least a portion of the function of the PTPase such as the phosphatase enzymatic activity or the binding of the extracellular domain to a ligand.
- a functional derivative consists essentially of either the intracellular or extracellular domain, or the amino acid sequence encoded by nucleotides 279 to 2874 of Figure la.
- fragment it is meant any subset of the molecule, that is, a shorter peptide.
- variant it is meant a molecule substantially similar to either the entire peptide or a fragment thereof.
- Natural variants include the homologs of PTP35 in different mammalian species other than mouse, that is the source from which PTP35 has been isolated:' Synthetic variants of a PTPase such as PTP35 may be obtained either by direct chemical synthesis using methods well-known in the art or inserting appropriate mutations in the DNA encoding the PTPase. The variants thus obtained include, for example, deletions, insertions or substitutions of residues as well as extensions.
- a variant is suitably at least 50%, at least 70%, at least 90%, at least 95% or at least 98% homologous to a PTPase such as PTP35 or fragment thereof.
- a deletion, insertion, extension or substitution may be N-terminal, C-terminal or internal and comprises one or more amino acids, suitably one, two, three, five, ten, twenty, thirty or forty amino acids.
- An extension may be much larger and may constitute a carrier protein.
- a substitution is generally a conservative substitution in which each substituted amino acid is replaced by a biologically similar amino acid. For example, each amino acid may be replaced by an amino acid of similar size, hydrophobicity, charge and/or chemical functionality.
- Candidate substitutions are:
- analog it is meant a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
- a "chemical derivative" of a PTPase such as PTP35 contains additional chemical moieties not normally a part of the peptide.
- chemical modifications include covalent modifications of the peptide, derivatization with bifunctional agents and amidation of the C-terminal carboxyl groups.
- derivatized moieties may improve the physico-chemical characteristics or the biological activity of the PTPase.
- a particularly preferred embodiment of the present invention is a protein having substantially the amino acid sequence shown in Figure la or lb.
- Another embodiment of the present invention is a DNA molecule consisting essentially of a nucleotide sequence encoding a PTPase or functional derivative thereof.
- the DNA sequence may be in the form of cDNA or genomic DNA.
- the sequence is suitably that of PTP35 or a derivative thereof.
- the recombinant DNA molecules of the present invention can be produced through any of a variety of means well known to the expert in the art and disclosed by, for example, Maniatis et al.. Molecular cloning: a laboratory manual. Second Edition, Cold
- Single stranded, fully degenerated oligonucleotide probes complementary to a conserved region of the PTPases family are useful for screening DNA, cDNA or RNA preparations derived from cell lines capable of expressing the PTPase gene.
- a preferred probe would be one directed to the nucleic acid sequence enconding at least 5 amino acid residues of the PTPase of the present invention.
- tissue-type plasminogen activator (Pennico, D. et al.. Nature 3.01:214-221 (1983)).
- Such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment.
- Examples of such methodologies are provided by Cohen et al. (U.S. Patent 4,237,224), Maniatis et al. (Molecular Clonin ⁇ : A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)) which references are herein incorporated by reference.
- PCR polymerase chain reaction
- the polymerase chain reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample.
- the method can be used to amplify either single- or double-stranded DNA.
- the essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.
- the invention provides a DNA molecule consisting essentially of a nucleotide sequence enconding a functional derivative of PTP35 as defined above.
- a DNA molecule may encode a variant of a PTPase of the invention.
- Such a DNA molecule is suitably at least 40%, at least 60%, at least 80%, at least 90%, at least 95% or at least 98% homologous to the sequence showin in Figure 1 or a fragment thereof.
- a DNA molecule may encode a PTPase of the invention having a deletion, insertion, extension or substitution as described above.
- Such a DNA molecule may be prepared by site-directed mutagenesis (as exemplified by Adelman et al.. DNA 2:183 (1983)) of nucleotides in the DNA encoding the polypeptide molecule, thereby producing DNA encoding the derivative, and thereafter expressing the DNA in recombinant cell culture.
- An expression vector is a vector which (due to the presence of transcriptional and translational control sequences) is capable of expressing a DNA molecule which has been cloned into the vector and of thereby producing a polypeptide.
- a vector may be a plasmid or viral vector. Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences, for example E. coli.
- the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequences.
- a yeast host may be employed, for example S. cerevisiae.
- insect cells may be used, in which case a baculovirus expression system may be appropriate.
- Another alternative host is a mammalic cell line, for example Chinese Hamster ovary cells.
- a DNA sequence encoding the PTPase of the present invention, or its functional derivatives may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis et al.. supra. and are well known in the art.
- a suitable expression vector contains transcriptional and translational regulatory information able to direct and control gene expression in an appropriate host. These sequences are operably linked to the gene sought to be expressed and include a promoter (which directs the initiation of RNA transcription) , the Shine-Dalgarno sequence, capping sequence, CAAT sequence and the like (which are involved with initiation of transcription and translation) .
- the promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral. Examples of suitable prokaryotic sequences include the P R and P L promoters of bacteriophage lambda (The Bacteriophage Lambda .
- Shine-Dalgarno sequence As far as the Shine-Dalgarno sequence is concerned.
- suitable regulatory sequences are represented by the Shine-Dalgarno of the replicase gene of the phage MS-2 and of the gene ell of bacteriophage lambda.
- the Shine-Dalgarno sequence may be directly followed by the DNA encoding PTP35 and result in the expression of the mature PTP35 protein.
- the DNA encoding PTP35 or genetic variants thereof may be preceded by a DNA sequence encoding a carrier peptide sequence.
- a fusion protein is produced in which the N-terminus of PTP35 is fused to a carrier peptide, which may help to increase the protein expression levels and intracellular stability, and provide simple means of purification.
- a preferred carrier peptide includes one or more of the IgG binding domains of Staphylococcus protein A. Fusion proteins comprising IgG binding domains of Protein A are easily purified to homogeneity by affinity chromatography e.g. on IgG- coupled Sepharose.
- a DNA sequence encoding a recognition site for a proteolyti ⁇ enzyme such as enterokinase, factor X or procollagenase may immediately precede the sequence for PTP35 or a variant thereof, to permit cleavage of the fusion protein to obtain the mature PTP35 protein.
- a suitable expression vector includes an appropriate marker which allows the screening of the transformed host cells.
- marker- is typically an antibiotic resistance gene which confers to the transformed host the ability to grow on a selective medium containing the antibiotic to which the gene confers resistance.
- the transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art described in Maniatis et al, supra.
- a further embodiment of the present invention is therefore a suitable expression vector containing the DNA sequence encoding the PTPase of the invention or a functional derivative thereof.
- the DNA sequence encoding PTP35 or variants thereof may be preceded by a sequence encoding a carrier peptide to obtain a fusion protein.
- One further embodiment of the invention is a host cell, either prokaryotic or eukaryotic, transformed with the said expression vector and able to produce, under appropriate culture conditions, the PTPase of the invention or a functional derivative thereof.
- the present invention is also directed to a process for preparing the PTPase protein of the invention or a functional derivative thereof, comprising:
- the host is suitably prepared by:
- the PTPases and functional derivatives of the invention are useful in methods for testing of compounds capable of enhancing or inhibiting the phosphatase activity.
- the invention is also useful in diagnosing disorders involving the PTPase of the invention.
- the ability of a compound under test to modify phosphatase activity can be tested in an in vitro system wherein the test compound is added to purified PTPase protein or enzymatically active derivatives thereof, and the effects on enzyme activity measured using standard enzymological procedures well known to those of skill in the art.
- the action of a compound on PTPase activity can be measured in a whole cell preparation using live or fixed cells, or a membrane fraction derived from live or fixed cells. This method is useful for screening compounds acting via the extracellular receptor portion of the protein, as well as compounds acting directly on the enzymatic portion of the protein.
- a test compound is incubated with cells, or with a membrane preparation derived therefrom, which express high amounts of the PTPase of this invention, such as NIH-3T3 cells.
- the amount of cellular phosphotyrosine is then measured, using methods well-known in the art (Honegger, A.M. et al.. Cell 51:199-209 (1987); Margolis, B. et al.. Cell 57:1101- 1107 (1989)) .
- the results are compared to results obtained in the absence of the test compound, or in the absence or presence of a known activator of R- TPase. In such studies, the action of the test compound in the presence of an activator of tyrosine kinase can also be measured.
- a compound which stimulates PTPase activity will result in a net decrease in the amount of phosphotyrosine, whereas a compound which inhibits PTPase activity will result in a net increase in the amount of phosphotyrosine.
- tyrosine kinases such as the receptors for epidermal growth factor (EGF) and for platelet-derived growth factor (PDGF)
- EGF epidermal growth factor
- PDGF platelet-derived growth factor
- tyrosine phosphorylation is linked to cell growth and to oncogenic transformation.
- Activation of a PTPase, leading to dephosphorylation would serve as a counterregulatory mechanism to prevent or inhibit growth, and might serve as an endogenous regulatory mechanism against cancer.
- mutation or disregulation of this receptor/enzyme system may promote susceptibility to cancer.
- the insulin receptor is also a tyrosine kinase, and phosphorylation of tyrosine in cells bearing insulin receptors would be associated with normal physiological function. In contrast to the case of cell growth and cancer, activation of a PTPase would counteract insulin effects. Subnormal PTPase levels or enzymatic activity would act to remove a normal counterregulatory mechanisms.
- susceptibility to diabetes may be associated with PTPase disregulation.
- recognition of tyrosine phosphatases expressed in pancreatic islets as autoantigens may lead to diabetes as a consequence of cells distruction by an autoimmune mechanism.
- the use of recombinant extracellular domain of such phosphatases may be used to block the binding of autoantibodies to phosphatases expressed on the surface of islet cells, thus providing a direct therapeutic effect.
- the methods of the present invention for measuring the amount or activity of PTPase associated with a cell or tissue can serve as methods for identifying susceptibility to cancer, diabetes, or other diseases associated with alterations in cellular phosphotyrosine metabolism.
- regulation of PTP35 activity may be important in neuronal cells differentiation and survival. Since it has been reported that insulin receptors distribution in brain corresponds to location of proteins phosphorylated in tyrosine residues, the action of tyrosine phosphatases which are specifically expressed in brain, such as PTP35, may be important in the regulation of the insulin receptor tyrosine kinase in this tissue.
- an embodiment of the present invention is a method for identifying a compound capable of stimulating or inhibiting the enzymatic activity of a protein according to the invention, comprising:
- step (b) incubating the mixture in step (a) for a sufficient interval;
- the invention includes an antibody capable of binding to a PTPase or derivative thereof according to the invention.
- the antibody may be monoclonal or polyclonal.
- the antibody is useful for diagnosing disorders involving a PTPase of the invention.
- a method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with a PTPase or derivative of the invention and isolating immunoglobulins from the immune serum. The animal may therefore be inoculated with the PTPase or derivative, blood subsequently removed from the animal and the IgG fraction purified.
- a method for producing a monoclonal antibody comprises immortalising cells which produce the desried antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein, Nature 256, 495-497, 1975).
- An immortalized cell producing the desired antibody may be selected by a conventional procedure.
- the hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host.
- Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.
- the experimental animal is suitably a goat, rabbit, rat or mouse.
- the PTPase or derivative of the invention may be administered as a conjugate in which the PTPase or derivative is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier polypeptide.
- the carrier molecule is typically a physiologically acceptable carrier.
- the antibody obtained may be isolated and, if desired, purified.
- the invention includes a method for the determination of a PTPase or derivative of the invention in a test sample, which method comprises contacting the test sample with an antibody of the invention, and determining the amount of antibody binding to the PTPase or derivative thereof.
- This method is useful in diagnosing disorders involving a PTPase of the invention.
- An ELISA (enzyme-linked immunoassay) method such as a non-competitive ELISA, is suitably used for the determination.
- an ELISA method comprises the steps of (i) immobilising on a solid support an unlabelled antibody in accordance with the invention, (ii) adding a test sample containing the PTPase or derivative thereof to be determined such that the PTPase or derivative thereof is captured by the unlabelled antibody, (iii) adding an antibody in accordance with the invention which has been labelled, and (iv) determining the amount of bound labelled antibody.
- Suitable antibody labels include biotin (which may be detected by avidin conjugated to peroxidase) and alkaline phosphatase.
- a Western blotting method may be used for determining a PTPase or derivative of the invention. Such a method can comprise the steps of
- the invention includes a method of determination of an mRNA encoding a PTPase or derivative thereof according to the invention in a test sample, which method comprises hybridising mRNA present in the test sample with a DNA probe consisting essentially of sequence shown in Figure 1, and determining the amount of resulting DNA:mRNA hybrids.
- a Northern Blotting method is used for determination of mRNA, which method may comprise (i) subjecting a test sample containing an mRNA encoding a PTPase or derivative thereof according to the invention to gel electrophoresis,
- RNA molecules in the gel onto a solid support (e.g. a nitrocellulse support) by blotting, (iii) allowing a DNA probe to hybridise to the mRNA, and (iv) measuring the amount of hybridised DNA probe.
- a solid support e.g. a nitrocellulse support
- Fig.l Sequence of PTP35 The sequences of the cDNA clones PTP35,11 ( Figure la) and PTP35, 12 ( Figure lb) are shown together with translation of the major open reading frame.
- DNA sequence the putative ATG (clone 11) and CTG (clone 12) start codons, and TGA termination codons are boxed.
- Protein sequence the RGD feature, the putative transmembrane peptide and the tyrosine phosphatase consensus motif are boxed.
- GPDH glyceraldeide-3-phosphate dehydrogenase
- Fig.3 Panel A Growth curve of Swiss NIH3T3 cells 3T3 cells were seeded at 10 3 cells/cm2 ⁇ .and cultured in DMEM medium supplemented with 10%FCS. Each dayiscells were trypsinized and counted. Panel
- Fig. 4 Panel A Kinetics of induction of PTP35 mRNA in 3T3 cells by bFGF Northern blot hybridization of total RNA from cells untreated (-) or treated with 30 ng/ml of bFGF (+) at different times after treatment with bFGF. PTP35 mRNA is maximally induced 24 hours after bFGF addition. Equal loading of all lanes was verifyed by rehybridization with an actin probe. The position of 28S and 18S ribosomal RNA is indicated. Panel B Specificity of induction of PTP 35 mRNA Northern blot hybridization of total RNA 24 hours after treatment. .
- Lane 1 control; lane 2, addition of 30 ng/ml bFGF; lane 3, addition of 30 ng/ml bFGF + 10 ⁇ g/ml cycloheximide; lane 4, addition of 10 ⁇ g/ml cycloheximide; lane 5, addition of 10 ng/ml PDGF; lane 6, addition of 10 ng/ml PDGF + 10 ⁇ g/ml cycloheximide. Equal loading of all lanes was verified by rehybridization with an actin probe.
- PTP35 intracellular domain Soluble cell extract after sonication was incubated with gluthatione agarose resin, extensively washed and then incubated with thrombin. Essentially pure, soluble PTP35 intracellular domain was collected in the medium after release from the resin. Lane 1, molecular weight markers; lane 2, soluble cell extract; lane 3, washing medium; lane 4, aliquot of the resin after thrombin addition; lane 5, soluble pure PTP35 intracellular domain. The arrows indicate GST-PTP35 fusion (lane 2) or mature PTP35 intracellular domain (lane 5) .
- RNA from actively growing Swiss 3T3 fibroblasts was prepared according to standard procedures (Maniatis, T. , Frisch, E'.F. and Sambrook, J.: Molecular cloning. A labouratory manual. Cold Spring Harbour labouratory, Cold Spring Harbour, NY 1989).
- First-strand cDNA was synthesized from 10 ⁇ g of total RNA using AMV reverse transcriptase (Boehringer Mannheim) and amplified by Polymerase Chain Reaction (PCR) using two degenerated primers encoding conserved phosphatase sequences.
- the sense primer 1 (5' AAG CTT CTG CAG GTC GAC TTT/C TGG GAA/G ATG GT/A/C/T/G TGG GA 3') harboured a Sail restriction site followed by a degenerated sequence encoding the conserved amino acids FWEMVWE.
- the complementary primer 4 (5' CGA ATT CGG TAC CGG ATC CTG CCC CGG CAC TGC AGT GCA C 3') harboured a sequence complementary to conserved amino acids VHCSAGA followed by an EcoRI restriction site.
- the two primers were used to amplify first strand cDNA from growing 3T3 cells using Taq Polymerase (Perkin-Elmer) .
- the annealing temperature was 55 C° and the polymerization reaction was done at 72 C°. 40 cycles of amplyfication were performed using a Perkin-Elmer thermal cycler.
- the amplified fragment with the expected size of about
- DNA of the recombinant pBluescript SK ⁇ plasmids was excided from the selected clones as described by the manufacturer (Stratagene, La Jolla, USA) and subjected to EcoRI digestion, to verify the presence and size of the inserts. Double stranded DNA of the different clones was directely sequenced using the dideoxynucleotide chain termination method (Sequenase, United States Biochemical; Proc. Natl. Acad. Sci 24. 5463-5476 (1977)) and the universal forward and reverse primers. The relative order and orientation of the EcoRI fragments in the recombinant plasmids was obtained by restriction mapping and sequence overlapping.
- Nucleotide sequence analysis was performed using the Geneworks program (Intelligenetics, CA) . Translation of the sequence contained in the cDNA clone PTP35,11 revealed the existance of a major open reading frame encoding 790 amino acids, assuming that translation initiates at nucleotide 505 (an in- frame stop codon is present at nucleotide 124, 381 nucleotides upstream) . On the basis of the presence of a hydrophobic stretch between amino acids 388 and 408, the protein is classified as transmembrane. The predicted intracellular portion of the protein consists of a unique domain displaying significant homology to the intracellular catalytic regions of the previously described transmembrane phosphatases.
- VHCSDG conserved sequence VHCSDG, which has been proposed to be part of the phosphatase catalytic site is present in PTP35. Since there is no duplication of the catalytic domain, protein PTP35 belongs to tyrosine phosphatases Class II.
- Nucleotide sequence analysis of clone PTP35,12 was performed using the Geneworks program (Intelligenetics, CA) . The sequence was found to correspond to that of clone 11 of PTP35 from nucleotide 279 to the end, while the two sequences diverge at their 5' ends, possibly due to alternative splicing.
- the mature PTP35 molecule encoded in clone PTP35,12 would then start at amino acid 18.
- the predicted intracellular portion, which corresponds to that encoded in clone PTP35,11, consists of a unique domain displaying significant homology to the intracellular catalytic regions of the previously described transmembrane phosphatases.
- the sequence IIVHCSDGAGRTG at amino acids 886 to 899 matches the consensus sequence for PTPase activity.
- RNA from mouse spleen, heart, brain and liver purchased from Clonetech, CA was performed according to standard procedures (Maniatis, T. , Frisch, E.F. and Sambrook, J. : Molecular cloning. A labouratory manual. Cold Spring Harbour labouratory, Cold Spring Harbour, NY 1989) , using 20 ⁇ g of RNA per lane.
- Hybridization was performed overnight at 42 ⁇ C in 50% formamide, 5 X SSC, 1 X Denhardt's, 0.1%SDS and 250 mg/ml of salmon sperm DNA, using the Sall/EcoRI fragment used for the library screening as a probe at 2 x 10 cpm/ml. Washing was done at 42°C in 1 X SSC, 0.1% SDS. Higher stringency washing did not noticeably affect the hybridization pattern.
- Fig. 2, panel A Northern analysis revealed a highly specific pattern of expression for PTP35 in mouse, essentially restricted to the brain. In this tissue, an mRNA of about 3200 bp was expressed at very high level, in addition to a second mRNA of smaller size, about 1400 bp, expressed in lower amounts. Analysis of PTP35 mRNA regulation
- rat cerebellum granules neurons express high levels of PTP35 mRNA of the same size, about 3200 bp, of that observed in mouse brain. Expression is higher in cells in advanced differentation state, with extended neurite outgrowth and cell to cell connnections, suggesting that PTP35 is involved in rat cerebellum granules neurons differentiation and survival.
- NIH3T3 cells were seeded at low density, 10 cells per cm , and cultivated for 7 days in DMEM medium (Gibco) supplemented with 10% Fetal Calf Serum (FCS) (Gibco) . Medium was changed every other day. Each day, cells from two dishes were trypsinized and counted. The growth curve obtained for 3T3 cells is shown in figure 3, panel A. In parallel, each day cells were harvested and total RNA was prepared and subjected to Northern blot analysis as described (Maniatis, T. , Frisch, E.F. and Sambrook, J.: Molecular cloning. A labouratory manual.
- RNA per lane 20 ⁇ g of RNA per lane.
- Hybridization was performed under standard conditions (see the above paragraph) using the Sall/EcoRI PTP35 fragment as a probe at 2 x 10 cpm/ml.
- the results of the Northern blot hybridization are shown in fig. 3, panel B.
- Analysis of mRNA steady-state levels obtained from cells at different days showed that PTP35 mRNA expression was directly correlated with cell growth. mRNA maximum levels were observed in actively growing cells and decreased until little or no expression was observed in confluent, quiescent 3T3 cells, suggesting that PTP35 is involved in the regulation of cell growth.
- bFGF basic Fibroblast Growth Factor
- the vector pRIT33 (commercially available from Pharmacia; see also Methods in Enzymology 185. 144-161 (1990)) is a derivative of plasmid pRIT2 and is designed for temperature- inducible expression of intracellular hybrid proteins in E. coli after induction of lambda P R promoter. Alternatively, expression can be obtained by induction of the lacUV5 promoter, which is inserted up-stream of the lambda P R promoter, with 1 mM IPTG.
- the construct contains the IgG-binding domains of Staphylococcal protein A which are under the control of the lambda P_ promoter. A multiple cloning site facilitates the insertion of foreign genes at the 3' side of protein A.
- the sequence encoding the recognition site for Factor X is present at the end of the Protein A coding sequence.
- the protein A transcription termination sequence is inserted immediately donwstream from the multiple cloning site.
- the sequence encoding PTP35 protein was inserted into plasmid pRIT33.
- the PTP35 sequence was retrieved from plasmid PTP35,4 which is one of the clones isolated after the screening of the library described in Example 1.
- PTP35,4 corresponds to plasmid pBluescript SK " containing the PTP35 coding sequence oriented in such way that its 5' end is preceded by the BamHI site of the polylinker and its 3' end is followed by the Sail site of the polylinker.
- a Stul-Sall fragment of about 3000 bp encompassing PTP35 coding sequence (nucleotides 590 to 3561 of clone 12, Figure lb) was isolated from plasmid PTP35, 4 and subcloned into plasmid pRIT33 cut with Smal and Sail. Smal and StuI blunt ends ligated together resulted in appropriate in-frame positioning of the PTP35 sequence immediately after the Factor X recognition sequence in pRIT33.
- the resulting plasmid pRIT33-PT35 carries an open reading frame, coding for a fusion protein of 120 kD, composed of protein A at the N-terminus followed by the PTP35 protein, separated be a specific sequence for proteolytic cleavage.
- plasmid PTP35,4 was cut with Ncol and the protruding ends were filled in by Klenow DNA polymerase to leave blunt ends. The plasmid was then cut with StuI and a 1200 bp fragment with blunt extremities (corresponding to nucleotides 590 to 1804 of clone PTP35,12) was subcloned into pRIT33 cut with Sma I and dephosphorylated.
- the resulting plasmid pRIT33-PTP35XC carries an open reading frame encoding a protein of about 75 kD composed of the protein A followed by the PTP35 extracellular domain amino acids, separated by a specific sequence for proteolytic cleavage.
- Expression of the Protein A-PTP35XC fusion protein was analyzed in HB101 E.Coli cells (J. Mol. Biol. 41, 454, 1969) cotransformed with a compatible plasmid, named pRITcl857, harbouring a termosensitive repressor of lambda P R promoter (Nilsson, B. and Abrah sen, L. Methods in Enzymology 185. 144- 161 , 1990 ) .
- Induction was obtained by a temperature shift (heat shock) from 30 ⁇ C to 42 ⁇ C, whereby the repressor is inactivated and transcription of the promoter is induced.
- expression was obtained after induction of the lacUV5 promoter with 1 mM IPTG. Since this promoter is never completely silent, high levels of protein are also expressed by extended growth of the transformed clones at 30".
- An example of the fusion protein levels after a 3 days growth at 30 ⁇ is shown in Figure 5. When grown in these conditions the amount of the Protein A-PTP35XC fusion protein represented around 20% of the total cellular protein.
- the apparent M r of the hybrid protein is, as expected, 75 Kd on SDS-PAGE.
- Plasmids of the pGEX series have been designed for the expression of recombinant proteins in E. coli as fusions with the C-terminus of Sj26, a 26- kD glutathione-S-transferase (GST) encoded by the parasitic helminth Schistosoma iaponicum.
- GST 26- kD glutathione-S-transferase
- These fusion proteins can be purified from crude bacterial lysates under non-denaturing conditions by affinity chromatography on immobilized glutathione.
- the vectors have been engineered so that the GST carrier can be cleaved from fusion proteins by digestion with site-specific proteases such as thrombin, yielding the mature recombinant polypeptide of interest in a practically pure form.
- the sequence encoding the putative intracellular domain (amino acids 583-961 of the protein encoded in clone PTP35,12) was amplified by PCR with 5' and 3' primers containing BamHI and EcoRI sites, respectively. The sequence was then inserted into plasmid pGEX- 2T cut with the same enzymes. The resulting plasmid, named pFC221, encodes a fusion protein of about 70 kD, composed of GST at the amino terminus followed by the putative PTPase domain of PTP35. High level expression of the fusion protein in the DH5 ⁇ strain after IPTG induction was obtained as described (Smith, D. and Kevin, J.
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EP93917704A EP0652959A1 (en) | 1992-07-31 | 1993-07-29 | Isolation and cloning of a protein with tyrosine-phosphatase activity |
AU47047/93A AU4704793A (en) | 1992-07-31 | 1993-07-29 | Isolation and cloning of a protein with tyrosine-phosphatase activity |
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GB9216328.6 | 1992-07-31 | ||
GB929216328A GB9216328D0 (en) | 1992-07-31 | 1992-07-31 | Isolation and cloning of a protein with tyrosine-phosphatase activity |
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EP (1) | EP0652959A1 (en) |
CN (1) | CN1082110A (en) |
AU (1) | AU4704793A (en) |
GB (1) | GB9216328D0 (en) |
IL (1) | IL106505A0 (en) |
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0684989A1 (en) * | 1993-02-10 | 1995-12-06 | New York University | Novel receptor-type phosphotyrosine phosphatase-beta |
WO1997032984A1 (en) * | 1996-03-06 | 1997-09-12 | Zymogenetics, Inc. | DIABETES MELLITUS 37 kD AUTOANTIGEN PROTEIN-TYROSINE PHOSPHATASE |
WO1998000552A2 (en) * | 1996-07-02 | 1998-01-08 | President And Fellows Of Harvard College | Receptor tyrosine phosphatase, and uses related thereto |
WO1998028408A1 (en) * | 1996-12-20 | 1998-07-02 | Novo Nordisk A/S | Peniophora phytase |
US6054306A (en) * | 1996-12-20 | 2000-04-25 | Novo Nordisk A/S | Peniophora phytase |
US6114140A (en) * | 1994-05-03 | 2000-09-05 | Cold Spring Harbor Laboratory | DNA encoding density enhanced protein tyrosine phosphatases |
WO2000053801A1 (en) * | 1999-03-08 | 2000-09-14 | Merck Frosst Canada & Co. | Intact cell assay for protein tyrosine phosphatases |
US6682905B1 (en) | 1990-07-11 | 2004-01-27 | New York University | Receptor-type phosphotyrosine phosphatase-alpha |
US7108994B2 (en) | 1990-07-11 | 2006-09-19 | New York University | Receptor-type phosphotyrosine phosphatase-alpha |
Families Citing this family (2)
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JP6311125B2 (en) * | 2014-11-26 | 2018-04-18 | パナソニックIpマネジメント株式会社 | Washing machine |
CN108823180A (en) * | 2018-06-29 | 2018-11-16 | 云南大学 | Make the dephosphorylized albumen of protein kinase B specific site and its nucleic acid |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992001050A1 (en) * | 1990-07-11 | 1992-01-23 | New York University | Novel receptor-type phosphotyrosine phosphatase |
-
1992
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-
1993
- 1993-07-27 CN CN93109073A patent/CN1082110A/en active Pending
- 1993-07-28 IL IL106505A patent/IL106505A0/en unknown
- 1993-07-29 ZA ZA935483A patent/ZA935483B/en unknown
- 1993-07-29 AU AU47047/93A patent/AU4704793A/en not_active Abandoned
- 1993-07-29 EP EP93917704A patent/EP0652959A1/en not_active Withdrawn
- 1993-07-29 MX MX9304580A patent/MX9304580A/en unknown
- 1993-07-29 WO PCT/EP1993/002036 patent/WO1994003610A2/en not_active Application Discontinuation
Patent Citations (1)
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---|---|---|---|---|
WO1992001050A1 (en) * | 1990-07-11 | 1992-01-23 | New York University | Novel receptor-type phosphotyrosine phosphatase |
Non-Patent Citations (2)
Title |
---|
EMBL Database entry HSICA512 Accession number X62899; 12 November 1991 RABIN, D. et al.:' An IDDM-specific islet autoantigen, ICA-512, is a putative tyrosine ŸBphosphatase' * |
EMBO JOURNAL vol. 9, no. 10 , October 1990 , EYNSHAM, OXFORD GB pages 3241 - 3252 KRUEGER, N. ET AL. 'Structural diversity and evolution of human receptor-like protein tyrosine phosphatases' cited in the application * |
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7108994B2 (en) | 1990-07-11 | 2006-09-19 | New York University | Receptor-type phosphotyrosine phosphatase-alpha |
US6682905B1 (en) | 1990-07-11 | 2004-01-27 | New York University | Receptor-type phosphotyrosine phosphatase-alpha |
EP0684989A4 (en) * | 1993-02-10 | 1998-06-03 | Univ New York | Novel receptor-type phosphotyrosine phosphatase-beta. |
EP0684989A1 (en) * | 1993-02-10 | 1995-12-06 | New York University | Novel receptor-type phosphotyrosine phosphatase-beta |
US6114140A (en) * | 1994-05-03 | 2000-09-05 | Cold Spring Harbor Laboratory | DNA encoding density enhanced protein tyrosine phosphatases |
US6552169B1 (en) | 1994-05-03 | 2003-04-22 | Cold Spring Harbor Laboratory | Density enhanced protein tyrosine phosphatases |
US7195762B2 (en) | 1994-05-03 | 2007-03-27 | Cold Spring Harbor Laboratory | Density enhanced protein tyrosine phosphatases |
US6300093B1 (en) | 1996-03-06 | 2001-10-09 | Zymogenetics, Inc. | Islet cell antigen 1851 |
WO1997032984A1 (en) * | 1996-03-06 | 1997-09-12 | Zymogenetics, Inc. | DIABETES MELLITUS 37 kD AUTOANTIGEN PROTEIN-TYROSINE PHOSPHATASE |
WO1998000552A3 (en) * | 1996-07-02 | 1998-03-12 | Harvard College | Receptor tyrosine phosphatase, and uses related thereto |
US6399326B1 (en) | 1996-07-02 | 2002-06-04 | President And Fellows Of Harvard College | Nucleic acids encoding neural/pancreatic receptor tyrosine phosphatase |
WO1998000552A2 (en) * | 1996-07-02 | 1998-01-08 | President And Fellows Of Harvard College | Receptor tyrosine phosphatase, and uses related thereto |
WO1998028408A1 (en) * | 1996-12-20 | 1998-07-02 | Novo Nordisk A/S | Peniophora phytase |
US6054306A (en) * | 1996-12-20 | 2000-04-25 | Novo Nordisk A/S | Peniophora phytase |
US6060298A (en) * | 1996-12-20 | 2000-05-09 | Novo Nordisk A/S | Peniophora phytase |
WO2000053801A1 (en) * | 1999-03-08 | 2000-09-14 | Merck Frosst Canada & Co. | Intact cell assay for protein tyrosine phosphatases |
US6329137B1 (en) | 1999-03-08 | 2001-12-11 | Merck Frosst Canada & Co. | Intact cell assay for protein tyrosine phosphatases using recombinant baculoviruses |
Also Published As
Publication number | Publication date |
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CN1082110A (en) | 1994-02-16 |
AU4704793A (en) | 1994-03-03 |
ZA935483B (en) | 1994-02-28 |
MX9304580A (en) | 1994-02-28 |
EP0652959A1 (en) | 1995-05-17 |
GB9216328D0 (en) | 1992-09-16 |
WO1994003610A3 (en) | 1994-03-31 |
IL106505A0 (en) | 1993-11-15 |
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