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WO2008044351A1 - Nouvelle protéine transporteuse chez les mammifères et son utilisation - Google Patents

Nouvelle protéine transporteuse chez les mammifères et son utilisation Download PDF

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Publication number
WO2008044351A1
WO2008044351A1 PCT/JP2007/057134 JP2007057134W WO2008044351A1 WO 2008044351 A1 WO2008044351 A1 WO 2008044351A1 JP 2007057134 W JP2007057134 W JP 2007057134W WO 2008044351 A1 WO2008044351 A1 WO 2008044351A1
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WIPO (PCT)
Prior art keywords
polypeptide
seq
polynucleotide
present
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/JP2007/057134
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English (en)
Japanese (ja)
Inventor
Yoshinori Moriyama
Hiroshi Omote
Masato Otsuka
Takuya Matsumoto
Miki Hiasa
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Okayama University NUC
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Okayama University NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/JP2006/320409 external-priority patent/WO2007043623A1/fr
Application filed by Okayama University NUC filed Critical Okayama University NUC
Priority to JP2008538574A priority Critical patent/JP5167547B2/ja
Publication of WO2008044351A1 publication Critical patent/WO2008044351A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • 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

Definitions

  • the present invention relates to a novel transporter protein and its use in mammals. More specifically, the present invention relates to a novel organic cation transporter protein that controls the final stage of excretion of drugs and Z or waste products, and the same. It is about use. Background art
  • Bacterial multidrug excretion pumps are available in several groups (eg, major facilitator superfamily (MSF), small multidrug resistance (SMR) family, resistance modulation cell division (RND) family 1 ⁇ , ATP binding).
  • MSF major facilitator superfamily
  • SMR small multidrug resistance
  • RTD resistance modulation cell division
  • Non-patent Document 11 genes encoding proteins that are likely to be classified as MATE family proteins exist in mammals.
  • Non-Patent Document 11 The deduced amino acid sequence of the gene described in Non-Patent Document 11 is classified as a MATE family protein with very low sequence homology with the Na + -dependent multidrug excretion transporter NorM of Vibrio, a prototype of the MATE family I can't imagine it being done.
  • the MATE family is a recently classified group of multidrug resistance acquisition proteins, some of which are known to excrete H + or Na + dependent cationic drugs in bacteria. There is only. In other words, the characteristics of the entire MATE family were not yet elucidated, and there was no accessory system for confirming that the target protein was a MATE family protein in mammals.
  • the present invention has been made in view of the above problems, and an object of the present invention is to provide a MATE family.
  • the aim is to establish an assembly system for confirming that it is a Liy protein, to find the MATE family protein in mammals, and to provide a technology that uses this function.
  • the polypeptide according to the present invention has a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 22, or one or several amino acids of the amino acid sequence represented by SEQ ID NO: 22 deleted, substituted or added.
  • a polypeptide comprising an amino acid sequence and having transporter activity in a cell membrane.
  • the polynucleotide according to the present invention is characterized by encoding the above-mentioned polypeptide.
  • the polynucleotide according to the present invention is a polynucleotide comprising the base sequence shown in SEQ ID NO: 21; one or several bases of the base sequence shown in SEQ ID NO: 21 are deleted, substituted or added A polynucleotide consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 21 and a polynucleotide hybridizing under stringent conditions; or a base sequence shown in SEQ ID NO: 21 A polynucleotide comprising a base sequence that is at least 80% identical.
  • a vector according to the present invention is characterized by containing the above-mentioned polynucleotide.
  • the transformant according to the present invention is characterized by containing the polynucleotide.
  • the antibody according to the present invention is characterized by specifically binding to the polypeptide.
  • a lipid membrane according to the present invention is characterized by containing the above-mentioned polypeptide.
  • the lipid membrane according to the present invention is preferably a cell membrane or a membrane vesicle when it is a naturally-derived lipid membrane, whether it is a naturally-derived lipid membrane or an artificial lipid membrane.
  • a human lipid membrane it is preferably a lipid flat membrane or a ribosome.
  • the transformant according to the present invention comprises a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 22, or one or several amino acids of the amino acid sequence represented by SEQ ID NO: 22 deleted, substituted or It is characterized in that a polynucleotide encoding a polypeptide comprising an added amino acid sequence and having transporter activity in the cell membrane is introduced.
  • the polypeptide is a polypeptide encoded by a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 21, or one or the number of the nucleotide sequence represented by SEQ ID NO: 21 It may be a polypeptide encoded by a polynucleotide consisting of a base sequence in which one base is deleted, substituted or added, and having transporter activity in the cell membrane.
  • the polypeptide is also a polypeptide encoded by a polynucleotide consisting of the base sequence shown in SEQ ID NO: 21 or a polypeptide consisting of the base sequence shown in SEQ ID NO: 21. It may be a polypeptide encoded by a polynucleotide capable of hybridizing with a nucleotide under stringent conditions and having transporter activity in a cell membrane.
  • the polypeptide is also a polypeptide encoded by a polynucleotide consisting of the base sequence shown in SEQ ID NO: 21 or a polypeptide consisting of the base sequence shown in SEQ ID NO: 21. It may be a polypeptide encoded by a polynucleotide having 80% or more homology with a nucleotide and having transporter activity in the cell membrane.
  • the ribosome composition according to the present invention comprises a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 22, or one or several amino acids of the amino acid sequence represented by SEQ ID NO: 22 deleted, substituted or added. It comprises a polypeptide comprising an amino acid sequence and having a transporter activity in the cell membrane.
  • the polypeptide is a polypeptide encoded by a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 21, or one or the number of the nucleotide sequence represented by SEQ ID NO: 21 It may be a polypeptide encoded by a polynucleotide having a base sequence in which one base is deleted, substituted or added and having a transporter activity in a cell membrane.
  • the polypeptide also comprises a polypeptide encoded by a polynucleotide consisting of the base sequence represented by SEQ ID NO: 21 or a base sequence represented by SEQ ID NO: 21 Encoded by a polynucleotide capable of hybridizing under stringent conditions with the polynucleotide and It may be a polypeptide having a sporter activity.
  • the polypeptide also comprises a polypeptide encoded by a polynucleotide consisting of the base sequence represented by SEQ ID NO: 21 or the base sequence represented by SEQ ID NO: 21. It may be a polypeptide encoded by a polynucleotide having 80% or more homology with the polynucleotide and having transporter activity in the cell membrane.
  • the ribosome composition according to the present invention may further contain H + -ATPase protein.
  • H + _ATPase protein is a protein that actively transports intracellular protons to the outside of the cell, and is also referred to as a proton pump.
  • the method for producing a lipid membrane according to the present invention is characterized by including a step of using a vector containing a polynucleotide encoding the following polypeptide in order to produce the lipid membrane: A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 22; or consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 22 have been deleted, substituted or added, and transporter activity in the cell membrane Polypeptide having
  • the lipid membrane production kit is characterized by comprising a vector containing a polynucleotide encoding the following polypeptide in order to produce the lipid membrane: SEQ ID NO: A polypeptide comprising the amino acid sequence shown in Fig. 22; or consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 22 have been deleted, substituted or added and exhibit transporter activity in the cell membrane. Having a polypeptide.
  • the test method according to the present invention is characterized by including the step of incubating the lipid membrane with the drug to be evaluated in order to test the nephrotoxicity and / or hepatotoxicity of the drug.
  • the test method according to the present invention includes tetraethylamine (TEA), 1_methyl_4_phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, corticostero in the step of incubating. , Rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenolecetin, rhodamine 6G, black mouth quinn, quinine, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papa It is preferable to coexist with belin or thiamine.
  • the test method according to the present invention includes a step of incubating the transformant with a substrate of a polypeptide having transporter activity in a cell membrane in order to test the nephrotoxicity and / or hepatotoxicity of the drug. It is characterized by that.
  • the substrate is tetraethylammonium (TEA), 1_methyl_4_phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticostero. , Rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenorecetin, rhodamine 6G, black mouth quinn, kyun, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, or thiamine .
  • the test method according to the present invention includes a step of incubating the ribosome composition with a substrate of a polypeptide having transporter activity in a cell membrane in order to test the nephrotoxicity and Z or hepatotoxicity of a drug. It is characterized by
  • the substrate includes tetraethylammonium (TEA), 1-methyl 4-phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, corti Costerone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenolecetin, rhodamine 6G, black mouth quinn, cune, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, or thiamine preferable.
  • TAA tetraethylammonium
  • MPP 1-methyl 4-phenylpyridinium
  • cimetidine quinidine
  • verapamil nicotine
  • corti Costerone rhodamine 123
  • testosterone melatonin
  • progesterone progesterone
  • androsterone kenolecetin
  • rhodamine 6G black mouth
  • a test kit according to the present invention is characterized by comprising the above lipid membrane in order to test the nephrotoxicity and / or hepatotoxicity of a drug.
  • the test kit comprises tetraethylammonium (TEA), 1_methyl_4-phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123 , Testosterone, melatonin, progesterone, androsterone, quercetin, rhodamine 6G, black mouth kin, kyun, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, or thiamine Yo!
  • TAA tetraethylammonium
  • MPP 1_methyl_4-phenylpyridinium
  • cimetidine quinidine
  • verapamil nicotine
  • conoleticosterone rhodamine 123
  • Testosterone melatonin
  • progesterone progesterone
  • androsterone quercetin
  • the test kit according to the present invention is for testing the nephrotoxicity and / or hepatotoxicity of a drug.
  • the above-mentioned transformant is provided.
  • the test kit according to the present invention comprises tetraethyl ammonium (TEA), 1-methyl-4-phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123. , Testosterone, melatonin, progesterone, androsterone, quercetin, rhodamine 6G, black mouth kin, kyun, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, or thiamine Is preferred.
  • TAA tetraethyl ammonium
  • MPP 1-methyl-4-phenylpyridinium
  • cimetidine quinidine
  • verapamil nicotine
  • conoleticosterone rhodamine 123.
  • Testosterone, melatonin, progesterone, androsterone quercetin, rh
  • a test kit according to the present invention comprises the above-mentioned ribosome composition in order to test the nephrotoxicity and / or hepatotoxicity of a drug.
  • the test kit comprises tetraethylammonium (TEA), 1_methyl_4-phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123 , Testosterone, melatonin, progesterone, androsterone, quercetin, rhodamine 6G, black mouth kin, kyun, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, or thiamine Is preferred.
  • TAA tetraethylammonium
  • MPP 1_methyl_4-phenylpyridinium
  • cimetidine quinidine
  • verapamil nicotine
  • conoleticosterone rhodamine 123
  • Testosterone melatonin
  • progesterone progesterone
  • androsterone quercetin
  • a screening method includes a step of incubating the lipid membrane with a candidate factor in order to screen a drug that regulates substance transport in the cell membrane.
  • the substance transport is preferably transport of nicotine, melatonin or steroid hormone, or excretion of drugs and / or waste products.
  • the screening method according to the present invention includes a step of incubating tetraethylammonium (TEA), 1-methyl-4-phenylpyridinium (MPP), cimetidine, quinidine, verapamil, Nicotine, corticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, quercetin, rhodamine 6G, black mouth quinn, kyun, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, Or it is preferable to make thiamine coexist.
  • TAA tetraethylammonium
  • MPP 1-methyl-4-phenylpyridinium
  • cimetidine quinidine
  • verapamil a step of incubating tetraethylammonium (TEA), 1-methyl-4-phenylpyridinium (MPP), ci
  • the screening method according to the present invention regulates excretion of drugs and Z or waste products.
  • the method includes a step of incubating the transformant with a substrate of a polypeptide having transporter activity in a cell membrane.
  • the substrate is tetraethylammonium.
  • TAA 1 _Methyl _4_Phenylpyridinium
  • MPP 1 _Methyl _4_Phenylpyridinium
  • cimetidine quinidine, verapaminole, nicotine, conoleticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenorecetin, rhodamine 6G , Kuroguchikin, Kyun, Pyrimethamine, Kuroguchi Promazine, Berberine, Cisplatin, Propranolol, Papaverine, or Thiamine.
  • the screening method according to the present invention comprises a step of incubating the above-mentioned ribosome composition with a substrate of a polypeptide having transporter activity in a cell membrane in order to screen a drug and an agent that regulates the excretion of Z or waste products.
  • the inclusion of is a special number.
  • the substrate is tetraethylammonium.
  • TAA 1-methyl 4-phenylpyridinium
  • MPP 1-methyl 4-phenylpyridinium
  • cimetidine quinidine, verapamil, nicotine, corticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenolecetin, rhodamine 6G
  • Preferred are black mouth quinn, kyun, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, or thiamine.
  • the screening method according to the present invention comprises a step of incubating the transformant with a substrate of a polypeptide having transporter activity in a cell membrane in order to screen a drug that regulates nicotine transport. It is characterized by including.
  • the substrate is tetraethylammonium.
  • TAA 1 _Methyl _4_Phenylpyridinium
  • MPP 1 _Methyl _4_Phenylpyridinium
  • cimetidine quinidine, verapaminole, nicotine, conoleticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenorecetin, rhodamine 6G , Kuroguchikin, Kyun, Pyrimethamine, Kuroguchi Promazine, Berberine, Cisplatin, Propranolol, Papaverine, or Thiamine.
  • the screening method according to the present invention comprises a step of incubating the above-mentioned ribosome composition with a substrate of a polypeptide having transporter activity in a cell membrane in order to screen a drug that regulates nicotine transport. It is characterized by including.
  • the substrate is tetraethylammonium.
  • TAA 1 _Methyl _4_Phenylpyridinium
  • MPP 1 _Methyl _4_Phenylpyridinium
  • cimetidine quinidine, verapaminole, nicotine, conoleticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenorecetin, rhodamine 6G , Kuroguchikin, Kyun, Pyrimethamine, Kuroguchi Promazine, Berberine, Cisplatin, Propranolol, Papaverine, or Thiamine.
  • the screening method according to the present invention includes a step of incubating the transformant with a substrate of a polypeptide having transporter activity in a cell membrane in order to screen for an agent that regulates melatonin transport. It is characterized by doing.
  • the substrate is tetraethylammonium.
  • TAA 1-methyl 4-phenylpyridinium
  • MPP 1-methyl 4-phenylpyridinium
  • cimetidine quinidine, verapamil, nicotine, corticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenolecetin, rhodamine 6G
  • Preferred are black mouth quinn, kyun, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, or thiamine.
  • the screening method according to the present invention includes a step of incubating the above-mentioned ribosome composition together with a substrate of a polypeptide having transporter activity in a cell membrane in order to screen a drug that regulates melatonin transport. It is characterized by
  • the substrate is tetraethylammonium.
  • TAA 1 _Methyl _4_Phenylpyridinium
  • MPP 1 _Methyl _4_Phenylpyridinium
  • cimetidine quinidine, verapaminole, nicotine, conoleticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenorecetin, rhodamine 6G , Black mouth kin, kyun, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, ma Or thiamin.
  • the screening method according to the present invention includes a step of incubating the transformant with a substrate of a polypeptide having transporter activity in a cell membrane in order to screen a drug that regulates steroid hormone transport. It is characterized by this.
  • the substrate is tetraethylammonium.
  • TAA 1 _Methyl _4_Phenylpyridinium
  • MPP 1 _Methyl _4_Phenylpyridinium
  • cimetidine quinidine, verapaminole, nicotine, conoleticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenorecetin, rhodamine 6G , Kuroguchikin, Kyun, Pyrimethamine, Kuroguchi Promazine, Berberine, Cisplatin, Propranolol, Papaverine, or Thiamine.
  • the screening method according to the present invention includes a step of incubating the ribosome composition described above with a substrate of a polypeptide having transporter activity in a cell membrane in order to screen a drug that regulates steroid hormone transport. It is characterized by this.
  • the substrate is tetraethylammonium.
  • TAA 1-methyl 4-phenylpyridinium
  • MPP 1-methyl 4-phenylpyridinium
  • cimetidine quinidine, verapamil, nicotine, corticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenolecetin, rhodamine 6G
  • Preferred are black mouth quinn, kyun, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, or thiamine.
  • a screening kit according to the present invention is characterized by comprising the lipid membrane described above in order to screen for a drug that regulates substance transport in a cell membrane.
  • the substance transport is preferably transport of nicotine, melatonin or steroid hormone, or excretion of drugs and / or waste products.
  • the screening kit according to the present invention includes tetraethylammonium (TEA), 1-methyl -4-phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicoti. , Corticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, quercetin, rhodamine 6G, black mouth kin, cun, pyrimethamine, chloropromazine, berberine, cisplatin, propranolol, papaverine, or thiamine You may have.
  • TAA tetraethylammonium
  • MPP 1-methyl -4-phenylpyridinium
  • cimetidine quinidine
  • verapamil nicoti.
  • Corticosterone rhodamine 123
  • testosterone melatonin
  • progesterone progesterone
  • androsterone quer
  • a screening kit according to the present invention is characterized by comprising the above-mentioned transformant in order to screen for a drug that regulates the excretion of drugs and / or waste products.
  • the screening kit comprises tetraethylammonium (TEA), 1-methinole_4_phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123, testosterone, It is further equipped with at least one of melatonin, progesterone, andorosterone, quercetin, rhodamine 6G, black mouth kin, kun, pyrimethamine, black mouth promadin, benorevelin, cisplatin, propranolol, papaverine, or thiamine It is preferable.
  • TAA tetraethylammonium
  • MPP 1-methinole_4_phenylpyridinium
  • cimetidine quinidine
  • verapamil nicotine
  • conoleticosterone nicotine
  • rhodamine 123 testosterone
  • It is further equipped with at least one of melatonin
  • the screening kit according to the present invention is characterized by comprising the above-mentioned ribosome composition in order to screen for drugs that regulate the excretion of drugs and / or waste products.
  • the screening kit according to the present invention comprises tetraethylammonium (TEA), 1-methinole — 4-phenylpyridinum (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123, testosterone. , Melatonin, progesterone, andorosterone, quercetin, rhodamine 6G, black mouth kin, kinin, pyrimethamine, black mouth promadin, benorevelin, cisplatin, propranolol, papaverine, or thiamine Preferably it is.
  • a screening kit according to the present invention comprises the above-described transformant in order to screen for a drug that regulates nicotine transport.
  • the screening kit comprises tetraethylammonium (TEA), 1-methinole_4_phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123, testosterone, Melatonin, progesterone, and mouth sterone, quercetin, rhodamine 6G, black mouth kin, kyun, pyrimethamine, black mouth promagic It is preferable to further comprise at least one of thiol, benovelin, cisplatin, propranolol, papaverine, or thiamine.
  • TAA tetraethylammonium
  • MPP 1-methinole_4_phenylpyridinium
  • cimetidine quinidine
  • verapamil nicotine
  • conoleticosterone rhodamine 123
  • testosterone Melatonin
  • progesterone progesterone
  • the screening kit according to the present invention is characterized by comprising the above-mentioned ribosome composition in order to screen a drug that regulates nicotine transport.
  • the screening kit according to the present invention includes tetraethylammonium (TEA), 1_methinole_4_phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123, testosterone, It is further equipped with at least one of melatonin, progesterone, andorosterone, quercetin, rhodamine 6G, black mouth kin, kun, pyrimethamine, black mouth promadin, benorevelin, cisplatin, propranolol, papaverine, or thiamine It is preferable.
  • TAA tetraethylammonium
  • MPP 1_methinole_4_phenylpyridinium
  • cimetidine quinidine
  • verapamil nicotine
  • conoleticosterone nicotine
  • rhodamine 123 testosterone
  • It is further equipped with at least
  • a screening kit according to the present invention comprises the above-described transformant in order to screen for a drug that regulates melatonin transport.
  • the screening kit according to the present invention comprises tetraethylammonium (TEA), 1-methinole — 4-phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123, testosterone, It is further equipped with at least one of melatonin, progesterone, andorosterone, quercetin, rhodamine 6G, black mouth kin, quinine, pyrimethamine, black mouth promadin, benorevelin, cisplatin, propranolol, papaverine, or thiamine It is preferable.
  • TAA tetraethylammonium
  • MPP 1-methinole — 4-phenylpyridinium
  • cimetidine quinidine
  • verapamil nicotine
  • conoleticosterone rhodamine 123
  • testosterone testosterone
  • a screening kit according to the present invention comprises the above-mentioned ribosome composition in order to screen for a drug that regulates melatonin transport.
  • the screening kit according to the present invention comprises tetraethylammonium (TEA), 1-methinole_4_phenylpyridinum (MPP), cimetidine, quinidine, verapamil, nicotine, conoreticosterone, rhodamine 123, testosterone, and melatonin. , Progesterone, Andorosterone, Quercetin, Rhodamine 6G, Black mouth kin, Kyun, Pyrimethamine, Black mouth promadin, Benolevelin, Cisplatin, Propranolol, Papaverine, or Thiamine Is preferred.
  • TAA tetraethylammonium
  • MPP 1-methinole_4_phenylpyridinum
  • cimetidine quinidine
  • verapamil nicotine
  • conoreticosterone rhodamine 123
  • testosterone testosterone
  • melatonin Progesterone, Andorosterone, Quercetin, Rho
  • a screening kit according to the present invention is characterized by comprising the transformant described above in order to screen for a drug that regulates steroid hormone transport.
  • the screening kit according to the present invention comprises tetraethylammonium (TEA), 1-methinole-4-phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123, testosterone.
  • TAA tetraethylammonium
  • MPP 1-methinole-4-phenylpyridinium
  • cimetidine quinidine
  • verapamil nicotine
  • nicotine conoleticosterone
  • rhodamine 123 testosterone.
  • the screening kit according to the present invention is characterized by comprising the ribosome composition described above in order to screen for a drug that regulates steroid hormone transport.
  • the screening kit comprises tetraethylammonium (TEA), 1-methinole_4_phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123, testosterone, It is further equipped with at least one of melatonin, progesterone, andorosterone, quercetin, rhodamine 6G, black mouth kin, quinine, pyrimethamine, black mouth promadin, benorevelin, cisplatin, propranolol, papaverine, or thiamine It is preferable.
  • TAA tetraethylammonium
  • MPP 1-methinole_4_phenylpyridinium
  • cimetidine quinidine
  • verapamil nicotine
  • conoleticosterone rhodamine 123
  • testosterone testosterone
  • It is further equipped with at least one of melatonin
  • the screening method according to the present invention includes a step of encapsulating the lipid membrane together with a candidate factor in order to screen for a substrate of a polypeptide having transporter activity in a cell membrane. .
  • tetraethylammonium (TEA) or 1-methyl-4-phenylpyridinium (MPP) preferably coexists in the incubation step.
  • TAA tetraethylammonium
  • MPP 1-methyl-4-phenylpyridinium
  • the transformant in order to screen for a substrate of a polypeptide having transporter activity in a cell membrane, the transformant is treated with tetraethylamine (TEA) or 1-methyl-4-phenylpyridinium. It includes the step of incubating with the MPP (MPP).
  • TAA tetraethylamine
  • MPP MPP
  • the ribosome composition in order to screen for a substrate of a polypeptide having a transporter activity in a cell membrane, is treated with tetraethylenmonium (TEA) or 1-methyl-4-phenylpyridinium. (MPP) together It is characterized by including the step of circulating.
  • TAA tetraethylenmonium
  • MPP 1-methyl-4-phenylpyridinium
  • a screening kit according to the present invention is characterized by comprising the above lipid membrane in order to screen a substrate of a polypeptide having transporter activity in a cell membrane.
  • the screening kit according to the present invention may further comprise tetraethylammonium (TEA) or 1_methyl_4-phenylpyridinium (MPP).
  • TAA tetraethylammonium
  • MPP 1_methyl_4-phenylpyridinium
  • the screening kit according to the present invention is characterized by comprising the above transformant in order to screen for a substrate of a polypeptide having transporter activity in a cell membrane.
  • the screening kit according to the present invention preferably further comprises tetraethylammonium (TEA) or 1-methyl_4-phenylpyridinium (MPP).
  • TAA tetraethylammonium
  • MPP 1-methyl_4-phenylpyridinium
  • the screening kit according to the present invention includes the above-mentioned ribosome composition in order to screen for a substrate of a polypeptide having transporter activity in a cell membrane.
  • the screening kit according to the present invention preferably further comprises tetraethylammonium (TEA) or 1-methyl-4-phenylpyridinium (MPP).
  • TAA tetraethylammonium
  • MPP 1-methyl-4-phenylpyridinium
  • the screening method according to the present invention includes a step of incubating the transformant with a candidate compound in order to screen for an inhibitor or activity enhancer for a polypeptide having transporter activity in a cell membrane. It is a feature.
  • a screening kit according to the present invention comprises the transformant described above in order to screen for an inhibitor or an activity enhancer for a polypeptide having transporter activity in a cell membrane. RU
  • the screening method according to the present invention includes a step of incubating the liposomal composition with a candidate compound in order to screen for an inhibitor or activity enhancer for a polypeptide having transporter activity in a cell membrane. It is a feature.
  • the screening kit according to the present invention is used for screening an inhibitor or an activity enhancer for a polypeptide having transporter activity in a cell membrane. It is characterized by having a posome composition.
  • an oligonucleotide consisting of at least 12 bases which is a fragment of the following polynucleotide or a complementary sequence thereof and is continuous is used.
  • a polynucleotide comprising a base sequence that is at least 80% identical to the base sequence shown in SEQ ID NO: 21.
  • the oligonucleotide is SEQ ID NO: 23 or SEQ ID NO: 2.
  • it consists of the base sequence shown in 4.
  • the diagnostic kit according to the present invention comprises an oligonucleotide consisting of at least 12 consecutive nucleotides, which is a fragment of the following polynucleotide or a complementary sequence thereof, in order to diagnose a disease caused by abnormal substance transport in the cell membrane. It is characterized by that:
  • a polynucleotide comprising a base sequence that is at least 80% identical to the base sequence shown in SEQ ID NO: 21.
  • the oligonucleotide is SEQ ID NO: 23 or SEQ ID NO:
  • It preferably consists of the base sequence shown in 24.
  • the diagnostic method according to the present invention uses an antibody that specifically binds to the polypeptide described below and a biological sample. It is characterized by including the step of cupping:
  • a polypeptide comprising an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 22 have been deleted, substituted or added, and having transporter activity in the cell membrane.
  • the antibody is preferably raised by a peptide consisting of the amino acid sequence IJ shown in SEQ ID NO: 25.
  • the diagnostic method according to the present invention is specific to a polypeptide having transporter activity in the cell membrane in order to diagnose a disease caused by a substance transport abnormality contributed by a polypeptide having transporter activity in the cell membrane.
  • a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22; or alternatively, one or several amino acids of the amino acid sequence set forth in SEQ ID NO: 22 Is a polypeptide comprising a deleted, substituted or added amino acid sequence and having a transporter activity in the cell membrane.
  • the diagnostic kit according to the present invention is characterized by comprising an antibody that specifically binds to the following polypeptide in order to diagnose a disease caused by a substance transport abnormality in a cell membrane:
  • a polypeptide comprising an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 22 have been deleted, substituted or added, and having transporter activity in the cell membrane.
  • the antibody is preferably raised by a peptide consisting of the amino acid sequence IJ shown in SEQ ID NO: 25.
  • the diagnostic kit according to the present invention is specific to a polypeptide having transporter activity in the cell membrane in order to diagnose a disease caused by a substance transport abnormality contributed by a polypeptide having transporter activity in the cell membrane.
  • the polypeptide comprises a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22; alternatively, one or several amino acids of the amino acid sequence shown in SEQ ID NO: 22 are missing. Lost It is characterized by being a polypeptide comprising a substituted or added amino acid sequence and having transporter activity in the cell membrane.
  • Fig. 1 is a diagram showing the positions and structures of the hMATEl gene, hMATE2 gene and hMATE3 gene on the chromosome.
  • FIG. 2 is a diagram showing an alignment of amino acid sequences lj of hMATEl, hMATE2 and hMATE3 with the amino acid sequence of NorM derived from bacteria. It can be seen that E273, which is important for transport activity, is stored in all.
  • Fig. 3 is a scheme showing the secondary structure of hMATEl's estimation.
  • the glutamic acid residue (E273) important for transport activity is circled.
  • FIG. 4 (a) is a diagram showing the results of Northern blot analysis examining the expression of hMATEl gene and hMATE2 gene in humans. It can be seen that the hMATEl gene is expressed mainly in the kidney, liver and skeletal muscle, and the hMATE2 gene is expressed in the kidney.
  • FIG. 4 (b) shows the results of Western blot analysis in which the expression of hMATEl was confirmed using an anti-hMATEl antibody.
  • the anti-hMATEl antibody specifically recognizes hMATEl expressed in HEK293 cells.
  • a protein of the same size is present in the kidney and is absorbed by the antigenic peptide (hMATEl N461-R546).
  • FIG. 4 (c) shows the results of immunohistochemistry of hMATEl in the kidney. Human specimen sections were stained by the HRP-DAB method. In the figure, PCT indicates the proximal tubule and DC T indicates the distal tubule. The bar is 100 ⁇ m.
  • FIG. 4 (d) shows the results of immunohistochemistry of hMATEl in the liver.
  • Fig. 5 shows the positions of mMATEl gene and mMATE2 gene on the chromosome. It is a figure which shows a structure.
  • FIG. 6 is a diagram showing an alignment between the amino acid sequence of mMATE 1 and mM ATE 2 and the amino acid sequence of hM ATE 1. In the figure, the same amino acid is marked with an asterisk.
  • E 273 is gnoretamic acid, an amino acid important for transport activity.
  • FIG. 7 (a) shows the results of Northern blot analysis for examining the expression of mMATEl gene and mMATE2 gene in various organs.
  • the mMATEl gene was mainly expressed in the kidney and liver, and the mMATE2 gene was expressed in the testis.
  • FIG. 7 (b) is a diagram showing the results of Western plot analysis in which the expression of mMATEl was confirmed using an anti-mMATEl antibody.
  • the anti-mMATEl antibody specifically recognizes mMATEl expressed in HEK293 cells.
  • proteins of the same size are present in the kidney and liver.
  • Fig. 7 (c) is a diagram showing the results of immunohistochemistry of mMATEl in the kidney.
  • Mouse specimen sections were stained by HRP-DAB method.
  • mMATEl is expressed in the renal cortex.
  • GL indicates glomeruli
  • PCT indicates proximal tubules
  • DCT indicates distal tubules
  • CCD indicates collecting ducts.
  • the bar is 100 / im.
  • FIG. 7 (d) is a diagram showing the results of immunohistochemistry of mMATEl in the kidney. Mouse specimen sections were stained by HRP-DAB method. mMATEl is expressed in the renal medulla.
  • Fig. 8 (a) is a diagram showing an immunoelectron microscopic image of mMATEl in the kidney.
  • mMATEl is present in the epidermis (membrane part) of the tubule.
  • BBM indicates brush border membrane
  • BM indicates basement membrane
  • M indicates mitochondria
  • L indicates lumen.
  • the bar is l x m.
  • Fig. 8 (b) is a diagram showing an indirect immunofluorescence microscopic image of mMATEl in the liver (capillary membrane).
  • Fig. 8 (c) shows an indirect immunofluorescence microscopic image of mMATEl in the liver (epidermal side of the bile duct membrane).
  • BD indicates the interlobular bile duct and HPV indicates the portal vein.
  • the bar is 10 ⁇ m.
  • Figure 9 (a) shows the wild type (Wild) and mutation of hMATEl expressed in HEK293 cells It is a figure which shows the immunofluorescence microscope image of a body (E273Q).
  • Fig. 9 (b) is a graph showing the results of observing the uptake of TEA (50 / i ⁇ ) at ⁇ 80 in the cells shown in Fig. 9 (a) over time. is there.
  • Fig. 9 (c) is a graph showing the results of observation of TEA transport at various concentrations.
  • the value in cells transfected with plasmid only (Mock) was calculated from the value in cells expressing the wild type of hM ATE1.
  • Fig. 9 (d) is a graph showing the results of observing TEA transport at various pH values.
  • FIG. 9 (e) is a graph showing the influence of Na + on TEA transport.
  • FIG. 9 (f) is a graph showing the results of observing the effect of pH on TEA excretion.
  • FIG. 10 is a graph showing that MATE1 functions as a nicotine transporter.
  • FIG. 11 is a diagram showing the results of CBB staining of a gel obtained by electrophoresis of sampnore in each purification step of hMATEl purified using a baculovirus system.
  • FIG. 12 is a graph showing changes in TEA transport over time measured using ribosomes containing MATE polypeptide.
  • Fig. 13 is a scheme showing that MATE1 is a multidrug transporter OC transporter responsible for the final stage of OC excretion.
  • MATE1 is a multidrug transporter OC transporter responsible for the final stage of OC excretion.
  • hepatocytes take up OC through the organic cation transporters OCT1 and OCT2 in the sinusoidal membrane and excrete them into bile through MATE1 and MDR1.
  • OC is taken up mainly by OCT2 in tubule cells and excreted via MATE1, MDR1, and OCTN2.
  • FIG. 14 shows the results of fluorescent staining of mMATEl in mouse alveolar epithelial cells.
  • FIG. 15 is a diagram showing the results of fluorescent staining of mMATEl in brain capillaries.
  • the bar is 10 x m.
  • FIG. 16 shows the results of staining of hMATEl in human alveolar epithelial cells by the HRP method.
  • the bar is 10 xm.
  • FIG. 17 is a diagram showing the localization of mMATEl in the skin. It can be seen that mMAT El exists in the sebaceous glands. In the figure, the bar is 10 / im.
  • FIG. 18 shows the localization of mMATEl in the pineal gland. It can be seen that m MATE1 is present in pineal cells. In the figure, the bar is 10 z m.
  • FIG. 19 is a diagram showing the localization of mMATE2 in the plasma membrane of testicular Leydig cells.
  • the bar is 10 x m.
  • FIG. 20 shows the expression of hMATE3 gene in the testis, skeletal muscle, kidney and liver examined by the RT-PCR method. It can be seen that the hMATE3 gene is expressed only in the testis as in the case of mMATE2.
  • FIG. 21 shows the expression of hMATEl gene, hMATE2 gene and hMATE3 gene in various organs examined by RT-PCR. It can be seen that the hMATE3 gene is expressed only in the testis and adrenal gland.
  • FIG. 22 shows the expression of hMATE3 gene in various organs examined by real-time PCR.
  • the hMATE3 gene is expressed only in the testis and adrenal glands.
  • FIG. 23 shows the results of fluorescent staining of HEK293 cells expressing the hMATE3 gene.
  • FIG. 24 (a) shows the results of immunostaining human adrenal cortex cells by the peroxidase method using hMATE3 antibody.
  • FIG. 24 (b) shows the results of immunostaining of human adrenal cortex cells by the peroxidase method using an absorptive antibody obtained by treating the hMATE3 antibody with an antigen peptide (hMATE3 I457 to Q559). It is.
  • FIG. 25 (a) is a graph showing the results of observing the uptake of TEA (50 ⁇ M) at pH 8.0 in the cells shown in FIG. 23 over time.
  • FIG. 25 (b) is a graph showing the results of observation of TEA transport at various concentrations.
  • the inventors of the present invention have the ability of a protein that is presumed to be encoded by a gene described in Non-Patent Document 11 to actually exist in a mammal, and that the protein is a MATE protein.
  • a protein that is presumed to be encoded by a gene described in Non-Patent Document 11 to actually exist in a mammal, and that the protein is a MATE protein.
  • RT-PCR using mammalian tissues was attempted by designing various primers based on the base sequence information of the above genes, but there was not much information on MATE family molecules, especially as specified. Because of the lack of the functional domain of MATE family molecules, there was no index for designing primers.
  • the present inventors have repeated trial and error in order to verify the certainty of the original point of focus, and as a result, have finally found that the target gene can be amplified using specific primers. .
  • the gene obtained on the basis of the inventors' original viewpoint and trial and error is present in humans and mice, and the protein encoded by the gene is expressed in the kidney. It was found in the membrane on the luminal side of tubules and bile ducts and functioning as a novel transporter.
  • the present inventors have identified a mammalian MATE polypeptide that is an ortholog in bacteria and MATE family of humans and mice, and the mammalian MATE polypeptide is expressed in the kidney and / or liver, It has been found that it is responsible for the final stage of excretion of the organic cationic compound by exchange transport with protons, and the present invention has been completed.
  • the present inventors have found that the toxic efflux transporter onorosolog found in bacteria exists in mammals, isolated its cDNA, and analyzed its function.
  • MATE polypeptide is used interchangeably with “MATE protein” or “MATE family protein” and is a polypeptide having MATE activity, i.e., organic through the cell membrane.
  • Polypeptides having transporter activity (transport activity) that transport cations (OC) are contemplated. That is, as used herein, the term “MATE activity”
  • ex is intended to mean transporter activity in the cell membrane (activity to transport substances through the cell membrane), more specifically, transporter activity (transport activity) to transport organic cations (OC) through the cell membrane. Intended.
  • polypeptide is used interchangeably with “peptide” or "protein”.
  • a “fragment” of a polypeptide is intended to be a partial fragment of the polypeptide.
  • the polypeptides according to the invention may also be chemically synthesized or isolated from natural sources.
  • isolated polypeptide or protein is intended to be a polypeptide or protein that has been removed from its natural environment.
  • recombinantly produced polypeptides and proteins expressed in a host cell can be isolated, as are natural or recombinant polypeptides and proteins that have been substantially purified by any suitable technique. It is thought that there is.
  • Polypeptides according to the present invention may comprise natural purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells, insect cells, and Including products produced by recombinant technology (including mammalian cells).
  • prokaryotic or eukaryotic hosts eg, bacterial cells, yeast cells, higher plant cells, insect cells, and Including products produced by recombinant technology (including mammalian cells).
  • the polypeptide according to the present invention is a state in which a polynucleotide according to the present invention (a gene encoding the polypeptide according to the present invention) described later is introduced into a host cell and the polypeptide is expressed in the cell. It may also be a case where it is isolated and purified from cells, tissues and the like.
  • polypeptide according to the present invention may include an additional peptide.
  • additional peptide examples include epitope labeled peptides such as His, Myc, and Flag.
  • the polypeptides according to the invention can be expressed recombinantly in a modified form such as a fusion protein.
  • additional amino acids, in particular charged amino acid regions, of the peptides according to the invention improve the stability and persistence in the host cell during purification or subsequent manipulation and storage. For this, it can be added to the N-terminus or C-terminus of the polypeptide.
  • the mammalian MATE polypeptide of the present invention comprises SEQ ID NOs: 2, 4, 6, 8, Or a polypeptide consisting of the amino acid sequence shown in 22 or a variant thereof.
  • the mammal is preferably a human or a mouse.
  • the term “variant” intends a polypeptide or protein having MATE activity.
  • HMATE3 polypeptide is a novel polypeptide that has never been known.
  • the polypeptide according to the present invention is a MATE polypeptide or a variant thereof, wherein the variant is a polypeptide having MATE activity, and is represented by SEQ ID NOs: 2, 4 6, 8 or 22 is preferably a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added.
  • Such variants include deletions, insertions, inversions, repeats, and type substitutions (e.g., replacement of a hydrophilic residue with another residue, usually a strong and hydrophilic residue). Are not substituted with strongly hydrophobic residues).
  • “neutral” amino acid substitutions in a polypeptide generally have little effect on the activity of the polypeptide.
  • any base of a polynucleotide encoding a polypeptide can be mutated.
  • a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide.
  • this specification Using the method described therein, it is possible to easily determine whether the produced mutant has the desired MATE activity.
  • Preferred variants have conservative or non-conservative amino acid substitutions, deletions, or additions. Silent substitution, addition, and deletion are preferred, and conservative substitution is particularly preferred. These do not change the MATE activity of the polypeptides according to the invention.
  • the polypeptide according to the present invention is a MATE polypeptide or a variant thereof, wherein the variant is a polypeptide having MATE activity, and has the sequence It is preferably encoded by a polynucleotide comprising a base sequence in which one or several bases of the base sequence shown in No. 1, 3, 5, 7 or 21 are missing, substituted or added.
  • the polypeptide according to the present invention is a MATE polypeptide or a variant thereof, wherein the variant is a polypeptide having MATE activity and is represented by SEQ ID NOs: 1, 3 It is preferably encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence shown in 5, 7 or 21.
  • the appropriate hybridization temperature varies depending on the base sequence and the length of the base sequence.For example, when a DNA fragment consisting of 18 bases encoding 6 amino acids is used as a probe, a temperature of 50 ° C or lower is required. preferable.
  • stringent conditions refers to a hybridization solution (50./. Formamide, 5 X SSC (150 mM NaCl). , (15 mM trisodium citrate), 50 mM sodium phosphate ( ⁇ 7 ⁇ 6), 5 X Denhart solution, 10% dextran sulfate, and 20 g / ml denatured sheared salmon sperm DNA) at 42 ° C It is intended to wash the filter in 0.1X SSC at approximately 65 ° C after incubation at 650 ° C.
  • a polynucleotide that hybridizes to a “portion” of a polynucleotide will cause at least about 15 nucleotides (nt) of the reference polynucleotide, more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably about 30n; polynucleotides that hybridize to longer polynucleotides (either DNA or RNA are intended.
  • Polynucleotides (oligonucleotides) that hybridize to “parts” of such polynucleotides are more It is also useful as a detection probe as discussed in detail.
  • the polypeptide according to the present invention is a MATE polypeptide or a variant thereof, wherein the variant is a polypeptide having MATE activity and is represented by SEQ ID NOs: 1, 3 5, 7 or 21 at least 80% identical, more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical It is preferably encoded by a polynucleotide consisting of a base sequence.
  • the target base sequence is the polynucleotide of the present invention. It is intended to be identical to the reference sequence, except that it may contain up to 5 mismatches per 100 nucleotides (bases) of the reference base sequence of the polynucleotide encoding the peptide.
  • the force S in order to obtain a polynucleotide having a base sequence that is at least 95% identical to the reference base sequence, the force S can be deleted up to 5% of the bases in the reference sequence, and the force that can be deleted or replaced with another base. ⁇ Or it can be inserted into the reference sequence as many as 5% of the total base in the reference sequence.
  • These discrepancies in the reference sequence are either dispersed individually at the 5 'or 3' end position of the reference base sequence or at the base in the reference sequence, or in one or more adjacent groups within the reference sequence. And can occur anywhere between these end portions.
  • the present invention also provides a MATE polynucleotide.
  • MATE polynucleotide refers to the base shown in SEQ ID NO: 1, 3, 5, 7, or 21.
  • a polynucleotide comprising the sequence or a variant thereof is contemplated.
  • variant intends a polynucleotide that encodes a polypeptide having MATE activity.
  • the base sequences of human MATE 1 polynucleotide, human MATE2 polynucleotide, mouse MATE1 polynucleotide, mouse MATE2 polynucleotide and human MATE3 polynucleotide are shown in SEQ ID NOs: 1, 3, 5, 7 and 21, respectively. .
  • the hMATE3 polynucleotide is a novel polynucleotide that has never been known.
  • polynucleotide is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides.
  • base coordination 1 is used interchangeably with “nucleic acid sequence” or “nucleotide coordination” and is abbreviated as deoxyribonucleotides (A, G, C, and T). Or as a sequence of ribonucleotides (C, A, G and U).
  • a polynucleotide or a fragment thereof containing the base sequence shown in SEQ ID NO: 1 means a polynucleotide or a polynucleotide containing the sequence shown by each of the deoxynucleotides A, G, C and / or T of SEQ ID NO: 1. That fragment is intended.
  • the polynucleotide according to the present invention may exist in the form of RNA (for example, mRNA) or in the form of DNA (for example, cDNA or genomic DNA).
  • DNA can be double-stranded or single-stranded.
  • Single-stranded DNA or RNA can be the force that can be the coding strand (also known as the sense strand) or the non-coding strand (also known as the antisense strand).
  • oligonucleotide is intended to be a combination of several or tens of nucleotides, and is used interchangeably with “polynucleotide”.
  • the short nucleotides are called dinucleotides (dimers) and trinucleotides (trimers), and the long ones are expressed as 30-mer or 100-mer.
  • the Oligonucleotides can be produced as fragments of longer polynucleotides or chemically synthesized.
  • polynucleotide according to the present invention can also be fused to the polynucleotide encoding the tag tag (tag sequence or marker arrangement IJ) described above on the 5 'side or 3' side.
  • the polynucleotide according to the present invention is a MATE polynucleotide.
  • the variant preferably encodes a MATE polypeptide and is a deviation of the following polynucleotides:
  • a polynucleotide comprising a base sequence in which one or several bases of the base sequence shown in SEQ ID NO: 1, 3, 5, 7 or 21 are deleted, substituted or added
  • the polynucleotide according to the present invention includes a primer pair consisting of a primer consisting of the base sequence shown in SEQ ID NO: 11 and a primer consisting of the base sequence shown in SEQ ID NO: 12, or SEQ ID NO: 13.
  • a polynucleotide amplified from a mouse cDN A library, and MA It is a polynucleotide encoding a polypeptide having TE activity.
  • the polynucleotide according to the present invention is amplified from a human cDNA library using a primer pair consisting of a primer consisting of the base sequence shown in SEQ ID NO: 23 and a primer consisting of the base sequence shown in SEQ ID NO: 24. It is characterized by being a polynucleotide.
  • the polynucleotide according to the present invention may contain a sequence such as an untranslated region (UTR) arrangement 1 or a vector sequence (including an expression vector sequence).
  • UTR untranslated region
  • vector sequence including an expression vector sequence
  • the source for obtaining the polynucleotide of the present invention is not particularly limited, but is preferably a biological material (for example, various organs such as human or mouse).
  • biological material refers to a biological sample (obtained from an organism). Tissue sample or cell sample).
  • a polypeptide or polynucleotide according to the present invention may be used as a tool for measuring MATE activity, as further described herein.
  • an object of the present invention is to provide a MATE polypeptide and a MATE polynucleotide, and resides in a polypeptide production method, a polynucleotide production method, and the like specifically described in the present specification. Nanare, Therefore, it should be noted that MATE polypeptides and MATE polynucleotides obtained by methods other than the above methods also belong to the technical scope of the present invention.
  • the present invention provides vectors used to produce MATE polypeptides.
  • the vector according to the present invention may be a vector used for in vitro translation or a vector used for recombinant expression.
  • the vector according to the present invention is not particularly limited as long as it contains the above-described polynucleotide according to the present invention.
  • a recombinant expression vector into which a cDNA of a polynucleotide (MATE polynucleotide) encoding a polypeptide having MATE activity has been inserted.
  • a method for producing a recombinant expression vector includes, but is not limited to, a method using a plasmid, phage, cosmid or the like.
  • the specific type of vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. That is, according to the type of host cell, a promoter sequence is appropriately selected in order to reliably express the polynucleotide of the present invention, and a vector in which this and the polynucleotide of the present invention are incorporated into various plasmids or the like is used as an expression vector. Use it. In addition, transformation of a host with an expression vector can also be performed according to a conventional technique.
  • the expression vector preferably contains at least one selectable marker.
  • One such force is the dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and the tetracytalin resistance gene or ampicillin resistance gene for culture in E. coli and other bacteria.
  • a MATE polynucleotide can be introduced into an organism or cell, and the MATE polypeptide can be expressed in the organism or cell. Furthermore, if the vector according to the present invention is used in a cell-free protein synthesis system, a MATE polypeptide can be synthesized.
  • the vector according to the present invention only needs to contain at least a polynucleotide encoding the polypeptide according to the present invention. That is, it should be noted that a vector other than the expression vector is also included in the technical scope of the present invention.
  • the present invention provides a lipid membrane containing a MATE polypeptide.
  • the lipid membrane according to the present invention may be a naturally derived lipid membrane or an artificial lipid membrane.
  • the lipid membrane according to the present invention may be a cell membrane or a membrane vesicle.
  • the lipid membrane according to the present invention is artificial, it may be a planar lipid membrane or a ribosome (ribosome composition). That's fine.
  • the lipid membrane according to the present invention may be a plasma membrane of a transformant into which a polynucleotide encoding a MATE polypeptide has been introduced.
  • transformant is intended to include individual organisms that are not only cells, tissues or organs.
  • the transformant in the present embodiment is characterized in that a plasma membrane containing a MATE polypeptide is formed.
  • the MATE polypeptide is stably expressed.
  • the transformant in the present embodiment is obtained by introducing a recombinant vector containing a MATE polynucleotide into an organism so that the MATE polypeptide can be expressed.
  • the transformant in this embodiment is a prokaryotic organism, Even eukaryotes. Since the analysis by the present inventors has shown that the MATE polypeptide is a membrane protein, in contrast to the transformant expressing the MATE polypeptide, a plasma membrane containing the MATE polypeptide is provided.
  • the method for introducing the expression vector into the host is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a ribosome method, or a DEAE dextran method is preferably used. Can do.
  • a conventionally known method such as an electroporation method, a calcium phosphate method, a ribosome method, or a DEAE dextran method is preferably used.
  • an expression system using baculovirus may be used.
  • the polynucleotide encoding the polypeptide according to the present invention may be introduced using the battery method or gene gun (particle bombardment method).
  • the transformant in the present embodiment only needs to have at least the plasma membrane containing the polypeptide according to the present invention. That is, it should be noted that a transformant produced by a known method other than the above method is also included in the technical scope of the present invention.
  • the lipid membrane according to the present invention may be a membrane vesicle obtained from a cell into which a polynucleotide encoding a MATE polypeptide has been introduced.
  • membrane vesicle intends a smaller vesicle formed by the plasma membrane of a cell that has been subjected to sonication or the like and destroyed.
  • the membrane vesicle according to the present embodiment only needs to be formed of at least a lipid membrane containing the polypeptide according to the present invention. That is, it should be noted that membrane vesicles including intracellular organelles that have lost their function are also included in the technical scope of the present invention.
  • MATE polypeptide is a transporter that transports various organic cations ((C).
  • the present inventors have previously reported what substances are transported by MATE polypeptides in HEK293 cells or Xenopus laevis oocytes in which MATE polypeptides are expressed using radiolabeled substrates. Investigated what will be done. Use such expressing cells In the system, measurement is performed under conditions where many cell-derived proteins coexist in addition to the MATE polypeptide, but interference due to many coexisting proteins is particularly difficult when using a highly hydrophobic transport substrate such as sex hormones. Especially problematic. That is, it is preferable to use a system in which no protein other than the MATE polypeptide is present in order to perform highly reliable measurement on a highly hydrophobic transport substrate.
  • the present inventors prepared an almost uniform MATE polypeptide in terms of protein science, prepared a ribosome incorporating the MATE polypeptide (re, so-called proteoribosome), and used this proteoliposome for transport experiments.
  • a transport measurement system for MATE polypeptide composed of a completely defined composition was completed.
  • the lipid membrane according to the present invention can be a ribosome composition containing a MATE polypeptide.
  • liposome composition intends a ribosome containing a particular substance. Ribosomes are artificial lipid membranes, also called vesicles, and can be produced by sonicating after a suspension of lipid (eg, phospholipid) is vigorously stirred and dispersed. Studies using ribosomes are widely performed, and are used as cell membrane models or as a means of drug delivery (DDS).
  • DDS drug delivery
  • the ribosome composition according to the present invention is characterized by containing a MATE polypeptide.
  • the ribosome composition according to the present invention may further contain H + _ATPase protein.
  • the H + _ATPase protein is a protein that actively transports intracellular protons to the outside of the cell, and is also referred to as a proton pump.
  • the purification method of the proton pump is not particularly limited, and Moriyama Y et al., J. Biol. Chem. 266, 2214 A conventionally known method such as 1-22146 (1991) may be suitably used.
  • the activity can be measured even if H + -ATPase protein does not coexist if it is a mammal-derived MATE polypeptide. .
  • the present invention it is possible to construct a system that does not require the use of a radiolabeled or fluorescently labeled substrate, so that a wide variety of drugs, toxicants, metabolic biological components, and the like can be obtained. It is possible to analyze whether or not the transporter protein is a transport target.
  • the present invention can solve the conventional problems and can understand the transport function of the MATE polypeptide more widely and deeply.
  • conventional labeled compounds can also be used with the present invention.
  • the ribosome composition according to the present invention only needs to contain at least the polypeptide according to the present invention. That is, it should be noted that a ribosome composition produced by a known method other than the above method is also included in the technical scope of the present invention.
  • the lipid membrane according to the present invention may be a planar lipid membrane containing a MATE polypeptide.
  • the lipid bilayer is a membrane-like structure in which polar lipids (particularly phospholipids) are bilayered.
  • the lipid bilayer structure stabilizes as a two-dimensional structure in a spherical shape, but it can become a planar structure if the end is isolated from water molecules.
  • the artificially produced lipid bilayer is called a ribosome, and the planar one is called a lipid planar membrane.
  • the MATE polypeptide may be embedded in an artificially formed lipid bilayer membrane.
  • the artificial lipid bilayer is a membrane tongue. It is used when measuring the activity of a protein (for example, a channel protein) in vitro, and any production method is well known in the art.
  • planar lipid membrane according to the present invention only needs to contain at least the polypeptide according to the present invention. That is, it should be noted that the planar lipid membrane produced by a known method other than the above method is also included in the technical scope of the present invention.
  • the MATE polypeptide when the vector according to the present invention is used, the MATE polypeptide can be expressed in an organism or cell introduced with the MATE polynucleotide. Since the cell membrane is supplied as a transformant or as a part of the transformant, a naturally occurring lipid membrane containing a MATE polypeptide can be obtained by using the vector according to the present invention. In order to obtain an artificial lipid membrane, a purified MATE polypeptide (either an expression system or a cell-free system) prepared using the above-described vector may be used.
  • the vector according to the present invention can be used as a tool for producing the lipid membrane according to the present invention. That is, the present invention provides a method and kit for producing a lipid membrane containing a MATE polypeptide.
  • the method for producing a lipid membrane containing a MATE polypeptide according to the present invention is characterized by including a step of producing a MATE polypeptide using a vector containing a MATE polynucleotide.
  • a step of producing a MATE polypeptide using a vector containing a MATE polynucleotide When producing a naturally occurring lipid membrane, it is not necessary to purify the produced MATE polypeptide, but when producing an artificial lipid membrane, the produced MATE polypeptide may be purified.
  • One of ordinary skill in the art having read this specification can readily produce a MATE polypeptide using a vector containing the MATE polynucleotide.
  • the kit for producing a lipid membrane containing the MATE polypeptide according to the present invention is characterized by comprising a vector containing the MATE polynucleotide.
  • the vector When producing a naturally-derived lipid membrane, the vector is a recombinant expression vector, and when producing an artificial lipid membrane, the vector may or may not be a recombinant expression vector.
  • the method and kit for producing a lipid membrane containing a MATE polypeptide according to the present invention may use at least a vector containing a MATE polynucleotide. That is, it should be noted that methods and kits applying known techniques other than those described above are also included in the technical scope of the present invention.
  • the present invention further provides use of the lipid membrane.
  • a lipid membrane will be described by taking a transformant or a ribosome composition as an example, but the use of the lipid membrane according to the present invention is not limited thereto.
  • MATE1 polypeptide in HEK293 cells resulted in H + dependent transport of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP).
  • TAA tetraethylammonium
  • MPP 1-methyl-4-phenylpyridinium
  • MATE1 is a multifunctional OC transporter that has been explored for a long time and excretes organic cations (OC) directly into urine or bile.
  • the MATE polypeptide has a transporter activity in the cell membrane. More specifically, the MATE polypeptide has a transporter activity (transport activity) that transports an organic cation (OC) through the cell membrane. Have.
  • transport activity transport activity
  • OC organic cation
  • the present invention provides methods and kits for further screening for substrates that are targets for such transport activity.
  • the screening method according to the present invention includes a step of incubating the transformant with tetraethylammonium (TEA) or 1-methyl-4-phenylpyridinium (MPP). It is said.
  • TEA tetraethylammonium
  • MPP 1-methyl-4-phenylpyridinium
  • the substrate candidate can be determined to be a new substrate for the MATE polypeptide
  • the screening method according to the present invention allows tetraethylammonium (TEA) and 1_methyl_4_phenylpyridini.
  • TAA tetraethylammonium
  • MPP rum
  • cimetidine, quinidine, verapamil, nicotine, corticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, quercetin, rhodamine 6G, black mouth quinine, quinine, pyrimethamine, black mouth promazine, Berberine, cisplatin, propranolol, papaverine, and thiamine are substrates for MATE polypeptides. That is, these compounds are transported by MATE polypeptides
  • one or more of the above compounds may be used in place of TEA or MPP, and the presence or absence of transport inhibition may be used as a criterion.
  • These compounds are preferably detectably labeled when used in the present invention. Preferred labels include, but are not limited to, radiolabels.
  • a screening kit according to the present invention is characterized by comprising the transformant.
  • the screening kit according to the present invention further comprises tetraethynolemonium (TEA) or 1-methyl-4-phenylpyridinium (MPP).
  • TEA tetraethynolemonium
  • MPP 1-methyl-4-phenylpyridinium
  • cimetidine quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123, testosterone, melatonin, progesterone, andorosterone, quercetin, rhodamine 6G, black mouth quinine, quinine, pyrimethamine, Use one or more of black mouth promadin, benovelin, cisplatin, propranolol, papaverine, or thiamine and use it as a criterion for determining whether or not its transport is blocked.
  • the present invention provides additional uses of transformants that express MATE polypeptides.
  • the present invention provides methods and kits for screening drugs and agents that modulate Z or waste product excretion.
  • the present invention provides methods and kits for testing drug nephrotoxicity and / or hepatotoxicity.
  • the present invention relates to monoamines, volatile organic cations, two Many biological components (especially those with hydrophobicity) such as absorption or excretion of cotin, secretion, absorption or excretion of melatonin, steroid hormones, sex hormones and their related preparations, concentration of plant alkanoides or phenols in plants
  • the present invention is characterized by using a transformant that expresses the MATE polypeptide, and preferably uses a substrate of the MATE polypeptide.
  • the screening kit according to the present invention can be used to examine whether any compound binds to the MATE polypeptide present in the cell membrane, screening for an inhibitor or activity enhancer for the MAT E polypeptide. Can do.
  • the present invention relates to a novel transporter that exists in abicular sites of kidney tubules (sites that contact raw urine) or liver micro-bile ducts (sites that excrete bile) using an unspecified number of organic cations as substrates.
  • a cultured cell line that constantly expresses was established. This cell line can be used as a measurement system for transport (excretion) testing of various pharmaceuticals or pesticides.
  • kits that include methods of inclusion and tools for generating transformants that express MATE polypeptides.
  • [B186] Use of a ribosome composition containing a MATE polypeptide
  • TAA tetraethylamine
  • MP P 1-methyl-4-phenylpyridinium
  • TAA tetraethylammonium
  • the substrate specificity of MATE1 was similar to the H + -dependent organic cation transporter present in the kidney or liver. Thus, it was revealed that MATE1 is a multifunctional 0C transporter that has been explored for a long time and excretes organic cation (OC) directly into urine or bile.
  • the MATE polypeptide has transporter activity in the cell membrane, and more specifically, transporter activity (transport activity) that transports an organic cation (0C) through the cell membrane. have.
  • transporter activity transport activity
  • the present invention provides methods and kits for further screening for substrates that are targets for such transport activity.
  • the screening method according to the present invention includes a step of incubating the ribosome composition together with tetraethylammonium (TEA) or 1-methyl-4-phenylpyridinium (MPP). It is a feature.
  • TEA tetraethylammonium
  • MPP 1-methyl-4-phenylpyridinium
  • the screening method according to the present invention allows for use of cimetidine, quinidine, verapamil. , Nicotine, corticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, quercetin, rhodamine 6G, black mouth kin, kyun, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, and Thiamine is a substrate for MATE polypeptide. That is, these compounds are transported by MATE polypeptides
  • a screening kit according to the present invention is characterized by comprising the ribosome composition.
  • the screening kit according to the present invention preferably further comprises tetraethylammonium (TEA) or 1-methyl-4-phenylpyridinium (MPP).
  • TEA tetraethylammonium
  • MPP 1-methyl-4-phenylpyridinium
  • cimetidine instead of TEA or MPP, cimetidine, quinidine, verapamil, nicotine, corticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenorecetin, rhodamine 6G, black mouth quin, cun
  • cimetidine instead of TEA or MPP, cimetidine, quinidine, verapamil, nicotine, corticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenorecetin, rhodamine 6G, black mouth quin, cun
  • pyrimethamine chloropromazine, berberine, cisplatin, propranolol, papaverine, or thiamine and use it as a criterion for its transport inhibition.
  • the present invention provides additional uses of ribosome compositions containing a MATE polypeptide.
  • the present invention provides methods and kits for screening for agents that modulate excretion of drugs and / or waste products.
  • the present invention provides methods and kits for testing drug nephrotoxicity and / or hepatotoxicity.
  • the present invention relates to monoamines, volatile organic chaotics, absorption or excretion of nicotine, secretion, absorption or excretion of melatonin, steroid hormones, sex hormones and their related preparations, plant alkanoides or phenols.
  • the present invention is characterized by using a ribosome composition containing a MATE polypeptide, preferably MATE A polypeptide substrate is utilized.
  • the screening kit according to the present invention can be used to examine what compounds bind to the MATE polypeptide present in the cell membrane, an inhibitor or activity enhancer for the MATE polypeptide can be screened. .
  • the present invention provides a novel transporter that exists in an abical site of the renal tubule (a site that contacts the original urine) or a micro bile duct of the liver (a site that excretes bile) using an unspecified number of organic cations as a substrate.
  • a ribosome composition containing it was established. This ribosome composition can be used as a measurement system for carrying out transport (excretion) tests of various pharmaceuticals or agricultural chemicals.
  • kits that further include methods and tools for making ribosome compositions containing MATE polypeptides.
  • the present invention provides an oligonucleotide consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7 or 21 or a fragment thereof or a complementary sequence thereof.
  • the oligonucleotide according to the present invention has at least 12 consecutive bases, preferably at least 15 salts of the base sequence shown in SEQ ID NO: 1, 3, 5, 7 or 21 or its complementary sequence. Groups, and more preferably fragments of a length of at least 20 bases, even more preferably at least 30 bases, and even more preferably at least 40 bases are contemplated.
  • the oligonucleotide according to the invention can be a DNA fragment based on SEQ ID NO: 1, 3, 5, 7 or 21.
  • the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7 or 21 is provided. It can be easily manufactured.
  • the oligonucleotide according to the present invention may be an oligonucleotide consisting of the nucleotide sequence shown in one of SEQ ID NOs: 11 to 18 or a complementary sequence thereof.
  • the oligonucleotide according to the present invention can be used for preparing the polypeptide according to the present invention as a primer for polymerase chain reaction (PCR). Further, the oligonucleotide according to the present invention can be used as a Northern blot analysis or PCR primer for detecting mRNA expression of a target gene in a specific tissue. That is, the oligonucleotide according to the present invention is used as a primer for amplifying a hybridization probe for detecting a polynucleotide encoding a MATE polypeptide or a polynucleotide encoding a MATE polypeptide. Makes it possible to easily detect an organism or tissue expressing a MATE polypeptide.
  • PCR polymerase chain reaction
  • polynucleotide or oligonucleotide according to the present invention it is possible to confirm the intensity of expression of a polynucleotide encoding a MATE polypeptide polypeptide, and thus diagnose diseases and disorders caused by abnormal substance transport. can do.
  • the present invention further provides a diagnostic method and a diagnostic kit using the oligonucleotide.
  • the oligonucleotide according to the present invention specifically hybridizes with a polynucleotide (MATE polynucleotide) encoding “MATE activity”, that is, a polypeptide having a transporter activity in the cell membrane (MATE polypeptide). To do. Therefore, by detecting the presence or absence of MATE polynucleotide in each biological tissue using the oligonucleotide according to the present invention, Diseases and disorders can be diagnosed.
  • MATE polynucleotide polynucleotide
  • MATE polypeptide polypeptide having a transporter activity in the cell membrane
  • a biological sample collected from a biological tissue by biopsy or the like (particularly, a specimen sample that is a tissue, cell, or body fluid obtained from a subject) and the adjusted mRNA, By reacting with the oligonucleotide, the expression level of the MATE polynucleotide in the biological sample can be determined.
  • the kit for diagnosing a disease caused by a substance transport abnormality contributed by the MATE polypeptide according to the present invention only needs to include at least the oligonucleotide according to the present invention.
  • the kit may further comprise a reagent for detecting this oligonucleotide, if necessary.
  • Substances transported by the MATE polypeptide include tetraethynole ammonium (TEA), 1-methyl-4-phenylpyridinium (MPP), cimetidine, kidinidine, verapaminole, nicotine, conoleticosterone, Rhodamine 123, testosterone, melatonin, progesterone, androsterone, kenorecetin, rhodamine 6G, black mouth quinine, quinine, pyrimethamine, black mouth promazine, berberine, cisplatin, propranolol, papaverine, and thiamine It is not limited.
  • TAA tetraethynole ammonium
  • MPP 1-methyl-4-phenylpyridinium
  • cimetidine kidinidine
  • verapaminole nicotine
  • conoleticosterone Rhodamine 123
  • testosterone melatonin
  • progesterone progesterone
  • kenorecetin
  • an object of the present invention is to provide the oligonucleotide according to the present invention and use thereof, and does not exist in the oligonucleotide production method or the like specifically described in the present specification. . Therefore, it should be noted that oligonucleotides obtained by methods other than the above methods also belong to the scope of the present invention.
  • the present invention provides antibodies that specifically bind to a MATE polypeptide.
  • the antibody according to the present invention is characterized in that it specifically binds to hMATEl polypeptide or mMATEl polypeptide.
  • the antibody according to the present embodiment is preferably raised by a peptide antigen consisting of the amino acid sequence shown in SEQ ID NO: 19 or a peptide antigen consisting of the amino acid sequence shown in SEQ ID NO: 20. Les, not limited to.
  • the antibody according to the present invention is characterized in that it specifically binds to an hMATE3 polypeptide.
  • the antibody according to the present embodiment is preferably raised by a peptide antigen consisting of the amino acid sequence represented by SEQ ID NO: 25. The law is not limited to this.
  • antibody refers to immunoglobulins (IgA, IgD, IgE, IgG, IgM and their Fab fragments, F (ab ') fragments, Fc fragments).
  • Examples include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, anti-idiotype antibodies, and humanized antibodies.
  • the antibody according to the present invention may be useful for selecting a biological material that expresses a MATE polypeptide, and is still useful for identifying an expression site.
  • an antibody that specifically binds to a MATE polypeptide refers to a complete antibody molecule and antibody fragment (eg, that binds specifically to a MATE polypeptide (eg, Fab and F (ab ') fragments).
  • Enzymes such as Fab fragments) or pepsin (which produces F (ab ') fragments)
  • MATE polypeptide binding fragments can be produced by the application of recombinant DNA techniques or by synthetic chemistry.
  • the antibody according to the present invention only needs to have at least an antibody fragment that recognizes the peptide antigen (for example, Fab and F (ab ') fragments).
  • an immunoglobulin comprising an antibody fragment that recognizes the peptide antigen and an Fc fragment of a different antibody molecule is also included in the present invention.
  • the present invention also provides a peptide antigen eliciting an antibody capable of detecting a mammalian MATE polypeptide. That is, the peptide antigen according to the present invention is useful in a method and a kit for producing an antibody effective for immunoassay.
  • the term “immune assay” is intended to be performed using an immunological binding reaction based on an antigen-antibody reaction. Assays that utilize immunological binding reactions include immunohistochemistry, immunoelectron microscopy, Western plots, immunoprecipitation, Sandwich ELISA assays, radioactive immunoassays, and immunodiffusion assays. Examples include double chromatography.
  • the above peptide antigen may be chemically synthesized or supplied naturally. It may be obtained using recombinant expression (eg, GST fusion protein) that may be isolated from the source.
  • the present invention further provides a diagnostic method and a diagnostic kit using the antibody.
  • the antibody according to the present invention specifically binds to a polypeptide having “MATE activity”, that is, transporter activity in the cell membrane (MATE polypeptide). Therefore, by detecting the presence or absence of the MATE polypeptide in each tissue of the living body using the antibody according to the present invention, it is possible to diagnose a disease or disorder caused by a substance transport abnormality. Specifically, biological samples collected from biological tissues by biopsy, etc. (especially, specimen samples that are tissues, cells, or body fluids obtained from a subject) and MATE polypeptides. By reacting with the bound antibody, the expression level of an antigen peptide (ie, MATE polypeptide) that binds to this antibody in a biological sample can be known.
  • MATE activity that is, transporter activity in the cell membrane
  • the kit for diagnosing a disease caused by a substance transport abnormality contributed by the MATE polypeptide according to the present invention includes at least an antibody that specifically binds to the MATE polypeptide. Or it is preferably an antibody against human MATE1 or MATE2. Further, the antibody is preferably an antibody against human MATE3. The kit may further include a reagent for detecting this antibody, if necessary.
  • Substances transported by the MATE polypeptide include tetraethylamine (TEA), 1_methyl_4_phenylpyridinium (MPP), cimetidine, quinidine, verapamil, nicotine, conoleticosterone, rhodamine 123 , Testosterone, melatonin, progesterone, androsterone, kenorecetin, rhodamine 6G, black mouth quinn, kinin, pyrimethamine, black mouth promazine, benovelin, cisplatin, propranolinole, papaverine, and thiamine. It is not limited.
  • the object of the present invention is to provide an antibody that recognizes the MATE polypeptide of the present invention and
  • the purpose of this is to provide each type of immunoglobulin (IgA, IgD, IgE, IgG or IgM) specifically described in the present specification, a method for producing a chimeric antibody, a method for producing a peptide antigen. It does n’t exist. Therefore, it should be noted that antibodies obtained by methods other than the above methods also belong to the scope of the present invention.
  • a knockout animal is a gene-deficient animal obtained by disrupting a specific gene.
  • a knockout animal (especially a mouse) performs gene disruption in an embryonic stem cell (ES cell) that has totipotency, selects an ES cell that has disrupted the target gene, and uses that cell and a mouse embryo to produce a chimeric individual. And is produced from a chimeric individual having germ cells derived from ES cells as an individual having a target disruption gene by homogenization of two or more generations.
  • Knockout animals are a very effective technique for creating new experimental animals, especially disease model animals.
  • the present invention provides a knockout animal in which the MATE gene is disrupted.
  • the knockout animal according to the present invention is preferably a mouse. Since methods for producing knockout animals are well known in the field, those skilled in the art can easily produce MATE gene knockout animals based on the information on the MATE gene provided by this specification.
  • the knockout animal according to the present invention By using the knockout animal according to the present invention, it is possible to screen for a drug that regulates substance transport in the cell membrane, or to screen for a substrate of a polypeptide having transporter activity in the cell membrane. Further, if the knockout animal according to the present invention is used, the nephrotoxicity and / or hepatotoxicity of the drug can be tested. Furthermore, the knockout animal according to the present invention can greatly contribute to disease treatment as a model animal related to a disease caused by abnormal substance transport in a cell membrane.
  • Human MATE1 (hMATEl: Accession No. NP—060712) cDNA from human brain
  • the extracted total RNA force was also cloned by RT-PCR.
  • the cDNA solution is diluted 10-fold and PCR containing 0.6 mM dNTPs (150 / i M of each dNTP) and 25 pmol of primer pair, 1.5 units of Ampli Taq polymerase (PerkinElmer) Added in buffer.
  • PCR amplification was performed as follows: denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 1 minute.
  • the amplified product (1804 base pairs) was analyzed by agarose gel electrophoresis.
  • the primer was amplified by Genbank accession number AK001709 (prepared based on this (sense primer: 5'-ggccggtacccgcgagtc acatggaagctc-3; non-sense sensor 1 ⁇ : 5-cacttctagacctgtgaattgtgtgta age- 3 ').
  • the DNA fragment was digested with restriction enzymes (Kpnl and Xbal) and inserted into pBlues criptKS (+)
  • the human MATE1 gene sequence was compared with the human genomic sequence to confirm that there were no errors.
  • a primer pair (sense: 5, — cacccggctccgggccctcggatggcgtg—3; antisense: 5′-tcattgaaaactcttaatgaaaattcc—3) was used to clone MATE3 (hMAT E3).
  • Non-Patent Document 12 a point mutation (E273Q) was introduced into human MATE1 using an oligonucleotide primer (5′-ggcccaccact gcatgcacagcatgagc-3 ′).
  • MTN Human and mouse multi-tissue northern blots
  • PCR N-terminal region of human MATE1 obtained by amplification (ntl0 to 601; 592 bp), C-terminal region of human MATE2 (ntl412 to 1712; 301 bp), C-terminal region of mouse MATE1 (ntl336 to: 1599; 264 bp) and mouse
  • the C-terminal region of MATE2 (nt1087 ⁇ : 1648; 562 bp) was labeled with 32 P-dCTP using a DNA labeling kit (Boehringer Mannheim) and used as a probe for Northern blotting.
  • the hybridization was performed for 1 hour at 68 ° C using Express Hyb hybridization buffer (Clontech), 50. Washed with C.
  • Usagi polyclonal antibodies specific for human MATE1 or mouse MATE1 were prepared using GST fusion protein in which amino acids 461 to 546 of human MATE1 or amino acids P495 to Q532 of mouse MATE1 were fused with GST as antigens.
  • Non-Patent Document 13 Gold colloidal silver sensitized electron microscopy was performed according to Non-Patent Document 13.
  • Anesthetized mice were perfused with physiological saline from the heart and then refluxed with 0.1 M sodium phosphate buffer (pH 7.4) in which 4% paraformaldehyde was dissolved.
  • Kidneys were isolated and washed with PBS.
  • a tissue infiltrated with a PBS solution containing 30% sucrose was sectioned (6 mm thickness), placed on a silanized slide glass, and encapsulated in an OTC compound (Sakura FineTek).
  • HEK293 cells were cultured at 37 ° C in 5% CO in Dulbecco's modified Eagle medium containing 10% urine fetal serum, penicillin and streptomycin according to Non-Patent Document 14.
  • PcDNA3.1 / hMATEl plasmid obtained by subcloning the cDNA encoding human MATE1 or mouse MATE1 into the expression vector pcDNA3.1 (+) (Invitrogen) was cultured 24 hours later using TransIT reagent (Mirus) The cells were transferred to HEK293 cells (number of cells: 1.5 ⁇ 10 6 cells / 10 cm dish).
  • the cells cultured for 2 days are collected, 125 mM sodium chloride, 4.8 mM potassium chloride, 5.6 mM D-glucose, 1.2 mM calcium chloride, 1.2 mM potassium dihydrogen phosphate, 1.2 mM magnesium sulfate and The suspension was suspended in a buffer for measuring activity (pH 8.0) consisting of 25 mM Tricine. Cells 37. Incubation was carried out for 5 minutes at C, and 50 ⁇ of RI-labeled TEA (5 kBq / Atsusei) (PerkinElmer Life Science, Inc.) was added to start the measurement of transport activity. Predetermined At the time, the reaction solution for activity measurement was collected at 200 / il each, filtered using a 0.45 / im HA membrane filter (Millipore), and the radioactivity remaining on the filter was measured.
  • Radionicotine 100 nCi, 2 ⁇ was added to the above activity measurement buffer in which the human MATE1-expressing HEK293 cells (8 ⁇ 10 5 cells) prepared in 8 above were suspended. 20 ⁇ ⁇ ⁇ . After incubating the cells at 37 ° C for 5 minutes, the suspension was centrifuged at 37 ° C at 5, OOOrpm. The cells and the supernatant were fractionated, and the radioactivity contained in each was measured with a liquid scintillation counter to measure the uptake of nicotine into the cells.
  • a proton pump FF protein was prepared according to the procedure described in Moriyama Y et al., J. Biol. Chem. 266, 22141—22146 (1991).
  • Escherichia coli DK8 containing the large-scale expression plasmid pBWU13 of FF was treated with 0.5% glyceride.
  • Tanaka medium containing rolls 34 mM potassium phosphate, 64 mM dipotassium phosphate, 20 mM ammonium sulfate, 0.3 mM magnesium chloride, 1 ⁇ ⁇ iron sulfate, 1 ⁇ calcium chloride, 1 ⁇ zinc chloride, 100 After culturing with ⁇ g / ml isoleucine, 100 ⁇ g / ⁇ valine, 2 ⁇ g / ml thiamin), the cells were collected. All subsequent preparations were performed at 4 ° C.
  • F F was solubilized. Centrifuge the solubilized solution at 260,000 Xg for 30 minutes and remove the supernatant fraction.
  • human MATE1 is an H + -dependent 0C transporter at the protein level, and investigated its expression and localization.
  • a 62 kDa band equal to the estimated molecular weight size was detected in the human kidney membrane fraction (FIG. 4 (b)).
  • Western ⁇ ⁇ sabiperoxidase (HRP) As a result of immunohistochemistry by DAB staining, human MATE1 protein is expressed in the epidermal cell site of the proximal and distal tubules of the kidney ( Figure 4). (c)), in particular, was found to be expressed in the biliary site of hepatocytes (Fig. 4 (d)).
  • MATE family analogs in mice were obtained for further analysis of the MATE family in mammals.
  • mMATEl accession number AAH31436
  • mMATE2 accession number XP_354611
  • mouse MATE1 gene was expressed as a 3.8 kb transcript mainly in the kidney, liver and heart.
  • mouse MATE2 gene was found to be expressed as a 3.3 kb transcript, particularly in the testis (Fig. 7 (a)).
  • mouse MATE1 As a result of immunoelectron microscopic analysis, the localization of mouse MATE1 was confirmed in the brush border membrane of the proximal tubule (Fig. 8 (a)). In the liver, mouse MATE1 was present in the capillary bile duct and distributed on the surface of the bile duct (Fig. 8 (b)). [0241] These results indicate that mammalian MATE1 is expressed mainly in the kidney (especially the brush border membrane of the proximal tubule) and the liver (capillary bile duct). This is consistent with the previously known exchange transport activity of H + / OC (see Non-Patent Documents 5 to 7).
  • Fig. 9 (e) In cells expressing wild-type MATE1, the transport activity is pH-dependent, with ⁇ 6.0 being less active and increasing with increasing pH, and with ⁇ ⁇ 8.0 to 8.5 (Fig. 9 (d)).
  • sodium ions were not required for this transport activity. That is, the addition of 5 ⁇ valinomycin in the presence of 65 mM potassium chloride did not affect the TEA uptake activity even when membrane depolarization was caused.
  • TEA uptake activity was inhibited by 60% by salt-ammonium.
  • TEA uptake activity decreased greatly when the pH gradient was extinguished by SF6847 (10 ⁇ ), which is the proton conductor, or by nailysine (5 ⁇ ) in the presence of potassium chloride (Fig. 9 (e)).
  • HEK293 cells expressing hMATEl or mMATEl, 50 ⁇ M RI-labeled tetraethylammonium (TEA) with or without the compounds listed in the table was analyzed at pH 8.0 (Table 1).
  • Rhodaminel 23 0.01 0.5 6 6.2 5.0 ⁇ 6.4
  • Salicylate 10 92.4 ⁇ 4.3 97.8 ⁇ 4.8
  • Rhodamine 6G 0.01 6.8 Sat 2.0 Chloroquine 0.01 56.9 Master 5.1
  • NMN N-methylnicotinamide.
  • the data are shown as the average soil standard deviation of 3-9 experiments. ⁇ ⁇ 0. 05, ⁇ ⁇ ⁇ 0. 001.
  • TEA uptake activity is inhibited by cimetidine, quinidine, verapamil, 1-methyl-4 phenolepyridinum (MPP), nicotine, corticosterone, rhodamine 123, testosterone, melatonin, progesterone, androsterone, and quercetin. That is, it was found that these compounds are transported by MATE1.
  • TEA uptake activity was strongly inhibited by cimetidine, quinidine or verapamil, and mildly inhibited by nicotine or choline.
  • MPP which is well known as a substrate for the proton-coupled OC transporter
  • Km value and Vmax 16 ⁇ and 170 pmol / min, respectively. / mg protein.
  • the transporter for nicotine was a force that was completely unknown until now. From experiments using radioactive nicotine, we found that the MATE1 protein functions as a nicotine transporter (Fig. 10). In the figure, the data are shown as the average soil standard deviation of three experiments.
  • HMATEl cDNA with 4 base pairs of CACC added to the 5 'end was mixed with TOPO vector, and these were introduced into a competent cell (DH5a) by the heat shock method.
  • Plasmid pENTER—hMATEl incorporating hMATEl was selected with kanamycin and recovered with the QIApre p Spin Miniprep Kit (QIAGEN).
  • the hMATEl cDNA in this plasmid was recombined into the expression vector pDESTIO (INVITROGEN) using LR recombinase (INVITROGEN).
  • the resulting plasmid was selected on ampicillin medium and recovered with the QIAprep Spin Miniprep Kit (QIAGEN).
  • DHlOBac (INVITROGEN) transformed with this plasmid was transformed into kanamycin, gentamicin, tetracycline, IPTG (isopropylthiogalactoside), X_gal (5_bromo_4_ Rho 3-Indolinole ⁇ D galactoside) was selected on medium.
  • DHlOBac cells have baculovirus genomic DNA, and the cDNA introduced on pDESTIO is automatically recombined onto the viral genome by the transposon. As a result, the galactosidase gene on the viral genome is destroyed and the purified colony becomes white. The white colony force on this medium was also recovered by the miniprep method according to the following procedure.
  • the transformed DHlOBac was suspended in 0.3 ml of Solution I (25 mM Tris-HCl (pH 8.0), 50 mM glucose, 10 mM ethylenediamine 4 acetic acid), and the same amount of solution was added to this suspension. 11 (0.2N NaOH, 1% sodium dodecyl sulfate) was added. After standing for 5 minutes, 0.3 ml of Solution III (3M potassium acetate (pH 5.2)) was added to the suspension. This suspension was centrifuged at 18,000 ⁇ g for 10 minutes, and 0.8 ml of isopropyl was added to the resulting supernatant.
  • Solution I 25 mM Tris-HCl (pH 8.0), 50 mM glucose, 10 mM ethylenediamine 4 acetic acid
  • 11 0.2N NaOH, 1% sodium dodecyl sulfate
  • Solution III 3M potassium acetate (pH 5.2)
  • a recombinant baculovirus was produced according to the following procedure. About 3 / il of the prepared bacmid was introduced into sf9 insect cells (INVITROG EN) using a self-ectin reagent (INVITROGEN). Cultured sf 9 with bacmid in TNM-FH medium (GIBCO) containing 10% urinary fetal serum, 100 / ig / ml benicillin, 100 / ig / ml streptomycin, 0.25 / ig / ml fungizone did.
  • TNM-FH medium containing 10% urinary fetal serum, 100 / ig / ml benicillin, 100 / ig / ml streptomycin, 0.25 / ig / ml fungizone did.
  • the culture supernatant was collected, and recombinant baculovirus was obtained from the culture supernatant.
  • the resulting recombinant baculovirus is infected again with sf 9 cells, and after culturing the infected cells in a 70-75 cm 2 flask for 4-7 days, the culture supernatant is recovered, Obtained.
  • Figure 11 shows each step of the hMATEl purification stage (S is the solubilized insect cell membrane fraction.
  • Soybean phospholipid (Sigma type IIS) was suspended in 10 mM MOPS-Tris (pH 7.0), 0.5 mM 0-cho (1011 ⁇ / 1111). This suspension was sonicated with an & _6 sonicator, and a homogeneous solution was dispensed and stored at 80 ° C.
  • This reconstituted ribosome is suspended in ice-cooled 20 mM MOPS—Tris ( ⁇ 7.0) 500 ⁇ l containing 0.1 M potassium acetate, 5 mM magnesium acetate, 0.5 mM DTT, and used for transport experiments. did.
  • ribosome (ribosome containing MATE polypeptide) 40 ⁇ K, equivalent to about 12 zg protein), was collected, and a buffer solution containing ImC 14 C_TEA (0.5 x Ci) (20 mM Tricine-NaOH) (pH 8.0), 0.1 M potassium acetate, 5 mM magnesium acetate) The measurement was started by adding 540 ⁇ 1.
  • Fig. 12 shows changes in transport over time.
  • pH of the external solution By changing the pH of the external solution, the magnitude of the pH gradient formed inside and outside the ribosome membrane can be changed.
  • uptake of TEA depending on pH was observed.
  • background level In the absence of the transporter MATE polypeptide, little radioactivity is detected (background level)
  • MATE1 can be said to be a proton-coupled OC transporter responsible for the final stage of OC excretion in the kidney and / or liver, which has been explored for a long time (Fig. 13). Based on the above results, the entire picture of the transporter involved in the mechanism of excretion of toxic OC from the living body can be clarified.
  • OC is taken up by liver organic cation transporter (OCT1) or OCT2 in renal tubules, but is excreted from cells by the concerted reaction of the taken up OC force MATE1 and P-glycoprotein. I found out. Competitive inhibition experiments also showed that MATE1 can use various simplifications as substrates.
  • MATE1 has the function.
  • MAT El can be said to be a very important protein clinically.
  • MATE polypeptide is expressed in the plasma membrane (apical side) and cerebral blood vessels of mouse alveolar epithelial cells ( Figures 14 and 15).
  • the expression of hMATEl has also been confirmed in human alveolar epithelial cells ( Figure 16).
  • MATE1 The MATE polypeptide was found to have functions other than excretion of waste products in various tissues such as skin and pineal gland.
  • MATE1 is expressed in sweat glands, sebaceous glands, etc., indicating that it is involved in percutaneous absorption of drugs and excretion of substances related to body odor (Fig. 17).
  • MATE1 was also expressed in the pineal gland that controls circadian rhythm through melatonin secretion (Fig. 18). This indirectly indicates that MATE1 force S melatonin is used as a transport substrate and supports the results shown in Table 1. Therefore, MATE1 is thought to be a melatonin transporter in pineal cells.
  • the homology between human MATE1 and mouse MATE1 is very high, so it is presumed that humans have the same role.
  • mouse MATE2 was found to be specifically expressed in testis Leydig cells that secrete steroid hormones such as testosterone in the testis ( Figure 19). This indirectly indicates that MATE2 transports steroid hormones and supports the results shown in Table 1. Therefore, MATE2 is considered to be a steroid hormone transporter. Since human MATE3 and mouse MATE2 have very high homology and expression sites are similar (Fig. 20), it is presumed that they play the same role in humans. .
  • quercetin a plant flavonoid
  • MATE1-like transporters function as plant flavonoid transporters in plants.
  • MATE1 and MATE2 other than the kidney and liver suggests that the MATE-type transporter has functions other than the OC transporter.
  • MAT E type transporters transport physiological metabolites with various sizes, structures, and hydrophobicity. It is thought to be responsible for the molecular mechanism that functionally controls and maintains the homeostasis of biological electrolytes.
  • RNA (1 ⁇ g per unit area) purchased by Clontech, 25 pmol random hexamer primer 1 ⁇ 1, 5 X RT buffer 4 ⁇ 1 (TOYOBO), DEPC (nacalai te sque)
  • TOYOBO 5 X RT buffer 4 ⁇ 1
  • DEPC nacalai te sque
  • Add the treated DDW, lOmM dNTP mix 2 / i 1 (TOYOBO) and MM LV-RT 1 ⁇ 1 (100 ⁇ / / ⁇ 1, TOYOBO) to a total reaction volume of 20 ⁇ 1 at 30 ° C. Incubated for 10 minutes at 42 ° C for 1 hour reverse transcription. The enzyme was inactivated by heating the reaction solution at 99 ° C for 5 minutes, and then stored at -20 ° C. The presence or absence of expression of hMATEl gene, hMATE2 gene and hMATE3 gene was examined by PCR using this RT reaction solution and the following primers
  • PCR reaction conditions were as follows: treatment at 95 ° C for 1 minute, denaturation reaction at 95 ° C for 30 seconds, annealing reaction at 62 ° C for 35 seconds, extension at 72 ° C for 20 seconds A reaction cycle consisting of a reaction is 35 cycles, and 72 more. The extension reaction was performed at C for 3 minutes. 5 '-CAGGACGGCGCTAAATT as a primer to detect hMATEl gene expression GTCCAG-3, (SEQ ID NO: 26), and 5'-TC ACTG AATTCTG AC ATAG AA TC-3 '(SEQ ID NO: 27) were used.
  • hMATE2 gene As primers for detecting the expression of hMATE2 gene, 5′-AGGGATGGCTGAAGGGGCAGG-3 ′ (SEQ ID NO: 28) and X 5′-AATAAATGAAAACTGGCCCTGC-3 ′ (SEQ ID NO: 29) were used. As primers for detecting the expression of the hM ATE3 gene, 5'-GCAATGTCCAC CGCTGGAGAGGTCC-3 '(SEQ ID NO: 30) and 5, -TCATTG AAAACTC TTAATG AAAATT-3' (SEQ ID NO: 31) were used. The expected size of each PCR product is 120 bps for the hMATEl gene, 143 bps for the hMATE2 gene, and 120 bps for the hMATE3 gene.
  • Figure 21 shows the results of the RT-PCR reaction. As shown in FIG. 21, it can be seen that the hMATE3 gene is expressed only in the adrenal gland and testis.
  • hMATE3 gene was examined by mRNA quantification by real-time PCR
  • the RT reaction 2 beta 1 as ⁇ Quantitative PCR was performed reactions.
  • the primer the same primer as that used in 5-1 above was used.
  • the total amount of the PCR reaction solution was 25 / l, including 10 pmol of the target gene amplification primer and 2 X SYBR Premix Ex Taq (5 U // i 1, manufactured by TaKaRa).
  • a reaction cycle consisting of denaturation reaction at 95 ° C for 15 seconds, annealing and extension reaction at 60 ° C for 30 seconds was performed 35 cycles. Relative quantification was performed using G3PDH as a housekeeping gene to correct the mRNA expression level.
  • Figure 22 shows the results of the real-time PCR reaction. As shown in FIG. 22, it can be seen that the hMATE3 gene is expressed only in the adrenal gland and testis.
  • hMATE3 amino acids I457 to Q559 (SEQ ID NO: 25) were expressed in Escherichia coli and purified by the column method.
  • the purified polypeptide is mixed with an adjuvant and then injected subcutaneously into Japanese rabbits (female) several times. Antiserum is obtained one month later. It was.
  • HEK293 cells expressing hMATE3 were immunostained and confirmed with a fluorescence microscope.
  • hMATE3-expressing HEK293 cells were obtained by incorporating hMATE3 cDNA into an expression plasmid and infecting HEK293 cells by the ribofusion method. The results are shown in FIG. As shown in FIG. 23, cells expressing hMATE3 were confirmed.
  • Fig. 24 (a) human adrenocortical cells were immunostained by the peroxidase method using the antibody described above. The results are shown in Fig. 24 (a). As shown in FIG. 24 (a), human adrenocortical cells were strongly stained. On the other hand, when human adrenocortical cells were similarly stained after the antibody was absorbed with the polypeptide for antigen, the positive reaction disappeared as shown in FIG. 24 (b). Therefore, it can be concluded that the positive reaction shown in Fig. 24 (a) is indeed due to the antibody reaction of hMATE3.
  • the transport activity of hMATE3 was measured in the same manner as the measurement of transport activity in hMATE1 in 1-8 above.
  • the plasmid used is the hMATE3 plasmid.
  • FIG. 25 (a) and (b) show the measurement results.
  • cells expressing hMATE3 showed time-dependent transport activity against TEA.
  • the transport activity was saturated in the cells expressing hMATE3, and the Km value for TEA was 370 ⁇ M.
  • ⁇ 293 cells expressing hMATE3 were used in the presence or absence of the compounds in Table 2 at 50 ⁇ RI-labeled tetraethylammonium (TEA). Uptake was analyzed at pH 8.0.
  • TAA tetraethylammonium
  • the present invention By using the present invention, it is possible to screen a drug (for example, pharmaceutical, agricultural chemical, etc.) that regulates the excretion of drugs and / or waste products.
  • the present invention can also be used to test drug nephrotoxicity and / or hepatotoxicity.
  • the present invention provides monoamines, volatile organic thiones, absorption or excretion of nicotine, secretion, absorption or excretion of melatonin, steroid hormones, sex hormones and related products, plant alkanoides or phenols.
  • Many biological components such as concentration in the body It can be used as an experimental system for measuring transport, secretion, accumulation or excretion.
  • the present invention it is possible to screen for drugs (for example, pharmaceuticals, agricultural chemicals, etc.) that regulate the excretion of drugs and / or waste products that could not be found so far. Can contribute. Moreover, since the nephrotoxicity and Z or hepatotoxicity of any drug can be tested by using the present invention, research in medical Z pharmacy can be promoted and contribute to the development of research tools.
  • drugs for example, pharmaceuticals, agricultural chemicals, etc.

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Abstract

La présente invention concerne une membrane lipidique qui contient un polypeptide présentant la séquence d'acides aminés décrite par SEQ ID NO:22. L'utilisation de la membrane lipidique permet de cribler une substance capable de réguler l'excrétion d'un agent médicinal et/ou d'une matière de déchet, de même qu'elle permet de déterminer la néphrotoxicité et/ou l'hépatotoxicité d'un agent médicinal.
PCT/JP2007/057134 2006-10-12 2007-03-30 Nouvelle protéine transporteuse chez les mammifères et son utilisation Ceased WO2008044351A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021100709A1 (fr) * 2019-11-19 2021-05-27 凸版印刷株式会社 Structure cellulaire, son procédé de production et procédé d'évaluation de l'hépatotoxicité d'une substance à tester
WO2022113336A1 (fr) * 2020-11-30 2022-06-02 学校法人昭和大学 Procédé d'immunocoloration, chambre d'échange d'échantillon et dispositif à faisceau de particules chargées

Citations (1)

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WO2002034783A2 (fr) * 2000-10-27 2002-05-02 Incyte Genomics, Inc. Proteines transmembranaires

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WO2002034783A2 (fr) * 2000-10-27 2002-05-02 Incyte Genomics, Inc. Proteines transmembranaires

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HVORUP R.N. ET AL.: "The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily", EUR. J. BIOCHEM., vol. 270, no. 5, 2003, pages 799 - 813, XP003015426 *
OTSUKA M. ET AL.: "A human transporter protein that mediates the final excretion step for toxic organic cations", PROC. NATL. ACAD. SCI. USA, vol. 102, no. 50, December 2005 (2005-12-01), pages 17923 - 17928, XP003015425 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021100709A1 (fr) * 2019-11-19 2021-05-27 凸版印刷株式会社 Structure cellulaire, son procédé de production et procédé d'évaluation de l'hépatotoxicité d'une substance à tester
JPWO2021100709A1 (fr) * 2019-11-19 2021-05-27
WO2022113336A1 (fr) * 2020-11-30 2022-06-02 学校法人昭和大学 Procédé d'immunocoloration, chambre d'échange d'échantillon et dispositif à faisceau de particules chargées
JPWO2022113336A1 (fr) * 2020-11-30 2022-06-02
JP7530441B2 (ja) 2020-11-30 2024-08-07 学校法人昭和大学 免疫染色方法、試料交換室、及び荷電粒子線装置

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