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WO2001098483A1 - Gene codant une nouvelle proteine de type protocadherine - Google Patents

Gene codant une nouvelle proteine de type protocadherine Download PDF

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Publication number
WO2001098483A1
WO2001098483A1 PCT/JP2001/005313 JP0105313W WO0198483A1 WO 2001098483 A1 WO2001098483 A1 WO 2001098483A1 JP 0105313 W JP0105313 W JP 0105313W WO 0198483 A1 WO0198483 A1 WO 0198483A1
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Prior art keywords
protein
dna
seq
protocadherin
amino acid
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PCT/JP2001/005313
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English (en)
Japanese (ja)
Inventor
Hisashi Koga
Naomi Takahashi
Noriko Okazaki
Yasuhiko Masuho
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Zoegene Corporation
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Priority to AU2001266320A priority Critical patent/AU2001266320A1/en
Publication of WO2001098483A1 publication Critical patent/WO2001098483A1/fr

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel Protocadherin-like proteins, proteins binding thereto, their genes, and their production and use.
  • the force doherin gene group has been identified as a molecule mainly involved in cell adhesion at the receptor present on the cell membrane.
  • cadherin genes have various functions, such as cell proliferation and neural network construction.
  • new members such as Protocadherin, FAT, and CNR (cadher in-related neuronal receptor) have also joined, and these now form a large group of genes called cadherins-perfamily ones.
  • E-Cadherin the most typical force-doherin, has three structures called cadherin lipids in its extracellular domain.
  • E-Cadherin is expressed on the cell membrane of epithelial cells, but expression of E-Cadherin is essential for the tissue organization of epithelial cells, and if it is lost, the adhesiveness between cells is lost. This is a particularly important finding in considering the pathology of the disease. For example, cancer cells in which the expression of E-Cadherin is reduced significantly enhance the metastatic potential.
  • the intracellular domain of E-Cadherin binds to /?-Catenin and negatively controls the cell proliferation and differentiation signals possessed by /?-Catenin.
  • E-cadherin usually has more force doherin repeats than classical force doherin, but its intracellular domain is quite different and does not have the ability to bind? -Catenin. Furthermore, its function is hardly clear, and it is only reported that PAPC (paraxial protocadherin) is involved in gastrulation morphogenesis.
  • FAT which has been identified as a Drosophila tumor suppressor gene, has extracellular EGF-like repeats in addition to 34 Dherin repeats.
  • Mouse homologs have already been identified, but whether they function as tumor suppressor genes in mice or the details of their mechanisms are not clear.
  • CNR is expressed in neurons as its name suggests, but its intracellular domain binds to Fyn, a thigh receptor-type tyrosine kinase, but not to -tenin.
  • the functional differences between CNR and the classical force doherin N-Cadherin expressed in neurons have not yet been clarified.
  • Protocadherin is attracting attention not only as a research target for elucidating life phenomena but also as a target for drug development. Disclosure of the invention
  • the present invention has been made in view of such circumstances, and an object of the present invention is to provide a novel Protocadherin-like protein, a binding protein thereof, a gene thereof, and a production method and use thereof.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, succeeded in isolating a gene encoding a novel Protocadherin-like protein by screening of a human kidney cDNA library.
  • the clone was named "Protocadherin LKC").
  • Protocadherin LKC When the expression of the Protocadherin LKC gene was analyzed, it was surprisingly found that the gene was expressed in normal tissues, whereas the expression was Expression was lost or reduced. Similarly, a decrease in its expression was observed in tumor samples from cancer patients. Furthermore, transformation of the Protocadherin LKC gene into a cancer cell line suppressed the proliferation ability of the cell. From these facts, it was suggested that the gene has a function of suppressing canceration in vivo, and that the abnormality contributes to canceration.
  • the present inventors have also identified a protein that binds to Protocadherin LKC using a two-hybrid system. Analysis by co-expression of the protein with Protocadherin LKC suggested that the protein had a function of transmitting a signal from Protocadherin LKC into cells.
  • Protocadherin LKC gene and its binding protein are important targets in drug development and therapy for cell proliferation-related diseases such as cancer.
  • the present invention relates to novel Protocadherin-like proteins, their binding proteins, their genes, and their production and use.
  • a protein comprising the amino acid sequence of SEQ ID NO: 2 having an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2, DNA that encodes a functionally equivalent protein.
  • DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 9, or a complementary strand thereof
  • a polynucleotide comprising at least 15 nucleotides complementary to
  • step (c) selecting a compound that promotes or inhibits the binding detected in step (b) as compared to the case where the compound is detected in the absence of the test sample.
  • a polynucleotide comprising at least 15 nucleotides complementary to MA or a complementary strand thereof, comprising the nucleotide sequence of SEQ ID NO: 1.
  • the present invention provides a novel Protocadherin-like protein.
  • the amino acid sequence of human Protocadherin LKC protein contained in the Protocadherin-like protein of the present invention is shown in SEQ ID NO: 2, and the nucleotide sequence of cDNA encoding the protein is shown in SEQ ID NO: 1.
  • the Protocadherin LKC gene encodes a 1310 amino acid protein having a structure characteristic of a cadherin superfamily protein involved in cell adhesion.
  • Protocadherin LKC gene was specifically expressed in liver, kidney, large intestine, small intestine, and testis in normal tissues. These tissues are organs whose main component is the epithelial tissue that forms the lumen, and the cells repeatedly divide and proliferate every day. The fact that these tissues do not grow indefinitely and maintain the organization of organs is mainly dependent on signals from cell-cell adhesion. When such signal of cell-cell adhesion disappears, cells are considered to repeat infinite proliferation and form a tumor. The gene is not only highly expressed in these tissues, but its expression is lost or decreased in cancer cells derived from these tissues, so it may be closely related to the regulation of cell growth based on tissue architecture High in nature.
  • Protocadherin LKC is considered to be a molecule that is expressed on the contact surface between cells and activates a signal to block contact. It is thought to form.
  • the Protocadherin LKC gene and its protein are important targets for the treatment of cancer.
  • it is important to monitor the expression of these molecules not only from the viewpoint of cancer diagnosis but also from the viewpoint of cancer metastasis.
  • the present invention also provides a MAST205 protein that binds to the Protocadherin-like protein of the present invention.
  • the amino acid sequence of human MAST205 protein isolated by the present inventors is shown in SEQ ID NO: 10, and the nucleotide sequence of DNA encoding the protein is shown in SEQ ID NO: 9. From a co-expression experiment of Protocadherin LKC protein and AST205 protein in MDCK cells, it was found that in the presence of Protocadherin LKC, human MAST205 changed its localization to the cell membrane where Protocadherin LKC was localized.
  • Human MAST205 is bound to Protocadherin LKC in the cell, and it is considered that human MAST205 is recruited to the cell membrane just below by Protocadherin LKC and a signal from cell adhesion by Protocadherin LKC is transmitted. ⁇
  • the Protocadherin LKC protein and MAST205 protein of the present invention can be prepared as a recombinant protein or as a natural protein.
  • the recombinant protein can be prepared, for example, by introducing a vector into which a DNA encoding these proteins has been inserted into an appropriate host cell and purifying the protein expressed in the transformant, as described later.
  • a natural protein can be prepared, for example, using an affinity column to which the antibody of the present invention described below is bound (Current Protocols in Molecinar Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 16. 16.19).
  • the antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody.
  • the present invention includes a protein functionally equivalent to the human-derived Protocadherin LKC protein or MAST205 protein identified in this Example.
  • proteins include, for example, human Protocadherin LKC protein of SEQ ID NO: 2 or a mutant, homolog, variant, etc. of the human MAST205 protein of SEQ ID NO: 10.
  • “functionally equivalent to human Protocadherin LKC protein” means that the target protein has a function related to the suppression of cell proliferation. Such a function is, for example, to forcibly express a target protein in a colon cancer cell line, and to detect whether or not the colon cancer cell line stops growing by contact with an adjacent cell. (See Example 5). Such a function can be inferred from the disappearance or decrease of its expression in cancer tissues as compared with normal tissues and the ability to bind to MAST205 protein.
  • “functionally equivalent to human MAST205 protein” means that the target protein has a function of binding to Protocadherin LKC in cells. Such a function can be detected using an immunoprecipitation method or a two-hybrid system (see Example 4).
  • proteins functionally equivalent to the proteins identified in this example, for example, by introducing a mutation into the amino acid sequence in the protein (for example, by site-directed mutagenesis (Current Protocols). (1987) Publish. John Wiley & Sons Section 8.1-8.5)) in molecular biology edit. Such proteins may also be caused by mutations in nature.
  • one or more amino acids in the amino acid sequence (SEQ ID NOs: 2, 10) may be substituted, deleted, or deleted as long as they have the same function as the protein identified in this example. Different proteins are included due to insertion and / or addition.
  • the number and location of amino acid mutations in proteins are not limited as long as their functions are maintained.
  • the number of mutations is typically within 30 amino acids, preferably 10 amino acids. It is within an acid, more preferably within 5 amino acids (for example, within 3 amino acids).
  • the substituted amino acid is preferably an amino acid having properties similar to the amino acid before substitution from the viewpoint of maintaining the function of the protein. For example, Ala, Val, Leu, I le, Pro, Met s Phe, Trp are all classified into non-polar amino acid, believed to have similar properties.
  • examples of the non-charger include Gly, Ser ⁇ Thr, Cys, Tyr, Asn, and Gin.
  • acidic amino acids include Asp and Glu
  • basic amino acids include Lys, Arg, and His.
  • Proteins functionally equivalent to the proteins identified in this example may be obtained using the known techniques known in the art (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.3-6.4) or gene amplification technology (PGR) (Current protocols in Molecular Biology edit.Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.4). It is possible. That is, those skilled in the art can use a DNA encoding the protein identified in this example (SEQ ID NOS: 1, 9) or a part thereof as a probe, or an oligonucleotide that hybridizes specifically with the DNA as a primer.
  • DNA that hybridizes with the DNA can be isolated. Further, based on the isolated DNA, a protein encoded by the DNA can be prepared.
  • the present invention includes proteins encoded by DNAs encoding these proteins and DNAs that hybridize with the DNAs, as long as they have the same function as the proteins identified in the present example. Examples of organisms for isolating functionally equivalent proteins include, but are not limited to, vertebrates such as humans, mice, rats, magpies, bushes, and magpies.
  • Stringent conditions for hybridization to isolate DNA encoding a functionally equivalent protein are usually ⁇ lxSSC, 0.1% SDS, about 37 ° Cj, and more stringent conditions are xSSC, 0.1% SDS, about 42 ° C ”, and more severe conditions are about“ 0.1xSS 0.1% SDS, about 65 ° C ”. As the conditions become more severe, isolation of DNA having higher homology to the probe sequence can be expected.
  • the above combinations of SSC, SDS and temperature conditions are examples, and those skilled in the art will appreciate that the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length) , Hybridization reaction time, etc.) as appropriate, it is possible to realize the same stringency as described above.
  • a protein isolated using such a hybridization technique or a gene amplification technique usually has a higher homology in its amino acid sequence as compared to the protein of the present invention described in SEQ ID NO: 2 or 10.
  • High homology refers to sequence identity of at least 50% or more, more preferably 70% or more, more preferably 90% or more (eg, 95% or more).
  • the present invention also provides a partial peptide of the Protocadherin-like protein or MAST205 protein of the present invention.
  • the partial peptide of the present invention can be used, for example, for preparing an antibody that binds to the Protocadherin-like protein or the MAST205 protein of the present invention.
  • the partial peptide of the present invention has at least 7 amino acids, preferably 9 amino acids or more, more preferably 12 amino acids or more, more preferably 15 amino acids or more. Become.
  • the partial peptide of the present invention can be produced, for example, by a genetic engineering technique, a known peptide synthesis method, or by cleaving the Protocadherin-like protein or MAST205 protein of the present invention with an appropriate peptidase.
  • the partial peptide of the Protocadherin-like protein of the present invention includes a partial peptide having a binding activity to MAST205 protein.
  • the partial peptides of the MAST205 protein of the present invention include partial peptides having a binding activity to a protocadherin-like protein (these partial peptides can be used for screening drug candidate compounds described below.
  • the present invention also provides a DNA encoding the Protocadherin-like protein or MAST205 protein of the present invention, as long as it can encode the Protocadherin-like protein or MAST205 protein of the present invention.
  • the form is not particularly limited and includes cDNA, genomic DNA, chemically synthesized DNA, etc.
  • the DNA encoding the Protocadherin-like protein of the present invention includes, as described above, the DNA sequence of SEQ ID NO: 1 or 9, Can be isolated by a conventional method such as a hybridization method using a part of the DNA as a probe or a gene amplification method (PCR) using primers designed based on the information of these DNA sequences.
  • PCR gene amplification method
  • the present invention also provides a vector into which a DNA encoding the Protocadherin-like protein or MAST205 protein of the present invention has been inserted.
  • the vector of the present invention is not particularly limited as long as it can stably maintain the inserted DNA.
  • Escherichia coli is used as a host, a pBluescript vector (Strage ne).
  • the expression vector is particularly useful.
  • pBEST vector Promega
  • PET vector Invitrogen
  • the PME18S vector (Mol Cell Biol. 8: 466-472 (1988)) can be suitably used. Insertion of a DNA encoding the Protocadhen-like protein of the present invention into a vector is carried out by a conventional method, for example, a ligase reaction using a restriction enzyme site (Curent protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).
  • the present invention also provides a transformant having a DNA encoding the Protocadherin-like protein or the MAST205 protein of the present invention or a vector into which the DNA has been inserted.
  • the host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells can be used depending on the purpose. Host cells can be used, for example, for the production of the proteins of the invention.
  • Production systems for protein production include in vitro and in vivo production systems.
  • the in vitro production system include a production system using eukaryotic cells and a production system using prokaryotic cells.
  • eukaryotic cells for example, animal cells, plant cells, and fungal cells can be used as hosts.
  • the host cells of the present invention also include cells intended for use in analyzing the function of Protocadherin LKC protein or MAST205 protein and screening for function inhibitors or function promoters utilizing these proteins.
  • calcium phosphate precipitation and electric pulse lumping L method (Current protocols in Molecular Biology edit. Ausubel et al. (198 7) Publish. John Wiley & Sons. Section 9.1-9.9) ) ⁇ It can be performed by a method such as the ribofectamine method (manufactured by GI BC0-BRL) or the microinjection method.
  • the Protocadherin LKC protein can be prepared from the transformant using a protein isolation / purification method known to those skilled in the art.
  • the present invention also provides a polynucleotide comprising at least 15 nucleotides complementary to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 9, or a complementary strand thereof.
  • the “complementary strand” refers to one strand of a double-stranded DNA composed of A: T and G: C base pairs with respect to the other strand.
  • “Complementary I is defined as at least 15 consecutive nucleotides.
  • the sequence is not limited to a completely complementary sequence in the nucleotide region, and it is sufficient that the homologous sequence has at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95% or more nucleotide sequence homology. .
  • the algorithm for determining homology may use the one described in the present specification.
  • Such a polynucleotide can be used as a probe for detecting and isolating a DNA encoding the Protocadherin-like protein or the MAST205 protein of the present invention, and as a primer for amplifying the DNA of the present invention. It is. When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp. When used as a probe, a DNA having at least a part or the entire sequence of the DNA of the present invention and having a chain length of at least 15 bp is used. When used as a primer, the 3 'region must be complementary, but a restriction enzyme recognition sequence, a tag, or the like can be added to the 5' region. These probes and primers are useful for diagnosing the cancer of the present invention.
  • the polynucleotide of the present invention includes an antisense for suppressing the expression of the Protocadherin LKC protein or MAST205 protein of the present invention.
  • the antisense has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more, and preferably has a chain length of 2000 bp or less in order to cause an antisense effect.
  • Such an antisense can be obtained, for example, by the phosphorothioate method (Stein, 1988 Physicochemical properties of phosphorothioate oligodeoxynuc leotides.Nucleic Acids Res 16, 3) based on the sequence information of DNA described in SEQ ID NO: 1 or 9. 209-21 (1988)).
  • the DNA of the present invention and its antisense can be applied to, for example, gene therapy.
  • a disease targeted for gene therapy using the DNA of the present invention for example, cancer (eg, colon cancer or liver cancer) is considered to be suitable.
  • the antisense of MA of the present invention is, for example, the case where the DNA of the present invention acquires an oncogene function due to a dominant negative mutation of the DNA of the present invention.
  • ex vivo methods such as viral vectors such as retroviral vectors, adenoviral vectors, and adeno-associated virus vectors, and non-viral vectors such as ribosomes are used. It may be administered to a patient by an in vivo method or the like.
  • the present invention also provides an antibody that binds to the Protocadherin-like protein or MAST205 protein of the present invention.
  • the form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. Also, all classes of antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.
  • the antibody of the present invention can be obtained by synthesizing an oligonucleotide corresponding to an amino acid sequence and immunizing a rabbit according to a conventional method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.12-; U.13)
  • mice are immunized using a protein expressed and purified in E. coli according to a conventional method. It can be obtained from hybridoma cells obtained by fusing spleen cells and myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).
  • Antibodies that bind to the Protocadherin-like protein or the MAST205 protein of the present invention can be used for, for example, testing and diagnosing abnormal expression or structural abnormality of these proteins in addition to purification of these proteins.
  • proteins are extracted from tissues, blood, cells, or the like, and the presence or absence of abnormalities in expression or structure is detected through detection of the Protocadherin-like protein of the present invention by Western blotting, immunoprecipitation, ELISA, or the like.
  • cancer for example, colon cancer and liver cancer is particularly suitable.
  • the antibody of the present invention relates to the Protocadherin-like protein or MAST205 protein of the present invention. It may be used for the purpose of treatment of the disease.
  • human antibodies or humanized antibodies are preferred because of their low immunogenicity.
  • Human antibodies include mice in which the immune system has been replaced by humans (eg, “Functional transplants of megabase human immunoglobulin loci recapitulates human anti body response in mice, Mendez, MJ et al. (1997) Nat. Genet. 15: 146 -156 ”).
  • a humanized antibody can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods in Enzymology 203, 99-121 (1991)).
  • the present invention also provides a method for screening a compound that binds to the Protocadherin-like protein or MAST205 protein of the present invention.
  • This screening method comprises: (a) a step of bringing a test sample into contact with the Protocadherin-like protein or MAST205 protein of the present invention or a partial peptide thereof; (b) a binding activity between the protein or a partial peptide thereof and a test sample. And (c) selecting a compound having an activity of binding to the protein or a partial peptide thereof.
  • Specific methods include, for example, a method of contacting and purifying a test sample with an affinity column of the Protocadherin-like protein or MAST205 protein of the present invention, a method of using a hybrid system, a West Western blotting method, Many known methods such as a method based on high throughput screening in a combinatorial chemistry technique can be used. Screening can also be performed by using a measuring device such as BIACORE (Pharmacia) to evaluate the binding of the test compound to the Protocadherin-like protein or MAST205 protein of the present invention.
  • the test sample used for screening is not limited to these, but includes, for example, a cell extract, an expression product of a gene library, a synthetic low molecular compound, a synthetic peptide, a natural compound, and the like.
  • Compounds isolated by this screening are compounds that promote or inhibit the activity of the Protocadherin-like protein or MAST205 protein of the present invention (agonists, angiotensins). Gonist). In addition, it is a candidate for a compound that inhibits the interaction between the Protocadherin-like protein of the present invention and the MAST205 protein in vivo.
  • the present invention also provides a method of screening for a compound that alters the binding of the Protocadhedn-like protein of the present invention to the MAST205 protein.
  • This screening method comprises the steps of: (a) contacting the Protocadherin-like protein or its partial peptide of the present invention with the MAST205 protein or its partial peptide in the presence of a test sample; (b) binding the two to each other. (C) selecting a compound that promotes or inhibits the binding detected in step (b) as compared with the case where detection is performed in the absence of the test sample.
  • test sample examples include, but are not limited to, a cell extract, an expression product of a gene library, a synthetic low molecular compound, a synthetic peptide, a natural compound, and the like. Further, a compound that binds to the Protocad herin-like protein or MAST205 protein of the present invention isolated by the above screening method can also be used as a test sample.
  • the Protocadherin-like protein and the MAST205 protein used in this screening may be a naturally-derived protein, a tagged protein or a fusion protein with another protein. It may be a purified protein or a culture supernatant of a transformed cell that secretes the recombinant protein. Further, a partial peptide having an activity of binding to the other may be used.
  • Specific methods include, for example, 1) immunoprecipitation using an antibody that binds to these proteins in the presence of a test sample to detect the binding of these proteins, 2) affinity to which one of these proteins is bound.
  • Many known methods can be used, such as a method of detecting the binding of these proteins by using EST Western plotting in the presence of a sample.
  • BIAC0HE Pier acia Screening can also be performed by evaluating the binding between the Protocadherin-like protein of the present invention and the MAST205 protein in the presence of a test sample using a measuring device such as the above.
  • a method for screening a compound that inhibits the binding of two proteins by the yeast two-hybrid system is shown.
  • a yeast strain having a tetracycline receptor (TetR) gene controlled by the LexA operator and a HIS3 gene controlled by the tetracycline (tet) operator is prepared.
  • TetR tetracycline receptor
  • tet tetracycline
  • the Protocadherin-like protein and the MAST205 protein bind
  • TetR is expressed by the yeast two-hybrid principle. This TetR binds to the tet operator and suppresses HIS3 expression. As a result, this yeast cannot grow on media without histidine.
  • test sample added to the culture medium inhibits the binding of the Protocadherin-like protein to the MAST205 protein
  • TetR expression is suppressed
  • HIS3 is expressed
  • yeast can grow on a histidine-free medium. become able to.
  • the Protocadherin-like protein, gene, vector or the compound isolated by the above-mentioned screening of the present invention may be used for controlling cell proliferation.
  • the drug itself can be used as a drug, but it can be used by a known pharmaceutical method. It can be used in the form of a formulation.
  • a pharmacologically acceptable carrier or medium specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent and the like.
  • Administration to a patient can be performed by a method known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection.
  • the dosage depends on the patient's weight and age, although it varies depending on the administration method and the like, those skilled in the art can appropriately select an appropriate dose.
  • the DNA When DNA is used as a therapeutic drug, the DNA may be incorporated into a gene therapy vector and administered to a patient.
  • the dose and administration method vary depending on the patient's weight, age, symptoms, and the like, but those skilled in the art can appropriately select the dose and administration method.
  • the present invention also provides a method for diagnosing cancer targeting the Protocadherin-like protein or gene of the present invention.
  • the method for diagnosing cancer according to the present invention comprises: (a) a step of preparing a cell sample from a subject; (b) a step of detecting expression or sequence of a DNA encoding a Protocadherin-like protein in the cell sample; (c) A step of determining that the subject is cancerous or at risk of cancer when the expression level of the MA is reduced or a mutation is present in the sequence as compared to that in a healthy subject , including.
  • the diagnosis of cancer of the present invention includes both a diagnosis using a transcription product of a DNA encoding a Protocadherin-like protein and a diagnosis using a translation product.
  • the cell sample prepared from the subject is not particularly limited, and for example, cells of a tissue obtained by colonoscopy or a tissue obtained by biopsy of liver or kidney can be suitably used. Therefore, the cancer diagnosis of the present invention is excellent in that analysis can be performed using cells of a small tissue obtained by a non-invasive method, and diagnosis can be performed without giving a subject the risk of surgery or the like.
  • gene amplification methods such as polymerase chain reaction (PCR) are used, it is possible to make diagnoses using samples such as bile (liver cancer), stool (colorectal cancer), and urine (kidney cancer). Conceivable.
  • Detection of the expression or sequence of DNA encoding a Protocadherin-like protein in a cell sample can be performed as follows.
  • abnormal expression can be examined by Northern hybridization using the polynucleotide of the present invention as a probe or RT-PCR using a primer as a primer, or the polynucleotide of the present invention can be used to detect the polynucleotide of the present invention.
  • the DNA encoding the protein of the present invention and its expression control region are amplified by genomic DNA-PCR or RT-PCR by the polymerase chain reaction used as the primer, and the sequence is analyzed by methods such as analysis, SSCP, and sequencing. Inspection and diagnosis of abnormalities.
  • proteins are extracted from cells collected from a subject, and expression and structural abnormalities are detected through detection of the protocadherin-like protein of the present invention by methods such as Western blotting, immunoprecipitation, and ELISA. Test for presence-can be diagnosed. When the expression level of the DNA encoding the Protocadherin-like protein in the subject is lower than that in the healthy subject, or when the sequence has a mutation, the subject has cancer. Or it is determined that there is a risk of cancer.
  • the antibody that binds to the Protocadherin-like protein of the present invention and the primer or probe that binds to the DNA encoding the Protocadherin-like protein of the present invention can be used as the cancer diagnostic agent of the present invention.
  • FIG. 1 is a diagram showing the results of a Pfam search analysis of the preservation status of force doherin repeats in Protocadherin LKC using Protoca dherin LKC as a query.
  • FIG. 2 is a continuation of FIG.
  • FIG. 3 is a continuation of FIG.
  • FIG. 4 is a photograph showing the result of detecting the expression of human Protocadherin LKC gene in normal tissues by RT-PCR.
  • FIG. 5 is a photograph showing the result of detecting the expression of human Protocadherin LKC gene in normal tissues by Northern plot.
  • FIG. 6 is a photograph showing the result of detecting the expression of the human Protocadherin LKC gene in a cancer cell line by RT-PCR.
  • FIG. 7 is a photograph showing the result of RT-PCR detecting expression of the human Protocadherin LKC gene in a cancer patient sample.
  • FIG. 8 is an electrophoretic photograph showing the result of confirming the specificity of the antibody obtained by immunizing Protocadherin LKC by Western blotting.
  • FIG. 9 is a micrograph showing the results of immunohistochemically detecting Protocadherin LKC in human colon, liver, and kidney tissues.
  • FIG. 10 is a micrograph showing the result of observing the intracellular localization of Protocadherin LKC in MDCK cells using a confocal laser microscope.
  • Figure 11 shows the results of the detection of whether the PDZ domain of human MAST205 and the C-terminal 4-amino acid (TTDL) of Protocadherin LKC SEQ ID NO: 13 are required for binding of these molecules by the two-hybrid method. is there.
  • TTDL C-terminal 4-amino acid
  • FIG. 12 is an electrophoresis photograph showing the result of detecting the binding between Protocadherin LKC and human MAST205 by Western blotting.
  • FIG. 13 is a photomicrograph showing the result of observing how intracellular localization of Protocadherin LKC and human MAST205 is changed by co-expression using a confocal laser microscope.
  • FIG. 14 is a micrograph showing the results of observing the effect of Protocadherin LKC expression on the phenotype of a colon cancer cell line.
  • FIG. 15 shows the results of measuring the saturation density of the Protocadherin LKC expression clone in the culture dish. Best shape bear for carrying out the invention
  • the human Protocadherin LKC gene was isolated as follows. First, 5, side bra The Ima was designed based on the sequence of the EST clone AA130780, while the 3 'primer was designed based on the sequence of the gene (FLJ20383) published on the public database overnight [2 948-5, -RACE-S (GCCTCCGTGGGMGGGGACACAGGT / SEQ ID NO: 3) and 2984-N3-AS
  • PCR polymerase chain reaction
  • PCR amplification was performed using TaKaRa LA Taq (TaKaRa, # RR002A). The reaction was performed at 98 ° C for 2.5 minutes, 5 cycles of “98 ° C for 5 seconds, 72 ° C for 4 minutes”, 5 cycles of “98 ° C for 5 seconds, 70 ° C for 4 minutes”, and “98 ° C for 5 seconds. 68 ° C for 4 minutes ”was performed under the condition of 25 cycles.
  • T that should be present in the 964-th from the 5 5 side not can only code 846 Amino acids for lacking, Protocadherin LKC further 5 5 side in addition to that comprise T 395 Due to the presence of the base, it encodes a 1310 amino acid containing the signal peptide required for the receptor.
  • the homology at the amino acid level between Protocadherin LKC and the known Protocadherin gene is about 9e-62 to le-31 in E value of the whole molecule.
  • the most homologous is the Fat gene, a tumor suppressor gene of Drosophila (Mahoney, PA, Weber 3 U., 0nofrechuk, P., Biessmann, H., Bryant, PJ and Goodman, CS
  • the fat at Tumor suppressor gene in Drosophila encodes a novel member of the cad herin gene superfamily Cell 67 (5), 853-868 (1991)), and the E value of the whole molecule was 6e-67.
  • Protocadherin LKC also reports on the preservation status of force doherin repeats in Protocadh Erin LKC was analyzed by Pf am search as a query. The results are shown in Figs. 1 to 3.
  • the c- cadherin repeat is a repeating structure consisting of about 110 amino acids, and is present in the force domain containing protocadherin.
  • Forcedherin is a Ca 2+ -dependent adhesion molecule.
  • Cadherin repeats contain an amino acid sequence such as DXD (N) XD (N) (D is aspartic acid, N is asparagine, and X is any amino acid). It is thought that Ca 2+ binds to this part.
  • Tissue-specific expression of Protocadherin LKC was analyzed by RT-PCR and Northern blot.
  • the expression distribution of the Protocadherin LKC gene was analyzed by RT-PCR using Multiple Tissue cDNA Panels (Clontech Cat. No. # K1420-1, # K1421-1). Using 5, -GAAGCTTCAAGCTATGAAGG-3, (SEQ ID NO: 5) as the feed primer and 5'-CCTTGATTTCCTGACTGTTC-3 '(SEQ ID NO: 6) as the reverse primer, and PCR using TaK aRa Ex Taq (Takara Shuzo) , # RP001A), a cycle of 95 ° C (30 seconds) / 52V (30 seconds) / 72 ° C (30 seconds) was repeated 29 times after 95 ° C (2.5 minutes).
  • Northern blot analysis was performed using a human MTN Blot (Clontech Cat. No. # 7760-1, # 7759-1) using the intracellular domain of Protocadherin LKC (437 bp fragment from 3552 to 3989) as a probe. went. Hybridization conditions were performed at 68 ° C for 1 hour using ExpressHyb Hybridization Solution (Clontech Cat. No. # 8015-1). Followinged by 2xSSC, 0.05% SDS room temperature After that, further washing was performed under the conditions of 0.1 ⁇ SSC, 0.01% SDS, and 50 ° C. After confirming that the back ground was sufficiently lowered, it was exposed to X-ray film.
  • Protocadherin LKC gene was compared between normal and cancer lesion samples obtained from the same patient with colorectal cancer or liver cancer.
  • the methods for cDNA synthesis and RT-PCR are as described above, but G3PDH, a houskeping gene, was also amplified and used as a control for the purpose of matching the RNA levels between normal and cancer lesions.
  • Protocadherin LKC A polyclonal antibody was prepared.
  • the peptide used as the antigen was the 19 amino acid at the C-terminal of Protocadherin LKC (SGQLEGPSYTNAGLDTTDL) SEQ ID NO: 11, and this synthetic peptide was subcutaneously subcutaneously injected into a heron three times at two-week intervals to obtain an antiserum.
  • the obtained antiserum was affinity-purified with a synthetic peptide used as an antigen to obtain a Protocadherin LKC-specific antibody.
  • Antibody specificity was determined by transfection of the Protocadherin LKC expression plasmid into the cell line HEK293 that does not express Protocadherin LKC, and Western blotting confirmed a band specific to only the transfected cells. (Fig. 8).
  • Western blotting Protocadherin LKC expression plasmid and Mock plasmid (pCEP4, invitrogen, # V044-50) were applied to 1.5 ⁇ 10 5 HEK293 cells using LipofectAMIN reagent (GIBCO, # 18324-012). 48 hours after transfection, cells were collected and analyzed by 7.5 SDS-PAGE.
  • a purified anti-Protocadherin LKC antibody (10000 dilution) was used as the primary antibody, and an anti-Egret Ig-HRP antibody (Amersham, Lake A934, 10.000 dilution) was used as the secondary antibody.
  • the size of the protein is shown on the left and the map of Protocadherin LKC expression plasmid is shown on the right.
  • 1.5x10 5 MDCK cells were transfected with Protocadherin LKC-expressing plasmid using LipofectAMIN reagent (GIBC0, # 18324-012), and the cells were fixed with 4% PFA in 48 hours after 48 hours. Staining was performed. Colored red using purified anti-Protocadherin LKC antibody (diluted 1.000-fold) as the primary antibody and anti-Egret IgG-Alexa Fluor 488 antibody (Molecular Probes, # A-1 transfer, diluted 200-fold) as the secondary antibody I let it.
  • FIG. 10 shows a vertical 'section' of a cell image-processed with a confocal laser microscope.
  • the lower arrow indicates an enlarged image of the microvillous processes observed in the high expression cells.
  • Mouse MAST205 ( ⁇ microtubule-associated in the spermatid manchette serine / threonine kinase- 205 kD) (Walden PD 3 illette) by screening 8 million human testis cDNA library (CL0NTECH, # HL4035AH) clones Human MAST205 which is a human homolog of CF. Biol Reprod. 1996 Nov; 55 (5): 1039-44., Walden PD, Cowan NJ. Mol Cell Biol.
  • Protocadherin LKC expression plasmid and human MAST205 expression plasmid with Flag-tag are used alone or mixed with 5 HEK293 cells using LipofectAMIN reagent (GIBC0, # 18324-012), and transfection is performed. The cells were dissolved in a buffer containing 0.1 TX100, and the supernatant was reacted with a purified anti-Protocadherin LKC antibody or anti-Flag antibody (KODAK, # IB13026) at 4 ° C for 30 minutes. 3. Add 20 ⁇ 1 of Protein G plus A agarose (Oncogene, # IP10-5ML). C ⁇ Reacted for 3 hours.
  • 2X Laemmli sample buffer 125 mM Tris-HCl (H6.8), 1 mM EDTA, 4% SDS, 20% glycerol, 0.2% bromophenol
  • the bound protein was eluted by adding 1 to the mixture at 100 ° C for 5 minutes. This sample was analyzed by 7.5% SDS-PAGE.
  • human MAST205 was diffusely present in the cytoplasm and Protocadherin in LKC was localized in the cell membrane as described above. However, when both of these molecules were co-expressed, it became clear that human MAST205 changed its localization to the cell membrane where Protocadherin LKC was localized (Fig. 13). The upper row of the figure shows cells expressed only with human MAST205 alone, the middle row shows cells expressed only with Protocadherin LKC, and the lower row shows cells co-expressed.
  • the saturation density in the culture dish was measured in order to confirm that the Protocadherin LKC-expressing clone stopped growing by contact with adjacent cells.
  • HCT116-B5 and -86 1.0 ⁇ 10 6 parent strains, control strains and Protocadherin LKC expression clones (HCT116-B5, -86) were spread on a culture dish having a diameter of 3.5 cm, and the number of viable cells was counted 10 days later.
  • HCT116-B5 and -B6 the proliferation stopped when the cells became confluent, but the parent strain and the control strain continued to grow thereafter, and the number of cells was extended to about three times. That is, the number of Protocadherin LKC expression clones was reduced to about 1/3 of that of the parent strain (FIG. 15).
  • triplicate experiments were performed for each cell, and the average was shown. bar Indicates the standard deviation.
  • Protocadherin LKC is a molecule that is expressed on the contact surface between cells and activates a signal of contact inhibition. This means that Protocadherin LKC is a very important molecule in the construction of normal tissues, and that in cancer tissues in which Protocadherin LKC has been lost, there is no signal to prevent contact, and cells grow indefinitely to form tumors. ing.
  • the Protocadherin LKC protein of the present invention is considered to be deeply involved in the regulation of cell growth, and are important targets for the development of therapeutics for diseases related to cell growth including cancer. . These molecules are also considered to be involved in the metastasis of cancer, and may serve as targets for diagnosis of not only cancer but also metastasis. That is, the present invention provided a novel target molecule for developing a method for treating or diagnosing cancer.

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Abstract

La présente invention concerne un gène codant une nouvelle protéine de type protocadhérine. Ce gène est isolé par criblage d'une banque d'ADN complémentaire de rein humain. Un gène codant une protéine se liant à la protéine susmentionnée est également isolé. De par leur apparente fonction anticancéreuse, ces gènes sont utilisés dans le traitement du cancer ou en tant que cibles diagnostiques.
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CN101492673B (zh) * 2008-01-25 2012-01-11 中国科学院上海生命科学研究院 非洲爪蟾xpapc基因启动子及其组织特异性增强子

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* Cited by examiner, † Cited by third party
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WO1999003990A1 (fr) * 1997-07-16 1999-01-28 Human Genome Sciences, Inc. Serie de 64 proteines humaines secretees

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999003990A1 (fr) * 1997-07-16 1999-01-28 Human Genome Sciences, Inc. Serie de 64 proteines humaines secretees

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
J. DUNNE ET AL.: "Molecular Cloning and Tissue Expression of FAT, the Human Homologue of the Drosophila fat Gene That is located on Chromosome 4q34-q35 and Encodes a Putative Adhesion Molecule.", GENOMICS, vol. 30, 1995, pages 207 - 223, XP002945860 *
Q. WU ET AL.: "A striking Organization of a Large Family of Human Neural Cadherin-like Cell Adhesion Genes.", CELL, vol. 97, 1999, pages 779 - 790, XP002945861 *
WALDEN, P.D. ET AL.: "A Novel 205-Kilodalton Testis-Specific Serine/Threonine Protein Kinase Associated with Microtubules of the Spermatid Manchette.", MOL.CELL.BIOL., vol. 13, 1993, pages 7625 - 7635, XP002945859 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101492673B (zh) * 2008-01-25 2012-01-11 中国科学院上海生命科学研究院 非洲爪蟾xpapc基因启动子及其组织特异性增强子

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