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WO2012029722A1 - Screening method - Google Patents

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Publication number
WO2012029722A1
WO2012029722A1 PCT/JP2011/069487 JP2011069487W WO2012029722A1 WO 2012029722 A1 WO2012029722 A1 WO 2012029722A1 JP 2011069487 W JP2011069487 W JP 2011069487W WO 2012029722 A1 WO2012029722 A1 WO 2012029722A1
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WIPO (PCT)
Prior art keywords
impa2
polypeptide
present
seq
polynucleotide
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Ceased
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PCT/JP2011/069487
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French (fr)
Japanese (ja)
Inventor
藤井　亮
直人 犬飼
久美子 波佐間
真人 湯上
壮 坂本
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Takeda Pharmaceutical Co Ltd
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Takeda Pharmaceutical Co Ltd
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Publication of WO2012029722A1 publication Critical patent/WO2012029722A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • 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
    • G01N33/5011—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 antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/164—Retinal disorders, e.g. retinopathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Definitions

  • the present invention relates to a screening method for a prophylactic / therapeutic agent for IMPA2-related peripheral diseases using IMPA2.
  • Non-patent document 1 Neuropsycho.
  • Non-patent literature 2 JOURNAL OF BIOLOGICAL CHEMISTRY Vol.282, No.1, 637-647
  • Non-patent literature 3 Neurosci Res. 2010 May; 67 (1): 86-94 ).
  • International Publication No. 2004/101779 Patent Document 1 describes a screening method for IMPA2 inhibitors targeting central diseases. This publication describes a measurement system for the activity of IMPA (inositol monophosphatase 1 and / or inositol monophosphatase 2) using radiolabeled inositol.
  • Patent Document 2 relates to a screening method for drugs that induce autophagy based on IMPA inhibitory activity. This publication describes that autophagy is induced by inhibiting IMPA using LiCl and L690330. However, in any of the above documents, there is a report that IMPA2 is related to peripheral diseases (for example, obesity, diabetes, hyperlipidemia, arteriosclerosis, chronic nephritis, diabetic nephropathy, ischemic heart disease, etc.) Not at all.
  • peripheral diseases for example, obesity, diabetes, hyperlipidemia, arteriosclerosis, chronic nephritis, diabetic nephropathy, ischemic heart disease, etc.
  • IMPA2 is a candidate gene involved in intracellular triglyceride accumulation and that expression of IMPA2 is suppressed.
  • TG triglyceride
  • IMPA2 inhibition leads to anti-obesity.
  • the present inventors have also found that the expression level of IMPA2 is involved in the regulation of intracellular mitochondrial metabolic activity.
  • the present inventors have found 6-phosphogluconic acid as a substrate having high specificity for IMPA2. As a result of further studies based on these findings, the present inventors have completed the present invention.
  • the present invention provides the following.
  • [1] The same or substantially the same amino acid sequence as shown in SEQ ID NO: 1 (human IMPA2), 3 (mouse IMPA2), 5 (rat IMPA2), 7 (bovine IMPA2), 9 (dog IMPA2) or 11 (monkey IMPA2)
  • a polypeptide containing the same amino acid sequence sometimes abbreviated as the polypeptide of the present invention in the present specification
  • a partial peptide or salt thereof or a cell producing the polypeptide or the partial peptide
  • a screening method for a prophylactic / therapeutic agent for an IMPA2-related peripheral disease is obesity.
  • Non-human mammal deficient in the expression of a polynucleotide wherein a polynucleotide comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12 is inactivated
  • a method for screening for an agent for promoting fat differentiation characterized in that [6]
  • Use of an exogenous non-human transgenic animal having a polynucleotide comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 A screening method for a prophylactic / therapeutic agent for IMPA2-related peripheral diseases.
  • a polypeptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 The method according to any one of [1] to [4] and [6], which is a compound or a salt thereof that promotes or inhibits activity.
  • a polypeptide comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, or a partial peptide thereof, or a salt thereof, or the polypeptide or a thereof A method for screening an IMPA2 inhibitor, comprising using a cell that produces a partial peptide, and 6-phosphogluconic acid or a similar compound thereof.
  • a prophylactic / therapeutic agent for IMPA2-related peripheral diseases comprising an IMPA2 inhibitor.
  • a method for preventing and treating an IMPA2-related peripheral disease comprising administering an effective amount of an IMPA2 inhibitor to a mammal.
  • the screening methods [1] to [7] above it is possible to efficiently search for preventive / therapeutic agents for IMPA2-related peripheral diseases (particularly obesity) and fat differentiation promoting agents.
  • the screening method of [8] above it is possible to efficiently search for an IMPA2 inhibitor.
  • the prophylactic / therapeutic agent according to [9] to [11] above containing an IMPA2 inhibitor is useful as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases (particularly obesity).
  • the horizontal axis indicates the number of days from the differentiation induction treatment, and the vertical axis indicates the relative amount of IMPA2 mRNA relative to the amount of 36B4 mRNA.
  • the diamond ( ⁇ ) shows the amount of mRNA (average value ⁇ standard deviation) of IMPA2 in the cells in which differentiation has not been induced, and the triangles ( ⁇ ) in cells in which differentiation has been induced.
  • Pio is 10 ⁇ M Pioglitazone (Pioglitazone), and 10% CS-DMEM is D-MEM medium containing 10% Calf Serum.
  • Fig. 2 shows the intracellular triglyceride accumulation inhibitory effect (Fig. 2a) and the mRNA expression inhibitory effect (Fig.
  • FIG. 2b of IMPA2 by siRNA specific to IMPA2.
  • the horizontal axis represents siRNA introduced into the cells, and the vertical axis represents FIG. 2a: fluorescence intensity (mean value ⁇ standard deviation), FIG. 2b: mRNA amount of IMPA2 (relative to 36B4 mRNA amount).
  • Non-target is Silencer Negative Control # 1 siRNA (Ambion, catalog number AM4611), siRNA1 is siRNA consisting of SEQ ID NOs: 16 and 17, and siRNA2 is siRNA consisting of SEQ ID NOs: 18 and 19.
  • the mouse IMPA2 mRNA expression level in the soleus and gastrocnemius muscles of normal (m + / m +) mice is shown.
  • Non-target is Silencer Negative Control # 1 siRNA (Ambion, catalog number AM4611), siRNA1 is siRNA consisting of SEQ ID NOs: 16 and 17, and siRNA2 is siRNA consisting of SEQ ID NOs: 18 and 19.
  • examples of the “IMPA2-related peripheral disease” include the following. (1) Diseases involving intracellular triglyceride accumulation (eg obesity, diabetes, hyperlipidemia, arteriosclerosis, chronic nephritis, diabetic nephropathy, fatty liver, hypertension, ischemic heart disease, cancer, etc.) (2) Diseases involving intracellular mitochondrial activity (eg obesity, retinal degenerative diseases, diabetic neuropathy, diabetic retinopathy, glomerulosclerosis, nephropathy, diabetic nephropathy, chronic nephropathy, insulin Resistant disease, hypertension, arteriosclerosis, diabetes, oxidative stress, ischemic heart disease, aging, myopathy, neuropathy, hearing loss, blindness, fatty liver, steatohepatitis, viral hepatitis, biliary cirrhosis, ischemia reperfusion Disorders, graft rejection, hemolytic anemia, cancer, etc.) Regarding the relationship between mitochondrial activity and diseases, for example
  • the present invention provides the following screening methods.
  • a polypeptide comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11, or a partial peptide thereof, or a salt thereof, or the polypeptide or a thereof
  • a screening method for a prophylactic / therapeutic agent for IMPA2-related peripheral diseases characterized by using cells that produce partial peptides.
  • the polypeptide of the present invention and its partial peptide, the polynucleotide of the present invention, the antibody of the present invention and the like used in the screening method of the present invention will be described.
  • the polypeptide of the present invention is preferably a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11, and represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11. More preferred is a polypeptide having an amino acid sequence, that is, IMPA2 of various mammals (human, mouse, rat, cow, dog, monkey). Of these, human IMPA2 (polypeptide having the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1) is preferred.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 includes the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11. About 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more homology or identity
  • NCBI BLAST National Center for Biotechnology Information Basic Local Alignment Search Tool
  • Amino acids are substituted with other amino acids, or (v) amino acid sequences in which (i) to (iv) above are combined.
  • the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.
  • polypeptide containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11 examples include the aforementioned SEQ ID NO: 1, 3, 5, 7, 9, or 11.
  • a polypeptide having substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11 and having substantially the same activity as the polypeptide comprising the amino acid sequence represented by SEQ ID NO: Peptides are preferred.
  • substantially the same quality of activity include intracellular triglyceride accumulation promoting activity (TG accumulation promoting activity) and phosphatase activity using inositol monophosphate, 6-phosphogluconic acid and the like as substrates.
  • Substantially homogeneous indicates that their properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, it is preferable that these activities are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times).
  • the quantitative factors such as the molecular weight of the polypeptide may be different.
  • the intracellular triglyceride accumulation-promoting activity of an object is measured, for example, by measuring the amount of intracellular triglyceride accumulated when cells (for example, differentiated adipocytes) are cultured in the presence and absence of the object. Can be evaluated (see also the measurement method described in Example 2 described later).
  • Non-Patent Document 1 (Cryns, K. et al., Neuropsycho-pharmacology 32, 881-891, 2007), Patent Document 2 (International Publication No. 2006 / 079792 pamphlet) or a method according thereto.
  • substantially homogeneous activity also includes intracellular mitochondrial metabolic activity or mitochondrial regulation.
  • substantially the same quality means that, as described above, these properties are qualitatively (for example, physiologically or pharmacologically) homogeneous. Therefore, it is preferable that these activities are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times).
  • the quantitative factors such as the molecular weight of the polypeptide may be different.
  • the intracellular mitochondrial metabolic activity or the amount of mitochondrial activity of an object is, for example, intracellular mitochondrial metabolic activity when cells (eg, differentiated adipocytes) are cultured in the presence and absence of the object, mitochondrial DNA It can be evaluated by measuring the amount and comparing the measured values.
  • the intracellular mitochondrial metabolic activity a known method, for example, can be measured by the method or methods analogous thereto described in Methods in Cell Biology (Volume 80, Mitochondrai 2 nd edition). The measurement method described in Example 5 or 6 described later can also be referred to.
  • 6-phosphogluconic acid is provided as a substrate having high specificity for the polypeptide of the present invention, preferably IMPA2.
  • 6-phosphogluconic acid or a similar compound can be used as a substrate.
  • 6-phosphogluconic acid is preferably used as a substrate.
  • a similar compound preferably 6-phosphogluconic acid
  • an effective IMPA2-related peripheral disease prophylactic / therapeutic agent with high IMPA2 inhibitory efficiency is obtained.
  • the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide designation.
  • the polypeptide of the present invention including the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11, has a C-terminus having a carboxyl group (—COOH), a carboxylate (—COO - ), Amide (-CONH 2 ) or ester (-COOR).
  • R in the ester for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc., for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, etc., for example, phenyl C 6-12 aryl groups such as ⁇ -naphthyl, C 7- such as phenyl-C 1-2 alkyl groups such as benzyl and phenethyl or ⁇ -naphthyl-C 1-2 alkyl groups such as ⁇ -naphthylmethyl 14 aralkyl group, pivaloyloxymethyl group is used.
  • a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc.
  • a C 3-8 cycloalkyl group such as
  • the polypeptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus
  • those in which the carboxyl group is amidated or esterified are also included in the polypeptide of the present invention.
  • the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
  • amino acid residues (eg, methionine residue) of the N-terminal amino group protecting group e.g., formyl group, such as C 1-6 alkanoyl such as acetyl group C 1-6 A group protected by an acyl group, an N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (eg, —OH, —SH) Amino group, imidazole group, indole group, guanidino group, etc.) are protected with an appropriate protecting group (for example, C 1-6 acyl group such as C 1-6 alkanoyl group such as formyl group, acetyl group, etc.) Or a complex protein such as a so-called glycoprotein having a sugar chain bound thereto.
  • an appropriate protecting group for example, C 1-6 acyl group such as C 1-6 alkanoyl group such as formyl group,
  • the partial peptide of the polypeptide of the present invention (sometimes abbreviated as the partial peptide of the present invention in the present specification) is the partial peptide of the polypeptide of the present invention described above, preferably the above-described present invention. It has the same property as the polypeptide.
  • the partial peptide of the present invention is used as an antigen for producing the antibody of the present invention described later, at least 20 or more, preferably 50 or more, more preferably among the constituent amino acid sequences of the polypeptide of the present invention.
  • the partial peptide of the present invention is one or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1) in the amino acid sequence of the corresponding polypeptide of the present invention.
  • amino acids or one or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1 to 6) in the amino acid sequence.
  • Amino acids or one or more amino acids in the amino acid sequence (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1 to 6)).
  • the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO—), an amide (—CONH 2 ), or an ester (—COOR).
  • the partial peptide of the present invention includes those having a carboxyl group (or carboxylate) in addition to the C-terminus, and an N-terminal amino acid residue (eg, methionine residue) as in the above-described polypeptide of the present invention.
  • Examples of the salt of the polypeptide of the present invention or a partial peptide thereof include physiologically acceptable metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, basic or acidic amino acids, And the like.
  • Preferable examples of the metal salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; aluminum salt and the like.
  • Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And the like.
  • Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like.
  • salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, propionic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid And salts with benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like.
  • salts with basic amino acids include salts with arginine, lysine, ornithine and the like
  • salts with acidic amino acids include salts with aspartic acid, glutamic acid and the like. Can be mentioned.
  • the polypeptide of the present invention or a partial peptide thereof or a salt thereof can be produced from a mammalian cell (human, mouse, rat, etc.) or tissue by a known purification method of the polypeptide, and encodes the polypeptide. It can also be produced by culturing a transformant containing DNA. Moreover, it can also manufacture according to the below-mentioned peptide synthesis method.
  • the polypeptide of the present invention or a partial peptide thereof is produced from mammalian tissues or cells, the tissues or cells of mammals are homogenized, followed by extraction with acid or the like, and the extract is subjected to reverse phase chromatography and ion exchange. It can be purified and isolated by combining chromatography such as chromatography.
  • the polypeptide of the present invention or a partial peptide thereof or a salt thereof can be produced according to a peptide synthesis method known per se.
  • a peptide synthesis method for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the polypeptide of the present invention is obtained by condensing a partial peptide or amino acid capable of constituting the polypeptide of the present invention or a partial peptide thereof and the remaining part, and removing the protective group when the product has a protective group.
  • a partial peptide thereof can be produced. Examples of known condensation methods and protecting group elimination include the methods described in the following (i) to (v). (I) M.
  • polypeptide of the present invention obtained by the above method or a partial peptide thereof is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, It can be converted into a free form or other salt by a known method or a method analogous thereto.
  • the polynucleotide of the present invention may be any polynucleotide as long as it contains a nucleotide sequence encoding the above-described polypeptide of the present invention or a partial peptide thereof, and is preferably DNA (herein In some cases, the DNA of the present invention may be abbreviated).
  • the DNA may be any of genomic DNA, genomic DNA library, cDNA derived from cells / tissues described later, cDNA library derived from cells / tissues described later, and synthetic DNA.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
  • RNA or mRNA fraction prepared from cells / tissues described later can be directly amplified by reverse transcription polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from cells / tissues described later.
  • RT-PCR method reverse transcription polymerase chain reaction
  • examples of the polynucleotide encoding the polypeptide of the present invention include SEQ ID NOs: 2, 4, 6, 8, 10, or 12.
  • a polynucleotide (preferably DNA) comprising the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 (preferably DNA), SEQ ID NO: 2. 4.
  • a polynucleotide preferably DNA
  • Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual. More preferably, it can be carried out according to highly stringent conditions.
  • the highly stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
  • polynucleotide encoding the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 is a polynucleotide (preferably DNA) comprising the base sequence represented by SEQ ID NO: 2
  • the polynucleotide encoding the polypeptide containing the amino acid sequence shown by No. 3 is a polynucleotide (preferably DNA) containing the base sequence shown by SEQ ID No. 4 or the like, and the amino acid shown by SEQ ID No.
  • a polynucleotide (preferably DNA) containing the base sequence shown by SEQ ID NO: 6 encodes a polypeptide containing the amino acid sequence shown by SEQ ID NO: 7.
  • the polynucleotide to be used is a polynucleotide comprising the base sequence represented by SEQ ID NO: 8.
  • a polynucleotide encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 9 such as a leotide (preferably DNA)
  • a polynucleotide comprising the base sequence represented by SEQ ID NO: 10 preferably DNA
  • a polynucleotide encoding the polypeptide containing the amino acid represented by SEQ ID NO: 11 is used.
  • Examples of the partial polynucleotide of the polynucleotide encoding the polypeptide of the present invention include a polynucleotide encoding the partial peptide of the present invention.
  • the polynucleotide encoding the partial peptide of the present invention may be any polynucleotide as long as it contains the base sequence encoding the partial peptide of the present invention described above. Further, any of genomic DNA, genomic DNA library, cDNA derived from cells / tissues described later, cDNA library derived from cells / tissues described later, and synthetic DNA may be used.
  • polynucleotide encoding the partial peptide of the present invention for example, a polynucleotide having a part of a polynucleotide (preferably DNA) comprising the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 (Preferably DNA), a polynucleotide (preferably DNA) having a part of a polynucleotide (preferably DNA) comprising the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, or A polypeptide comprising a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 under highly stringent conditions and having substantially the same quality of activity as the polypeptide of the present invention.
  • a polynucleotide comprising a portion of a polynucleotide (preferably DNA) encoding a peptide (preferably a DNA) DNA) and the like can be used.
  • the polynucleotide (preferably DNA) that hybridizes with the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 has the same meaning as described above.
  • a part of the base sequence encoding the peptide of the present invention is used.
  • the hybridization method can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989).
  • the DNA base sequence is converted by PCR, a known kit such as Mutan TM -super Express Km (Takara Shuzo), Mutan TM -K (Takara Shuzo), etc., using the ODA-LA PCR method, It can be carried out according to a method known per se such as the Gapped duplex method and the Kunkel method or a method analogous thereto.
  • the DNA encoding the cloned peptide can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired.
  • the DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
  • the peptide of the present invention is produced by transforming a suitable host with the expression vector of the peptide of the present invention, culturing the obtained transformant, and then separating and purifying the peptide of the present invention from the obtained culture.
  • the peptide expression vector of the present invention can be obtained by, for example, (i) cutting out the target DNA fragment from the DNA encoding the peptide of the present invention, and (ii) linking the DNA fragment downstream of the promoter in an appropriate expression vector. Can be manufactured.
  • plasmids derived from E. coli eg, pBR322, pBR325, pUC12, pUC13
  • plasmids derived from Bacillus subtilis eg, pUB110, pTP5, pC194
  • yeast-derived plasmids eg, pSH19, pSH15
  • Bacteriophage such as ⁇ phage
  • animal viruses such as retrovirus, vaccinia virus, baculovirus, and the like, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like.
  • Examples of the promoter described above include SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like when animal cells are used as a host. Of these, it is preferable to use a CMV (cytomegalovirus) promoter, SR ⁇ promoter, or the like.
  • CMV cytomegalovirus
  • SR ⁇ promoter or the like.
  • the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P L promoter, lpp promoter, T7 promoter, etc.
  • the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc.
  • the host is yeast
  • the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable.
  • the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
  • an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like is used as desired. it can.
  • selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Amp r ), neomycin resistance gene (hereinafter sometimes abbreviated as Neo r, G418 resistance).
  • the target gene can also be selected by a medium not containing thymidine.
  • a signal sequence suitable for the host is added to the N-terminal side of the peptide of the present invention.
  • the host is Escherichia
  • the host is Bacillus
  • MF ⁇ • signal sequence, SUC2 • signal sequence, etc. can be used.
  • insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule / signal sequence, etc. can be used.
  • a transformant can be produced using a vector containing the DNA encoding the peptide of the present invention thus constructed.
  • Escherichia for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
  • Specific examples of the genus Escherichia include, for example, Escherichia coli K12 ⁇ DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] Etc. are used.
  • Bacillus bacterium examples include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)].
  • yeast examples include Saccharomyces cerevisiae AH22, AH22R ⁇ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Used.
  • insect cells for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five TM derived from eggs of Trichoplusia ni Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • larvae-derived cell lines Spodoptera frugiperda cells; Sf cells
  • MG1 cells derived from the midgut of Trichoplusia ni High Five TM derived from eggs of Trichoplusia ni Cells
  • cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • BmNPV sputum-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used.
  • Sf cells examples include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like.
  • insects examples include silkworm larvae [Maeda et al., Nature, 315, 592 (1985)].
  • animal cells examples include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁇ ) cells), mouse L Cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells, human 293 cells and the like are used.
  • CHO cells Chinese hamster cells CHO
  • CHO (dhfr ⁇ ) cells examples include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁇ ) cells), mouse L Cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells, human 293 cells and the like are used
  • Transformation of Escherichia can be performed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982). Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, 168, 111 (1979). In order to transform yeast, for example, the method described in Methods in Enzymology, 194, 182-287 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like can be performed. it can. Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • a liquid medium is suitable as a medium used for the culture, and a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, and potato extract.
  • inorganic or organic substances and inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • a medium for culturing Escherichia for example, M9 medium containing glucose and casamino acid (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972) is preferable. .
  • a drug such as 3 ⁇ -indolylacrylic acid can be added to make the promoter work efficiently if necessary.
  • the host is an Escherichia bacterium
  • the culture is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation can be added.
  • the host is Bacillus
  • the culture is usually performed at about 30 to 40 ° C.
  • examples of the medium include a Burkholder minimum medium [Bostian, KL et al., Proc. Natl. Acad. Sci. USA, vol. 77, 4505 (1980)]. And SD medium containing 0.5% casamino acid [Bitter, GA et al. Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)].
  • the pH of the medium is preferably adjusted to about 5-8. Cultivation is usually carried out at about 20 ° C. to 35 ° C. for about 24 to 72 hours, with aeration and agitation as necessary.
  • an appropriate medium such as 10% bovine serum that has been immobilized on Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)) is used as the medium. Additions etc. are used.
  • the pH of the medium is preferably adjusted to about 6.2 to 6.4.
  • the culture is usually carried out at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.
  • examples of the medium include MEM medium (Science, Vol.
  • the peptide of the present invention can be produced in the cell, in the cell membrane or outside of the transformant.
  • Separation and purification of the peptide of the present invention from the culture can be performed, for example, by the following method.
  • the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freeze-thaw, etc.
  • a method of obtaining a crude peptide extract by centrifugation or filtration after destroying cells or cells by the method is appropriately used.
  • the buffer may contain a protein (peptide) denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM .
  • the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.
  • the peptide contained in the culture supernatant or the extract thus obtained can be purified by appropriately combining known separation / purification methods.
  • These known separation and purification methods include mainly molecular weights such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • Method using difference in charge method using difference in charge such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and difference in hydrophobicity such as reverse phase high performance liquid chromatography
  • a method using a difference in isoelectric point such as a method, isoelectric focusing method, or the like is used.
  • the peptide thus obtained when obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when obtained as a salt, a method known per se or a method analogous thereto Can be converted to the free form or other salts.
  • the peptide produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by allowing an appropriate protein modifying enzyme to act before or after purification.
  • the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
  • the presence of the peptide of the present invention thus produced can be measured by enzyme immunoassay or Western blotting using an antibody that specifically binds to the peptide of the present invention.
  • the antibody of this invention is an antibody couple
  • the antibody of the present invention can be produced according to a method for producing an antibody or antiserum known per se, using the peptide of the present invention as an antigen.
  • Monoclonal antibodies can be produced according to a method known per se.
  • the peptide of the present invention is administered to a warm-blooded animal itself or a carrier and a diluent at a site where antibody production is possible by administration.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens, and mice and rats are preferably used.
  • an individual having an antibody titer is selected from a warm-blooded animal immunized with an antigen, such as a mouse, and spleen or lymph nodes are collected 2 to 5 days after the final immunization, and antibody-producing cells contained therein are collected.
  • Monoclonal antibody-producing hybridomas can be prepared by fusing with myeloma cells from the same or different animals.
  • the antibody titer in the antiserum can be measured, for example, by reacting a labeled peptide described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
  • PEG polyethylene glycol
  • myeloma cells include warm-blooded animal myeloma cells such as NS-1, P3U1, SP2 / 0, AP-1, and P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%.
  • the cell fusion can be efficiently carried out by incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes.
  • the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which peptide antigens are adsorbed directly or together with a carrier, and then radioactive substances or An anti-immunoglobulin antibody labeled with an enzyme or the like (when the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A, and a method for detecting a monoclonal antibody bound to a solid phase;
  • a method for detecting a monoclonal antibody bound to a solid phase examples include a method in which a hybridoma culture supernatant is added to a solid phase on which a globulin antibody or protein A is adsorbed, a peptide labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected.
  • the selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium may be used as long as the hybridoma can grow.
  • RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal calf serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1 to 10% fetal calf serum, or serum-free for hybridoma culture A medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used.
  • the culture temperature is usually 20 to 40 ° C., preferably about 37 ° C.
  • the culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.
  • Separation and purification of the monoclonal antibody can be performed by a method known per se, for example, an immunoglobulin separation and purification method (eg, salting out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchanger (eg, DEAE)).
  • an immunoglobulin separation and purification method eg, salting out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchanger (eg, DEAE)
  • an immunoglobulin separation and purification method eg, salting out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchanger (eg, DEAE)
  • a polyclonal antibody can be produced according to a method known per se or a method analogous thereto.
  • an immune antigen peptide antigen
  • a warm-blooded animal is immunized in the same manner as in the case of the above monoclonal antibody. It can be produced by collecting the product and performing separation and purification of the antibody.
  • the type of carrier peptide and the mixing ratio of carrier and hapten are effective for antibodies against hapten immunized by cross-linking to carrier.
  • any substance may be cross-linked at any ratio.
  • bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are about 0.1 to 20, preferably about 1 by weight ratio to hapten 1.
  • a method of coupling at a rate of ⁇ 6 is used.
  • various condensing agents can be used for coupling of the hapten and the carrier, but active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and dithiobilidyl group are used.
  • the condensation product is administered to a warm-blooded animal by itself, together with a carrier and a diluent, at a site where antibody production is possible.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually about once every 2 to 6 weeks, about 3 to 10 times in total.
  • Polyclonal antibodies can be collected from blood, ascites, etc., preferably from blood of warm-blooded animals immunized by the above method.
  • the polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as in the case of the monoclonal antibody described above.
  • a cell producing the polypeptide of the present invention or its partial peptide is used.
  • Such cells include, for example, cells originally having the polypeptide of the present invention or a partial peptide thereof (preferably animal cells), or a vector containing a DNA encoding the polypeptide of the present invention or a partial peptide thereof.
  • a transformed host (transformant) or the like is used.
  • mammalian cells such as brown adipocytes, white adipocytes, hepatocytes, myoblasts, visceral adipocytes, kidney tubules, macrophages, dendritic cells, cardiomyocytes are preferably used.
  • mammalian cells such as brown adipocytes, white adipocytes, hepatocytes, myoblasts, visceral adipocytes, kidney tubules, macrophages, dendritic cells, cardiomyocytes are preferably used.
  • Other cell lines can also be used.
  • test target substances to be screened include peptides, proteins, antibodies, non-peptidic compounds, and synthetic compounds. , Fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like. Whether or not a test compound affects the amount of intracellular triglyceride accumulated can be confirmed, for example, by measuring the amount of intracellular triglyceride accumulated when cells (eg, adipocytes) are cultured in the presence of the test compound. Can be evaluated. For example, the ability of a test compound to change intracellular triglyceride accumulation can be evaluated according to the method described in Example 2 below.
  • the polypeptide of the present invention is involved in fatty acid ⁇ -oxidation through phosphatase activity. By inhibiting this phosphatase activity, intracellular fatty acid ⁇ oxidation can be promoted.
  • the fatty acid ⁇ -oxidation activity of a test compound can be evaluated, for example, by culturing cells (for example, brown adipocytes) in the presence of the test compound and measuring the intracellular fatty acid ⁇ -oxidation activity.
  • the ability of the test compound to change intracellular fatty acid ⁇ -oxidation activity can be evaluated according to the measurement method described in Example 7 described later. It can also be evaluated by measuring the amount of ATP produced by oxidative phosphorylation accompanying the increase in fatty acid ⁇ oxidation in cells, or by increasing the oxygen consumption rate.
  • examples of the active ingredient of the preventive / therapeutic agent for IMPA2-related peripheral diseases include substances that promote or inhibit the activity of IMPA2, and substances that promote or inhibit the expression of IMPA2.
  • a substance that inhibits the activity or expression of IMPA2 (sometimes abbreviated as “IMPA2 inhibitor” in the present specification) is desirable.
  • a test compound that promotes or inhibits the phosphatase activity, intracellular triglyceride accumulation effect or fatty acid ⁇ -oxidation effect possessed by the polypeptide of the present invention preferably IMPA2
  • the test compound obtainable by the screening method of the present invention is useful as a low-toxic preventive or therapeutic agent for the above-mentioned IMPA2-related peripheral diseases (particularly obesity).
  • the compound that suppresses the IMPA2 activity obtained by the screening method of the present invention has a phosphatase inhibitory activity, an intracellular triglyceride accumulation lowering action or a fatty acid ⁇ oxidation promoting action
  • the prophylactic agent for the above-mentioned IMPA2-related peripheral diseases -It is useful as a therapeutic agent.
  • a test compound that promotes or inhibits the effect of regulating the mitochondrial activity or mitochondrial amount possessed by the polypeptide of the present invention, IMPA2 can be obtained. Due to these actions, the compound obtainable by the screening method of the present invention is useful as a low-toxic prophylactic or therapeutic agent for the above-mentioned IMPA2-related peripheral diseases. For example, those skilled in the art can measure the amount of intracellular mitochondrion as described in Example 5 or 6 of the present specification.
  • the compound that suppresses the IMPA2 activity obtained by the screening method of the present invention is useful as a prophylactic or therapeutic agent for the above-mentioned IMPA2-related peripheral diseases due to its action to suppress mitochondrial activity or to reduce the amount of mitochondria.
  • the compound that promotes the IMPA2 activity obtained by the screening method of the present invention is useful as a fat differentiation promoter by, for example, an intracellular triglyceride accumulation promoting action, a mitochondrial activity promoting action, or a mitochondrial amount increasing action.
  • Examples of the substrate used for measuring the activity of the polypeptide of the present invention include 6-phosphogluconic acid having a high substrate specificity for IMPA2, and the above-mentioned analogs of 6-phosphogluconic acid. Of these, 6-phosphogluconic acid is preferable.
  • the screening method of the present invention makes it possible to more efficiently screen for an IMPA2 inhibitor, preferably an IMPA2 inhibitor that specifically inhibits IMPA2, by using 6-phosphogluconic acid or a similar compound of 6-phosphogluconic acid. It shall be Moreover, the screening method of the present invention can make screening for an IMPA2 inhibitor that specifically inhibits IMPA2 more efficient by using IMPA1 as a control.
  • phosphatase activity by IMPA2 and IMPA1 for example, human IMPA1 described in WO 2006/079792 (amino acid sequence: NP — 005527.1, GI: 5031789)
  • a test solution using 6-phosphogluconic acid I
  • the phosphatase activity of the polypeptide of the present invention can be measured by the above-mentioned known methods.
  • Such test compounds can include, for example, analogs of substrates that are highly specific for IMPA2.
  • a substrate having high specificity for IMPA2 is preferably dephosphorylated by IMPA2.
  • An example of a substrate having high specificity for IMPA2 is 6-phosphogluconic acid.
  • the polypeptide of the present invention or a partial peptide thereof or a salt thereof can be obtained by a known method, for example, using the antibody of the present invention, the polypeptide present in a cell extract or the like by Western analysis, ELISA method, particularly sandwich immunization. It can be quantified with high sensitivity according to a method such as a measurement method or a method analogous thereto.
  • one antibody is an antibody that recognizes the N-terminal part of the polypeptide of the present invention or a partial peptide thereof, and the other antibody is the C-terminal part of the polypeptide of the present invention or its partial peptide It is desirable that the antibody be reactive with.
  • the polypeptide of the present invention can be quantified using a monoclonal antibody against the polypeptide of the present invention or a partial peptide thereof (hereinafter sometimes referred to as the monoclonal antibody of the present invention). Polypeptide detection can also be performed.
  • the antibody molecule itself may be used, or F (ab ′) 2 , Fab ′ or Fab fraction of the antibody molecule may be used.
  • the method for quantifying the polypeptide of the present invention using the antibody of the present invention is not particularly limited, and an antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (eg, amount of polypeptide) in the solution to be measured.
  • Any measurement method may be used as long as it is a measurement method in which the amount of the body is detected by a chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen.
  • nephrometry, competition method, immunometric method and sandwich method are preferably used, but the sandwich method described later is particularly preferable in view of sensitivity and specificity.
  • Examples of the labeling agent used for labeling the polypeptide of the present invention or a partial peptide thereof and the antibody of the present invention include radioisotopes (eg, [ 125 I], [ 131 I], [ 3 H], [ 14 C], [ 32 P], [ 33 P], [ 35 S], etc.), fluorescent substance [eg, cyanine fluorescent dye (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (made by Amersham Biosciences), etc.) ), Fluorescamine, fluorescein isothiocyanate, NBD (7-nitrobenz-2-oxa-1,3-diazol), etc.], enzymes (eg, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malic acid) Dehydrogenase, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), biotin, lanthanide
  • insolubilization of an antigen or antibody physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a polypeptide or an enzyme may be used.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further labeled with another monoclonal antibody of the present invention (secondary reaction), and then on the insolubilized carrier.
  • the polypeptide of the present invention in the test solution can be quantified.
  • the primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times.
  • the labeling agent and the insolubilization method can be the same as those described above.
  • the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and two or more types are used for the purpose of improving measurement sensitivity. A mixture of antibodies may be used.
  • the monoclonal antibody of the present invention used for the primary reaction and the secondary reaction is preferably an antibody having a different site to which the polypeptide of the present invention or its partial peptide binds.
  • the antibody used in the secondary reaction recognizes the C-terminal part of the polypeptide of the present invention or a partial peptide thereof
  • the antibody used in the primary reaction preferably has a non-C-terminal part, for example, an N-terminal part. Recognizing antibody.
  • the monoclonal antibody of the present invention can be used for measurement methods other than the sandwich method, for example, competition method, immunometric method, nephrometry and the like.
  • competition method the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified.
  • a soluble antibody is used as an antibody
  • B / F separation is performed using polyethylene glycol
  • a liquid phase method using a second antibody against the antibody and a solid-phased antibody is used as the first antibody
  • a soluble method is used, and a solid phase method using a solid phase antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are competitively reacted with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or After reacting the antigen with an excess amount of labeled antibody, and then adding a solid-phased antigen to bind unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the test solution.
  • nephrometry the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained.
  • the polypeptide of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • the screening method of [1] above [1] A polypeptide comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, or a partial peptide thereof, or a salt thereof, or the polypeptide or a peptide thereof
  • Examples of the screening method for a prophylactic / therapeutic agent for IMPA2-related peripheral diseases characterized by using cells that produce partial peptides include the following screening methods [1a] to [1d].
  • [1a] The phosphatase activity of a polypeptide comprising the amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11 or a partial peptide thereof or a salt thereof (i) (1) The screening method according to [1], comprising measuring in the presence of the test compound and (ii) in the absence of the test compound, and comparing (i) and (ii). [1b] Measuring the amount of triglyceride accumulation or fatty acid ⁇ -oxidation activity in the cells (i) in the presence of the test compound and (ii) in the absence of the test compound, and comparing (i) and (ii) The screening method according to [1].
  • the screening method according to [1] comprising measuring at least one item in (i) administration of a test compound and (ii) non-administration of a test compound, and comparing (i) and (ii) .
  • the phosphatase activity of the polypeptide of the present invention or a partial peptide thereof or a salt thereof can be determined by a known method, for example, the measurement method described in Example 4 or 9 described later, Non-Patent Document 1 (Cryns, K. et al. , Neuropsycho-pharmacology 32, 881-891, 2007), Patent Document 2 (International Publication No. 2006/079792 pamphlet) or a method analogous thereto.
  • the phosphatase activity of the polypeptide of the present invention or a partial peptide thereof or a salt thereof in the presence of a test compound is determined in the presence of (i) the test compound and (ii) the absence of the test compound.
  • a compound that significantly lowers the cases of (i) and (ii) can be selected, preferably about 20% or more, about 30% or more, more preferably about 50% or more, More preferably, a compound that decreases about 60% or more can be selected as an agent for preventing / treating IMPA2-related peripheral diseases.
  • the amount of the polypeptide of the present invention or a partial peptide thereof or a salt thereof can be determined by a known method, for example, Western blotting analysis or enzyme immunoassay method for the polypeptide present in a cell extract using the antibody of the present invention. It can measure according to the method of following or the method according to it.
  • the polypeptide of the present invention or a partial peptide thereof or a salt thereof used in the screening method of the present invention is involved in, for example, intracellular triglyceride accumulation action through phosphatase activity. By inhibiting this phosphatase activity, the amount of intracellular triglyceride accumulation can be reduced.
  • the amount of intracellular triglyceride accumulated in a test compound can be evaluated, for example, by measuring the amount of intracellular triglyceride accumulated when cells (for example, differentiated adipocytes) are cultured in the presence of the test compound. For example, the ability of the test compound to change the amount of intracellular triglyceride accumulation can be evaluated according to the measurement method described in Example 2 described later.
  • the polypeptide of the present invention or a partial peptide thereof or a salt thereof is involved in, for example, fatty acid ⁇ oxidation through phosphatase activity. By inhibiting this phosphatase activity, intracellular fatty acid ⁇ oxidation can be promoted.
  • the fatty acid ⁇ -oxidation activity of the test compound can be determined, for example, by culturing cells (for example, brown adipocytes) that produce the polypeptide of the present invention or a partial peptide thereof, It can be evaluated by measuring in the presence and (ii) in the absence of the test compound and comparing these measurements. For example, the ability of the test compound to change intracellular fatty acid ⁇ -oxidation activity can be evaluated according to the measurement method described in Example 7 described later.
  • test compounds that can be significantly reduced can be selected, preferably test compounds that reduce about 20% or more, about 30% or more, more preferably about 50% or more, and even more preferably about 60% or more of the present invention. It can be selected as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases.
  • intracellular fatty acid ⁇ -oxidation activity is measured in the presence of (i) the test compound and (ii) in the absence of the test compound, and the cases of (i) and (ii) Test compounds that can be significantly elevated relative to each other can be selected, and preferably test compounds that promote about 20% or more, about 30% or more, more preferably about 50% or more, and even more preferably about 60% or more, It can be selected as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases.
  • the expression level of the polypeptide of the present invention or a partial peptide thereof correlates with intracellular mitochondrial metabolic activity or mitochondrial level. Therefore, a prophylactic / therapeutic agent for IMPA2-related peripheral diseases can also be screened by measuring and comparing intracellular mitochondrial metabolic activity or mitochondrial amount in the presence and absence of a test compound.
  • a method for measuring intracellular mitochondrial metabolic activity a known method, for example, Methods in Cell Biology (Volume 80 , Mitochondrai 2 nd edition) is described, the activity of the respiratory chain complexes, ATP, lactic, pyruvic acid production And methods for measuring the amount, enzyme activity of the TCA cycle, oxygen consumption, and the like.
  • intracellular mitochondrial metabolic activity or mitochondrial amount is measured in the presence of (i) the presence of the test compound and (ii) in the absence of the test compound, and (i) and ( A test compound can be selected that significantly increases compared to ii), preferably about 20% or more, about 30% or more, more preferably about 50% or more, even more preferably about 60% or more.
  • the test compound to be reduced can be selected as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases.
  • a test compound is administered to a test animal (mouse, rat, rabbit, dog, pig, monkey, etc.) having cells producing the polypeptide of the present invention or a partial peptide thereof, At least one item selected from the group consisting of triglyceride accumulation amount, body fat amount, blood glucose level, blood lipid level, blood cholesterol level, systemic energy consumption, oxygen consumption, respiratory quotient and blood pressure in the subject animal Can be screened for prophylactic / therapeutic agents for IMPA2-related peripheral diseases.
  • test compound as a prophylactic / therapeutic agent for a disease involving the above-mentioned intracellular triglyceride accumulation amount or a disease involving intracellular mitochondrial activity
  • test compound a test compound
  • a non-administered group or a placebo-administered group can be evaluated by measuring the severity of each disease.
  • Standards known in the art can be used for the severity of each of the above-mentioned diseases and the evaluation method thereof.
  • the systemic energy consumption or oxygen consumption of (i) the test compound administration group and (ii) the test compound non-administration group or placebo administration group is measured,
  • a test compound that significantly increases the i) and (ii) groups can be selected as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases.
  • the amount of triglyceride accumulated, body fat mass, blood glucose level, blood lipid level, blood of (i) test compound administration group and (ii) non-test compound administration group or placebo administration group Test compounds that measure medium cholesterol level, respiratory quotient or blood pressure and significantly lower (i) and (ii) groups can be selected as prophylactic / therapeutic agents for IMPA2-related peripheral diseases .
  • Test compounds that measure medium cholesterol level, respiratory quotient or blood pressure and significantly lower (i) and (ii) groups can be selected as prophylactic / therapeutic agents for IMPA2-related peripheral diseases .
  • a method for administering the test compound to the test animal for example, oral administration, intravenous injection and the like are used, and can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like.
  • the dosage of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
  • the amount of triglyceride accumulated is determined by measuring the triglyceride concentration in serum with various reagent kits and clinical test kits (for example, Wako Pure Chemicals, “L-type Wako TG / M”, etc.). This can be evaluated.
  • the body fat mass can be measured using, for example, a body fat scale.
  • the blood glucose level can be measured by a dedicated medical blood glucose level measuring device, a blood glucose level measuring kit (for example, glucose CII-Test Wako reagent, etc.) and the like.
  • the blood lipid level and blood cholesterol level can be measured according to a known method, for example, using a commercially available measurement kit.
  • the whole-body energy consumption is measured by, for example, a direct method in which the amount of generated heat is directly measured using water or the like, or an indirect method in which the amount of heat is indirectly estimated from the amount of oxygen consumed in the body.
  • the oxygen consumption is measured, for example, by analysis of exhaled gas.
  • the respiratory quotient is derived, for example, by measuring oxygen consumption and carbon dioxide output by exhaled gas analysis, and the main energy source is one of caloric sources such as carbohydrates, lipids, and polypeptides. Can be estimated indirectly.
  • the blood pressure is measured using, for example, a sphygmomanometer.
  • the present invention also provides the following screening method [5].
  • [5] Non-human mammal deficient in the expression of a polynucleotide, wherein a polynucleotide comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12 is inactivated
  • a method for screening for an agent for promoting fat differentiation characterized in that A polynucleotide comprising the same or substantially the same base sequence as shown in SEQ ID NO: 4, 6, 8, 10 or 12 (in this specification, it may be abbreviated as the non-human polynucleotide of the present invention) ) Is inactivated, and the non-human mammal incapable of expressing the polynucleotide (in the present specification, may be abbreviated as a non-human mammal deficient in the expression of the polynucleotide of the present invention) according to a method known per se For example, introducing a mutation into
  • the non-human polynucleotide of the present invention includes (a) a polynucleotide (preferably DNA) encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, 5, 7, 9 or 11, or (b) A polynucleotide (preferably DNA) containing the base sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12 is preferred, and (c) the amino acid sequence represented by SEQ ID NO: 3, 5, 7, 9 or 11 More preferred is a polynucleotide (preferably DNA) encoding a polypeptide consisting of: (d) a polynucleotide comprising the base sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12 (preferably DNA) .
  • polynucleotide containing the nucleotide sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12 include the nucleotide sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12.
  • a polynucleotide preferably DNA. Specifically, it is about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 99 with the base sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12.
  • a polynucleotide preferably DNA
  • a polynucleotide preferably DNA
  • examples of the non-human mammal used for producing the non-human mammal deficient in polynucleotide expression of the present invention include mouse, rat, guinea pig, hamster, cow, pig, sheep, goat, rabbit, dog and cat. .
  • rodents, especially mice for example, C57BL / 6 strain, DBA2 strain, etc. as pure strains, etc.
  • mice for example, C57BL / 6 strain, DBA2 strain, etc. as pure strains, etc.
  • test compound examples include peptides, proteins, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like.
  • the non-human mammal deficient in polynucleotide expression of the present invention can be used for screening for an agent for promoting fat differentiation.
  • the test compound is administered to the non-human mammal deficient in polynucleotide expression of the present invention (the administration method can be appropriately selected according to the symptom of the test animal, the nature of the test compound, etc. , Oral administration, intravenous injection, intramuscular injection, etc.) and observing / measuring changes in the animals (for example, treating the animals with untreated control animals and changes in the symptoms of each organ, tissue, disease, etc.) By comparison), it is possible to screen a fat differentiation promoter.
  • Evaluation as a fat differentiation promoter is, for example, fat compared with a control cell or subject in a cell or an individual that does not express the polypeptide of the present invention or a partial peptide thereof when the test compound of the above screening method is used. It can be evaluated based on the accumulation promoting action, blood glucose level, blood lipid level and / or blood cholesterol level change.
  • the compound obtained by such a method can be obtained by, for example, triglyceride accumulation, body fat mass, blood glucose level, blood lipid level, blood cholesterol level, systemic cholesterol, as described in the above [1d] method for test animals.
  • the ability to change energy consumption, oxygen consumption, respiratory quotient and blood pressure can be evaluated.
  • a fat differentiation promoter increases normal small fat cells and decreases hypertrophic fat cells by promoting differentiation from enlarged fat cells to fat cells. Therefore, it is considered to have a prophylactic / therapeutic effect on lifestyle-related diseases such as diabetes, hyperlipidemia, hypertension, obesity, immune abnormalities, cancer and the like caused by an increase in enlarged fat cells.
  • the present invention also provides the following screening method [6].
  • An exogenous polynucleotide comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 (in the present specification, the foreign molecule of the present invention) IMPA2-related peripheral disease, characterized by using a non-human transgenic animal (sometimes abbreviated as a polynucleotide-transferred animal of the present invention) having a non-human transgenic animal having a Screening method for prophylactic / therapeutic agents.
  • the polynucleotide-transferred animal of the present invention can be produced according to a method for producing a non-human transgenic animal known per se.
  • the exogenous polynucleotide of the present invention can be obtained by subjecting an unfertilized egg, a fertilized egg, a sperm, and a starting material thereof by the calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, etc.
  • Produced by metastasis preferably to embryonic cells containing cells, preferably at the stage of embryonic development in non-human mammal development (more preferably at the single cell or fertilized egg cell stage and generally prior to the 8-cell stage). can do.
  • the exogenous polynucleotide of the present invention is not a polynucleotide originally possessed by a non-human mammal, but a polynucleotide once isolated and extracted from a mammal.
  • the exogenous polynucleotide of the present invention includes (a) a polynucleotide (preferably DNA) encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11, or ( b) A polynucleotide (preferably DNA) comprising the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 is preferred, and (c) SEQ ID NO: 1, 3, 5, 7, 9 or A polynucleotide (preferably DNA) encoding a polypeptide consisting of the amino acid sequence shown by 11 or (d) a polynucleotide consisting of the base sequence shown by SEQ ID NO: 2, 4, 6, 8, 10 or 12 ( Preferably, DNA is more preferable.
  • polynucleotide comprising a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12
  • the polynucleotide represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 is used.
  • a nucleotide sequence that hybridizes under high stringency conditions with a nucleotide sequence that is substantially the same as a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 And a polynucleotide (preferably, DNA) encoding a polypeptide having the above.
  • the exogenous polynucleotide of the present invention to be transferred in the polynucleotide-transferred animal of the present invention may be derived from a mammal of the same species as that of the transferred animal or from a heterologous mammal.
  • the polynucleotide-transferred animal of the present invention is produced using a non-human mammal.
  • the non-human mammal to be used is a non-human non-human expression of the polynucleotide of the present invention described in the above screening method [5].
  • the thing similar to what was used with the mammal is mentioned. Examples of the test compound include the same compounds as those described in the above screening method [5].
  • the polynucleotide-transferred animal of the present invention When the polynucleotide-transferred animal of the present invention highly expresses IMPA2, the transferred animal is considered to develop IMPA2 hyperfunction. In that case, the polynucleotide-transferred animal of the present invention can be used as a disease state model animal for an IMPA2-related peripheral disease caused by the enhanced function of IMPA2. Therefore, the polynucleotide-transferred animal of the present invention can be used for screening for preventive / therapeutic agents for IMPA2-related peripheral diseases (particularly obesity). In addition, using the polynucleotide-transferred animal of the present invention, it is also possible to elucidate the pathologic mechanism of IMPA2-related peripheral diseases and to examine methods for treating these diseases.
  • test compound can be administered to the polynucleotide-transferred animal of the present invention, and the prophylactic / therapeutic agent for IMPA2-related peripheral diseases can be screened by observing and measuring changes in the animal.
  • Test compounds selected by such screening can improve the pathology of IMPA2-related peripheral diseases resulting from hyperactivity of IMPA2.
  • an organ, tissue or cell collected from the polynucleotide-transferred animal of the present invention can be used.
  • the present invention also provides the following screening method [8].
  • a polypeptide comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, or a partial peptide thereof, or a salt thereof, or the polypeptide or a thereof
  • a method for screening an IMPA2 inhibitor comprising using a cell that produces a partial peptide, and 6-phosphogluconic acid or a similar compound thereof.
  • the screening method [8] can be carried out in the same manner as described in the screening method of the present invention.
  • a prophylactic / therapeutic agent for IMPA2-related peripheral diseases comprising an IMPA2 inhibitor.
  • the prophylactic / therapeutic agent of the present invention is desirably an IMPA2 inhibitor obtained by the screening method of the present invention described above.
  • Examples of the IMPA2 inhibitor obtained from the screening method of the present invention include peptides, proteins, polypeptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like. These compounds may be novel compounds or known compounds.
  • the IMPA2 inhibitor may form a salt, and examples of such a salt include those exemplified as the salt of the polynucleotide of the present invention or a partial peptide thereof.
  • the IMPA2 inhibitor include the following (a) to (e).
  • siRNA is more preferable. The above (a) to (e) will be described in detail below.
  • the compound that binds to the polypeptide of the present invention and inhibits the phosphatase activity of the polypeptide of the present invention includes a compound that binds to IMPA2 obtained by the screening method of the present invention and directly inhibits the activity of IMPA2.
  • Known compounds that bind to IMPA2 and directly inhibit the activity of IMPA2 include lithium or a salt thereof.
  • examples of the lithium salt include lithium carbonate and lithium citrate.
  • the IMPA2 inhibitor preferably used in the present invention is an inhibitor specific to IMPA2. For example, in Example 4 of this specification, 6-phosphogluconic acid is described as a substrate having high specificity for IMPA2.
  • This 6-phosphogluconic acid or its related compounds can be used to screen for IMPA2 inhibitors.
  • an inhibitor specific for IMPA2 can be isolated by using IMPA1 or the like as a control.
  • a novel compound can be designed using a known drug design technique. As such a technique, for example, Japanese Patent No. 3256307, Japanese Patent No. 3669704, Japanese Patent No. 3747048, and the like can be referred to.
  • a compound having advantages such as fewer side effects can be designed.
  • These designed compounds are evaluated by the screening method of the present invention for their ability to accumulate intracellular glycerol, regulate intracellular mitochondrial activity, etc., as well as the degree of specificity of the target polypeptide. be able to.
  • Neutralizing antibody against the polypeptide of the present invention includes, for example, an antibody that binds to the polypeptide of the present invention and neutralizes phosphatase activity.
  • (C) Compound that inhibits the expression of the polypeptide of the present invention includes, for example, an intracellular signal transduction mechanism and an intracellular transport mechanism obtained in the screening method of the present invention. Examples include compounds that inhibit the expression of IMPA2 by inhibiting intracellular expression of IMPA2 via, for example.
  • the compound that inhibits the expression of the polypeptide of the present invention is preferably a compound that inhibits the expression of the gene encoding the polypeptide of the present invention.
  • Examples of the method for evaluating the expression level of IMPA2 include the above-described method for quantifying the polypeptide of the present invention, a method for measuring the mRNA level of IMPA2, and a method for measuring the activity level of IMPA2.
  • the amount of mRNA of IMPA2 is measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 2, 4, 6, 8, 10, 12 or a part thereof as a probe, or SEQ ID NO: as a primer. : Measured according to a quantitative PCR method using a nucleic acid containing 2, 4, 6, 8, 10, 12, or a part thereof, or a method analogous thereto.
  • the expression level of the polypeptide of the present invention is measured in the presence of (i) the test compound and (ii) in the absence, and (i) and (ii) are compared to about 20% or more, preferably 30 %, More preferably about 50%, even more preferably about 60% or more of the test compound that inhibits the expression can be selected as a compound that inhibits the expression of the polypeptide of the present invention.
  • An antisense polynucleotide for a polynucleotide encoding the polypeptide of the present invention A base sequence complementary to or substantially complementary to the base sequence of the polynucleotide (preferably DNA) of the present invention or a part thereof
  • the antisense polynucleotide having (sometimes abbreviated as the antisense polynucleotide of the present invention in the present specification) is a base sequence complementary to or substantially complementary to the base sequence of the polynucleotide of the present invention
  • any antisense polynucleotide may be used as long as it has a part and has an action capable of suppressing the expression of the polynucleotide, but antisense DNA is preferred.
  • the base sequence substantially complementary to the base sequence of the polynucleotide of the present invention is, for example, all bases of the base sequence complementary to the base sequence of the polynucleotide of the present invention (for example, the complementary strand of the DNA of the present invention). About 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more homology or identity with a sequence or partial base sequence And a nucleotide sequence having the property.
  • the base sequence of the portion encoding the N-terminal site of the polypeptide of the present invention for example, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99%
  • the antisense polynucleotide having the above homology or identity is (ii) an antisense polynucleotide directed to RNA degradation by RNase H
  • the antisense polynucleotide has about 70% of the complementary strand of the entire base sequence of the DNA of the present invention including introns.
  • % Or more about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of an antisense polynucleotide having homology or identity Each suitable That.
  • a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide containing the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, or a part thereof An antisense polynucleotide having a nucleotide sequence, preferably having a nucleotide sequence complementary to the nucleotide sequence of a polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12, or a part thereof, for example Antisense polynucleotide (more preferably, an antisense polynucleotide having a base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 or a part thereof.
  • the antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.
  • the phosphate residue (phosphate) of each nucleotide constituting the antisense polynucleotide is a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, phosphorodithionate, etc. May be substituted.
  • the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base part (pyrimidine, purine) is also chemically modified. As long as it hybridizes to DNA containing the base sequence represented by SEQ ID NO: 2, 6, 8, 10 or 12, any of them may be used.
  • These antisense polynucleotides can be produced using a known DNA synthesizer.
  • An antisense polynucleotide (nucleic acid) corresponding to the gene capable of inhibiting replication or expression of the gene encoding the polypeptide of the present invention is a cloned or determined DNA sequence encoding the polypeptide Design and synthesize based on information.
  • Such an antisense polynucleotide can hybridize with RNA of a gene encoding the polypeptide of the present invention, and can inhibit the synthesis or function of the RNA.
  • the above-described antisense polynucleotide is useful for regulating / controlling the expression of a gene encoding the polypeptide of the present invention in vivo and in vitro, and also useful for treatment or diagnosis of diseases.
  • corresponding means homologous to or complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes.
  • Corresponding between a nucleotide, base sequence or nucleic acid and polypeptide usually refers to the amino acid of the polypeptide (instruction) derived from the nucleotide (nucleic acid) sequence or its complement.
  • 5 'end hairpin loop of gene encoding polypeptide 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation initiation codon, polypeptide coding region, ORF translation stop codon, 3' end non A translation region, 3′-end palindromic region, 3′-end hairpin loop, or the like can be selected as a preferred target region, but any region in a gene encoding a polypeptide can be selected as a target.
  • the target nucleic acid and a polynucleotide complementary to at least a part of the target region if the target nucleic acid can hybridize to the target region, the target nucleic acid It can be said that it is “antisense”.
  • Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, non-polynucleotides, Other polymers with a nucleotide backbone (eg, commercially available polypeptide nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a base as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a base).
  • They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Such as polypeptides (eg, nucleases, nuclease inhibitors, toxins, antibodies, One having a side chain group such as a guanal peptide, poly-L-lysine, etc., sugar (eg, monosaccharide, etc.), one having an intercurrent compound
  • nucleoside may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens or aliphatic groups, or converted to functional groups such as ethers, amines, etc. May have been.
  • the antisense polynucleotide of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA).
  • modified nucleic acids include nucleic acid sulfur derivatives, thiophosphate derivatives, polynucleoside amides and oligonucleoside amides that are resistant to degradation.
  • the antisense polynucleotide of the present invention can be designed, for example, as follows. That is, to make the antisense polynucleotide in the cell more stable, to increase the cell permeability of the antisense polynucleotide, to increase the affinity for the target sense strand, and to reduce the toxicity In some cases, the antisense polynucleotide is less toxic.
  • the antisense polynucleotide of the present invention may be altered and may contain modified sugars, bases and bonds, and may be provided in special forms such as liposomes, microspheres, or by gene therapy. It can be applied or given in an added form.
  • the additional form can be used as a polycationic substance such as polylysine that works to neutralize the charge of the phosphate group skeleton, a lipid that enhances the interaction with the cell membrane or increases the uptake of nucleic acid ( Examples include hydrophobic ones such as phospholipid and cholesterol.
  • Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 ′ end or 5 ′ end of the nucleic acid, and can be attached via a base, sugar, intramolecular nucleoside bond.
  • the other group include a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid and for preventing degradation by a nuclease such as exonuclease or RNase.
  • capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • Double-stranded RNA containing a part of the RNA encoding the polypeptide of the present invention is publicly known. In accordance with the method (eg, Nature, 411, 494, 2001), it can be designed and produced based on the sequence of the polynucleotide of the present invention.
  • IMPA2 inhibitors are safe and have low toxicity
  • mammals eg, humans, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats
  • IMPA2-related peripheral diseases e.g., Dogs, monkeys, etc .; humans are preferred
  • IMPA2 inhibitors are useful for the prevention and / or treatment of IMPA2-related peripheral diseases such as obesity. It is also useful for the prevention and / or treatment of IMPA2-related central diseases (eg, neurodegenerative diseases, schizophrenia).
  • the dose of the IMPA2 inhibitor varies depending on its action, target disease, administration subject, administration route, etc., but when an IMPA2 inhibitor is orally administered for the purpose of treating obesity or the like, generally an adult (with a body weight of 60 kg) In this case, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of IMPA2 inhibitor is administered per day.
  • the single dose of the IMPA2 inhibitor varies depending on the administration subject, the target disease, etc.
  • the IMPA2 inhibitor is usually administered in the form of an injection for the purpose of treating obesity or the like.
  • IMPA2 inhibitor When administered (with a body weight of 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of IMPA2 inhibitor is administered by injection per day. Is convenient. In the case of other animals, an amount converted per 60 kg body weight can be administered.
  • the prophylactic / therapeutic agent for IMPA2-related peripheral diseases can be produced by a method known per se as a pharmaceutical composition containing an IMPA2 inhibitor as an active ingredient together with a pharmaceutically acceptable carrier and the like.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like.
  • Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
  • injections for example, injections, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections. Includes dosage forms such as agents.
  • Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the IMPA2 inhibitor in a sterile aqueous or oily liquid usually used for injections.
  • aqueous solution for injection for example, isotonic solutions containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) and the like may be used in combination.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
  • the prepared injection solution is usually filled in a suitable amp
  • the IMPA2 inhibitor is an antisense polynucleotide, siRNA or shRNA
  • a suitable vector such as a retroviral vector, adenoviral vector, adenoviral associated viral vector, etc.
  • a mammal eg, , Human, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc .; human is preferred
  • the antisense polynucleotide or the like can be formulated as it is or with a physiologically recognized carrier such as an auxiliary agent for promoting intake, and can be administered by a gene gun or a catheter such as a hydrogel catheter.
  • the above-mentioned antisense polynucleotides or the like are formulated (injection) alone or together with a carrier such as liposomes, and are administered intravenously, subcutaneously, etc. It may be administered.
  • the present invention also provides a kit used in the screening of the present invention (sometimes abbreviated as the screening kit of the present invention).
  • the screening kit of the present invention contains a polypeptide used in the present invention, a cell having the ability to produce the polypeptide of the present invention, or a buffer as necessary.
  • the screening kit of the present invention may also contain a phosphatase substrate compound of the polypeptide of the present invention.
  • Examples of such compounds include 6-phosphogluconic acid and its related compounds such as myo-inositol-1-phosphate, myo-inositol-4-phosphate, myo-inositol-1,3-diphosphate, glucose-1- Phosphate, ⁇ -glycerophosphate, myo-inositol-1,4-diphosphate, myo-inositol-3-phosphate, 6-phosphoglucono-d-lactone, ribulose 5-phosphate, xylose 5-phosphate, erythrose 4- Examples thereof include, but are not limited to, phosphoric acid, 2,3-diphospho-D-glyceric acid, and glycerol-3-phosphate.
  • the screening kit of the present invention contains 6-phosphogluconic acid.
  • the screening kit of the present invention can contain a reagent used in the above screening method and a test compound as a control.
  • the compound obtained by using the screening method of the present invention or the screening kit of the present invention or a salt thereof is a test compound as described above, for example, a peptide, a polypeptide, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, A compound selected from plant extracts, animal tissue extracts, plasma and the like, or a salt thereof, which inhibits the activity of the polypeptide of the present invention (eg, phosphatase activity, intracellular triglyceride accumulation promoting activity, etc.), A compound or a salt thereof that inhibits gene expression or inhibits expression of the polypeptide of the present invention.
  • a compound or salt thereof that inhibits the activity of the polypeptide of the present invention, a compound or salt thereof that inhibits the expression of the gene encoding the polypeptide of the present invention, a compound or salt thereof that inhibits the expression of the polypeptide of the present invention It has low toxicity and is useful as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases (for example, diseases involving intracellular triglyceride accumulation or diseases involving intracellular mitochondrial activity: especially obesity).
  • IMPA2-related peripheral diseases for example, diseases involving intracellular triglyceride accumulation or diseases involving intracellular mitochondrial activity: especially obesity.
  • the polypeptide of the present invention is involved in intracellular triglyceride accumulation action or intracellular ⁇ -oxidation activity through phosphatase activity, early diagnosis of IMPA2-related peripheral diseases, determination of symptom severity, disease progression Useful as a marker for prediction. Accordingly, a compound or a salt thereof that inhibits the activity of the polypeptide of the present invention, an antibody against the polypeptide of the present invention, an antisense polynucleotide of a gene encoding the polypeptide of the present invention, etc. Can be used to diagnose and predict these diseases.
  • the polypeptide of the present invention is also involved in intracellular mitochondrial activity or mitochondrial mass, it is useful as a marker for early diagnosis of IMPA2-related peripheral diseases, determination of the severity of symptoms, and prediction of disease progression. Therefore, the compounds or salts thereof that inhibit the activity of the polypeptide of the present invention, the antibody of the present invention, the antisense polynucleotide of the present invention, etc. are used as diagnostic agents for the above diseases, for example, to diagnose and predict these diseases. can do.
  • the present invention also relates to a method for measuring obesity or an IMPA2-related peripheral disease characterized by measuring the expression of the polypeptide of the present invention or a gene encoding the same in a cell-containing sample collected from a mammal.
  • a diagnostic method is also provided. For example, when the gene encoding the polypeptide of the present invention is overexpressed, it is expected that the amount of intracellular triglyceride accumulation is high, so that the degree of obesity is high or the patient is suffering from an IMPA2-related peripheral disease Or it can be predicted that there is a high possibility of being affected. Therefore, it can be said that measuring the expression of the polypeptide of the present invention or a gene encoding the same in the cell-containing sample is useful for predicting obesity or diagnosing / predicting IMPA2-related peripheral diseases.
  • the present invention also provides a method for diagnosing an IMPA2-related peripheral disease, which comprises measuring the expression of the polypeptide of the present invention or a gene encoding the same in a cell-containing sample collected from a mammal.
  • a method for diagnosing an IMPA2-related peripheral disease comprises measuring the expression of the polypeptide of the present invention or a gene encoding the same in a cell-containing sample collected from a mammal.
  • intracellular mitochondrial activity is expected to be high, so that the patient is or is likely to suffer from an IMPA2-related peripheral disease. Can be predicted. Therefore, it can be said that measuring the expression of the polypeptide of the present invention or a gene encoding the same in the cell-containing sample is useful for the diagnosis / prediction of IMPA2-related peripheral diseases.
  • a sample for example, blood, cells, tissue, etc. collected from a patient
  • the expression level of the polypeptide of the present invention is measured (quantified).
  • the antibody of the present invention can be used for detecting the polypeptide of the present invention present in a subject such as a body fluid or tissue.
  • the antibody of the present invention can be prepared by preparing an antibody column used for purifying the polypeptide of the present invention, detecting the polypeptide of the present invention in each fraction during purification, and detecting the polypeptide of the present invention in a test cell. It can be used for analysis of peptide behavior.
  • the DNA of the present invention can be used as, for example, a mammal, such as a mammal (eg, human, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey, Since abnormality (gene abnormality) of DNA or mRNA encoding the polypeptide of the present invention or a partial peptide thereof (genetic abnormality) in chimpanzees and the like (preferably human) can be detected, for example, damage, mutation or expression of the DNA or mRNA It is useful as a genetic diagnostic agent for reduction, increase or overexpression of the DNA or mRNA. Such a gene diagnostic agent is included in the present invention.
  • a mammal eg, human, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey
  • the above gene diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879, 1989, Proceedings of the National Academy of Sciences of the United States). States of America, 86, 2766-2770, 1989). For example, if an overexpression is detected by Northern hybridization, or if a DNA mutation is detected by the PCR-SSCP method, the subject is affected by the IMPA2-related peripherality or affected by these diseases. Can be diagnosed as being at high risk.
  • bases, amino acids and the like are indicated by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below.
  • optical isomers with respect to amino acids L form is shown unless otherwise specified.
  • DNA deoxyribonucleic acid
  • cDNA complementary deoxyribonucleic acid
  • A adenine T: thymine
  • G guanine
  • C cytosine RNA: ribonucleic acid
  • mRNA messenger ribonucleic acid
  • dATP deoxyadenosine triphosphate
  • dTTP deoxythymidine triphosphate
  • dGTP deoxyguanosine tri
  • Phosphate dCTP deoxycytidine triphosphate ATP: adenosine triphosphate
  • EDTA ethylenediaminetetraacetic acid
  • SDS sodium dodecyl sulfate
  • Gly glycine Ala: alanine Val: valine Leu: leucine
  • Ile isoleucine
  • Ser threonine
  • CyrM threonine
  • Methionine Glu Glutamic acid
  • Asp Aspa Gin acid
  • sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • Example 1 Increase in IMPA2 expression accompanying differentiation in C3H10T1 / 2 differentiated adipocytes
  • Mouse C3H10T1 / 2 cells were cultured in D-MEM medium containing 10% CS. Differentiation into adipocytes was induced by the following method.
  • C3H10T1 / 2 cells were seeded in a 96-well Collagen type 1 coated plate (IWAKI) at 10,000 cells / well and cultured at 37 ° C. in a 0.5% CO 2 atmosphere. After overnight culture, the cells were replaced with 10% FBS-containing D-MEM medium containing 10 ⁇ M pioglitazone to induce differentiation into adipocytes.
  • IWAKI Collagen type 1 coated plate
  • RNA amount of IMPA2 was quantified from the prepared cDNA sample by TaqMan PCR according to the attached protocol.
  • Example 2 Inhibition of adipocyte triglyceride (TG) accumulation by suppression of IMPA2 expression using siRNA siRNA against IMPA2 was applied to C3H10T1 / 2 differentiated adipocytes induced by the same method as in Example 1 by the following method. Introduced. Using the transfection reagent HiperFect (Qiagen), siRNAs of SEQ ID NOs: 16 and 17, or SEQ ID NOs: 18 and 19 were introduced into differentiated adipocytes on the second day after induction of fat differentiation according to the attached protocol. On the fourth day after siRNA introduction, intracellular triglyceride accumulation was quantified using the following method.
  • TG adipocyte triglyceride
  • the cells were treated with 2.5% glutaraldehyde for 15 minutes and then treated with AdipoRed solution (CAMBREX) diluted 100-fold with PBS for 20 minutes to stain intracellular triglycerides. Stained triglycerides were detected with EnVision (485 nm / 535 nm, PerkinElmer). Furthermore, using the cells into which siRNA was introduced under the same conditions, the amount of mRNA of IMPA2 was quantified in the same manner as in Example 1, and the gene expression suppression effect by siRNA was evaluated. As a result, it was clarified that the suppression of IMPA2 gene expression using siRNA suppresses triglyceride accumulation in adipocytes (FIG. 2).
  • Example 3 Large-scale preparation of human IMPA2 and human IMPA1 protein (polypeptide)
  • the human IMPA2 gene expression vector pcDNA3.1F-hIMPA2 was prepared as follows. First, a pcDNA3.1FF vector in which a FLAG sequence was introduced into pcDNA3.1 (+) (Invitrogen) was constructed using a synthetic DNA having a FLAG sequence.
  • FLAGNHENOT-F 5'-CTAGCGCCACCATGGACTACAAGGACGACGATGACAAGGGATCCGACTACAAGGACGACGATGACAAGTGAGC-3 '(SEQ ID NO: 26) and FLAGNHENOT-R 5'-GGCCGCTCACTTGTCATCGTCGTCCTTGTAGTCTCGATCTGTTTGCATCGTCTC Slow cooling and annealing.
  • a vector pcDNA3.1F-hIMPA2 for expressing a protein (polypeptide) in which FLAG was fused to the N-terminal side of human IMPA2 was constructed by the following method. That is, the IMPA2 fragment was amplified by PCR using a primer with the BAMHI at the 5 'end of IMPA2 and the XhoI recognition sequence added at the 3' end from the entry vector pENTR TM -IMPA2 (Invitrogen) purchased from Invitrogen. Using the / XhoI restriction site, it was inserted into pcDNA3.1FF, and the resulting plasmid was transformed into DH5 ⁇ strain (Invitrogen).
  • Plasmid DNA was extracted from the obtained clone with QIAprep spin Miniprep kit (50) (QIAGEN), and sequence confirmation (ABI PRISM 3130xl Genetic Analyzer, ABI) was performed.
  • a vector pcDNA3.1F-hIMPA1 for expressing a protein (polypeptide) in which FLAG was fused to the N-terminal side of human IMPA1 was constructed by the following method.
  • Total RNA was extracted from HEK293 cells using RNeasy mini kit (QIAGEN), and the extracted total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (ABI PRISM) to obtain cDNA.
  • the IMPA1 fragment was amplified by PCR using primers with BglII at the 5 'end and XhoI recognition sequence added at the 3' end, and the amplified fragment was converted into pcDNA3.1FF using BamHI / XhoI restriction sites.
  • the DH5 ⁇ strain (Invitrogen) was transformed with the resulting plasmid. Positive clones were selected by colony PCR using T7 forward primer, BGH reverse primer, Insert check Ready (TOYOBO), and plasmid DNA was extracted with QIAprep spin Miniprep kit (50) (QIAGEN) for sequence confirmation.
  • QIAprep spin Miniprep kit 50
  • QIAGEN QIAGEN
  • pcDNA3.1F-hIMPA2 and pcDNA3.1F-hIMPA1 were introduced into FreeStyle293 cells, respectively, and human IMPA2 protein (polypeptide) and human IMPA1 protein (polypeptide) were prepared in large quantities by affinity purification using anti-FLAG antibody. That is, FreeStyle293 was seeded in 90 mL of FreeStyle293 expression medium (Invitrogen) at a cell concentration of 0.6 ⁇ 10 6 cells / mL and cultured overnight. On the next day, the number of cells was counted, and the cells were diluted with a medium so as to be 1.1 ⁇ 10 6 cells / mL.
  • FreeStyle293 FreeStyle293 expression medium (Invitrogen) at a cell concentration of 0.6 ⁇ 10 6 cells / mL and cultured overnight.
  • the number of cells was counted, and the cells were diluted with a medium so as to be 1.1 ⁇ 10 6 cells / mL.
  • pcDNA3.1F-hIMPA2 or pcDNA3.1F-hIMPA190 ⁇ g was added to a 50 mL tube (Falcon), and 3 ml of Opti-MEM (Invitrogen) was added thereto and mixed.
  • Opti-MEM Invitrogen
  • 120 ⁇ l of 293fectin and 3 mL of Opti-MEM were added and mixed.
  • the DNA diluted solution and the 293fectin diluted solution were mixed and reacted at room temperature for 20 minutes. After 20 minutes, the DNA-293fectin reaction solution was added to the culture solution, and then the cells were cultured for 72 hours. After 72 hours, cells were collected and stored at -80 ° C.
  • Lysis buffer (20 mM TrisHCl (pH 7.5), 0.25 M sucrose, 1.2 mM EGTA, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM NaF, 1% Triton X-100 , 0.5% Nonidet P-40, 150 mM NaCl).
  • Example 4 Phosphatase activity of human IMPA2
  • the standard measurement system was 50 mM Tris-HCl (pH 8.0), 0.1 mM EGTA, 1 mM substrate (compound described in Table 1 below), IMPA1 obtained in the above Example 3 or IMPA2 70 ng (40 ⁇ l) was used at 37 ° C. for 24 hours.
  • To 20 ⁇ l of each reaction solution add 30 ⁇ L of 50 mM Tris-HCl (pH 8.0) 0.1 mM EGTA buffer and 100 ⁇ L of Biomol Green reagent (BIOMOL Research Lab., Plymouth Meeting, PA), and measure the absorbance at 600 nm
  • the relative activity of IMPA2 to IMPA1 is shown in Table 1.
  • 6-phosphogluconic acid was identified as a substrate compound having a different reactivity from IMPA1 and having a high relative activity to IMPA2.
  • This 6-phosphogluconic acid is a novel substrate for IMPA2 and can be a substrate although it is weak against IMPA1, but it clearly has a high substrate specificity for IMPA2. Therefore, this 6-phosphogluconic acid related compound is very likely to be an IMPA2-specific substrate.
  • Example 5 IMPA2 mRNA expression in mouse muscle tissue From normal (m + / m +) mice (BKS.Cg-m + / m + / Jcl, 9 weeks old, male, 5 mice), the soleus and gastrocnemius red The dark part and the thin part were collected separately. For each tissue, total RNA was prepared with Isogen (Nippon Gene) and RNeasy mini (Qiagen) according to the manual. From 1.35 ⁇ g of this total RNA, cDNA was synthesized according to the manual using High Capacity cDNA Reverse Transcription Kit (ABI). Sequence Detection System Prism 7900 (ABI) was used to measure the expression level of mouse IMPA2 mRNA.
  • Isogen Nippon Gene
  • RNeasy mini Qiagen
  • RNA expression level of IMPA2 in the obtained various mouse tissues was calculated as the copy number per 10 ng of total RNA (FIG. 3). Since the red part of the soleus or gastrocnemius muscle is known to have a large amount of mitochondria and high mitochondrial activity, the results shown in Fig. 3 indicate that IMPA2 is strongly involved in mitochondrial function or activity. Strongly suggest.
  • Example 6 IMPA2 mRNA expression in rat adipose tissue Brown adipose tissue, subcutaneous fat, mesenteric fat and epididymal fat were separately collected from Wistar rats (9 weeks old, male). For each tissue, total RNA was prepared by ISOGEN (Nippon Gene) according to the manual. CDNA was synthesized from 750 ng of this total RNA using the High Capacity cDNA Reverse Transcription kit according to the manual. For the expression level of rat IMPA2 mRNA, Sequence Detection System Prism 7900 (ABI) was used.
  • 5'-GTGGCATTGTGATTGACACCTC-3 '(SEQ ID NO: 23), 5'-GAGCTATGAGCACTGCCATCTC-3' (SEQ ID NO: 24) as primers for amplification and detection of rat IMPA2, and 5 '-(FAM)-as TaqMan probe AGCCACCACTCTGCACGACATGAGGT- (TAMRA) -3 ′ (SEQ ID NO: 25) was used.
  • Express qPCR Supermix Universal (Invitrogen) 5.0 ⁇ l, 0.1 ⁇ l of 100 ⁇ M primer solution, 1 ⁇ l of 5 ⁇ M TaqMan probe, and 2 ⁇ l of the cDNA solution prepared above were added, and the total reaction volume was adjusted to 10 ⁇ l with distilled water.
  • RNA expression level of IMPA2 in the obtained rat tissues was calculated as the copy number per 10 ng of total RNA (FIG. 4). Since brown fat is known to have a large amount of mitochondria and high mitochondrial activity, the results shown in FIG. 4 strongly suggest that IMPA2 is strongly involved in mitochondrial function or activity.
  • Example 7 Increase in fatty acid ⁇ oxidation activity by suppression of IMPA2 expression using siRNA siRNA against IMPA2 was introduced into C3H10T1 / 2 cells cultured in D-MEM medium containing 10% CS in the same manner as in Example 2. The same siRNA as in Example 2 was used. 5 days after introduction, reaction medium (5 nCi / ml [ 3 H] -oleic acid (GE Healthcare, TRK140), 0.1 mM oleic acid (Wako), 1.1% ethanol, 0.12% BSA (Sigma) -containing DMEM (Invitrogen) ) And cultured for 6 hours.
  • reaction medium 5 nCi / ml [ 3 H] -oleic acid (GE Healthcare, TRK140), 0.1 mM oleic acid (Wako), 1.1% ethanol, 0.12% BSA (Sigma) -containing DMEM (Invitrogen)
  • reaction medium 5 nCi / ml [ 3 H] -ole
  • the culture supernatant was passed through a MultiScreen DV 0.65 mm plate (Millipore) filled with Norit (Wako), and the radioactivity of the water-soluble fraction containing tritium water produced by fatty acid oxidation was measured with TopCount (PerkinElmer) (FIG. 5). , P ⁇ 0.05).
  • suppression of intracellular IMPA2 expression using siRNA1 and siRNA2 of IMPA2 resulted in an increase in intracellular fatty acid ⁇ -oxidation activity. This reduces the amount of intracellular triglycerides, suggesting that IMPA2 inhibitors are useful as prophylactic / therapeutic agents for IMPA2-related peripheral diseases, particularly diseases related to triglyceride accumulation such as obesity.
  • Example 8 Large-scale preparation of human IMPA2 and human IMPA1 protein (polypeptide) pcDNA3.1F-hIMPA2 and pcDNA3.1F-hIMPA1 were introduced into FreeStyle293 cells, respectively, and human IMPA2 protein ( Polypeptide) and human IMPA1 protein (polypeptide) were prepared in large quantities. That is, FreeStyle293 was seeded in 90 mL of FreeStyle293 expression medium (Invitrogen) at a cell concentration of 0.6 ⁇ 10 6 cells / mL and cultured overnight. The next day, the number of cells was counted, and the cells were diluted with a medium so as to be 1.1 ⁇ 10 6 cells / mL.
  • FreeStyle293 FreeStyle293 expression medium (Invitrogen) at a cell concentration of 0.6 ⁇ 10 6 cells / mL and cultured overnight. The next day, the number of cells was counted, and the cells were diluted with a medium so as to be 1.1 ⁇
  • pcDNA3.1F-hIMPA2 90 ⁇ g of pcDNA3.1F-hIMPA2 (or pcDNA3.1F-hIMPA1) was added to a 50 mL tube (Falcon), and 3 ml of Opti-MEM (Invitrogen) was added thereto and mixed. Then, 120 ⁇ l of 293fectin and 3 mL of Opti-MEM were added to another 50 mL falcon tube and mixed. The obtained DNA diluted solution and 293fectin diluted solution were mixed and further reacted at room temperature for 20 minutes. The obtained DNA-293fectin reaction solution was added to the culture solution, the cells were cultured for 72 hours, and the cells were collected and stored at ⁇ 80 ° C.
  • the absorbance at a wavelength of 280 nm for the flow-through was measured, and washing was performed four times until the absorbance was 0.05 or less. Thereafter, the target FLAG fusion protein (polypeptide) bound to the resin was competitively eluted using an Elution buffer (TBS, 10% Glycerol, 1 mM DTT, 200 ng / ⁇ L 3 ⁇ FLAG peptide (Sigma)).
  • Elution buffer TBS, 10% Glycerol, 1 mM DTT, 200 ng / ⁇ L 3 ⁇ FLAG peptide (Sigma)
  • the obtained human IMPA2 protein (polypeptide) and human IMPA1 protein (polypeptide) were prepared with Elution buffer to a protein concentration of 2 mg / ml and stored at -80 ° C.
  • Example 9 Human IMPA2 phosphatase activity measurement system used for screening and selectivity test 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 2 mM MgCl 2 , 0.01% BSA, 1 mM DTT, 0.01% CHAPS, 1 Reaction was performed at room temperature for 50 minutes using mM 6-phosphogluconic acid, 27 ng (9 ⁇ l) of IMPA2 obtained in Example 8 above, and 3 ⁇ M (or each concentration of 0.3-100 ⁇ M selectivity test). went.
  • a prophylactic / therapeutic agent for IMPA2-related peripheral diseases (particularly obesity) can be efficiently searched.
  • the preventive / therapeutic agent for IMPA2-related peripheral diseases comprising an IMPA2 inhibitor is useful as a preventive / therapeutic agent for the above-mentioned IMPA2-related peripheral diseases (particularly obesity), and has low toxicity and safety. It is.

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Abstract

The purpose of the present invention is to provide a novel therapeutic agent for IMPA2-related peripheral diseases including obesity and diabetes and a method for screening for such a therapeutic agent. A compound of the present invention, which inhibits the activity of a polypeptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11, or a salt and the like of the compound can be used as a prophylactic or therapeutic agent for IMPA2-related peripheral diseases such as diseases associated with intracellular accumulation of triglycerides (for example, obesity, diabetes, hyperlipemia, arteriosclerosis, chronic nephropathy, diabetic nephropathy, fatty liver, hypertension, ischemic heart disease and cancer) and diseases associated with intracellular mitochondrial activity (for example, obesity, retinal degenerative disease, diabetic neuropathy, diabetic retinopathy, renal glomerular sclerosis, nephropathy, diabetic nephropathy, chronic nephropathy, insulin resistance disease, hypertension, arteriosclerosis, diabetes, oxidative stress, ischemic heart disease, aging, muscle disorder, nerve disorder, hearing loss, blindness, fatty liver, steatohepatitis, viral hepatitis, biliary liver cirrhosis, ischemia reperfusion injury, graft rejection, hemolytic anemia and cancer).

Description

スクリーニング方法Screening method

　本発明は、ＩＭＰＡ２を用いるＩＭＰＡ２関連末梢性疾患の予防・治療剤のスクリーニング方法等に関する。 The present invention relates to a screening method for a prophylactic / therapeutic agent for IMPA2-related peripheral diseases using IMPA2.

発明の背景Background of the Invention

　ＩＭＰＡ２（（ミオ）イノシトール－１（または－４）モノホスファターゼ２）は、イノシトールの代謝に関係することから、ＩＭＰＡ２は鬱病等の中枢性疾患に関与すると考えられている（非特許文献１：Neuropsycho-pharmacology 32, 881-891, 2007、非特許文献２：JOURNAL OF BIOLOGICAL CHEMISTRY Vol.282, No.1, 637-647、非特許文献３：Neurosci Res. 2010 May ;67(1):86-94）。国際公開第2004/101779号（特許文献１）には、中枢性疾患を対象とするＩＭＰＡ２阻害薬のスクリーニング法が記載されている。この公報には、放射性ラベルされたイノシトールを用いて、ＩＭＰＡ（イノシトールモノホスファターゼ１および／またはイノシトールモノホスファターゼ２）の活性の測定系が記載されている。 Since IMPA2 ((myo) inositol-1 (or -4) monophosphatase 2) is involved in the metabolism of inositol, it is considered that IMPA2 is involved in central diseases such as depression (Non-patent document 1: Neuropsycho). -pharmacology 32, 881-891, 2007, Non-patent literature 2: JOURNAL OF BIOLOGICAL CHEMISTRY Vol.282, No.1, 637-647, Non-patent literature 3: Neurosci Res. 2010 May; 67 (1): 86-94 ). International Publication No. 2004/101779 (Patent Document 1) describes a screening method for IMPA2 inhibitors targeting central diseases. This publication describes a measurement system for the activity of IMPA (inositol monophosphatase 1 and / or inositol monophosphatase 2) using radiolabeled inositol.

　また、国際公開第2006/079792号（特許文献２）は、ＩＭＰＡ阻害活性に基づくオートファジーを誘導するような薬剤のスクリーニング法に関する。この公報には、LiClおよびL690330を用いてＩＭＰＡを阻害することによりオートファジーが誘導されることが記載されている。
　しかしながら、上記いずれの文献にも、ＩＭＰＡ２が末梢性疾患（例えば、肥満症、糖尿病、高脂血症、動脈硬化、慢性腎炎、糖尿病性腎症、虚血性心疾患等）に関係するといった報告は一切されていない。 International Publication No. 2006/079792 (Patent Document 2) relates to a screening method for drugs that induce autophagy based on IMPA inhibitory activity. This publication describes that autophagy is induced by inhibiting IMPA using LiCl and L690330.
However, in any of the above documents, there is a report that IMPA2 is related to peripheral diseases (for example, obesity, diabetes, hyperlipidemia, arteriosclerosis, chronic nephritis, diabetic nephropathy, ischemic heart disease, etc.) Not at all.

国際公開第2004/101779号パンフレットInternational Publication No. 2004/101779 Pamphlet 国際公開第2006/079792号パンフレットInternational Publication No. 2006/079792 Pamphlet

Neuropsycho-pharmacology 32, 881-891, 2007Neuropsycho-pharmacology 32, 881-891, 2007 JOURNAL OF BIOLOGICAL CHEMISTRY Vol.282, No.1, 637-647JOURNAL OF BIOLOGICAL CHEMISTRY Vol.282, No.1, 637-647 Neurosci Res. 2010 May; 67(1):86-94Neurosci Res. 2010 May; 67 (1): 86-94

　肥満症や糖尿病等を含むＩＭＰＡ２関連末梢性疾患の新たな治療薬やそのような治療薬を探索する方法を開発することが望まれている。 It is desired to develop new therapeutic agents for IMPA2-related peripheral diseases including obesity and diabetes and a method for searching for such therapeutic agents.

　本発明者らは、siRNAライブラリーを用いて脂肪細胞におけるトリグリセリド（TG）蓄積に関与する遺伝子を探索した結果、ＩＭＰＡ２が細胞内トリグリセリドの蓄積に関与する候補遺伝子であること、およびＩＭＰＡ２の発現抑制が細胞内の脂肪酸β酸化活性の上昇に繋がることを見出した。（この脂肪酸β酸化促進作用は細胞内のトリグリセリド蓄積量の抑制と関係することから、ＩＭＰＡ２阻害が抗肥満に繋がることが直接的に示された。）
　また、本発明者らは、ＩＭＰＡ２の発現量が細胞内のミトコンドリア代謝活性の調節に関与することを見出した。
　さらに、本発明者らは、ＩＭＰＡ２に特異性が高い基質として６－ホスホグルコン酸を見出した。本発明者らは、これらの知見に基づき、さらに研究をすすめた結果、本発明を完成するに至った。 As a result of searching for a gene involved in triglyceride (TG) accumulation in adipocytes using the siRNA library, the present inventors have found that IMPA2 is a candidate gene involved in intracellular triglyceride accumulation and that expression of IMPA2 is suppressed. Was found to lead to an increase in intracellular fatty acid β-oxidation activity. (Since this fatty acid β-oxidation promoting action is related to the suppression of intracellular triglyceride accumulation, it was directly shown that IMPA2 inhibition leads to anti-obesity.)
The present inventors have also found that the expression level of IMPA2 is involved in the regulation of intracellular mitochondrial metabolic activity.
Furthermore, the present inventors have found 6-phosphogluconic acid as a substrate having high specificity for IMPA2. As a result of further studies based on these findings, the present inventors have completed the present invention.

　すなわち、本発明は、以下を提供する。
〔１〕　配列番号：１（ヒトＩＭＰＡ２）、３（マウスＩＭＰＡ２）、５（ラットＩＭＰＡ２）、７（ウシＩＭＰＡ２）、９（イヌＩＭＰＡ２）または１１（サルＩＭＰＡ２）で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むポリペプチド（本明細書中、本発明のポリペプチドと略記する場合がある）もしくはその部分ペプチドまたはその塩、あるいは該ポリペプチドまたはその部分ペプチドを産生する細胞を用いることを特徴とする、ＩＭＰＡ２関連末梢性疾患の予防・治療剤のスクリーニング方法。
〔２〕　ＩＭＰＡ２関連末梢性疾患が肥満症である、〔１〕記載の方法。
〔３〕　さらに６－ホスホグルコン酸またはその類縁化合物を用いる、〔１〕または〔２〕に記載の方法。
〔４〕　配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むポリペプチドをコードするポリヌクレオチドまたはその部分ポリヌクレオチド（本明細書中、本発明のポリヌクレオチドと略記する場合がある）、あるいは該ポリペプチドまたはその部分ペプチドに対する抗体（本明細書中、本発明の抗体と略記する場合がある）をさらに用いることを特徴とする、〔１〕または〔２〕に記載の方法。
〔５〕　配列番号：４、６、８、１０または１２で示される塩基配列と同一または実質的に同一な塩基配列を含むポリヌクレオチドが不活性化された、該ポリヌクレオチド発現不全非ヒト哺乳動物を用いることを特徴とする、脂肪分化促進剤のスクリーニング方法。
〔６〕　外来性の、配列番号：２、４、６、８、１０または１２で示される塩基配列と同一または実質的に同一な塩基配列を含むポリヌクレオチドを有する非ヒトトランスジェニック動物を用いることを特徴とする、ＩＭＰＡ２関連末梢性疾患の予防・治療剤のスクリーニング方法。
〔７〕　ＩＭＰＡ２関連末梢性疾患の予防・治療剤が、配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含有するポリペプチドの活性を促進または阻害する化合物またはその塩である、〔１〕～〔４〕および〔６〕のいずれか１項記載の方法。
〔８〕　配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むポリペプチドもしくはその部分ペプチドまたはその塩、あるいは該ポリペプチドまたはその部分ペプチドを産生する細胞、および６－ホスホグルコン酸またはその類縁化合物を用いることを特徴とする、ＩＭＰＡ２阻害薬のスクリーニング方法。
〔９〕　ＩＭＰＡ２阻害薬を含有してなるＩＭＰＡ２関連末梢性疾患の予防・治療剤。
〔１０〕　ＩＭＰＡ２関連末梢性疾患が肥満症である、〔９〕記載の剤。
〔１１〕　ＩＭＰＡ２阻害薬がsiRNAである、〔９〕または〔１０〕記載の剤。
〔１２〕　哺乳動物に対して、ＩＭＰＡ２阻害薬の有効量を投与することを特徴とするＩＭＰＡ２関連末梢性疾患の予防・治療方法。
〔１３〕　ＩＭＰＡ２関連末梢性疾患の予防・治療のための、ＩＭＰＡ２阻害薬。 That is, the present invention provides the following.
[1] The same or substantially the same amino acid sequence as shown in SEQ ID NO: 1 (human IMPA2), 3 (mouse IMPA2), 5 (rat IMPA2), 7 (bovine IMPA2), 9 (dog IMPA2) or 11 (monkey IMPA2) Using a polypeptide containing the same amino acid sequence (sometimes abbreviated as the polypeptide of the present invention in the present specification) or a partial peptide or salt thereof, or a cell producing the polypeptide or the partial peptide A screening method for a prophylactic / therapeutic agent for an IMPA2-related peripheral disease.
[2] The method according to [1], wherein the IMPA2-related peripheral disease is obesity.
[3] The method according to [1] or [2], further using 6-phosphogluconic acid or an analogous compound thereof.
[4] A polynucleotide encoding a polypeptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11 or a partial polynucleotide thereof (herein And may be abbreviated as the polynucleotide of the present invention), or an antibody against the polypeptide or a partial peptide thereof (may be abbreviated as the antibody of the present invention in the present specification). [1] or [2].
[5] Non-human mammal deficient in the expression of a polynucleotide, wherein a polynucleotide comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12 is inactivated A method for screening for an agent for promoting fat differentiation, characterized in that
[6] Use of an exogenous non-human transgenic animal having a polynucleotide comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 A screening method for a prophylactic / therapeutic agent for IMPA2-related peripheral diseases.
[7] A polypeptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 The method according to any one of [1] to [4] and [6], which is a compound or a salt thereof that promotes or inhibits activity.
[8] A polypeptide comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, or a partial peptide thereof, or a salt thereof, or the polypeptide or a thereof A method for screening an IMPA2 inhibitor, comprising using a cell that produces a partial peptide, and 6-phosphogluconic acid or a similar compound thereof.
[9] A prophylactic / therapeutic agent for IMPA2-related peripheral diseases, comprising an IMPA2 inhibitor.
[10] The agent according to [9], wherein the IMPA2-related peripheral disease is obesity.
[11] The agent according to [9] or [10], wherein the IMPA2 inhibitor is siRNA.
[12] A method for preventing and treating an IMPA2-related peripheral disease, comprising administering an effective amount of an IMPA2 inhibitor to a mammal.
[13] An IMPA2 inhibitor for prevention / treatment of IMPA2-related peripheral diseases.

　上記〔１〕～〔７〕のスクリーニング方法によれば、ＩＭＰＡ２関連末梢性疾患（特に肥満症）の予防・治療剤や、脂肪分化促進剤を効率よく探索することができる。
　上記〔８〕のスクリーニング方法によれば、ＩＭＰＡ２阻害薬を効率よく探索することができる。
　また、ＩＭＰＡ２阻害薬を含有してなる、上記〔９〕～〔１１〕の予防・治療剤は、ＩＭＰＡ２関連末梢性疾患（特に肥満症）の予防・治療剤として有用である。 According to the screening methods [1] to [7] above, it is possible to efficiently search for preventive / therapeutic agents for IMPA2-related peripheral diseases (particularly obesity) and fat differentiation promoting agents.
According to the screening method of [8] above, it is possible to efficiently search for an IMPA2 inhibitor.
In addition, the prophylactic / therapeutic agent according to [9] to [11] above containing an IMPA2 inhibitor is useful as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases (particularly obesity).

横軸は分化誘導処理からの日数を示し、縦軸は36B4のmRNA量に対する相対的なＩＭＰＡ２のmRNA量を示す。ダイヤ（◆）が分化誘導をしていない細胞、三角（▲）が分化誘導した細胞におけるＩＭＰＡ２のmRNA量（平均値±標準偏差）を示す。Pioは10μM ピオグリタゾン（Pioglitazone）、10％ CS-DMEMは10％ Calf Serum含有D-MEM培地を示す。The horizontal axis indicates the number of days from the differentiation induction treatment, and the vertical axis indicates the relative amount of IMPA2 mRNA relative to the amount of 36B4 mRNA. The diamond (♦) shows the amount of mRNA (average value ± standard deviation) of IMPA2 in the cells in which differentiation has not been induced, and the triangles (▲) in cells in which differentiation has been induced. Pio is 10 μM Pioglitazone (Pioglitazone), and 10% CS-DMEM is D-MEM medium containing 10% Calf Serum. ＩＭＰＡ２に特異的なsiRNAによる細胞内トリグリセリド蓄積抑制効果（図２a）とＩＭＰＡ２のmRNA発現抑制効果（図２b）を示す。横軸は細胞に導入したsiRNAを示し、縦軸は、図２a：蛍光強度（平均値±標準偏差）、図２b：ＩＭＰＡ２のmRNA量（36B4 mRNA量との相対値）を示す。Non-targetはSilencer Negative Control ＃1 siRNA（Ambion、カタログ番号AM4611）を示し、siRNA1は配列番号：16と17とからなるsiRNA、siRNA2は配列番号：18と19とからなるsiRNAを示す。Fig. 2 shows the intracellular triglyceride accumulation inhibitory effect (Fig. 2a) and the mRNA expression inhibitory effect (Fig. 2b) of IMPA2 by siRNA specific to IMPA2. The horizontal axis represents siRNA introduced into the cells, and the vertical axis represents FIG. 2a: fluorescence intensity (mean value ± standard deviation), FIG. 2b: mRNA amount of IMPA2 (relative to 36B4 mRNA amount). Non-target is Silencer Negative Control # 1 siRNA (Ambion, catalog number AM4611), siRNA1 is siRNA consisting of SEQ ID NOs: 16 and 17, and siRNA2 is siRNA consisting of SEQ ID NOs: 18 and 19. 正常（ｍ＋／ｍ＋）マウスのヒラメ筋、腓腹筋におけるマウスIMPA2 mRNA発現量を示す。ミトコンドリア含量が高いヒラメ筋と、腓腹筋のうち、赤色が濃くミトコンドリア含量の多いとされる部分と、赤色の薄い部分の３箇所について発現量（n=2-4の平均値＋標準偏差）を示す。The mouse IMPA2 mRNA expression level in the soleus and gastrocnemius muscles of normal (m + / m +) mice is shown. The expression level (average value of n = 2-4 + standard deviation) is shown in three parts of the soleus muscle with high mitochondrial content, the gastrocnemius muscle, the portion that is red and rich in mitochondrial content, and the light red portion . Wistarラットの各脂肪組織におけるラットIMPA2 mRNAの発現量（褐色脂肪についてはn=2、それ以外の組織についてはn=４の平均値＋標準偏差）を示す。The expression level of rat IMPA2 mRNA in each adipose tissue of Wistar rat (n = 2 for brown fat and n = 4 for other tissues + standard deviation) is shown. ＩＭＰＡ２に特異的なsiRNAによる細胞内脂肪酸β酸化活性の促進効果を示す。横軸は細胞に導入したsiRNAを示し、縦軸は放射活性（cpm, n=3の平均値±標準偏差）を示す（p<0.05）。Non-targetはSilencer Negative Control ＃1 siRNA（Ambion、カタログ番号AM4611）を示し、siRNA1は配列番号：16と17とからなるsiRNA、siRNA2は配列番号：18と19とからなるsiRNAを示す。It shows the effect of promoting intracellular fatty acid β oxidation activity by siRNA specific to IMPA2. The horizontal axis represents siRNA introduced into cells, and the vertical axis represents radioactivity (cpm, mean value of n = 3 ± standard deviation) (p <0.05). Non-target is Silencer Negative Control # 1 siRNA (Ambion, catalog number AM4611), siRNA1 is siRNA consisting of SEQ ID NOs: 16 and 17, and siRNA2 is siRNA consisting of SEQ ID NOs: 18 and 19.

　本明細書において「ＩＭＰＡ２関連末梢性疾患」として、以下のものが挙げられる。
（１）細胞内トリグリセリド蓄積量が関与する疾患（例えば、肥満症、糖尿病、高脂血症、動脈硬化、慢性腎炎、糖尿病性腎症、脂肪肝、高血圧、虚血性心疾患、癌など）
（２）細胞内ミトコンドリア活性が関与する疾患（例えば、肥満症、網膜変性疾患、糖尿病性神経障害、糖尿病性網膜症、腎糸球体硬化症、腎症、糖尿病性腎症、慢性腎症、インスリン抵抗性疾患、高血圧、動脈硬化、糖尿病、酸化ストレス、虚血性心疾患、老化、筋障害、神経障害、難聴、失明、脂肪肝、脂肪性肝炎、ウイルス性肝炎、胆汁性肝硬変、虚血再灌流障害、移植片拒絶、溶血性貧血、癌など）
　なお、ミトコンドリアの活性と疾患との関係については、例えば、Exp Mol Path83(2007) 〔例、Experimental and  Molecular Pathology 83 (2007):84-92〕、Diabetes Care〔例、Diabetes Care 31 (Suppl. 2):S170-S180, 2008〕、Curr.Opin.Pharm〔例、Current Opinion in Pharmacology 2009; (9): 780-786〕、Curr Med Chem. 2010 May 24. [Epub ahead of print]、Targeting Mitochondria: A New Promising Approach for the Treatment of Liver Diseases（Curr Med Chem. 2010;17(22):2325-37.）を参照することができる。
　ＩＭＰＡ２関連末梢性疾患としては、肥満症が好ましい。本発明において、肥満症は末梢性の肥満症が好ましい。 In the present specification, examples of the “IMPA2-related peripheral disease” include the following.
(1) Diseases involving intracellular triglyceride accumulation (eg obesity, diabetes, hyperlipidemia, arteriosclerosis, chronic nephritis, diabetic nephropathy, fatty liver, hypertension, ischemic heart disease, cancer, etc.)
(2) Diseases involving intracellular mitochondrial activity (eg obesity, retinal degenerative diseases, diabetic neuropathy, diabetic retinopathy, glomerulosclerosis, nephropathy, diabetic nephropathy, chronic nephropathy, insulin Resistant disease, hypertension, arteriosclerosis, diabetes, oxidative stress, ischemic heart disease, aging, myopathy, neuropathy, hearing loss, blindness, fatty liver, steatohepatitis, viral hepatitis, biliary cirrhosis, ischemia reperfusion Disorders, graft rejection, hemolytic anemia, cancer, etc.)
Regarding the relationship between mitochondrial activity and diseases, for example, Exp Mol Path83 (2007) [eg, Experimental and Molecular Pathology 83 (2007): 84-92], Diabetes Care [eg, Diabetes Care 31 (Suppl. 2) ): S170-S180, 2008], Curr.Opin.Pharm (eg, Current Opinion in Pharmacology 2009; (9): 780-786), Curr Med Chem. 2010 May 24. [Epub ahead of print], Targeting Mitochondria: A New Promising Approach for the Treatment of Liver Diseases (Curr Med Chem. 2010; 17 (22): 2325-37.) Can be referred to.
As the IMPA2-related peripheral disease, obesity is preferable. In the present invention, obesity is preferably peripheral obesity.

〔本発明のスクリーニング方法〕
　本発明は、以下のスクリーニング方法を提供する。
〔１〕配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むポリペプチドもしくはその部分ペプチドまたはその塩、あるいは該ポリペプチドまたはその部分ペプチドを産生する細胞を用いることを特徴とする、ＩＭＰＡ２関連末梢性疾患の予防・治療剤のスクリーニング方法。
　以下、本発明のスクリーニング方法において用いられる、本発明のポリペプチドおよびその部分ペプチド、本発明のポリヌクレオチド、本発明の抗体などについて説明する。 [Screening method of the present invention]
The present invention provides the following screening methods.
[1] A polypeptide comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11, or a partial peptide thereof, or a salt thereof, or the polypeptide or a thereof A screening method for a prophylactic / therapeutic agent for IMPA2-related peripheral diseases, characterized by using cells that produce partial peptides.
Hereinafter, the polypeptide of the present invention and its partial peptide, the polynucleotide of the present invention, the antibody of the present invention and the like used in the screening method of the present invention will be described.

　本発明のポリペプチドとしては、配列番号：１、３、５、７、９または１１で示されるアミノ酸配列を含むポリペプチドが好ましく、配列番号：１、３、５、７、９または１１で示されるアミノ酸配列からなるポリペプチド、すなわち、各種哺乳動物（ヒト、マウス、ラット、ウシ、イヌ、サル）のＩＭＰＡ２がより好ましい。なかでもヒトのＩＭＰＡ２（配列番号：１で示されるアミノ酸配列と同一のアミノ酸配列からなるポリペプチド）が好ましい。 The polypeptide of the present invention is preferably a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11, and represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11. More preferred is a polypeptide having an amino acid sequence, that is, IMPA2 of various mammals (human, mouse, rat, cow, dog, monkey). Of these, human IMPA2 (polypeptide having the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1) is preferred.

　配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と実質的に同一のアミノ酸配列としては、配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と約６０％以上、約７０％以上、約８０％以上、約９０％以上、約９５％以上、約９６％以上、約９７％以上、約９８％以上または約９９％以上の相同性または同一性を有するアミノ酸配列などが挙げられる。
　アミノ酸配列の相同性は、相同性計算アルゴリズムNCBI BLAST（National Center for Biotechnology Information Basic Local Alignment Search Tool）を用い、以下の条件（期待値＝10；ギャップを許す；マトリクス＝BLOSUM62；フィルタリング＝OFF）にて計算することができる。 The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 includes the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11. About 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more homology or identity An amino acid sequence having
The homology of the amino acid sequence was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering = OFF) Can be calculated.

　また、配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と実質的に同一のアミノ酸配列として、例えば、（i）配列番号：１、３、５、７、９または１１で示されるアミノ酸配列中の１または２個以上（例えば１～１００個程度、好ましくは１～３０個程度、好ましくは１～１０個程度、さらに好ましくは数（１～６）個）のアミノ酸が欠失したアミノ酸配列、（ii）配列番号：１、３、５、７、９または１１で示されるアミノ酸配列に１または２個以上（例えば１～１００個程度、好ましくは１～３０個程度、好ましくは１～１０個程度、さらに好ましくは数（１～６）個）のアミノ酸が付加したアミノ酸配列、（iii）配列番号：１、３、５、７、９または１１で示されるアミノ酸配列に１または２個以上（例えば１～１００個程度、好ましくは１～３０個程度、好ましくは１～１０個程度、さらに好ましくは数（１～６）個）のアミノ酸が挿入されたアミノ酸配列、（iv）配列番号：１、３、５、７、９または１１で示されるアミノ酸配列中の１または２個以上（例えば１～１００個程度、好ましくは１～３０個程度、好ましくは１～１０個程度、さらに好ましくは数（１～６）個）のアミノ酸が他のアミノ酸で置換されたアミノ酸配列、または（v）前記（i）～（iv）を組み合わせたアミノ酸配列が挙げられる。
　上記のようにアミノ酸配列が挿入、欠失または置換されている場合、その挿入、欠失または置換の位置としては、とくに限定されない。 Moreover, as an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11, for example, (i) SEQ ID NO: 1, 3, 5, 7, 9, or 11 1 or 2 (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably several (1 to 6)) amino acids in the amino acid sequence represented by A deleted amino acid sequence, (ii) one or more (for example, about 1 to 100, preferably about 1 to 30) in the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11; Preferably, about 1 to 10, more preferably several (1 to 6) amino acids are added, (iii) the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 1 or 2 or more (eg 1 to 10) (Iv) SEQ ID NOs: 1, 3, 5 (about 4), preferably about 1-30, preferably about 1-10, and more preferably several (1-6) amino acids. , 7, 9 or 11 (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably a number (1 to 6). )) Amino acids are substituted with other amino acids, or (v) amino acid sequences in which (i) to (iv) above are combined.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.

　配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と実質的に同一のアミノ酸配列を含むポリペプチドとしては、前記した配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と実質的に同一のアミノ酸配列を含有し、配列番号：１、３、５、７、９または１１で示されるアミノ酸配列を含むポリペプチドと実質的に同質の活性を有するポリペプチドなどが好ましい。
　実質的に同質の活性としては、例えば、細胞内トリグリセリド蓄積促進活性（ＴＧ蓄積促進活性）、およびイノシトールモノリン酸、６－ホスホグルコン酸等を基質とするホスファターゼ活性が挙げられる。実質的に同質とは、それらの性質が性質的に（例、生理学的に、または薬理学的に）同質であることを示す。したがって、これらの活性が同等（例、約０．０１～１００倍、好ましくは約０．１～１０倍、より好ましくは０．５～２倍）であることが好ましいが、これらの活性の程度、ポリペプチドの分子量などの量的要素は異なっていてもよい。
　対象物の細胞内トリグリセリド蓄積促進活性は、例えば、その対象物の存在下および非存在下に細胞（例えば、分化脂肪細胞）を培養した際の細胞内トリグリセリド蓄積量を測定し、それらの測定値を比較することによって評価することができる（後述の実施例２に記載の測定法も参照）。本発明のポリペプチドのホスファターゼ活性の測定は、公知の方法、例えば、非特許文献１（Cryns, K.ら、Neuropsycho-pharmacology 32, 881-891, 2007）、特許文献２（国際公開第2006/079792号パンフレット）に記載の方法またはそれに準ずる方法によって測定することができる。 Examples of the polypeptide containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11 include the aforementioned SEQ ID NO: 1, 3, 5, 7, 9, or 11. A polypeptide having substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11 and having substantially the same activity as the polypeptide comprising the amino acid sequence represented by SEQ ID NO: Peptides are preferred.
Examples of substantially the same quality of activity include intracellular triglyceride accumulation promoting activity (TG accumulation promoting activity) and phosphatase activity using inositol monophosphate, 6-phosphogluconic acid and the like as substrates. Substantially homogeneous indicates that their properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, it is preferable that these activities are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). The quantitative factors such as the molecular weight of the polypeptide may be different.
The intracellular triglyceride accumulation-promoting activity of an object is measured, for example, by measuring the amount of intracellular triglyceride accumulated when cells (for example, differentiated adipocytes) are cultured in the presence and absence of the object. Can be evaluated (see also the measurement method described in Example 2 described later). The phosphatase activity of the polypeptide of the present invention can be measured by a known method, for example, Non-Patent Document 1 (Cryns, K. et al., Neuropsycho-pharmacology 32, 881-891, 2007), Patent Document 2 (International Publication No. 2006 / 079792 pamphlet) or a method according thereto.

　また、実質的に同質の活性としては、細胞内ミトコンドリア代謝活性またはミトコンドリア量の調節作用も挙げられる。実質的に同質とは、上記と同様に、それらの性質が性質的に（例、生理学的に、または薬理学的に）同質であることを示す。したがって、これらの活性が同等（例、約０．０１～１００倍、好ましくは約０．１～１０倍、より好ましくは０．５～２倍）であることが好ましいが、これらの活性の程度、ポリペプチドの分子量などの量的要素は異なっていてもよい。
　対象物の細胞内ミトコンドリア代謝活性またはミトコンドリア量の調節作用は、例えば、その対象物の存在下および非存在下において細胞（例えば、分化脂肪細胞）を培養した際の細胞内ミトコンドリア代謝活性、ミトコンドリアＤＮＡ量などを測定し、それらの測定値を比較することによって評価できる。この細胞内ミトコンドリア代謝活性は、公知の方法、例えば、Methods in Cell Biology (Volume 80, Mitochondrai 2ndedition)に記載の方法またはそれに準ずる方法によって測定することができる。後述の実施例５または６に記載の測定法も参照できる。 In addition, the substantially homogeneous activity also includes intracellular mitochondrial metabolic activity or mitochondrial regulation. The term “substantially the same quality” means that, as described above, these properties are qualitatively (for example, physiologically or pharmacologically) homogeneous. Therefore, it is preferable that these activities are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). The quantitative factors such as the molecular weight of the polypeptide may be different.
The intracellular mitochondrial metabolic activity or the amount of mitochondrial activity of an object is, for example, intracellular mitochondrial metabolic activity when cells (eg, differentiated adipocytes) are cultured in the presence and absence of the object, mitochondrial DNA It can be evaluated by measuring the amount and comparing the measured values. The intracellular mitochondrial metabolic activity, a known method, for example, can be measured by the method or methods analogous thereto described in Methods in Cell Biology (Volume 80, Mitochondrai 2 nd edition). The measurement method described in Example 5 or 6 described later can also be referred to.

　本発明において、本発明のポリペプチド、好ましくはＩＭＰＡ２に特異性が高い基質として、６－ホスホグルコン酸が提供される。前記した本発明のポリペプチドの活性、例えば、ホスファターゼ活性を測定する場合、６－ホスホグルコン酸、もしくはその類縁化合物（ミオイノシトール－１－リン酸、ミオイノシトール－４－リン酸、ミオイノシトール－１，３－二リン酸、グルコース－１－リン酸、β－グリセロリン酸、ミオイノシトール－１，４－二リン酸、ミオイノシトール－３－リン酸、グリセロール－３－リン酸、６－ホスホグルコノ－ｄ－ラクトン、リブロース５－リン酸、キシロース５－リン酸、エリトロース４－リン酸、または２，３－ジホスホ－Ｄ－グリセリン酸など）を基質として用いることができる。
　なかでも、６－ホスホグルコン酸を基質として用いることが好ましい。
　本発明のスクリーニング方法において、６－ホスホグルコン酸またはその類縁化合物（好ましくは、６－ホスホグルコン酸）を用いることにより、ＩＭＰＡ２阻害効率が高く、有効なＩＭＰＡ２関連末梢性疾患の予防・治療剤をスクリーニングすることができる。　 In the present invention, 6-phosphogluconic acid is provided as a substrate having high specificity for the polypeptide of the present invention, preferably IMPA2. When the activity of the above-described polypeptide of the present invention, for example, phosphatase activity is measured, 6-phosphogluconic acid or a similar compound (myoinositol-1-phosphate, myoinositol-4-phosphate, myoinositol-1 , 3-diphosphate, glucose-1-phosphate, β-glycerophosphate, myo-inositol-1,4-diphosphate, myo-inositol-3-phosphate, glycerol-3-phosphate, 6-phosphoglucono-d -Lactose, ribulose 5-phosphate, xylose 5-phosphate, erythrose 4-phosphate, or 2,3-diphospho-D-glyceric acid) can be used as a substrate.
Of these, 6-phosphogluconic acid is preferably used as a substrate.
In the screening method of the present invention, by using 6-phosphogluconic acid or a similar compound (preferably 6-phosphogluconic acid), an effective IMPA2-related peripheral disease prophylactic / therapeutic agent with high IMPA2 inhibitory efficiency is obtained. Can be screened.

　本発明のポリペプチドは、ペプチド標記の慣例に従って左端がＮ末端（アミノ末端）、右端がＣ末端（カルボキシル末端）である。配列番号：１、３、５、７、９または１１で示されるアミノ酸配列を含むポリペプチドをはじめとする、本発明のポリペプチドは、Ｃ末端がカルボキシル基（-COOH）、カルボキシレート(-COO-）、アミド（-CONH2）またはエステル（-COOR）の何れであってもよい。
　ここでエステルにおけるＲとしては、例えば、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチルなどのＣ1-6アルキル基、例えば、シクロペンチル、シクロヘキシルなどのＣ3-8シクロアルキル基、例えば、フェニル、α－ナフチルなどのＣ6-12アリール基、例えば、ベンジル、フェネチルなどのフェニル－Ｃ1-2アルキル基もしくはα－ナフチルメチルなどのα－ナフチル－Ｃ1-2アルキル基などのＣ7-14アラルキル基、ピバロイルオキシメチル基などが用いられる。
　本発明のポリペプチドがＣ末端以外にカルボキシル基（またはカルボキシレート）を有している場合、カルボキシル基がアミド化またはエステル化されているものも本発明のポリペプチドに含まれる。この場合のエステルとしては、例えば上記したＣ末端のエステルなどが用いられる。
　さらに、本発明のポリペプチドには、Ｎ末端のアミノ酸残基（例、メチオニン残基）のアミノ基が保護基（例えば、ホルミル基、アセチル基などのＣ1-6アルカノイルなどのＣ1-6アシル基など）で保護されているもの、生体内で切断されて生成するＮ末端のグルタミン残基がピログルタミン酸化したもの、分子内のアミノ酸の側鎖上の置換基（例えば－ＯＨ、－ＳＨ、アミノ基、イミダゾール基、インドール基、グアニジノ基など）が適当な保護基（例えば、ホルミル基、アセチル基などのＣ1-6アルカノイル基などのＣ1-6アシル基など）で保護されているもの、あるいは糖鎖が結合したいわゆる糖タンパク質などの複合タンパク質なども含まれる。 In the polypeptide of the present invention, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide designation. The polypeptide of the present invention, including the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11, has a C-terminus having a carboxyl group (—COOH), a carboxylate (—COO - ), Amide (-CONH 2 ) or ester (-COOR).
Here, as R in the ester, for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc., for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, etc., for example, phenyl C 6-12 aryl groups such as α-naphthyl, C 7- such as phenyl-C 1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C 1-2 alkyl groups such as α-naphthylmethyl 14 aralkyl group, pivaloyloxymethyl group is used.
When the polypeptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the polypeptide of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
Further, the polypeptides of the present invention, amino acid residues (eg, methionine residue) of the N-terminal amino group protecting group (e.g., formyl group, such as C 1-6 alkanoyl such as acetyl group C 1-6 A group protected by an acyl group, an N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (eg, —OH, —SH) Amino group, imidazole group, indole group, guanidino group, etc.) are protected with an appropriate protecting group (for example, C 1-6 acyl group such as C 1-6 alkanoyl group such as formyl group, acetyl group, etc.) Or a complex protein such as a so-called glycoprotein having a sugar chain bound thereto.

　本発明のポリペプチドの部分ペプチド（本明細書中、本発明の部分ペプチドと略記する場合がある）としては、前記した本発明のポリペプチドの部分ペプチドであって、好ましくは、前記した本発明のポリペプチドと同様の性質を有するものである。
　本発明の部分ペプチドは、後述する本発明の抗体を作製するための抗原として用いられる場合には、本発明のポリペプチドの構成アミノ酸配列のうち少なくとも２０個以上、好ましくは５０個以上、さらに好ましくは７０個以上、より好ましくは１００個以上、最も好ましくは２００個以上のアミノ酸配列を含むポリペプチドである。
　また、本発明の部分ペプチドは、対応する本発明のポリペプチドのアミノ酸配列中の１または２個以上（好ましくは１～２０個程度、より好ましくは１～１０個程度、さらに好ましくは数（１～６）個）のアミノ酸が欠失し、または、そのアミノ酸配列に１または２個以上（好ましくは１～２０個程度、より好ましくは１～１０個程度、さらに好ましくは数（１～６）個）のアミノ酸が付加し、または、そのアミノ酸配列に１または２個以上（好ましくは１～２０個程度、より好ましくは１～１０個程度、さらに好ましくは数（１～６）個）のアミノ酸が挿入され、または、そのアミノ酸配列中の１または２個以上（好ましくは１～２０個程度、より好ましくは１～１０個程度、さらに好ましくは数（１～６）個）のアミノ酸が他のアミノ酸で置換されていてもよい。 The partial peptide of the polypeptide of the present invention (sometimes abbreviated as the partial peptide of the present invention in the present specification) is the partial peptide of the polypeptide of the present invention described above, preferably the above-described present invention. It has the same property as the polypeptide.
When the partial peptide of the present invention is used as an antigen for producing the antibody of the present invention described later, at least 20 or more, preferably 50 or more, more preferably among the constituent amino acid sequences of the polypeptide of the present invention. Is a polypeptide comprising an amino acid sequence of 70 or more, more preferably 100 or more, most preferably 200 or more.
In addition, the partial peptide of the present invention is one or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1) in the amino acid sequence of the corresponding polypeptide of the present invention. To 6) amino acids) or one or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1 to 6) in the amino acid sequence. Amino acids) or one or more amino acids in the amino acid sequence (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1 to 6)). Or one or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 6)) amino acids in the amino acid sequence amino acid It may be substituted.

　また、本発明の部分ペプチドはＣ末端がカルボキシル基（-COOH）、カルボキシレート（-COO-）、アミド（-CONH2）またはエステル（-COOR）の何れであってもよい。
　さらに、本発明の部分ペプチドには、前記した本発明のポリペプチドと同様に、Ｃ末端以外にカルボキシル基（またはカルボキシレート）を有しているもの、Ｎ末端のアミノ酸残基（例、メチオニン残基）のアミノ基が保護基で保護されているもの、Ｎ端側が生体内で切断され生成したグルタミン残基がピログルタミン酸化したもの、分子内のアミノ酸の側鎖上の置換基が適当な保護基で保護されているもの、あるいは糖鎖が結合したいわゆる糖ペプチドなどの複合ペプチドなども含まれる。 In the partial peptide of the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO—), an amide (—CONH 2 ), or an ester (—COOR).
Furthermore, the partial peptide of the present invention includes those having a carboxyl group (or carboxylate) in addition to the C-terminus, and an N-terminal amino acid residue (eg, methionine residue) as in the above-described polypeptide of the present invention. Group) whose amino group is protected with a protecting group, whose N-terminal side is cleaved in vivo and the glutamine residue produced is pyroglutamine oxidized, and the substituent on the side chain of the amino acid in the molecule is appropriately protected Also included are those protected by a group, or complex peptides such as so-called glycopeptides to which sugar chains are bound.

　本発明のポリペプチドまたはその部分ペプチドの塩としては、生理学的に許容される金属塩、アンモニウム塩、有機塩基との塩、無機酸との塩、有機酸との塩、塩基性または酸性アミノ酸との塩などが挙げられる。金属塩の好適な例としては、例えばナトリウム塩、カリウム塩などのアルカリ金属塩；カルシウム塩、マグネシウム塩、バリウム塩などのアルカリ土類金属塩；アルミニウム塩などが挙げられる。有機塩基との塩の好適な例としては、例えばトリメチルアミン、トリエチルアミン、ピリジン、ピコリン、２，６－ルチジン、エタノールアミン、ジエタノールアミン、トリエタノールアミン、シクロヘキシルアミン、ジシクロヘキシルアミン、Ｎ，Ｎ'－ジベンジルエチレンジアミンなどとの塩が挙げられる。無機酸との塩の好適な例としては、例えば塩酸、臭化水素酸、硝酸、硫酸、リン酸などとの塩が挙げられる。有機酸との塩の好適な例としては、例えばギ酸、酢酸、トリフルオロ酢酸、プロピオン酸、フタル酸、フマル酸、シュウ酸、酒石酸、マレイン酸、クエン酸、コハク酸、リンゴ酸、メタンスルホン酸、安息香酸、ベンゼンスルホン酸、ｐ－トルエンスルホン酸などとの塩が挙げられる。塩基性アミノ酸との塩の好適な例としては、例えばアルギニン、リジン、オルニチンなどとの塩が挙げられ、酸性アミノ酸との塩の好適な例としては、例えばアスパラギン酸、グルタミン酸などとの塩などが挙げられる。 Examples of the salt of the polypeptide of the present invention or a partial peptide thereof include physiologically acceptable metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, basic or acidic amino acids, And the like. Preferable examples of the metal salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; aluminum salt and the like. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And the like. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Preferable examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, propionic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid And salts with benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Preferable examples of salts with basic amino acids include salts with arginine, lysine, ornithine and the like, and preferable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid and the like. Can be mentioned.

　本発明のポリペプチドもしくはその部分ペプチドまたはその塩は、哺乳動物（ヒト、マウス、ラットなど）の細胞または組織から自体公知のポリペプチドの精製方法によって製造することもできるし、ポリペプチドをコードするＤＮＡを含有する形質転換体を培養することによっても製造することができる。また、後述のペプチド合成法に準じて製造することもできる。
　哺乳動物の組織または細胞から本発明のポリペプチドもしくはその部分ペプチドを製造する場合、哺乳動物の組織または細胞をホモジナイズした後、酸などで抽出を行ない、該抽出液を逆相クロマトグラフィー、イオン交換クロマトグラフィーなどのクロマトグラフィーを組み合わせることにより精製単離することができる。 The polypeptide of the present invention or a partial peptide thereof or a salt thereof can be produced from a mammalian cell (human, mouse, rat, etc.) or tissue by a known purification method of the polypeptide, and encodes the polypeptide. It can also be produced by culturing a transformant containing DNA. Moreover, it can also manufacture according to the below-mentioned peptide synthesis method.
When the polypeptide of the present invention or a partial peptide thereof is produced from mammalian tissues or cells, the tissues or cells of mammals are homogenized, followed by extraction with acid or the like, and the extract is subjected to reverse phase chromatography and ion exchange. It can be purified and isolated by combining chromatography such as chromatography.

　本発明のポリペプチドもしくはその部分ペプチドまたはこれらの塩は、自体公知のペプチドの合成法に従って製造することができる。ペプチドの合成法としては、例えば、固相合成法、液相合成法のいずれによっても良い。すなわち、本発明のポリペプチドもしくはその部分ペプチドを構成し得る部分ペプチドもしくはアミノ酸と残余部分とを縮合させ、生成物が保護基を有する場合は保護基を脱離することにより、本発明のポリペプチドもしくはその部分ペプチドを製造することができる。公知の縮合方法や保護基の脱離としては、例えば、以下の（i）～（v）に記載された方法が挙げられる。
（i）M. Bodanszky およびM.A. Ondetti、ペプチド・シンセシス (Peptide Synthesis), Interscience Publishers, New York (1966年)
（ii）SchroederおよびLuebke、ザ・ペプチド(The Peptide), Academic Press, New York (1965年)
（iii）泉屋信夫他、ペプチド合成の基礎と実験、 丸善(株) （1975年）
（iv）矢島治明 および榊原俊平、生化学実験講座 1、 タンパク質の化学IV、 205、(1977年)
（v）矢島治明監修、続医薬品の開発、第14巻、ペプチド合成、広川書店
　また、反応後は通常の精製法、例えば、溶媒抽出・蒸留・カラムクロマトグラフィー・液体クロマトグラフィー・再結晶などを組み合わせて本発明のポリペプチドもしくはその部分ペプチドを精製単離することができる。上記方法で得られる本発明のポリペプチドもしくはその部分ペプチドが遊離体である場合は、公知の方法あるいはそれに準じる方法によって適当な塩に変換することができるし、逆に塩で得られた場合は、公知の方法あるいはそれに準じる方法によって遊離体または他の塩に変換することができる。 The polypeptide of the present invention or a partial peptide thereof or a salt thereof can be produced according to a peptide synthesis method known per se. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the polypeptide of the present invention is obtained by condensing a partial peptide or amino acid capable of constituting the polypeptide of the present invention or a partial peptide thereof and the remaining part, and removing the protective group when the product has a protective group. Alternatively, a partial peptide thereof can be produced. Examples of known condensation methods and protecting group elimination include the methods described in the following (i) to (v).
(I) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(Ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(Iii) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(Iv) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
(V) Supervised by Haruaki Yajima, Development of follow-up drugs, Volume 14, Peptide synthesis, Hirokawa Shoten Also, after the reaction, usual purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. In combination, the polypeptide of the present invention or a partial peptide thereof can be purified and isolated. When the polypeptide of the present invention obtained by the above method or a partial peptide thereof is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, It can be converted into a free form or other salt by a known method or a method analogous thereto.

　本発明のポリヌクレオチドとして、前述した本発明のポリペプチドまたはその部分ペプチドをコードする塩基配列を含むポリヌクレオチドであれば、いかなるポリヌクレオチドであってもよいが、好ましくはＤＮＡである（本明細書中、本発明のＤＮＡと略記する場合がある）。
　ＤＮＡとしては、ゲノムＤＮＡ、ゲノムＤＮＡライブラリー、後述する細胞・組織由来のｃＤＮＡ、後述する細胞・組織由来のｃＤＮＡライブラリー、合成ＤＮＡのいずれでもよい。
　ライブラリーに使用するベクターは、バクテリオファージ、プラスミド、コスミド、ファージミドなどいずれであってもよい。また、後述する細胞・組織よりtotalＲＮＡまたはｍＲＮＡ画分を調製したものを用いて直接 Reverse Transcription Polymerase Chain Reaction（以下、ＲＴ－ＰＣＲ法と略称する）によって増幅することもできる。
　本発明のポリヌクレオチドとしては、本発明のポリペプチドをコードするポリヌクレオチドが好ましく、本発明のポリペプチドをコードするポリヌクレオチドとしては、例えば、配列番号：２、４、６、８、１０または１２で示される塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）、配列番号：２、４、６、８、１０または１２で示される塩基配列からなるポリヌクレオチド（好ましくは、ＤＮＡ）、配列番号：２、４、６、８、１０または１２で示される塩基配列とハイストリンジェントな条件下でハイブリダイズする塩基配列を含有し、前記した配列番号：１、３、５、７、９または１１で示されるアミノ酸配列を含むポリペプチドと実質的に同質の性質を有するポリペプチドをコードするポリヌクレオチド（好ましくは、ＤＮＡ）が挙げられる。 The polynucleotide of the present invention may be any polynucleotide as long as it contains a nucleotide sequence encoding the above-described polypeptide of the present invention or a partial peptide thereof, and is preferably DNA (herein In some cases, the DNA of the present invention may be abbreviated).
The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from cells / tissues described later, cDNA library derived from cells / tissues described later, and synthetic DNA.
The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Further, it can be directly amplified by reverse transcription polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from cells / tissues described later.
As the polynucleotide of the present invention, a polynucleotide encoding the polypeptide of the present invention is preferable. Examples of the polynucleotide encoding the polypeptide of the present invention include SEQ ID NOs: 2, 4, 6, 8, 10, or 12. A polynucleotide (preferably DNA) comprising the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 (preferably DNA), SEQ ID NO: 2. 4. A nucleotide sequence that hybridizes with a nucleotide sequence represented by 4, 6, 8, 10, or 12 under highly stringent conditions, and represented by the aforementioned SEQ ID NO: 1, 3, 5, 7, 9, or 11. A polynucleotide encoding a polypeptide having preferably substantially the same properties as the polypeptide comprising the amino acid sequence (preferably D A), and the like.

　配列番号：２、４、６、８、１０または１２で示される塩基配列とハイストリンジェントな条件下でハイブリダイズするポリヌクレオチドとしては、例えば、配列番号：２、４、６、８、１０または１２で示される塩基配列と約６０％以上、約７０％以上、約８０％以上、約９０％以上、９５％、約９６％、約９７％、約９８％、約９９％以上の相同性を有する塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）などが用いられる。
　塩基配列の相同性は、相同性計算アルゴリズムNCBI BLAST（National Center for Biotechnology Information Basic Local Alignment Search Tool）を用い、以下の条件（期待値＝10；ギャップを許す；フィルタリング＝ON；マッチスコア＝1；ミスマッチスコア＝-3）にて計算することができる。
　ハイブリダイゼーションは、自体公知の方法あるいはそれに準じる方法、例えば、Molecular Cloning 2nd（J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989）に記載の方法などに従って行なうことができる。また、市販のライブラリーを使用する場合、添付の使用説明書に記載の方法に従って行なうことができる。より好ましくは、ハイストリンジェントな条件に従って行なうことができる。
　ハイストリンジェントな条件とは、例えば、ナトリウム濃度が約１９～４０ｍＭ、好ましくは約１９～２０ｍＭで、温度が約５０～７０℃、好ましくは約６０～６５℃の条件を示す。特に、ナトリウム濃度が約１９ｍＭで温度が約６５℃の場合が最も好ましい。 Examples of the polynucleotide that hybridizes with the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 under highly stringent conditions include, for example, SEQ ID NO: 2, 4, 6, 8, 10, or About 60% or more, about 70% or more, about 80% or more, about 90% or more, 95%, about 96%, about 97%, about 98%, about 99% or more A polynucleotide (preferably DNA) containing a nucleotide sequence having it is used.
The homology of the base sequence was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; filtering = ON; match score = 1) It can be calculated by mismatch score = -3).
Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual. More preferably, it can be carried out according to highly stringent conditions.
The highly stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.

　より具体的には、配列番号：１で示されるアミノ酸配列を含むポリペプチドをコードするポリヌクレオチドとしては、配列番号：２で示される塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）などが、配列番号：３で示されるアミノ酸配列を含むポリペプチドをコードするポリヌクレオチドとしては、配列番号：４で示される塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）などが、配列番号：５で示されるアミノ酸配列を含むポリペプチドをコードするポリヌクレオチドとしては、配列番号：６で示される塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）などが、配列番号：７で示されるアミノ酸配列を含むポリペプチドをコードするポリヌクレオチドとしては、配列番号：８で示される塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）などが、配列番号：９で示されるアミノ酸配列を含むポリペプチドをコードするポリヌクレオチドとしては、配列番号：１０で示される塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）などが、配列番号：１１で示されるアミノ酸を含むポリペプチドをコードするポリヌクレオチドとしては、配列番号：１２で示される塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）などが用いられる。 More specifically, the polynucleotide encoding the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 is a polynucleotide (preferably DNA) comprising the base sequence represented by SEQ ID NO: 2, The polynucleotide encoding the polypeptide containing the amino acid sequence shown by No. 3 is a polynucleotide (preferably DNA) containing the base sequence shown by SEQ ID No. 4 or the like, and the amino acid shown by SEQ ID No. 5 As the polynucleotide encoding the polypeptide containing the sequence, a polynucleotide (preferably DNA) containing the base sequence shown by SEQ ID NO: 6 encodes a polypeptide containing the amino acid sequence shown by SEQ ID NO: 7. The polynucleotide to be used is a polynucleotide comprising the base sequence represented by SEQ ID NO: 8. As a polynucleotide encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 9, such as a leotide (preferably DNA), a polynucleotide comprising the base sequence represented by SEQ ID NO: 10 (preferably DNA) As the polynucleotide encoding the polypeptide containing the amino acid represented by SEQ ID NO: 11, a polynucleotide (preferably, DNA) containing the base sequence represented by SEQ ID NO: 12 is used.

　本発明のポリペプチドをコードするポリヌクレオチドの部分ポリヌクレオチドとしては、例えば、本発明の部分ペプチドをコードするポリヌクレオチドが挙げられる。
　本発明の部分ペプチドをコードするポリヌクレオチドとしては、前述した本発明の部分ペプチドをコードする塩基配列を含むものであればいかなるものであってもよい。また、ゲノムＤＮＡ、ゲノムＤＮＡライブラリー、後述する細胞・組織由来のｃＤＮＡ、後述する細胞・組織由来のｃＤＮＡライブラリー、合成ＤＮＡのいずれでもよい。
　本発明の部分ペプチドをコードするポリヌクレオチドとしては、例えば、配列番号：２、４、６、８、１０または１２で示される塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）の一部分を有するポリヌクレオチド（好ましくは、ＤＮＡ）、配列番号：２、４、６、８、１０または１２で示される塩基配列からなるポリヌクレオチド（好ましくは、ＤＮＡ）の一部分を有するポリヌクレオチド（好ましくは、ＤＮＡ）、または配列番号：２、４、６、８、１０または１２で示される塩基配列とハイストリンジェントな条件下でハイブリダイズする塩基配列を含み、本発明のポリペプチドと実質的に同質の活性を有するポリペプチドをコードするポリヌクレオチド（好ましくは、ＤＮＡ）の一部分を含むポリヌクレオチド（好ましくは、ＤＮＡ）などが用いられる。
　配列番号：２、４、６、８、１０または１２で示される塩基配列とハイブリダイズするポリヌクレオチド（好ましくは、ＤＮＡ）は、前記と同意義を示す。 Examples of the partial polynucleotide of the polynucleotide encoding the polypeptide of the present invention include a polynucleotide encoding the partial peptide of the present invention.
The polynucleotide encoding the partial peptide of the present invention may be any polynucleotide as long as it contains the base sequence encoding the partial peptide of the present invention described above. Further, any of genomic DNA, genomic DNA library, cDNA derived from cells / tissues described later, cDNA library derived from cells / tissues described later, and synthetic DNA may be used.
As the polynucleotide encoding the partial peptide of the present invention, for example, a polynucleotide having a part of a polynucleotide (preferably DNA) comprising the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 (Preferably DNA), a polynucleotide (preferably DNA) having a part of a polynucleotide (preferably DNA) comprising the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, or A polypeptide comprising a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 under highly stringent conditions and having substantially the same quality of activity as the polypeptide of the present invention. A polynucleotide comprising a portion of a polynucleotide (preferably DNA) encoding a peptide (preferably a DNA) DNA) and the like can be used.
The polynucleotide (preferably DNA) that hybridizes with the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 has the same meaning as described above.

　本発明のポリペプチドまたは部分ペプチド（以下、これらを単に本発明のペプチドと略記する場合がある）を完全にコードするＤＮＡのクローニングの手段としては、本発明のペプチドをコードする塩基配列の一部分を有する合成ＤＮＡプライマーを用いてＰＣＲ法によって増幅するか、または適当なベクターに組み込んだＤＮＡを本発明のペプチドの一部あるいは全領域をコードするＤＮＡ断片もしくは合成ＤＮＡを用いて標識したものとのハイブリダイゼーションによって選別することができる。ハイブリダイゼーションの方法は、例えば、Molecular Cloning 2nd（J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989）に記載の方法などに従って行なうことができる。また、市販のライブラリーを使用する場合、添付の使用説明書に記載の方法に従って行なうことができる。
　ＤＮＡの塩基配列の変換は、ＰＣＲ、公知のキット、例えば、MutanTM-super Express Km（宝酒造（株））、MutanTM-K（宝酒造（株））等を用いて、ODA-LA PCR法、Gapped duplex法、Kunkel法等の自体公知の方法あるいはそれらに準じる方法に従って行なうことができる。
　クローン化されたペプチドをコードするＤＮＡは目的によりそのまま、または所望により制限酵素で消化したり、リンカーを付加したりして使用することができる。該ＤＮＡはその５’末端側に翻訳開始コドンとしてのＡＴＧを有し、また３’末端側には翻訳終止コドンとしてのＴＡＡ、ＴＧＡまたはＴＡＧを有していてもよい。これらの翻訳開始コドンや翻訳終止コドンは、適当な合成ＤＮＡアダプターを用いて付加することもできる。
　本発明のペプチドは、適当な宿主を、本発明のペプチドの発現ベクターで形質転換し、得られた形質転換体を培養後、得られた培養物から本発明のペプチドを分離精製することによって製造することもできる。
　本発明のペプチドの発現ベクターは、例えば、（i）本発明のペプチドをコードするＤＮＡから目的とするＤＮＡ断片を切り出し、（ii）該ＤＮＡ断片を適当な発現ベクター中のプロモーターの下流に連結することにより製造することができる。 As a means for cloning DNA that completely encodes the polypeptide or partial peptide of the present invention (hereinafter sometimes simply referred to as the peptide of the present invention), a part of the base sequence encoding the peptide of the present invention is used. Amplified by a PCR method using a synthetic DNA primer or a DNA fragment that has been incorporated into an appropriate vector and labeled with a DNA fragment or synthetic DNA encoding a part or all of the peptide of the present invention. It can be selected by hybridization. The hybridization method can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.
The DNA base sequence is converted by PCR, a known kit such as Mutan ™ -super Express Km (Takara Shuzo), Mutan ™ -K (Takara Shuzo), etc., using the ODA-LA PCR method, It can be carried out according to a method known per se such as the Gapped duplex method and the Kunkel method or a method analogous thereto.
The DNA encoding the cloned peptide can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
The peptide of the present invention is produced by transforming a suitable host with the expression vector of the peptide of the present invention, culturing the obtained transformant, and then separating and purifying the peptide of the present invention from the obtained culture. You can also
The peptide expression vector of the present invention can be obtained by, for example, (i) cutting out the target DNA fragment from the DNA encoding the peptide of the present invention, and (ii) linking the DNA fragment downstream of the promoter in an appropriate expression vector. Can be manufactured.

　本発明のペプチドの発現ベクターとしては、大腸菌由来のプラスミド（例、ｐＢＲ３２２，ｐＢＲ３２５，ｐＵＣ１２，ｐＵＣ１３）、枯草菌由来のプラスミド（例、ｐＵＢ１１０，ｐＴＰ５，ｐＣ１９４）、酵母由来プラスミド（例、ｐＳＨ１９，ｐＳＨ１５）、λファージなどのバクテリオファージ、レトロウイルス，ワクシニアウイルス，バキュロウイルスなどの動物ウイルスなどの他、ｐＡ１－１１、ｐＸＴ１、ｐＲｃ／ＣＭＶ、ｐＲｃ／ＲＳＶ、ｐｃＤＮＡＩ／Ｎｅｏなどが用いられる。 As an expression vector for the peptide of the present invention, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15). ), Bacteriophage such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, and the like, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like.

　前記したプロモーターとして、例えば、動物細胞を宿主として用いる場合は、ＳＲαプロモーター、ＳＶ４０プロモーター、ＬＴＲプロモーター、ＣＭＶプロモーター、ＨＳＶ-ＴＫプロモーターなどが挙げられる。これらのうち、ＣＭＶ（サイトメガロウイルス）プロモーター、ＳＲαプロモーターなどを用いるのが好ましい。
　宿主がエシェリヒア属菌である場合は、ｔｒｐプロモーター、ｌａｃプロモーター、ｒｅｃＡプロモーター、λＰＬプロモーター、ｌｐｐプロモーター、Ｔ７プロモーターなどが、宿主がバチルス属菌である場合は、ＳＰＯ１プロモーター、ＳＰＯ２プロモーター、ｐｅｎＰプロモーターなど、宿主が酵母である場合は、ＰＨＯ５プロモーター、ＰＧＫプロモーター、ＧＡＰプロモーター、ＡＤＨプロモーターなどが好ましい。宿主が昆虫細胞である場合は、ポリヘドリンプロモーター、Ｐ１０プロモーターなどが好ましい。 Examples of the promoter described above include SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like when animal cells are used as a host. Of these, it is preferable to use a CMV (cytomegalovirus) promoter, SRα promoter, or the like.
When the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P L promoter, lpp promoter, T7 promoter, etc. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

　発現ベクターには、以上の他に、所望によりエンハンサー、スプライシングシグナル、ポリＡ付加シグナル、選択マーカー、ＳＶ４０複製オリジン（以下、ＳＶ４０ｏｒｉと略称する場合がある）などを含有しているものを用いることができる。選択マーカーとしては、例えば、ジヒドロ葉酸還元酵素（以下、ｄｈｆｒと略称する場合がある）遺伝子〔メソトレキセート（ＭＴＸ）耐性〕、アンピシリン耐性遺伝子（以下、Ａｍｐｒと略称する場合がある）、ネオマイシン耐性遺伝子（以下、Ｎｅｏｒと略称する場合がある、Ｇ４１８耐性）等が挙げられる。特に、ｄｈｆｒ遺伝子欠損チャイニーズハムスター細胞を用いてｄｈｆｒ遺伝子を選択マーカーとして使用する場合、目的遺伝子をチミジンを含まない培地によっても選択できる。
　また、必要に応じて、宿主に合ったシグナル配列を、本発明のペプチドのＮ端末側に付加する。宿主がエシェリヒア属菌である場合は、PhoA・シグナル配列、OmpA・シグナル配列などが、宿主がバチルス属菌である場合は、α－アミラーゼ・シグナル配列、サブチリシン・シグナル配列などが、宿主が酵母である場合は、MFα・シグナル配列、SUC2・シグナル配列などが、宿主が動物細胞である場合には、インシュリン・シグナル配列、α－インターフェロン・シグナル配列、抗体分子・シグナル配列などがそれぞれ利用できる。
　このようにして構築された本発明のペプチドをコードするＤＮＡを含有するベクターを用いて、形質転換体を製造することができる。 In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like is used as desired. it can. Examples of selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Amp r ), neomycin resistance gene (hereinafter sometimes abbreviated as Neo r, G418 resistance). In particular, when a dhfr gene-deficient Chinese hamster cell is used and the dhfr gene is used as a selection marker, the target gene can also be selected by a medium not containing thymidine.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the peptide of the present invention. When the host is Escherichia, the PhoA signal sequence, OmpA, signal sequence, etc., and when the host is Bacillus, the α-amylase signal sequence, subtilisin signal sequence, etc. In some cases, MFα • signal sequence, SUC2 • signal sequence, etc. can be used. When the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used.
A transformant can be produced using a vector containing the DNA encoding the peptide of the present invention thus constructed.

　宿主としては、例えば、エシェリヒア属菌、バチルス属菌、酵母、昆虫細胞、昆虫、動物細胞などが用いられる。
　エシェリヒア属菌の具体例としては、例えば、エシェリヒア・コリ（Escherichia coli）Ｋ１２・ＤＨ１〔Proc. Natl. Acad. Sci. USA, 60巻, 160(1968)〕，ＪＭ１０３〔Nucleic Acids Research,9巻,309(1981)〕，ＪＡ２２１〔Journal of Molecular Biology,120巻,517(1978)〕，ＨＢ１０１〔Journal of Molecular Biology,41巻,459(1969)〕，Ｃ６００〔Genetics,39巻,440(1954)〕などが用いられる。
　バチルス属菌としては、例えば、バチルス・サブチルス（Bacillus subtilis）ＭＩ１１４〔Gene,24巻,255(1983)〕，２０７－２１〔Journal of Biochemistry,95巻,87(1984)〕などが用いられる。
　酵母としては、例えば、サッカロマイセス　セレビシエ（Saccharomyces cerevisiae）ＡＨ２２，ＡＨ２２Ｒ－，ＮＡ８７－１１Ａ，ＤＫＤ－５Ｄ，２０Ｂ－１２、シゾサッカロマイセス　ポンベ（Schizosaccharomyces pombe）ＮＣＹＣ１９１３，ＮＣＹＣ２０３６、ピキア　パストリス（Pichia pastoris）ＫＭ７１などが用いられる。
　昆虫細胞としては、例えば、ウイルスがＡｃＮＰＶの場合は、夜盗蛾の幼虫由来株化細胞（Spodoptera frugiperda cell；Ｓｆ細胞）、Trichoplusia niの中腸由来のＭＧ１細胞、Trichoplusia niの卵由来のHigh FiveTM細胞、Mamestra brassicae由来の細胞またはEstigmena acrea由来の細胞などが用いられる。ウイルスがＢｍＮＰＶの場合は、蚕由来株化細胞（Bombyx mori N 細胞；ＢｍＮ細胞）などが用いられる。該Ｓｆ細胞としては、例えば、Ｓｆ９細胞（ATCC CRL1711）、Ｓｆ２１細胞（以上、Vaughn, J.L.ら、In Vivo,13, 213-217,(1977)）などが用いられる。
　昆虫としては、例えば、カイコの幼虫などが用いられる〔前田ら、Nature,315巻,592(1985)〕。
　動物細胞としては、例えば、サル細胞ＣＯＳ－７，Ｖｅｒｏ，チャイニーズハムスター細胞ＣＨＯ（以下、ＣＨＯ細胞と略記），ｄｈｆｒ遺伝子欠損チャイニーズハムスター細胞ＣＨＯ（以下、ＣＨＯ（ｄｈｆｒ－）細胞と略記），マウスＬ細胞，マウスＡｔＴ－２０，マウスミエローマ細胞，マウスＡＴＤＣ５細胞，ラットＧＨ３，ヒトＦＬ細胞、ヒト293細胞などが用いられる。 As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Specific examples of the genus Escherichia include, for example, Escherichia coli K12 · DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] Etc. are used.
Examples of the Bacillus bacterium include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)].
Examples of the yeast include Saccharomyces cerevisiae AH22, AH22R − , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Used.
As insect cells, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five ™ derived from eggs of Trichoplusia ni Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, sputum-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like.
Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr − ) cells), mouse L Cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells, human 293 cells and the like are used.

　エシェリヒア属菌を形質転換するには、例えば、Proc. Natl. Acad. Sci. USA, 69巻, 2110(1972)やGene, 17巻, 107(1982)などに記載の方法に従って行なうことができる。
　バチルス属菌を形質転換するには、例えば、Molecular & General Genetics,168巻,111(1979)などに記載の方法に従って行なうことができる。
　酵母を形質転換するには、例えば、Methods in Enzymology,194巻,182-187(1991)、Proc. Natl. Acad. Sci. USA, 75巻, 1929(1978）などに記載の方法に従って行なうことができる。
　昆虫細胞または昆虫を形質転換するには、例えば、Bio/Technology,6, 47-55(1988)などに記載の方法に従って行なうことができる。
　動物細胞を形質転換するには、例えば、細胞工学別冊８　新細胞工学実験プロトコール.263-267(1995)（秀潤社発行）、Virology,52巻,456(1973)に記載の方法に従って行なうことができる。
　このようにして、ペプチドをコードするＤＮＡを含有する発現ベクターで形質転換された形質転換体を得ることができる。 Transformation of Escherichia can be performed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982).
Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, 168, 111 (1979).
In order to transform yeast, for example, the method described in Methods in Enzymology, 194, 182-287 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like can be performed. it can.
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
In order to transform animal cells, for example, cell engineering separate volume 8 New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973) Can do.
In this way, a transformant transformed with an expression vector containing DNA encoding the peptide can be obtained.

　宿主がエシェリヒア属菌、バチルス属菌である形質転換体を培養する際、培養に使用される培地としては液体培地が適当であり、その中には該形質転換体の生育に必要な炭素源、窒素源、無機物その他が含有せしめられる。炭素源としては、例えば、グルコース、デキストリン、可溶性澱粉、ショ糖など、窒素源としては、例えば、アンモニウム塩類、硝酸塩類、コーンスチープ・リカー、ペプトン、カゼイン、肉エキス、大豆粕、バレイショ抽出液などの無機または有機物質、無機物としては、例えば、塩化カルシウム、リン酸二水素ナトリウム、塩化マグネシウムなどが挙げられる。また、酵母エキス、ビタミン類、生長促進因子などを添加してもよい。培地のｐＨは約５～８が望ましい。
　エシェリヒア属菌を培養する際の培地としては、例えば、グルコース、カザミノ酸を含むＭ９培地〔ミラー（Miller），Journal of Experiments in Molecular Genetics,431-433,Cold Spring Harbor Laboratory, New York 1972〕が好ましい。ここに必要によりプロモーターを効率よく働かせるために、例えば、３β－インドリルアクリル酸のような薬剤を加えることができる。
　宿主がエシェリヒア属菌の場合、培養は通常約１５～４３℃で約３～２４時間行ない、必要により、通気や撹拌を加えることもできる。
　宿主がバチルス属菌の場合、培養は通常約３０～４０℃で約６～２４時間行ない、必要により通気や撹拌を加えることもできる。
　宿主が酵母である形質転換体を培養する際、培地としては、例えば、バークホールダー（Burkholder）最小培地〔Bostian, K. L. ら、Proc. Natl. Acad. Sci. USA, 77巻, 4505(1980)〕や０.５％カザミノ酸を含有するＳＤ培地〔Bitter, G. A. らProc. Natl. Acad. Sci. USA, 81巻, 5330(1984）〕が挙げられる。培地のｐＨは約５～８に調整するのが好ましい。培養は通常約２０℃～３５℃で約２４～７２時間行ない、必要に応じて通気や撹拌を加える。
　宿主が昆虫細胞または昆虫である形質転換体を培養する際、培地としては、Grace's Insect Medium（Grace, T.C.C., Nature,195,788(1962)）に非動化した１０％ウシ血清等の添加物を適宜加えたものなどが用いられる。培地のｐＨは約６．２～６．４に調整するのが好ましい。培養は通常約２７℃で約３～５日間行ない、必要に応じて通気や撹拌を加える。
　宿主が動物細胞である形質転換体を培養する際、培地としては、例えば、約５～２０％の胎児牛血清を含むＭＥＭ培地〔Science,122巻,501(1952)〕，ＤＭＥＭ培地〔Virology,8巻,396(1959)〕，ＲＰＭＩ １６４０培地〔The Journal of the American Medical Association 199巻,519(1967)〕，１９９培地〔Proceeding of the Society for the Biological Medicine,73巻,1(1950)〕などが用いられる。ｐＨは約６～８であるのが好ましい。培養は通常約３０℃～４０℃で約１５～６０時間行ない、必要に応じて通気や撹拌を加える。
　以上のようにして、形質転換体の細胞内、細胞膜または細胞外に本発明のペプチドを生成せしめることができる。 When culturing a transformant whose host is an Escherichia bacterium or Bacillus genus, a liquid medium is suitable as a medium used for the culture, and a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, and potato extract. Examples of inorganic or organic substances and inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.
As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972) is preferable. . If necessary, a drug such as 3β-indolylacrylic acid can be added to make the promoter work efficiently if necessary.
When the host is an Escherichia bacterium, the culture is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation can be added.
When the host is Bacillus, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration or agitation can be added.
When cultivating a transformant whose host is yeast, examples of the medium include a Burkholder minimum medium [Bostian, KL et al., Proc. Natl. Acad. Sci. USA, vol. 77, 4505 (1980)]. And SD medium containing 0.5% casamino acid [Bitter, GA et al. Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. Cultivation is usually carried out at about 20 ° C. to 35 ° C. for about 24 to 72 hours, with aeration and agitation as necessary.
When cultivating a transformant whose host is an insect cell or an insect, an appropriate medium such as 10% bovine serum that has been immobilized on Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)) is used as the medium. Additions etc. are used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually carried out at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.
When cultivating a transformant whose host is an animal cell, examples of the medium include MEM medium (Science, Vol. 122, 501 (1952)) containing about 5 to 20% fetal bovine serum, DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], etc. Is used. The pH is preferably about 6-8. Cultivation is usually carried out at about 30 ° C. to 40 ° C. for about 15 to 60 hours, with aeration and agitation as necessary.
As described above, the peptide of the present invention can be produced in the cell, in the cell membrane or outside of the transformant.

　上記培養物から本発明のペプチドを分離精製するには、例えば、下記の方法により行なうことができる。
　本発明のペプチドを培養菌体あるいは細胞から抽出するに際しては、培養後、公知の方法で菌体あるいは細胞を集め、これを適当な緩衝液に懸濁し、超音波、リゾチームおよび／または凍結融解などによって菌体あるいは細胞を破壊したのち、遠心分離やろ過によりペプチドの粗抽出液を得る方法などが適宜用いられる。緩衝液の中に尿素や塩酸グアニジンなどのタンパク質（ペプチド）変性剤や、トリトンＸ－１００TMなどの界面活性剤が含まれていてもよい。培養液中にペプチドが分泌される場合には、培養終了後、それ自体公知の方法で菌体あるいは細胞と上清とを分離し、上清を集める。
　このようにして得られた培養上清、あるいは抽出液中に含まれるペプチドの精製は、自体公知の分離・精製法を適切に組み合わせて行なうことができる。これらの公知の分離、精製法としては、塩析や溶媒沈澱法などの溶解度を利用する方法、透析法、限外ろ過法、ゲルろ過法、およびＳＤＳ－ポリアクリルアミドゲル電気泳動法などの主として分子量の差を利用する方法、イオン交換クロマトグラフィーなどの荷電の差を利用する方法、アフィニティークロマトグラフィーなどの特異的親和性を利用する方法、逆相高速液体クロマトグラフィーなどの疎水性の差を利用する方法、等電点電気泳動法などの等電点の差を利用する方法などが用いられる。 Separation and purification of the peptide of the present invention from the culture can be performed, for example, by the following method.
When extracting the peptide of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freeze-thaw, etc. For example, a method of obtaining a crude peptide extract by centrifugation or filtration after destroying cells or cells by the method is appropriately used. The buffer may contain a protein (peptide) denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ™ . When the peptide is secreted into the culture solution, after completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.
The peptide contained in the culture supernatant or the extract thus obtained can be purified by appropriately combining known separation / purification methods. These known separation and purification methods include mainly molecular weights such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method using difference in charge, method using difference in charge such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and difference in hydrophobicity such as reverse phase high performance liquid chromatography A method using a difference in isoelectric point, such as a method, isoelectric focusing method, or the like is used.

　かくして得られるペプチドが遊離体で得られた場合には、自体公知の方法あるいはそれに準じる方法によって塩に変換することができ、逆に塩で得られた場合には自体公知の方法あるいはそれに準じる方法により、遊離体または他の塩に変換することができる。
　なお、組換え体が産生するペプチドを、精製前または精製後に適当なタンパク質修飾酵素を作用させることにより、任意に修飾を加えたり、ポリペプチドを部分的に除去したりすることもできる。タンパク質修飾酵素としては、例えば、トリプシン、キモトリプシン、アルギニルエンドペプチダーゼ、プロテインキナーゼ、グリコシダーゼなどが用いられる。
　かくして生成する本発明のペプチドの存在は、本発明のペプチドに特異的に結合する抗体を用いたエンザイムイムノアッセイやウエスタンブロッティングなどにより測定することができる。 When the peptide thus obtained is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when obtained as a salt, a method known per se or a method analogous thereto Can be converted to the free form or other salts.
The peptide produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by allowing an appropriate protein modifying enzyme to act before or after purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The presence of the peptide of the present invention thus produced can be measured by enzyme immunoassay or Western blotting using an antibody that specifically binds to the peptide of the present invention.

　本発明の抗体は、本発明のペプチドに結合する抗体であれば、ポリクローナル抗体、モノクローナル抗体の何れであってもよい。
　本発明の抗体は、本発明のペプチドを抗原として用い、自体公知の抗体または抗血清の製造法に従って製造することができる。 As long as the antibody of this invention is an antibody couple | bonded with the peptide of this invention, any of a polyclonal antibody and a monoclonal antibody may be sufficient.
The antibody of the present invention can be produced according to a method for producing an antibody or antiserum known per se, using the peptide of the present invention as an antigen.

（ａ）モノクローナル抗体の作製
　モノクローナル抗体は、自体公知の方法に従って製造することができる。
　まず、本発明のペプチドを、温血動物に対して投与により抗体産生が可能な部位にそれ自体あるいは担体、希釈剤とともに投与する。投与に際して抗体産生能を高めるため、完全フロイントアジュバントや不完全フロイントアジュバントを投与してもよい。投与は通常２～６週毎に１回ずつ、計２～１０回程度行われる。用いられる温血動物としては、例えば、サル、ウサギ、イヌ、モルモット、マウス、ラット、ヒツジ、ヤギ、ニワトリが挙げられるが、マウスおよびラットが好ましく用いられる。
　次に、抗原で免疫された温血動物、例えばマウスから抗体価の認められた個体を選択し、最終免疫の２～５日後に脾臓またはリンパ節を採取し、それらに含まれる抗体産生細胞を同種または異種動物の骨髄腫細胞と融合させることにより、モノクローナル抗体産生ハイブリドーマを調製することができる。抗血清中の抗体価の測定は、例えば、後記の標識化したペプチドと抗血清とを反応させたのち、抗体に結合した標識剤の活性を測定することにより行なうことができる。融合操作は既知の方法、例えば、ケーラーとミルスタインの方法〔Nature、256、495 (1975)〕に従い実施することができる。融合促進剤としては、例えば、ポリエチレングリコール（ＰＥＧ）やセンダイウイルスなどが挙げられるが、好ましくはＰＥＧが用いられる。
　骨髄腫細胞としては、例えば、ＮＳ－１、Ｐ３Ｕ１、ＳＰ２／０、ＡＰ－１などの温血動物の骨髄腫細胞が挙げられるが、Ｐ３Ｕ１が好ましく用いられる。用いられる抗体産生細胞（脾臓細胞）数と骨髄腫細胞数との好ましい比率は１：１～２０：１程度であり、ＰＥＧ（好ましくはＰＥＧ１０００～ＰＥＧ６０００）が１０～８０％程度の濃度で添加され、２０～４０℃、好ましくは３０～３７℃で１～１０分間インキュベートすることにより効率よく細胞融合を実施できる。 (A) Production of monoclonal antibody Monoclonal antibodies can be produced according to a method known per se.
First, the peptide of the present invention is administered to a warm-blooded animal itself or a carrier and a diluent at a site where antibody production is possible by administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens, and mice and rats are preferably used.
Next, an individual having an antibody titer is selected from a warm-blooded animal immunized with an antigen, such as a mouse, and spleen or lymph nodes are collected 2 to 5 days after the final immunization, and antibody-producing cells contained therein are collected. Monoclonal antibody-producing hybridomas can be prepared by fusing with myeloma cells from the same or different animals. The antibody titer in the antiserum can be measured, for example, by reacting a labeled peptide described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
Examples of myeloma cells include warm-blooded animal myeloma cells such as NS-1, P3U1, SP2 / 0, AP-1, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. The cell fusion can be efficiently carried out by incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes.

　モノクローナル抗体産生ハイブリドーマのスクリーニングには種々の方法が使用できるが、例えば、ペプチド抗原を直接あるいは担体とともに吸着させた固相（例、マイクロプレート）にハイブリドーマ培養上清を添加し、次に放射性物質や酵素などで標識した抗免疫グロブリン抗体（細胞融合に用いられる細胞がマウスの場合、抗マウス免疫グロブリン抗体が用いられる）またはプロテインＡを加え、固相に結合したモノクローナル抗体を検出する方法、抗免疫グロブリン抗体またはプロテインＡを吸着させた固相にハイブリドーマ培養上清を添加し、放射性物質や酵素などで標識したペプチドを加え、固相に結合したモノクローナル抗体を検出する方法などが挙げられる。 Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which peptide antigens are adsorbed directly or together with a carrier, and then radioactive substances or An anti-immunoglobulin antibody labeled with an enzyme or the like (when the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A, and a method for detecting a monoclonal antibody bound to a solid phase; Examples include a method in which a hybridoma culture supernatant is added to a solid phase on which a globulin antibody or protein A is adsorbed, a peptide labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected.

　モノクローナル抗体の選別は、自体公知あるいはそれに準じる方法に従って行なうことができる。通常ＨＡＴ（ヒポキサンチン、アミノプテリン、チミジン）を添加した動物細胞用培地で行なうことができる。選別および育種用培地としては、ハイブリドーマが生育できるものならばどのような培地を用いても良い。例えば、１～２０％、好ましくは１０～２０％の牛胎児血清を含むＲＰＭＩ １６４０培地、１～１０％の牛胎児血清を含むＧＩＴ培地（和光純薬工業（株））あるいはハイブリドーマ培養用無血清培地（ＳＦＭ－１０１、日水製薬（株））などを用いることができる。培養温度は、通常２０～４０℃、好ましくは約３７℃である。培養時間は、通常５日～３週間、好ましくは１週間～２週間である。培養は、通常５％炭酸ガス下で行なうことができる。ハイブリドーマ培養上清の抗体価は、上記の抗血清中の抗体価の測定と同様にして測定できる。 The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium may be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal calf serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1 to 10% fetal calf serum, or serum-free for hybridoma culture A medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culture temperature is usually 20 to 40 ° C., preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.

　モノクローナル抗体の分離精製は、自体公知の方法、例えば、免疫グロブリンの分離精製法〔例、塩析法、アルコール沈殿法、等電点沈殿法、電気泳動法、イオン交換体（例、ＤＥＡＥ）による吸脱着法、超遠心法、ゲルろ過法、抗原結合固相あるいはプロテインＡあるいはプロテインＧなどの活性吸着剤により抗体のみを採取し、結合を解離させて抗体を得る特異的精製法〕に従って行なうことができる。 Separation and purification of the monoclonal antibody can be performed by a method known per se, for example, an immunoglobulin separation and purification method (eg, salting out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchanger (eg, DEAE)). Absorption / desorption method, ultracentrifugation method, gel filtration method, specific purification method in which only the antibody is collected with an antigen-binding solid phase or an active adsorbent such as protein A or protein G, and the binding is dissociated to obtain the antibody. Can do.

（ｂ）ポリクローナル抗体の作製
　ポリクローナル抗体は、それ自体公知あるいはそれに準じる方法に従って製造することができる。例えば、免疫抗原（ペプチド抗原）自体、あるいはそれとキャリアーペプチドとの複合体をつくり、上記のモノクローナル抗体の場合と同様に温血動物に免疫を行ない、得られる免疫動物から本発明のペプチドに対する抗体含有物を採取して、抗体の分離精製を行なうことにより製造することができる。
　温血動物を免疫するために用いられる免疫抗原とキャリアーペプチドとの複合体に関し、キャリアーペプチドの種類およびキャリアーとハプテンとの混合比は、キャリアーに架橋させて免疫したハプテンに対して抗体が効率良くできれば、どの様なものをどの様な比率で架橋させてもよいが、例えば、ウシ血清アルブミンやウシサイログロブリン、ヘモシアニン等を重量比でハプテン１に対し、約０．１～２０、好ましくは約１～６の割合でカプルさせる方法が用いられる。
　また、ハプテンとキャリアーのカプリングには、種々の縮合剤を用いることができるが、グルタルアルデヒドやカルボジイミド、マレイミド活性エステル、チオール基、ジチオビリジル基を含有する活性エステル試薬等が用いられる。
　縮合生成物は、温血動物に対して、抗体産生が可能な部位にそれ自体あるいは担体、希釈剤とともに投与される。投与に際して抗体産生能を高めるため、完全フロイントアジュバントや不完全フロイントアジュバントを投与してもよい。投与は、通常約２～６週毎に１回ずつ、計約３～１０回程度行なわれる。
　ポリクローナル抗体は、上記の方法で免疫された温血動物の血液、腹水など、好ましくは血液から採取することができる。
　抗血清中のポリクローナル抗体価の測定は、上記の抗血清中の抗体価の測定と同様にして測定できる。ポリクローナル抗体の分離精製は、上記のモノクローナル抗体の場合と同様の免疫グロブリンの分離精製法に従って行なうことができる。 (B) Preparation of polyclonal antibody A polyclonal antibody can be produced according to a method known per se or a method analogous thereto. For example, an immune antigen (peptide antigen) itself or a complex of it and a carrier peptide is prepared, and a warm-blooded animal is immunized in the same manner as in the case of the above monoclonal antibody. It can be produced by collecting the product and performing separation and purification of the antibody.
Regarding the complex of immunizing antigen and carrier peptide used to immunize warm-blooded animals, the type of carrier peptide and the mixing ratio of carrier and hapten are effective for antibodies against hapten immunized by cross-linking to carrier. As long as it is possible, any substance may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are about 0.1 to 20, preferably about 1 by weight ratio to hapten 1. A method of coupling at a rate of ˜6 is used.
In addition, various condensing agents can be used for coupling of the hapten and the carrier, but active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and dithiobilidyl group are used.
The condensation product is administered to a warm-blooded animal by itself, together with a carrier and a diluent, at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually about once every 2 to 6 weeks, about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, etc., preferably from blood of warm-blooded animals immunized by the above method.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as in the case of the monoclonal antibody described above.

　本発明のスクリーニング方法において、上記した本発明のポリペプチドおよびその部分ペプチド、本発明のポリヌクレオチドおよび本発明の抗体などの他に、本発明のポリペプチドまたはその部分ペプチドを産生する細胞を使用することもできる。
　このような細胞としては、例えば、本発明のポリペプチドまたはその部分ペプチドを本来有する細胞（好ましくは、動物細胞）、または本発明のポリペプチドまたはその部分ペプチドをコードするＤＮＡを含有するベクターで形質転換された宿主（形質転換体）などが用いられる。宿主としては、例えば、褐色脂肪細胞、白色脂肪細胞、肝細胞、筋芽細胞、内臓脂肪細胞、腎臓尿細管、マクロファージ、樹状細胞、心筋細胞などの哺乳動物細胞が好ましく用いられる。また、例えば、サル細胞COS-7、Vero、CHO細胞、CHO(dhfr-)細胞、マウスＬ細胞、マウスAtT-20、マウスミエローマ細胞、マウスATDC5細胞、ラットGH3、ヒトFL細胞、ヒト293細胞などの細胞株も使用できる。 In the screening method of the present invention, in addition to the polypeptide of the present invention and its partial peptide, the polynucleotide of the present invention and the antibody of the present invention, a cell producing the polypeptide of the present invention or its partial peptide is used. You can also.
Such cells include, for example, cells originally having the polypeptide of the present invention or a partial peptide thereof (preferably animal cells), or a vector containing a DNA encoding the polypeptide of the present invention or a partial peptide thereof. A transformed host (transformant) or the like is used. As the host, for example, mammalian cells such as brown adipocytes, white adipocytes, hepatocytes, myoblasts, visceral adipocytes, kidney tubules, macrophages, dendritic cells, cardiomyocytes are preferably used. Further, for example, monkey cell COS-7, Vero, CHO cell, CHO (dhfr − ) cell, mouse L cell, mouse AtT-20, mouse myeloma cell, mouse ATDC5 cell, rat GH3, human FL cell, human 293 cell, etc. Other cell lines can also be used.

　上記した本発明のスクリーニング方法において、スクリーニングの対象となる試験対象物質（本明細書中、試験化合物と略記する場合がある）としては、例えば、ペプチド、タンパク質、抗体、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液、血漿などが挙げられる。
　試験化合物が、細胞内トリグリセリド蓄積量に影響するかどうかの確認は、例えば、その試験化合物の存在下に細胞（例えば、脂肪細胞など）を培養した場合の細胞内トリグリセリド蓄積量を測定することによって評価することができる。例えば、後述の実施例２に記載の方法に従って、細胞内トリグリセリド蓄積量を変化させる試験化合物の能力を評価することができる。
　また、トリグリセリドをリポ蛋白リパーゼで水解して生成したグリセロールを比色法やUV法で測定することも可能である。
　本発明のポリペプチドは、ホスファターゼ活性を介して脂肪酸β酸化作用に関与する。そしてこのホスファターゼ活性を阻害することによって細胞内脂肪酸β酸化を促進させることができる。試験化合物の脂肪酸β酸化活性は、例えば、その試験化合物の存在下に細胞（例えば、褐色脂肪細胞）を培養して、細胞内の脂肪酸β酸化活性を測定することによって評価することができる。例えば、後述の実施例７に記載の測定法に従って、細胞内脂肪酸β酸化活性を変化させる試験化合物の能力を評価することができる。
　また、細胞内での脂肪酸β酸化上昇に伴う酸化的リン酸化によるATP産生量の測定、あるいは酸素消費速度の上昇によっても評価できる。 In the above-described screening method of the present invention, examples of test target substances to be screened (sometimes abbreviated as test compounds in the present specification) include peptides, proteins, antibodies, non-peptidic compounds, and synthetic compounds. , Fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like.
Whether or not a test compound affects the amount of intracellular triglyceride accumulated can be confirmed, for example, by measuring the amount of intracellular triglyceride accumulated when cells (eg, adipocytes) are cultured in the presence of the test compound. Can be evaluated. For example, the ability of a test compound to change intracellular triglyceride accumulation can be evaluated according to the method described in Example 2 below.
It is also possible to measure glycerol produced by hydrolyzing triglycerides with lipoprotein lipase by a colorimetric method or UV method.
The polypeptide of the present invention is involved in fatty acid β-oxidation through phosphatase activity. By inhibiting this phosphatase activity, intracellular fatty acid β oxidation can be promoted. The fatty acid β-oxidation activity of a test compound can be evaluated, for example, by culturing cells (for example, brown adipocytes) in the presence of the test compound and measuring the intracellular fatty acid β-oxidation activity. For example, the ability of the test compound to change intracellular fatty acid β-oxidation activity can be evaluated according to the measurement method described in Example 7 described later.
It can also be evaluated by measuring the amount of ATP produced by oxidative phosphorylation accompanying the increase in fatty acid β oxidation in cells, or by increasing the oxygen consumption rate.

　本明細書において、ＩＭＰＡ２関連末梢性疾患の予防・治療剤の有効成分としては、ＩＭＰＡ２の活性を促進または阻害する物質や、ＩＭＰＡ２の発現を促進または阻害する物質が挙げられるが、後述する〔本発明の予防・治療剤〕にあるように、ＩＭＰＡ２の活性または発現を阻害する物質（本明細書中「ＩＭＰＡ２阻害薬」と略記する場合がある。）が望ましい。本発明のスクリーニング方法において、本発明のポリペプチド（好ましくはＩＭＰＡ２）が有するホスファターゼ活性、または細胞内トリグリセリド蓄積作用もしくは脂肪酸β酸化作用を促進または阻害する試験化合物を取得することができる。これら活性または作用によって、本発明のスクリーニング方法によって取得できる試験化合物は、上記のＩＭＰＡ２関連末梢性疾患（特に肥満症）の低毒性な予防剤または治療剤として有用である。
　例えば、本発明のスクリーニング方法によって得られるＩＭＰＡ２活性を抑制する化合物は、ホスファターゼ阻害活性、または細胞内トリグリセリド蓄積量低下作用もしくは脂肪酸β酸化促進作用を有するため、上記のＩＭＰＡ２関連末梢性疾患の予防剤・治療剤として有用である。
　あるいは、本発明のスクリーニング方法において、本発明のポリペプチドであるＩＭＰＡ２が有するミトコンドリア活性またはミトコンドリア量の調節作用を促進または阻害する試験化合物を取得することができる。これら作用によって、本発明のスクリーニング方法によって取得できる化合物は、上記のＩＭＰＡ２関連末梢性疾患の低毒性な予防剤または治療剤として有用である。例えば、当業者は、本明細書の実施例５または６に記載のように、細胞内ミトコンドリア量を測定することができる
　例えば、本発明のスクリーニング方法によって得られるＩＭＰＡ２活性を抑制する化合物は、細胞内ミトコンドリア活性抑制作用、またはミトコンドリア量低下作用によって、上記のＩＭＰＡ２関連末梢性疾患の予防剤または治療剤として有用である。
　あるいは、本発明のスクリーニング方法によって得られるＩＭＰＡ２活性を促進する化合物は、例えば、細胞内トリグリセリド蓄積促進作用、ミトコンドリア活性促進作用、またはミトコンドリア量上昇作用などによって、脂肪分化促進剤として有用である。 In this specification, examples of the active ingredient of the preventive / therapeutic agent for IMPA2-related peripheral diseases include substances that promote or inhibit the activity of IMPA2, and substances that promote or inhibit the expression of IMPA2. As described in the preventive / therapeutic agent of the invention], a substance that inhibits the activity or expression of IMPA2 (sometimes abbreviated as “IMPA2 inhibitor” in the present specification) is desirable. In the screening method of the present invention, a test compound that promotes or inhibits the phosphatase activity, intracellular triglyceride accumulation effect or fatty acid β-oxidation effect possessed by the polypeptide of the present invention (preferably IMPA2) can be obtained. Due to these activities or actions, the test compound obtainable by the screening method of the present invention is useful as a low-toxic preventive or therapeutic agent for the above-mentioned IMPA2-related peripheral diseases (particularly obesity).
For example, since the compound that suppresses the IMPA2 activity obtained by the screening method of the present invention has a phosphatase inhibitory activity, an intracellular triglyceride accumulation lowering action or a fatty acid β oxidation promoting action, the prophylactic agent for the above-mentioned IMPA2-related peripheral diseases -It is useful as a therapeutic agent.
Alternatively, in the screening method of the present invention, a test compound that promotes or inhibits the effect of regulating the mitochondrial activity or mitochondrial amount possessed by the polypeptide of the present invention, IMPA2, can be obtained. Due to these actions, the compound obtainable by the screening method of the present invention is useful as a low-toxic prophylactic or therapeutic agent for the above-mentioned IMPA2-related peripheral diseases. For example, those skilled in the art can measure the amount of intracellular mitochondrion as described in Example 5 or 6 of the present specification. For example, the compound that suppresses the IMPA2 activity obtained by the screening method of the present invention It is useful as a prophylactic or therapeutic agent for the above-mentioned IMPA2-related peripheral diseases due to its action to suppress mitochondrial activity or to reduce the amount of mitochondria.
Alternatively, the compound that promotes the IMPA2 activity obtained by the screening method of the present invention is useful as a fat differentiation promoter by, for example, an intracellular triglyceride accumulation promoting action, a mitochondrial activity promoting action, or a mitochondrial amount increasing action.

　本発明のポリペプチドの活性を測定する場合に用いられる基質としては、ＩＭＰＡ２に対する基質特異性の高い６－ホスホグルコン酸、および前記した６－ホスホグルコン酸の類縁化合物が挙げられる。なかでも、６－ホスホグルコン酸が好ましい。 Examples of the substrate used for measuring the activity of the polypeptide of the present invention include 6-phosphogluconic acid having a high substrate specificity for IMPA2, and the above-mentioned analogs of 6-phosphogluconic acid. Of these, 6-phosphogluconic acid is preferable.

　本発明のスクリーニング方法は、６－ホスホグルコン酸または前記した６－ホスホグルコン酸の類縁化合物を用いることによって、ＩＭＰＡ２阻害薬、好ましくはＩＭＰＡ２を特異的に阻害するＩＭＰＡ２阻害薬のスクリーニングをより効率的なものとする。また、本発明のスクリーニング方法は、ＩＭＰＡ１を対照として用いることにより、ＩＭＰＡ２を特異的に阻害するＩＭＰＡ２阻害薬のスクリーニングをより効率的なものとすることができる。例えば、６－ホスホグルコン酸を用いて、被検液中のＩＭＰＡ２およびＩＭＰＡ１（例えば、国際公開第2006/079792号に記載のヒトＩＭＰＡ１（アミノ酸配列：NP_005527.1、GI:5031789））によるホスファターゼ活性を(i)試験化合物の存在下および(ii)試験化合物の非存在下においてそれぞれ測定すること、ならびに(i)試験化合物の存在下においてＩＭＰＡ２を含む場合のホスファターゼ活性を阻害する試験化合物を単離することを含む方法が挙げられる。
　本発明のポリペプチドのホスファターゼ活性の測定は、上記の公知の方法などにより測定することができる。このような試験化合物は、例えばＩＭＰＡ２に特異性の高い基質の類縁体を含み得る。
　ＩＭＰＡ２に特異性の高い基質は、ＩＭＰＡ２によって脱リン酸化されることが好ましい。ＩＭＰＡ２に特異性の高い基質としては、６－ホスホグルコン酸が挙げられる。 The screening method of the present invention makes it possible to more efficiently screen for an IMPA2 inhibitor, preferably an IMPA2 inhibitor that specifically inhibits IMPA2, by using 6-phosphogluconic acid or a similar compound of 6-phosphogluconic acid. It shall be Moreover, the screening method of the present invention can make screening for an IMPA2 inhibitor that specifically inhibits IMPA2 more efficient by using IMPA1 as a control. For example, phosphatase activity by IMPA2 and IMPA1 (for example, human IMPA1 described in WO 2006/079792 (amino acid sequence: NP — 005527.1, GI: 5031789)) in a test solution using 6-phosphogluconic acid (I) in the presence of the test compound and (ii) in the absence of the test compound, respectively, and (i) isolation of a test compound that inhibits phosphatase activity when containing IMPA2 in the presence of the test compound The method including doing is mentioned.
The phosphatase activity of the polypeptide of the present invention can be measured by the above-mentioned known methods. Such test compounds can include, for example, analogs of substrates that are highly specific for IMPA2.
A substrate having high specificity for IMPA2 is preferably dephosphorylated by IMPA2. An example of a substrate having high specificity for IMPA2 is 6-phosphogluconic acid.

　本発明のポリペプチドもしくはその部分ペプチドまたはその塩は、公知の方法、例えば、本発明の抗体を用いて、細胞抽出液中などに存在する前記ポリペプチドを、ウェスタン解析、ＥＬＩＳＡ法、特にサンドイッチ免疫測定法などの方法またはそれに準じる方法に従い高感度に定量することができる。
　すなわち、本明細書において、
（ｉ）本発明の抗体と、被検液および標識化された本発明のポリペプチドもしくはその部分ペプチドとを競合的に反応させ、該抗体に結合した標識化された本発明のポリペプチドもしくはその部分ペプチドの割合を測定することを特徴とする被検液中の本発明のポリペプチドの定量法、および
（ii）被検液と担体上に不溶化した本発明の抗体および標識化された本発明の別の抗体とを同時あるいは連続的に反応させたのち、不溶化担体上の標識剤の活性を測定することを特徴とする被検液中の本発明のポリペプチドの定量法が提供される。
　上記（ii）の定量法においては、一方の抗体が本発明のポリペプチドもしくはその部分ペプチドのＮ端部を認識する抗体で、他方の抗体が本発明のポリペプチドもしくはその部分ペプチドのＣ端部に反応する抗体であることが望ましい。
　また、本発明のポリペプチドもしくはその部分ペプチドに対するモノクローナル抗体（以下、本発明のモノクローナル抗体と称する場合がある）を用いて本発明のポリペプチドの定量を行なえるほか、組織染色等による本発明のポリペプチドの検出を行なうこともできる。これらの目的には、抗体分子そのものを用いてもよく、また、抗体分子のF(ab')2 、Fab'またはFab画分を用いてもよい。
　本発明の抗体を用いる本発明のポリペプチドの定量法は、特に制限されるべきものではなく、被測定液中の抗原量（例えば、ポリペプチド量）に対応した抗体、抗原もしくは抗体－抗原複合体の量を化学的または物理的手段により検出し、これを既知量の抗原を含む標準液を用いて作製した標準曲線より算出する測定法であれば、いずれの測定法を用いてもよい。例えば、ネフロメトリー、競合法、イムノメトリック法およびサンドイッチ法が好適に用いられるが、感度、特異性の点で、後述するサンドイッチ法を用いるのが特に好ましい。 The polypeptide of the present invention or a partial peptide thereof or a salt thereof can be obtained by a known method, for example, using the antibody of the present invention, the polypeptide present in a cell extract or the like by Western analysis, ELISA method, particularly sandwich immunization. It can be quantified with high sensitivity according to a method such as a measurement method or a method analogous thereto.
That is, in this specification,
(I) The labeled polypeptide of the present invention or the labeled polypeptide bound to the antibody by competitively reacting the antibody of the present invention with the test solution and the labeled polypeptide of the present invention or a partial peptide thereof A method for quantifying the polypeptide of the present invention in a test solution, characterized by measuring the proportion of a partial peptide, and (ii) the antibody of the present invention insolubilized on the test solution and a carrier and the labeled present invention There is provided a method for quantifying the polypeptide of the present invention in a test solution, wherein the activity of a labeling agent on an insolubilized carrier is measured after reacting with another antibody simultaneously or continuously.
In the quantification method of (ii) above, one antibody is an antibody that recognizes the N-terminal part of the polypeptide of the present invention or a partial peptide thereof, and the other antibody is the C-terminal part of the polypeptide of the present invention or its partial peptide It is desirable that the antibody be reactive with.
Further, the polypeptide of the present invention can be quantified using a monoclonal antibody against the polypeptide of the present invention or a partial peptide thereof (hereinafter sometimes referred to as the monoclonal antibody of the present invention). Polypeptide detection can also be performed. For these purposes, the antibody molecule itself may be used, or F (ab ′) 2 , Fab ′ or Fab fraction of the antibody molecule may be used.
The method for quantifying the polypeptide of the present invention using the antibody of the present invention is not particularly limited, and an antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (eg, amount of polypeptide) in the solution to be measured. Any measurement method may be used as long as it is a measurement method in which the amount of the body is detected by a chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but the sandwich method described later is particularly preferable in view of sensitivity and specificity.

　本発明のポリペプチドもしくはその部分ペプチドおよび本発明の抗体の標識化に用いられる標識剤としては、例えば、放射性同位元素（例、〔125I〕、〔131I〕、〔3H〕、〔14C〕、〔32P〕、〔33P〕、〔35S〕など）、蛍光物質〔例、シアニン蛍光色素（例、Cy2、Cy3、Cy5、Cy5.5、Cy7（アマシャムバイオサイエンス社製）など）、フルオレスカミン、フルオレッセンイソチオシアネート、NBD (7-nitrobenz-2-oxa-1,3-diazol)など〕、酵素（例、β－ガラクトシダーゼ、β－グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素など）、発光物質（例、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなど）、ビオチン、ランタニド元素などが用いられる。さらに、抗体または抗原と標識剤との結合にビオチン－アビジン系を用いることもできる。 Examples of the labeling agent used for labeling the polypeptide of the present invention or a partial peptide thereof and the antibody of the present invention include radioisotopes (eg, [ 125 I], [ 131 I], [ 3 H], [ 14 C], [ 32 P], [ 33 P], [ 35 S], etc.), fluorescent substance [eg, cyanine fluorescent dye (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (made by Amersham Biosciences), etc.) ), Fluorescamine, fluorescein isothiocyanate, NBD (7-nitrobenz-2-oxa-1,3-diazol), etc.], enzymes (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malic acid) Dehydrogenase, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), biotin, lanthanide elements, etc. are used. Furthermore, a biotin-avidin system can also be used for binding an antibody or antigen to a labeling agent.

　抗原あるいは抗体の不溶化に当っては、物理吸着を用いてもよく、また通常ポリペプチドあるいは酵素等を不溶化、固定化するのに用いられる化学結合を用いる方法でもよい。担体としては、アガロース、デキストラン、セルロースなどの不溶性多糖類、ポリスチレン、ポリアクリルアミド、シリコン等の合成樹脂、あるいはガラス等が挙げられる。
　サンドイッチ法においては不溶化した本発明のモノクローナル抗体に被検液を反応させ（１次反応）、さらに標識化した別の本発明のモノクローナル抗体を反応させ（２次反応）たのち、不溶化担体上の標識剤の活性を測定することにより被検液中の本発明のポリペプチドを定量することができる。１次反応と２次反応は逆の順序に行っても、また、同時に行なってもよいし時間をずらして行なってもよい。標識化剤および不溶化の方法は前記のそれらに準じることができる。また、サンドイッチ法による本発明のポリペプチドの測定法において、固相用抗体あるいは標識用抗体に用いられる抗体は必ずしも１種類である必要はなく、測定感度を向上させる等の目的で２種類以上の抗体の混合物を用いてもよい。
　サンドイッチ法による本発明のポリペプチドの測定法において、１次反応と２次反応に用いられる本発明のモノクローナル抗体としては、本発明のポリペプチドもしくはその部分ペプチドの結合する部位が相異なる抗体が好ましく用いられる。例えば、２次反応で用いられる抗体が、本発明のポリペプチドもしくはその部分ペプチドのＣ端部を認識する場合、１次反応で用いられる抗体は、好ましくはＣ端部以外、例えばＮ端部を認識する抗体である。 For insolubilization of an antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a polypeptide or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further labeled with another monoclonal antibody of the present invention (secondary reaction), and then on the insolubilized carrier. By measuring the activity of the labeling agent, the polypeptide of the present invention in the test solution can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times. The labeling agent and the insolubilization method can be the same as those described above. In the method for measuring the polypeptide of the present invention by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and two or more types are used for the purpose of improving measurement sensitivity. A mixture of antibodies may be used.
In the method for measuring the polypeptide of the present invention by the sandwich method, the monoclonal antibody of the present invention used for the primary reaction and the secondary reaction is preferably an antibody having a different site to which the polypeptide of the present invention or its partial peptide binds. Used. For example, when the antibody used in the secondary reaction recognizes the C-terminal part of the polypeptide of the present invention or a partial peptide thereof, the antibody used in the primary reaction preferably has a non-C-terminal part, for example, an N-terminal part. Recognizing antibody.

　本発明のモノクローナル抗体をサンドイッチ法以外の測定法、例えば、競合法、イムノメトリック法あるいはネフロメトリーなどに用いることができる。
　競合法では、被検液中の抗原と標識抗原とを抗体に対して競合的に反応させたのち、未反応の標識抗原（Ｆ）と、抗体と結合した標識抗原（Ｂ）とを分離し（Ｂ／Ｆ分離）、Ｂ，Ｆいずれかの標識量を測定し、被検液中の抗原量を定量する。本反応法には、抗体として可溶性抗体を用い、Ｂ／Ｆ分離をポリエチレングリコール、前記抗体に対する第２抗体などを用いる液相法、および、第１抗体として固相化抗体を用いるか、あるいは、第１抗体は可溶性のものを用い第２抗体として固相化抗体を用いる固相化法とが用いられる。
　イムノメトリック法では、被検液中の抗原と固相化抗原とを一定量の標識化抗体に対して競合反応させた後、固相と液相を分離するか、あるいは、被検液中の抗原と過剰量の標識化抗体とを反応させ、次に固相化抗原を加え未反応の標識化抗体を固相に結合させたのち、固相と液相を分離する。次に、いずれかの相の標識量を測定し被検液中の抗原量を定量する。
　また、ネフロメトリーでは、ゲル内あるいは溶液中で抗原抗体反応の結果生じた不溶性の沈降物の量を測定する。被検液中の抗原量が僅かであり、少量の沈降物しか得られない場合にもレーザーの散乱を利用するレーザーネフロメトリーなどが好適に用いられる。 The monoclonal antibody of the present invention can be used for measurement methods other than the sandwich method, for example, competition method, immunometric method, nephrometry and the like.
In the competitive method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid-phased antibody is used as the first antibody, or As the first antibody, a soluble method is used, and a solid phase method using a solid phase antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are competitively reacted with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or After reacting the antigen with an excess amount of labeled antibody, and then adding a solid-phased antigen to bind unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the test solution.
In nephrometry, the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained.

　これら個々の測定法を本発明のポリペプチドの定量法に適用するにあたっては、特別の条件、操作等の設定は必要とされない。それぞれの方法における通常の条件、操作法に当業者の通常の技術的配慮を加えて本発明のポリペプチドの測定系を構築すればよい。これらの一般的な技術手段の詳細については、　例えば、入江　寛編「ラジオイムノアッセイ」（講談社、昭和４９年発行）、入江　寛編「続ラジオイムノアッセイ」（講談社、昭和５４年発行）、石川栄治ら編「酵素免疫測定法」（医学書院、昭和５３年発行）、石川栄治ら編「酵素免疫測定法」（第２版）（医学書院、昭和５７年発行）、石川栄治ら編「酵素免疫測定法」（第３版）（医学書院、昭和６２年発行）、「Methods in ENZYMOLOGY」 Vol. 70(Immunochemical Techniques(Part A))、 同書 Vol. 73(Immunochemical Techniques(Part B))、 同書 Vol. 74(Immunochemical Techniques(Part C))、 同書 Vol. 84(Immunochemical Techniques(Part D:Selected Immunoassays))、 同書 Vol. 92(Immunochemical Techniques(Part E:Monoclonal Antibodies and General Immunoassay Methods))、同書 Vol. 121(Immunochemical Techniques(Part I:Hybridoma Technology and Monoclonal Antibodies))（以上、アカデミックプレス社発行）などを参照することができる。
　以上のようにして、本発明の抗体を用いることによって、本発明のポリペプチドを感度良く定量することができる。 In applying these individual measurement methods to the polypeptide quantification method of the present invention, special conditions, operations, and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of the polypeptide of this invention, adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, see, for example, Hiroshi Irie “Radioimmunoassay” (Kodansha, issued in 1974), Hiroshi Irie “Continue Radioimmunoassay” (Kodansha, issued in 1974), Eiji Ishikawa et al. Edited by “Enzyme Immunoassay” (Medical School, published in 1978), edited by Eiji Ishikawa et al. “Enzyme Immunoassay” (2nd edition) (Medical School, published in 1982), edited by Eiji Ishikawa et al. "Method" (3rd edition) (Medical Shoin, published in 1987), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), ibid. Vol. 73 (Immunochemical Techniques (Part B)), ibid. Vol. 74 (Immunochemical Techniques (Part C)), ibid. Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid. (Immunochemical Techniques (Part I: Hybridoma Technology and Mono clonal Antibodies)) (published by Academic Press).
As described above, the polypeptide of the present invention can be quantified with high sensitivity by using the antibody of the present invention.

　本発明のスクリーニング方法の内、前記〔１〕のスクリーニング方法、すなわち、
〔１〕　配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むポリペプチドもしくはその部分ペプチドまたはその塩、あるいは該ポリペプチドまたはその部分ペプチドを産生する細胞を用いることを特徴とする、ＩＭＰＡ２関連末梢性疾患の予防・治療剤のスクリーニング方法
の実施態様として、以下の〔１ａ〕～〔１ｄ〕のスクリーニング方法が挙げられる。
〔１ａ〕　配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むポリペプチドもしくはその部分ペプチドまたはその塩のホスファターゼ活性を、(i)試験化合物の存在下および(ii)試験化合物の非存在下において測定すること、ならびに(i)および(ii)を比較することを含む、〔１〕記載のスクリーニング方法。
〔１ｂ〕　前記細胞内のトリグリセリド蓄積量または脂肪酸β酸化活性を(i)試験化合物の存在下および(ii)試験化合物の非存在下において測定すること、ならびに(i)および(ii)を比較することを含む、〔１〕記載のスクリーニング方法。
〔１ｃ〕　前記細胞内のミトコンドリア代謝活性またはミトコンドリア量を(i)試験化合物の存在下および(ii)試験化合物の非存在下において測定すること、ならびに(i)および(ii)を比較することを含む、〔１〕記載のスクリーニング方法。
〔１ｄ〕　前記細胞を有する動物におけるトリグリセリド蓄積量、体脂肪量、血糖値、血中脂質レベル、血中コレステロールレベル、全身のエネルギー消費量、酸素消費量、呼吸商および血圧からなる群より選択される少なくとも１つの項目を、(i)試験化合物の投与および(ii)試験化合物の非投与において測定すること、ならびに(i)および(ii)を比較することを含む、〔１〕記載のスクリーニング方法。 Among the screening methods of the present invention, the screening method of [1] above,
[1] A polypeptide comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, or a partial peptide thereof, or a salt thereof, or the polypeptide or a peptide thereof Examples of the screening method for a prophylactic / therapeutic agent for IMPA2-related peripheral diseases characterized by using cells that produce partial peptides include the following screening methods [1a] to [1d].
[1a] The phosphatase activity of a polypeptide comprising the amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11 or a partial peptide thereof or a salt thereof (i) (1) The screening method according to [1], comprising measuring in the presence of the test compound and (ii) in the absence of the test compound, and comparing (i) and (ii).
[1b] Measuring the amount of triglyceride accumulation or fatty acid β-oxidation activity in the cells (i) in the presence of the test compound and (ii) in the absence of the test compound, and comparing (i) and (ii) The screening method according to [1].
[1c] Measuring the intracellular mitochondrial metabolic activity or mitochondrial amount in the cells (i) in the presence of the test compound and (ii) in the absence of the test compound, and comparing (i) and (ii) The screening method according to [1].
[1d] selected from the group consisting of triglyceride accumulation amount, body fat amount, blood glucose level, blood lipid level, blood cholesterol level, whole body energy consumption, oxygen consumption, respiratory quotient and blood pressure in the animal having the cells The screening method according to [1], comprising measuring at least one item in (i) administration of a test compound and (ii) non-administration of a test compound, and comparing (i) and (ii) .

　前記のとおり、本発明のポリペプチドもしくはその部分ペプチドまたはその塩のホスファターゼ活性は、公知の方法、例えば、後述の実施例４もしくは９に記載の測定法、非特許文献１（Cryns, K.ら、Neuropsycho-pharmacology 32, 881-891, 2007）、特許文献２（国際公開第2006/079792号パンフレット）に記載の方法またはそれに準ずる方法によって測定することができる。
　本発明のスクリーニング方法において、試験化合物が存在する場合の本発明のポリペプチドもしくはその部分ペプチドまたはその塩のホスファターゼ活性を、(i)試験化合物の存在下および(ii)試験化合物の非存在下において測定し、(i)と(ii)との場合を比較して有意に低下させる化合物を選択することができ、好ましくは約２０％以上、約３０％以上、より好ましくは約５０％以上、さらにより好ましくは約６０％以上減少させる化合物を、ＩＭＰＡ２関連末梢性疾患の予防・治療剤として選択することができる。
　本発明のポリペプチドもしくはその部分ペプチドまたはその塩の量は、公知の方法、例えば、本発明の抗体を用いて、細胞抽出液中などに存在する前記ポリペプチドを、ウエスタンブロッティング解析、エンザイムイムノアッセイ法などの方法またはそれに準じる方法に従い測定することができる。 As described above, the phosphatase activity of the polypeptide of the present invention or a partial peptide thereof or a salt thereof can be determined by a known method, for example, the measurement method described in Example 4 or 9 described later, Non-Patent Document 1 (Cryns, K. et al. , Neuropsycho-pharmacology 32, 881-891, 2007), Patent Document 2 (International Publication No. 2006/079792 pamphlet) or a method analogous thereto.
In the screening method of the present invention, the phosphatase activity of the polypeptide of the present invention or a partial peptide thereof or a salt thereof in the presence of a test compound is determined in the presence of (i) the test compound and (ii) the absence of the test compound. And a compound that significantly lowers the cases of (i) and (ii) can be selected, preferably about 20% or more, about 30% or more, more preferably about 50% or more, More preferably, a compound that decreases about 60% or more can be selected as an agent for preventing / treating IMPA2-related peripheral diseases.
The amount of the polypeptide of the present invention or a partial peptide thereof or a salt thereof can be determined by a known method, for example, Western blotting analysis or enzyme immunoassay method for the polypeptide present in a cell extract using the antibody of the present invention. It can measure according to the method of following or the method according to it.

　本発明のスクリーニング方法において使用される、本発明のポリペプチドもしくはその部分ペプチドまたはその塩は、ホスファターゼ活性を介して例えば細胞内トリグリセリド蓄積作用に関与する。そしてこのホスファターゼ活性を阻害することによって細胞内トリグリセリド蓄積量を低下させることができる。試験化合物の細胞内トリグリセリド蓄積量は、例えば、その試験化合物の存在下に細胞（例えば、分化脂肪細胞）を培養した際の細胞内トリグリセリド蓄積量を測定することによって評価することができる。例えば、後述の実施例２に記載の測定法などに従って、細胞内トリグリセリド蓄積量を変化させる試験化合物の能力を評価することができる。
　本発明のポリペプチドもしくはその部分ペプチドまたはその塩は、ホスファターゼ活性を介して例えば脂肪酸β酸化作用に関与する。そしてこのホスファターゼ活性を阻害することによって細胞内脂肪酸β酸化を促進させることができる。試験化合物の脂肪酸β酸化活性は、例えば、本発明のポリペプチドまたはその部分ペプチドを産生する細胞（例えば、褐色脂肪細胞）を培養して、細胞内の脂肪酸β酸化活性を(i)試験化合物の存在下および(ii)試験化合物の非存在下において測定し、これらの測定値を比較することによって評価することができる。例えば、後述の実施例７に記載の測定法に従って、細胞内脂肪酸β酸化活性を変化させる試験化合物の能力を評価することができる。
　本発明のスクリーニング方法において、細胞内トリグリセリド蓄積量を、(i)試験化合物の存在下および(ii)試験化合物の非存在下において測定し、(i)と(ii)との場合を比較して有意に低下させる試験化合物を選択することができ、好ましくは約２０％以上、約３０％以上、より好ましくは約５０％以上、さらにより好ましくは約６０％以上低下させる試験化合物を、本発明のＩＭＰＡ２関連末梢性疾患の予防・治療剤として選択することができる。
　または、本発明のスクリーニング方法において、細胞内脂肪酸β酸化活性を、(i)試験化合物の存在下および(ii)試験化合物の非存在下において測定し、(i)と(ii)との場合を比較して有意に上昇させる試験化合物を選択することができ、好ましくは約２０％以上、約３０％以上、より好ましくは約５０％以上、さらにより好ましくは約６０％以上促進させる試験化合物を、ＩＭＰＡ２関連末梢性疾患の予防・治療剤として選択することができる。 The polypeptide of the present invention or a partial peptide thereof or a salt thereof used in the screening method of the present invention is involved in, for example, intracellular triglyceride accumulation action through phosphatase activity. By inhibiting this phosphatase activity, the amount of intracellular triglyceride accumulation can be reduced. The amount of intracellular triglyceride accumulated in a test compound can be evaluated, for example, by measuring the amount of intracellular triglyceride accumulated when cells (for example, differentiated adipocytes) are cultured in the presence of the test compound. For example, the ability of the test compound to change the amount of intracellular triglyceride accumulation can be evaluated according to the measurement method described in Example 2 described later.
The polypeptide of the present invention or a partial peptide thereof or a salt thereof is involved in, for example, fatty acid β oxidation through phosphatase activity. By inhibiting this phosphatase activity, intracellular fatty acid β oxidation can be promoted. The fatty acid β-oxidation activity of the test compound can be determined, for example, by culturing cells (for example, brown adipocytes) that produce the polypeptide of the present invention or a partial peptide thereof, It can be evaluated by measuring in the presence and (ii) in the absence of the test compound and comparing these measurements. For example, the ability of the test compound to change intracellular fatty acid β-oxidation activity can be evaluated according to the measurement method described in Example 7 described later.
In the screening method of the present invention, intracellular triglyceride accumulation was measured in the presence of (i) the test compound and (ii) in the absence of the test compound, and the cases of (i) and (ii) were compared. Test compounds that can be significantly reduced can be selected, preferably test compounds that reduce about 20% or more, about 30% or more, more preferably about 50% or more, and even more preferably about 60% or more of the present invention. It can be selected as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases.
Alternatively, in the screening method of the present invention, intracellular fatty acid β-oxidation activity is measured in the presence of (i) the test compound and (ii) in the absence of the test compound, and the cases of (i) and (ii) Test compounds that can be significantly elevated relative to each other can be selected, and preferably test compounds that promote about 20% or more, about 30% or more, more preferably about 50% or more, and even more preferably about 60% or more, It can be selected as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases.

　本発明のポリペプチドまたはその部分ペプチドの発現量は、細胞内ミトコンドリア代謝活性またはミトコンドリア量と相関する。したがって、試験化合物の存在下および非存在下において、細胞内ミトコンドリア代謝活性またはミトコンドリア量を測定し、比較することによっても、ＩＭＰＡ２関連末梢性疾患の予防・治療剤をスクリーニングすることができる。細胞内ミトコンドリア代謝活性を測定する方法としては、公知の方法、例えば、Methods in Cell Biology(Volume 80, Mitochondrai 2ndedition)に記載される、呼吸鎖複合体の活性、ATP、乳酸、ピルビン酸産生量、TCA回路の酵素活性、酸素消費量などを測定する方法が挙げられる。また、細胞内ミトコンドリア量を測定する方法としては、例えばミトコンドリアＤＮＡ量を定量的ＰＣＲなどによって測定する方法が挙げられる。
　より詳細には、本発明のスクリーニング方法において、細胞内ミトコンドリアの代謝活性またはミトコンドリア量を、(i)試験化合物の存在下および(ii)試験化合物の非存在下において測定し、(i)と(ii)との場合を比較して有意に上昇させる試験化合物を選択することができ、好ましくは約２０％以上、約３０％以上、より好ましくは約５０％以上、さらにより好ましくは約６０％以上低下させる試験化合物を、ＩＭＰＡ２関連末梢性疾患の予防・治療剤として選択することができる。 The expression level of the polypeptide of the present invention or a partial peptide thereof correlates with intracellular mitochondrial metabolic activity or mitochondrial level. Therefore, a prophylactic / therapeutic agent for IMPA2-related peripheral diseases can also be screened by measuring and comparing intracellular mitochondrial metabolic activity or mitochondrial amount in the presence and absence of a test compound. As a method for measuring intracellular mitochondrial metabolic activity, a known method, for example, Methods in Cell Biology (Volume 80 , Mitochondrai 2 nd edition) is described, the activity of the respiratory chain complexes, ATP, lactic, pyruvic acid production And methods for measuring the amount, enzyme activity of the TCA cycle, oxygen consumption, and the like. Moreover, as a method for measuring the amount of intracellular mitochondria, for example, a method of measuring the amount of mitochondrial DNA by quantitative PCR or the like can be mentioned.
More specifically, in the screening method of the present invention, intracellular mitochondrial metabolic activity or mitochondrial amount is measured in the presence of (i) the presence of the test compound and (ii) in the absence of the test compound, and (i) and ( A test compound can be selected that significantly increases compared to ii), preferably about 20% or more, about 30% or more, more preferably about 50% or more, even more preferably about 60% or more. The test compound to be reduced can be selected as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases.

　また、本発明のスクリーニング方法において、試験化合物を、本発明のポリペプチドまたはその部分ペプチドを産生する細胞を有する、被験動物（マウス、ラット、ウサギ、イヌ、ブタ、サルなど）に投与して、該被験動物におけるトリグリセリド蓄積量、体脂肪量、血糖値、血中脂質レベル、血中コレステロールレベル、全身のエネルギー消費量、酸素消費量、呼吸商および血圧からなる群より選択される少なくとも１つの項目を測定することによって、ＩＭＰＡ２関連末梢性疾患の予防・治療剤をスクリーニングすることができる。例えば、上記の細胞内トリグリセリド蓄積量が関与する疾患または細胞内ミトコンドリア活性が関与する疾患の予防・治療剤としての試験化合物の能力については、これら各疾患のモデル動物を用いて(i)試験化合物を投与した群と、(ii)非投与群またはプラセボ投与群と比較して、各疾患の重篤度を測定することによって評価できる。上記の各疾患の重篤度およびその評価方法については、当該分野で公知の基準を用いることができる。
　具体的には、本発明のスクリーニング方法において、(i)試験化合物の投与群および(ii)試験化合物の非投与群またはプラセボ投与群の、全身のエネルギー消費量または酸素消費量を測定し、(i)と(ii)との群を比較して有意に上昇させる試験化合物を、ＩＭＰＡ２関連末梢性疾患の予防・治療剤として選択することができる。あるいは、本発明のスクリーニング方法において、(i)試験化合物の投与群および(ii)試験化合物の非投与群またはプラセボ投与群の、トリグリセリド蓄積量、体脂肪量、血糖値、血中脂質レベル、血中コレステロールレベル、呼吸商または血圧を測定し、(i)と(ii)との群を比較して有意に下降させる試験化合物を、ＩＭＰＡ２関連末梢性疾患の予防・治療剤として選択することができる。
　被検動物に試験化合物を投与する方法としては、例えば、経口投与、静脈注射などが用いられ、被検動物の症状、試験化合物の性質などにあわせて適宜選択することができる。また、試験化合物の投与量は、投与方法、試験化合物の性質などにあわせて適宜選択することができる。
　上記の測定される項目のうち、例えば、トリグリセリド蓄積量は、血清中のトリグリセリド濃度を種々の試薬キット、臨床検査キット（例えば、和光純薬、「Ｌタイプワコー　ＴＧ・Ｍ」など）で測定することにより、評価することができる。
　体脂肪量は、例えば体脂肪計を用いて測定することができる。
　血糖値は、専用の医療用血糖値測定機器、血糖値測定キット（例えば、グルコースＣII－テストワコー試薬など）などによって測定することができる。
　血中脂質レベルおよび血中コレステロールレベルは、例えば、市販の測定キットを利用するなど、公知の方法に従って測定することができる。
　全身のエネルギー消費量は、例えば、発生した熱量を水などを用いて直接測定する直接法、体内で利用した酸素の消費量から間接的に熱量を推定する間接法などによって測定される。
　酸素消費量は、例えば、呼気ガスの分析によって測定される。
　呼吸商は、例えば、呼気ガス分析によって酸素消費量と二酸化炭素産出量とを測定することによって導出され、主なエネルギー源となる物質が糖質、脂質、ポリペプチドなどのカロリー源のいずれかを間接的に推定できる。血圧は、例えば血圧計を用いて測定される。 In the screening method of the present invention, a test compound is administered to a test animal (mouse, rat, rabbit, dog, pig, monkey, etc.) having cells producing the polypeptide of the present invention or a partial peptide thereof, At least one item selected from the group consisting of triglyceride accumulation amount, body fat amount, blood glucose level, blood lipid level, blood cholesterol level, systemic energy consumption, oxygen consumption, respiratory quotient and blood pressure in the subject animal Can be screened for prophylactic / therapeutic agents for IMPA2-related peripheral diseases. For example, with regard to the ability of a test compound as a prophylactic / therapeutic agent for a disease involving the above-mentioned intracellular triglyceride accumulation amount or a disease involving intracellular mitochondrial activity, (i) a test compound And (ii) a non-administered group or a placebo-administered group can be evaluated by measuring the severity of each disease. Standards known in the art can be used for the severity of each of the above-mentioned diseases and the evaluation method thereof.
Specifically, in the screening method of the present invention, the systemic energy consumption or oxygen consumption of (i) the test compound administration group and (ii) the test compound non-administration group or placebo administration group is measured, A test compound that significantly increases the i) and (ii) groups can be selected as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases. Alternatively, in the screening method of the present invention, the amount of triglyceride accumulated, body fat mass, blood glucose level, blood lipid level, blood of (i) test compound administration group and (ii) non-test compound administration group or placebo administration group Test compounds that measure medium cholesterol level, respiratory quotient or blood pressure and significantly lower (i) and (ii) groups can be selected as prophylactic / therapeutic agents for IMPA2-related peripheral diseases .
As a method for administering the test compound to the test animal, for example, oral administration, intravenous injection and the like are used, and can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dosage of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
Among the items to be measured, for example, the amount of triglyceride accumulated is determined by measuring the triglyceride concentration in serum with various reagent kits and clinical test kits (for example, Wako Pure Chemicals, “L-type Wako TG / M”, etc.). This can be evaluated.
The body fat mass can be measured using, for example, a body fat scale.
The blood glucose level can be measured by a dedicated medical blood glucose level measuring device, a blood glucose level measuring kit (for example, glucose CII-Test Wako reagent, etc.) and the like.
The blood lipid level and blood cholesterol level can be measured according to a known method, for example, using a commercially available measurement kit.
The whole-body energy consumption is measured by, for example, a direct method in which the amount of generated heat is directly measured using water or the like, or an indirect method in which the amount of heat is indirectly estimated from the amount of oxygen consumed in the body.
The oxygen consumption is measured, for example, by analysis of exhaled gas.
The respiratory quotient is derived, for example, by measuring oxygen consumption and carbon dioxide output by exhaled gas analysis, and the main energy source is one of caloric sources such as carbohydrates, lipids, and polypeptides. Can be estimated indirectly. The blood pressure is measured using, for example, a sphygmomanometer.

　また、本発明は、以下の〔５〕のスクリーニング方法も提供する。
〔５〕　配列番号：４、６、８、１０または１２で示される塩基配列と同一または実質的に同一な塩基配列を含むポリヌクレオチドが不活性化された、該ポリヌクレオチド発現不全非ヒト哺乳動物を用いることを特徴とする、脂肪分化促進剤のスクリーニング方法。
　配列番号：４、６、８、１０または１２で示される塩基配列と同一または実質的に同一な塩基配列を含むポリヌクレオチド（本明細書中、本発明の非ヒトポリヌクレオチドと略記する場合がある）が不活性化された、該ポリヌクレオチドが発現不能な非ヒト哺乳動物（本明細書中、本発明のポリヌクレオチド発現不全非ヒト哺乳動物と略記する場合がある）は、自体公知の方法に従って作出できるが、例えば、当該ポリヌクレオチドを含む遺伝子またはそれに関係するプロモーターなどの遺伝子中に、突然変異を導入すること（当該ポリヌクレオチドの塩基配列の一部又は全部の削除、他のポリヌクレオチドを挿入または置換させることにより、コドンの読み取り枠をずらしたり、プロモーターあるいはエキソンの機能を破壊したりすること等）によって作出することができる。 The present invention also provides the following screening method [5].
[5] Non-human mammal deficient in the expression of a polynucleotide, wherein a polynucleotide comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12 is inactivated A method for screening for an agent for promoting fat differentiation, characterized in that
A polynucleotide comprising the same or substantially the same base sequence as shown in SEQ ID NO: 4, 6, 8, 10 or 12 (in this specification, it may be abbreviated as the non-human polynucleotide of the present invention) ) Is inactivated, and the non-human mammal incapable of expressing the polynucleotide (in the present specification, may be abbreviated as a non-human mammal deficient in the expression of the polynucleotide of the present invention) according to a method known per se For example, introducing a mutation into a gene such as a gene containing the polynucleotide or a promoter related thereto (deletion of a part or all of the nucleotide sequence of the polynucleotide, insertion of another polynucleotide) Or, by substituting, the reading frame of the codon is shifted, or the function of the promoter or exon is destroyed. ) It can be produced by.

　本発明の非ヒトポリヌクレオチドとしては、（ａ）配列番号：３、５、７、９または１１で示されるアミノ酸配列を含むポリペプチドをコードするポリヌクレオチド（好ましくは、ＤＮＡ）、または（ｂ）配列番号：４、６、８、１０または１２で示される塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）が好ましく、（ｃ）配列番号：３、５、７、９または１１で示されるアミノ酸配列からなるポリペプチドをコードするポリヌクレオチド（好ましくは、ＤＮＡ）、または（ｄ）配列番号：４、６、８、１０または１２で示される塩基配列からなるポリヌクレオチド（好ましくは、ＤＮＡ）がより好ましい。
　配列番号：４、６、８、１０または１２で示される塩基配列と実質的に同一な塩基配列を含むポリヌクレオチドとしては、配列番号：４、６、８、１０または１２で示される塩基配列とハイストリンジェントな条件下でハイブリダイズする塩基配列を含有し、配列番号：３、５、７、９または１１で示されるアミノ酸配列を含むポリペプチドと実質的に同質の性質を有するポリペプチドをコードするポリヌクレオチド（好ましくは、ＤＮＡ）などが挙げられる。具体的には、配列番号：４、６、８、１０または１２で示される塩基配列と約６０％以上、約７０％以上、約８０％以上、約９０％以上、約９５％以上、約９９％以上の相同性を有する塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）が挙げられる。
　本発明のポリヌクレオチド発現不全非ヒト哺乳動物を作出するのに用いられる非ヒト哺乳動物としては、マウス、ラット、モルモット、ハムスター、ウシ、ブタ、ヒツジ、ヤギ、ウサギ、イヌ、ネコなどが挙げられる。なかでも、病体動物モデル系の作成の面から個体発生および生物サイクルが比較的短く、また、繁殖が容易なゲッ歯動物、とりわけマウス（例えば、純系として、Ｃ５７ＢＬ／６系統，ＤＢＡ２系統など、交雑系として、Ｂ６Ｃ３Ｆ１系統，ＢＤＦ１系統，Ｂ６Ｄ２Ｆ１系統，ＢＡＬＢ／ｃ系統，ＩＣＲ系統など）またはラット（例えば、Ｗｉｓｔａｒ，ＳＤなど）などが好ましい。
　試験化合物としては、例えば、ペプチド、タンパク質、抗体、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液、血漿などが挙げられる。 The non-human polynucleotide of the present invention includes (a) a polynucleotide (preferably DNA) encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, 5, 7, 9 or 11, or (b) A polynucleotide (preferably DNA) containing the base sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12 is preferred, and (c) the amino acid sequence represented by SEQ ID NO: 3, 5, 7, 9 or 11 More preferred is a polynucleotide (preferably DNA) encoding a polypeptide consisting of: (d) a polynucleotide comprising the base sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12 (preferably DNA) .
Examples of the polynucleotide containing the nucleotide sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12 include the nucleotide sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12. A polypeptide comprising a base sequence that hybridizes under highly stringent conditions and having substantially the same quality as a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, 5, 7, 9 or 11 And a polynucleotide (preferably DNA). Specifically, it is about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 99 with the base sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12. And a polynucleotide (preferably DNA) containing a base sequence having a homology of at least%.
Examples of the non-human mammal used for producing the non-human mammal deficient in polynucleotide expression of the present invention include mouse, rat, guinea pig, hamster, cow, pig, sheep, goat, rabbit, dog and cat. . Among them, rodents, especially mice (for example, C57BL / 6 strain, DBA2 strain, etc. as pure strains, etc.) that are relatively short in ontogenesis and biological cycle from the viewpoint of creating a disease animal model system and that are easy to reproduce. As the system, B6C3F 1 system, BDF 1 system, B6D2F 1 system, BALB / c system, ICR system, etc.) or rats (for example, Wistar, SD, etc.) are preferable.
Examples of the test compound include peptides, proteins, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like.

　本発明のポリヌクレオチド発現不全非ヒト哺乳動物は、脂肪分化促進剤のスクリーニングに用いることができる。具体的には、本発明のポリヌクレオチド発現不全非ヒト哺乳動物に、試験化合物を投与（投与方法としては、試験動物の症状、試験化合物の性質などにあわせて適宜選択することができるが、例えば、経口投与、静脈注射、筋肉注射などにより投与）し、該動物の変化を観察・測定すること（例えば、該動物を無処理の対照動物と、各器官・組織・疾病の症状などの変化を比較すること）により、脂肪分化促進剤をスクリーニングすることができる。
　脂肪分化促進剤としての評価は、例えば、上記スクリーニング方法の試験化合物を用いた場合の本発明のポリペプチドまたはその部分ペプチドを発現しない細胞内または個体における、対照の細胞または被験体と比較した脂肪蓄積促進作用、血糖値、血中脂質レベルおよび／または血中コレステロールレベルの変化などに基づき評価することができる。このような方法によって得られる化合物は、上記〔１ｄ〕の被験動物における方法において説明されるように、例えばトリグリセリド蓄積量、体脂肪量、血糖値、血中脂質レベル、血中コレステロールレベル、全身のエネルギー消費量、酸素消費量、呼吸商および血圧などを変化させる能力について評価できる。
　なお、脂肪分化促進剤は、肥大化脂肪細胞から脂肪細胞への分化を促進することにより、正常の小型脂肪細胞を増やし、肥大化脂肪細胞を減少させる。従って、肥大化脂肪細胞の増加に伴い引き起こされる糖尿病、高脂血症、高血圧、肥満、免疫異常、ガン等の生活習慣病に対して、予防・治療効果を示すものと考えられる。  The non-human mammal deficient in polynucleotide expression of the present invention can be used for screening for an agent for promoting fat differentiation. Specifically, the test compound is administered to the non-human mammal deficient in polynucleotide expression of the present invention (the administration method can be appropriately selected according to the symptom of the test animal, the nature of the test compound, etc. , Oral administration, intravenous injection, intramuscular injection, etc.) and observing / measuring changes in the animals (for example, treating the animals with untreated control animals and changes in the symptoms of each organ, tissue, disease, etc.) By comparison), it is possible to screen a fat differentiation promoter.
Evaluation as a fat differentiation promoter is, for example, fat compared with a control cell or subject in a cell or an individual that does not express the polypeptide of the present invention or a partial peptide thereof when the test compound of the above screening method is used. It can be evaluated based on the accumulation promoting action, blood glucose level, blood lipid level and / or blood cholesterol level change. The compound obtained by such a method can be obtained by, for example, triglyceride accumulation, body fat mass, blood glucose level, blood lipid level, blood cholesterol level, systemic cholesterol, as described in the above [1d] method for test animals. The ability to change energy consumption, oxygen consumption, respiratory quotient and blood pressure can be evaluated.
In addition, a fat differentiation promoter increases normal small fat cells and decreases hypertrophic fat cells by promoting differentiation from enlarged fat cells to fat cells. Therefore, it is considered to have a prophylactic / therapeutic effect on lifestyle-related diseases such as diabetes, hyperlipidemia, hypertension, obesity, immune abnormalities, cancer and the like caused by an increase in enlarged fat cells.

　本発明は、以下の〔６〕のスクリーニング方法も提供する。
〔６〕　外来性の、配列番号：２、４、６、８、１０または１２で示される塩基配列と同一または実質的に同一な塩基配列を含むポリヌクレオチド（本明細書中、本発明の外来性ポリヌクレオチドと略記する場合がある）を有する非ヒトトランスジェニック動物（本明細書中、本発明のポリヌクレオチド転移動物と略記する場合がある）を用いることを特徴とする、ＩＭＰＡ２関連末梢性疾患の予防・治療剤のスクリーニング方法。
　本発明のポリヌクレオチド転移動物は、自体公知の非ヒトトランスジェニック動物の作製法に従って作出できる。例えば、本発明の外来性ポリヌクレオチドを、リン酸カルシウム法、電気パルス法、リポフェクション法、凝集法、マイクロインジェクション法、パーティクルガン法、ＤＥＡＥ－デキストラン法などにより、未受精卵、受精卵、精子およびその始原細胞を含む胚芽細胞などに対して、好ましくは、非ヒト哺乳動物の発生における胚発生の段階（さらに好ましくは、単細胞または受精卵細胞の段階でかつ一般に８細胞期以前）に、転移することによって作出することができる。 The present invention also provides the following screening method [6].
[6] An exogenous polynucleotide comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 (in the present specification, the foreign molecule of the present invention) IMPA2-related peripheral disease, characterized by using a non-human transgenic animal (sometimes abbreviated as a polynucleotide-transferred animal of the present invention) having a non-human transgenic animal having a Screening method for prophylactic / therapeutic agents.
The polynucleotide-transferred animal of the present invention can be produced according to a method for producing a non-human transgenic animal known per se. For example, the exogenous polynucleotide of the present invention can be obtained by subjecting an unfertilized egg, a fertilized egg, a sperm, and a starting material thereof by the calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, etc. Produced by metastasis, preferably to embryonic cells containing cells, preferably at the stage of embryonic development in non-human mammal development (more preferably at the single cell or fertilized egg cell stage and generally prior to the 8-cell stage). can do.

　本発明の外来性ポリヌクレオチドは、非ヒト哺乳動物が本来有しているポリヌクレオチドではなく、いったん哺乳動物から単離・抽出されたポリヌクレオチドである。
　本発明の外来性ポリヌクレオチドとしては、（ａ）配列番号：１、３、５、７、９または１１で示されるアミノ酸配列を含むポリペプチドをコードするポリヌクレオチド（好ましくは、ＤＮＡ）、または（ｂ）配列番号：２、４、６、８、１０または１２で示される塩基配列を含むポリヌクレオチド（好ましくは、ＤＮＡ）が好ましく、（ｃ）配列番号：１、３、５、７、９または１１で示されるアミノ酸配列からなるポリペプチドをコードするポリヌクレオチド（好ましくは、ＤＮＡ）、または（ｄ）配列番号：２、４、６、８、１０または１２で示される塩基配列からなるポリヌクレオチド（好ましくは、ＤＮＡ）がより好ましい。
　配列番号：２、４、６、８、１０または１２で示される塩基配列と実質的に同一な塩基配列を含むポリヌクレオチドとしては、配列番号：２、４、６、８、１０または１２で示される塩基配列とハイストリンジェントな条件下でハイブリダイズする塩基配列を含有し、配列番号：１、３、５、７、９または１１で示されるアミノ酸配列を含むポリペプチドと実質的に同質の性質を有するポリペプチドをコードするポリヌクレオチド（好ましくは、ＤＮＡ）などが挙げられる。
　本発明のポリヌクレオチド転移動物において転移される、本発明の外来性ポリヌクレオチドは、該転移動物と同種の哺乳動物由来のものであっても、異種の哺乳動物由来のものであってもよい。
　本発明のポリヌクレオチド転移動物は、非ヒト哺乳動物を用いて作出されるが、用いられる非ヒト哺乳動物としては、前記〔５〕のスクリーニング方法で説明した、本発明のポリヌクレオチド発現不全非ヒト哺乳動物で用いたものと同様のものが挙げられる。
　試験化合物としては、前記〔５〕のスクリーニング方法で説明したものと同様のものが挙げられる。 The exogenous polynucleotide of the present invention is not a polynucleotide originally possessed by a non-human mammal, but a polynucleotide once isolated and extracted from a mammal.
The exogenous polynucleotide of the present invention includes (a) a polynucleotide (preferably DNA) encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11, or ( b) A polynucleotide (preferably DNA) comprising the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 is preferred, and (c) SEQ ID NO: 1, 3, 5, 7, 9 or A polynucleotide (preferably DNA) encoding a polypeptide consisting of the amino acid sequence shown by 11 or (d) a polynucleotide consisting of the base sequence shown by SEQ ID NO: 2, 4, 6, 8, 10 or 12 ( Preferably, DNA is more preferable.
As a polynucleotide comprising a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12, the polynucleotide represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 is used. A nucleotide sequence that hybridizes under high stringency conditions with a nucleotide sequence that is substantially the same as a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 And a polynucleotide (preferably, DNA) encoding a polypeptide having the above.
The exogenous polynucleotide of the present invention to be transferred in the polynucleotide-transferred animal of the present invention may be derived from a mammal of the same species as that of the transferred animal or from a heterologous mammal.
The polynucleotide-transferred animal of the present invention is produced using a non-human mammal. The non-human mammal to be used is a non-human non-human expression of the polynucleotide of the present invention described in the above screening method [5]. The thing similar to what was used with the mammal is mentioned.
Examples of the test compound include the same compounds as those described in the above screening method [5].

　本発明のポリヌクレオチド転移動物が、ＩＭＰＡ２を高発現している場合、該転移動物はＩＭＰＡ２の機能亢進症を発症すると考えられる。その場合、本発明のポリヌクレオチド転移動物は、ＩＭＰＡ２の機能亢進に起因するＩＭＰＡ２関連末梢性疾患の病態モデル動物として利用することができる。そのため、本発明のポリヌクレオチド転移動物は、ＩＭＰＡ２関連末梢性疾患（特に肥満症）の予防・治療剤のスクリーニングに用いることができる。他にも、本発明のポリヌクレオチド転移動物を用いて、ＩＭＰＡ２関連末梢性疾患の病態機序の解明や、これらの疾患の治療方法の検討を行なうことも可能である。
　具体的には、本発明のポリヌクレオチド転移動物に、試験化合物を投与し、該動物の変化を観察・測定することにより、ＩＭＰＡ２関連末梢性疾患の予防・治療剤をスクリーニングすることができる。このようなスクリーニングによって選択される試験化合物は、ＩＭＰＡ２の機能亢進に起因するＩＭＰＡ２関連末梢性疾患の病態を改善し得る。
　本発明のスクリーニング方法には、本発明のポリヌクレオチド転移動物から採取した器官、組織または細胞を使用することができる。 When the polynucleotide-transferred animal of the present invention highly expresses IMPA2, the transferred animal is considered to develop IMPA2 hyperfunction. In that case, the polynucleotide-transferred animal of the present invention can be used as a disease state model animal for an IMPA2-related peripheral disease caused by the enhanced function of IMPA2. Therefore, the polynucleotide-transferred animal of the present invention can be used for screening for preventive / therapeutic agents for IMPA2-related peripheral diseases (particularly obesity). In addition, using the polynucleotide-transferred animal of the present invention, it is also possible to elucidate the pathologic mechanism of IMPA2-related peripheral diseases and to examine methods for treating these diseases.
Specifically, the test compound can be administered to the polynucleotide-transferred animal of the present invention, and the prophylactic / therapeutic agent for IMPA2-related peripheral diseases can be screened by observing and measuring changes in the animal. Test compounds selected by such screening can improve the pathology of IMPA2-related peripheral diseases resulting from hyperactivity of IMPA2.
In the screening method of the present invention, an organ, tissue or cell collected from the polynucleotide-transferred animal of the present invention can be used.

　本発明はまた、以下の〔８〕のスクリーニング方法も提供する。
〔８〕　配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むポリペプチドもしくはその部分ペプチドまたはその塩、あるいは該ポリペプチドまたはその部分ペプチドを産生する細胞、および６－ホスホグルコン酸またはその類縁化合物を用いることを特徴とする、ＩＭＰＡ２阻害薬のスクリーニング方法。
　〔８〕のスクリーニング方法は、前述の本発明のスクリーニング方法で説明したのと同じようにして実施することができる。 The present invention also provides the following screening method [8].
[8] A polypeptide comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, or a partial peptide thereof, or a salt thereof, or the polypeptide or a thereof A method for screening an IMPA2 inhibitor, comprising using a cell that produces a partial peptide, and 6-phosphogluconic acid or a similar compound thereof.
The screening method [8] can be carried out in the same manner as described in the screening method of the present invention.

〔本発明の予防・治療剤〕
　本発明は、以下の予防・治療剤を提供する。
〔９〕ＩＭＰＡ２阻害薬を含有してなるＩＭＰＡ２関連末梢性疾患の予防・治療剤。
　本発明の予防・治療剤は、前記した本発明のスクリーニング方法によって得られるＩＭＰＡ２阻害薬であるのが望ましい。
　本発明のスクリーニング方法より得られるＩＭＰＡ２阻害薬は、例えば、ペプチド、タンパク質、ポリペプチド、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液、血漿などが挙げられ、これら化合物は新規な化合物であってもよいし、公知の化合物であってもよい。
　ＩＭＰＡ２阻害薬は、塩を形成していてもよく、このような塩としては、前記した本発明のポリヌクレオチドまたはその部分ペプチドの塩として例示したものが挙げられる。 [Prophylactic / therapeutic agent of the present invention]
The present invention provides the following preventive / therapeutic agents.
[9] A prophylactic / therapeutic agent for IMPA2-related peripheral diseases, comprising an IMPA2 inhibitor.
The prophylactic / therapeutic agent of the present invention is desirably an IMPA2 inhibitor obtained by the screening method of the present invention described above.
Examples of the IMPA2 inhibitor obtained from the screening method of the present invention include peptides, proteins, polypeptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like. These compounds may be novel compounds or known compounds.
The IMPA2 inhibitor may form a salt, and examples of such a salt include those exemplified as the salt of the polynucleotide of the present invention or a partial peptide thereof.

　ＩＭＰＡ２阻害薬の具体例としては、以下の（ａ）～（ｅ）が挙げられる。
（ａ）本発明のポリペプチドに結合して本発明のポリペプチドのホスファターゼ活性を阻害する化合物
（ｂ）本発明のポリペプチドに対する中和抗体
（ｃ）本発明のポリペプチドの発現を阻害する化合物
（ｄ）本発明のポリペプチドをコードするポリヌクレオチドに対するアンチセンスポリヌクレオチド、あるいは
（ｅ）本発明のポリペプチドをコードするポリヌクレオチドに対するsiRNAまたはshRNA
　上記（ａ）～（ｅ）のなかでも、（ａ）または（ｅ）が好ましく、siRNAがさらに好ましい。
　以下、上記（ａ）～（ｅ）について詳述する。 Specific examples of the IMPA2 inhibitor include the following (a) to (e).
(A) a compound that binds to the polypeptide of the present invention and inhibits the phosphatase activity of the polypeptide of the present invention (b) a neutralizing antibody against the polypeptide of the present invention (c) a compound that inhibits the expression of the polypeptide of the present invention (D) an antisense polynucleotide against a polynucleotide encoding a polypeptide of the present invention, or (e) an siRNA or shRNA against a polynucleotide encoding a polypeptide of the present invention
Among the above (a) to (e), (a) or (e) is preferable, and siRNA is more preferable.
The above (a) to (e) will be described in detail below.

（ａ）本発明のポリペプチドに結合して本発明のポリペプチドのホスファターゼ活性を阻害する化合物としては、本発明のスクリーニング方法によって取得されるＩＭＰＡ２に結合してＩＭＰＡ２の活性を直接阻害する化合物が挙げられる。
　ＩＭＰＡ２に結合してＩＭＰＡ２の活性を直接阻害する化合物で公知のものとしては、リチウムまたはその塩などが挙げられる。ここで、リチウムの塩としては、炭酸リチウム、クエン酸リチウムなどが挙げられる。本発明において好ましく用いられるＩＭＰＡ２阻害薬は、ＩＭＰＡ２に特異的阻害剤である。例えば、本明細書の実施例４においてＩＭＰＡ２に特異性の高い基質として６－ホスホグルコン酸が記載されている。この６－ホスホグルコン酸またはその類縁化合物を用いて、ＩＭＰＡ２阻害薬をスクリーニングすることができる。例えば、本発明のスクリーニング方法において、ＩＭＰＡ１などを対照として用いることにより、ＩＭＰＡ２に特異的な阻害薬を単離することができる。
　さらに、ＩＭＰＡ２の構造情報に基づき、公知のドラッグデザインの手法を用いて新規の化合物を設計することもできる。このような手法としては、例えば、特許第3256307号、特許第3669704号、特許第3747048号などを参照できる。このような手法によって、より副作用が少ないなどの利点を有する化合物を設計することができる。それら設計された化合物については、本発明のスクリーニング方法によって、細胞内グリセロール蓄積作用、細胞内ミトコンドリア活性調節作用などについての能力、ならびにその化合物が対象とするポリペプチドの特異性の程度などについて評価することができる。 (A) The compound that binds to the polypeptide of the present invention and inhibits the phosphatase activity of the polypeptide of the present invention includes a compound that binds to IMPA2 obtained by the screening method of the present invention and directly inhibits the activity of IMPA2. Can be mentioned.
Known compounds that bind to IMPA2 and directly inhibit the activity of IMPA2 include lithium or a salt thereof. Here, examples of the lithium salt include lithium carbonate and lithium citrate. The IMPA2 inhibitor preferably used in the present invention is an inhibitor specific to IMPA2. For example, in Example 4 of this specification, 6-phosphogluconic acid is described as a substrate having high specificity for IMPA2. This 6-phosphogluconic acid or its related compounds can be used to screen for IMPA2 inhibitors. For example, in the screening method of the present invention, an inhibitor specific for IMPA2 can be isolated by using IMPA1 or the like as a control.
Furthermore, based on the structural information of IMPA2, a novel compound can be designed using a known drug design technique. As such a technique, for example, Japanese Patent No. 3256307, Japanese Patent No. 3669704, Japanese Patent No. 3747048, and the like can be referred to. By such a method, a compound having advantages such as fewer side effects can be designed. These designed compounds are evaluated by the screening method of the present invention for their ability to accumulate intracellular glycerol, regulate intracellular mitochondrial activity, etc., as well as the degree of specificity of the target polypeptide. be able to.

（ｂ）本発明のポリペプチドに対する中和抗体
　本発明のポリペプチドに対する中和抗体として、例えば、本発明のポリペプチドに結合して、ホスファターゼ活性を中和する抗体が挙げられる。 (B) Neutralizing antibody against the polypeptide of the present invention The neutralizing antibody against the polypeptide of the present invention includes, for example, an antibody that binds to the polypeptide of the present invention and neutralizes phosphatase activity.

（ｃ）本発明のポリペプチドの発現を阻害する化合物
　本発明のポリペプチドの発現を阻害する化合物としては、本発明のスクリーニング方法において取得される、例えば、細胞内シグナル伝達機構、細胞内輸送機構などを介してＩＭＰＡ２の細胞内発現を阻害することによって、ＩＭＰＡ２の発現を阻害する化合物が挙げられる。本発明のポリペプチドの発現を阻害する化合物としては、好ましくは本発明のポリペプチドをコードする遺伝子の発現を阻害する化合物である。ＩＭＰＡ２の発現量の評価方法としては、前記した本発明のポリペプチドの定量法、ＩＭＰＡ２のｍＲＮＡ量を測定する方法、ＩＭＰＡ２の活性量を測定する方法などが挙げられる。
　ＩＭＰＡ２のｍＲＮＡ量の測定は、公知の方法、例えば、プローブとして配列番号：２、４、６、８、１０、１２またはそれらの一部を含有する核酸を用いるノーザンハイブリダイゼーション、あるいはプライマーとして配列番号：２、４、６、８、１０、１２またはそれらの一部を含有する核酸を用いる定量的ＰＣＲ法またはそれに準じる方法に従い測定することができる。例えば、(i)試験化合物の存在下および(ii)非存在下において本発明のポリペプチドの発現量を測定し、(i)と(ii)とを比較して約２０％以上、好ましくは３０％以上、より好ましくは約５０％、さらにより好ましくは約６０％以上発現を阻害する試験化合物を、本発明のポリペプチドの発現を阻害する化合物として選択することができる。 (C) Compound that inhibits the expression of the polypeptide of the present invention The compound that inhibits the expression of the polypeptide of the present invention includes, for example, an intracellular signal transduction mechanism and an intracellular transport mechanism obtained in the screening method of the present invention. Examples include compounds that inhibit the expression of IMPA2 by inhibiting intracellular expression of IMPA2 via, for example. The compound that inhibits the expression of the polypeptide of the present invention is preferably a compound that inhibits the expression of the gene encoding the polypeptide of the present invention. Examples of the method for evaluating the expression level of IMPA2 include the above-described method for quantifying the polypeptide of the present invention, a method for measuring the mRNA level of IMPA2, and a method for measuring the activity level of IMPA2.
The amount of mRNA of IMPA2 is measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 2, 4, 6, 8, 10, 12 or a part thereof as a probe, or SEQ ID NO: as a primer. : Measured according to a quantitative PCR method using a nucleic acid containing 2, 4, 6, 8, 10, 12, or a part thereof, or a method analogous thereto. For example, the expression level of the polypeptide of the present invention is measured in the presence of (i) the test compound and (ii) in the absence, and (i) and (ii) are compared to about 20% or more, preferably 30 %, More preferably about 50%, even more preferably about 60% or more of the test compound that inhibits the expression can be selected as a compound that inhibits the expression of the polypeptide of the present invention.

（ｄ）本発明のポリペプチドをコードするポリヌクレオチドに対するアンチセンスポリヌクレオチド
　本発明のポリヌクレオチド（好ましくはＤＮＡ）の塩基配列に相補的な、または実質的に相補的な塩基配列またはその一部を有するアンチセンスポリヌクレオチド（本明細書中、本発明のアンチセンスポリヌクレオチドと略記する場合がある）としては、本発明のポリヌクレオチドの塩基配列に相補的な、または実質的に相補的な塩基配列またはその一部を有し、該ポリヌクレオチドの発現を抑制し得る作用を有するものであれば、いずれのアンチセンスポリヌクレオチドであってもよいが、アンチセンスＤＮＡが好ましい。 (D) An antisense polynucleotide for a polynucleotide encoding the polypeptide of the present invention A base sequence complementary to or substantially complementary to the base sequence of the polynucleotide (preferably DNA) of the present invention or a part thereof The antisense polynucleotide having (sometimes abbreviated as the antisense polynucleotide of the present invention in the present specification) is a base sequence complementary to or substantially complementary to the base sequence of the polynucleotide of the present invention Alternatively, any antisense polynucleotide may be used as long as it has a part and has an action capable of suppressing the expression of the polynucleotide, but antisense DNA is preferred.

　本発明のポリヌクレオチドの塩基配列に実質的に相補的な塩基配列とは、例えば、本発明のポリヌクレオチドの塩基配列に相補的な塩基配列（例えば、本発明のＤＮＡの相補鎖）の全塩基配列あるいは部分塩基配列と約70％以上、約80％以上、約90％以上、約95％以上、約96％以上、約97％以上、約98％以上または約99％以上の相同性または同一性を有する塩基配列などが挙げられる。特に、本発明のＤＮＡの相補鎖の全塩基配列うち、（i）翻訳阻害を指向したアンチセンスポリヌクレオチドの場合は、本発明のポリペプチドのＮ末端部位をコードする部分の塩基配列（例えば、開始コドン付近の塩基配列など）の相補鎖と約70％以上、約80％以上、約90％以上、約95％以上、約96％以上、約97％以上、約98％以上または約99％以上の相同性または同一性を有するアンチセンスポリヌクレオチドが、（ii）ＲＮａｓｅＨによるＲＮＡ分解を指向するアンチセンスポリヌクレオチドの場合は、イントロンを含む本発明のＤＮＡの全塩基配列の相補鎖と約70％以上、約80％以上、約90％以上、約95％以上、約96％以上、約97％以上、約98％以上または約99％以上の相同性または同一性を有するアンチセンスポリヌクレオチドがそれぞれ好適である。
　具体的には、配列番号：２、４、６、８、１０または１２で示される塩基配列を含有するポリヌクレオチドの塩基配列に相補的な、もしくは実質的に相補的な塩基配列、またはその一部分を有するアンチセンスポリヌクレオチド、好ましくは例えば、配列番号：２、４、６、８、１０または１２で示される塩基配列を含有するポリヌクレオチドの塩基配列に相補的な塩基配列、またはその一部分を有するアンチセンスポリヌクレオチド（より好ましくは、配列番号：２、４、６、８、１０または１２で示される塩基配列を含有するＤＮＡの塩基配列に相補的な塩基配列、またはその一部分を有するアンチセンスポリヌクレオチド）が挙げられる。
　アンチセンスポリヌクレオチドは通常、１０～４０個程度、好ましくは１５～３０個程度の塩基から構成される。
　ヌクレアーゼなどの加水分解酵素による分解を防ぐために、アンチセンスポリヌクレオチドを構成する各ヌクレオチドのリン酸残基（ホスフェート）は、例えば、ホスホロチオエート、メチルホスホネート、ホスホロジチオネートなどの化学修飾リン酸残基に置換されていてもよい。また、各ヌクレオチドの糖（デオキシリボース）は、２’－Ｏ－メチル化などの化学修飾糖構造に置換されていてもよいし、塩基部分（ピリミジン、プリン）も化学修飾を受けたものであってもよく、配列番号：２、６、８、１０または１２で示される塩基配列を含有するＤＮＡにハイブリダイズするものであればいずれのものでもよい。これらのアンチセンスポリヌクレオチドは、公知のＤＮＡ合成装置などを用いて製造することができる。 The base sequence substantially complementary to the base sequence of the polynucleotide of the present invention is, for example, all bases of the base sequence complementary to the base sequence of the polynucleotide of the present invention (for example, the complementary strand of the DNA of the present invention). About 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more homology or identity with a sequence or partial base sequence And a nucleotide sequence having the property. In particular, among the entire base sequences of the complementary strand of the DNA of the present invention, (i) in the case of an antisense polynucleotide directed to translation inhibition, the base sequence of the portion encoding the N-terminal site of the polypeptide of the present invention (for example, About 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% When the antisense polynucleotide having the above homology or identity is (ii) an antisense polynucleotide directed to RNA degradation by RNase H, the antisense polynucleotide has about 70% of the complementary strand of the entire base sequence of the DNA of the present invention including introns. % Or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of an antisense polynucleotide having homology or identity Each suitable That.
Specifically, a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide containing the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, or a part thereof An antisense polynucleotide having a nucleotide sequence, preferably having a nucleotide sequence complementary to the nucleotide sequence of a polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12, or a part thereof, for example Antisense polynucleotide (more preferably, an antisense polynucleotide having a base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 or a part thereof. Nucleotide).
The antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.
In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense polynucleotide is a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, phosphorodithionate, etc. May be substituted. In addition, the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base part (pyrimidine, purine) is also chemically modified. As long as it hybridizes to DNA containing the base sequence represented by SEQ ID NO: 2, 6, 8, 10 or 12, any of them may be used. These antisense polynucleotides can be produced using a known DNA synthesizer.

　本発明のポリペプチドをコードする遺伝子の複製または発現を阻害することのできる該遺伝子に対応するアンチセンスポリヌクレオチド（核酸）は、クローン化した、あるいは決定されたポリペプチドをコードするＤＮＡの塩基配列情報に基づき設計し、合成しうる。かかるアンチセンスポリヌクレオチドは、本発明のポリペプチドをコードする遺伝子のＲＮＡとハイブリダイズすることができ、該ＲＮＡの合成または機能を阻害することができる。
　上記のアンチセンスポリヌクレオチドは、生体内および生体外で本発明のポリペプチドをコードする遺伝子の発現を調節・制御するのに有用であり、また病気などの治療または診断に有用である。用語「対応する」とは、遺伝子を含めたヌクレオチド、塩基配列または核酸の特定の配列に相同性を有するあるいは相補的であることを意味する。ヌクレオチド、塩基配列または核酸とポリペプチドとの間で「対応する」とは、ヌクレオチド（核酸）の配列またはその相補体から誘導される（指令にある）ポリペプチドのアミノ酸を通常指している。ポリペプチドをコードする遺伝子の５’端ヘアピンループ、５’端６－ベースペア・リピート、５’端非翻訳領域、ポリペプチド翻訳開始コドン、ポリペプチドコード領域、ＯＲＦ翻訳終止コドン、３’端非翻訳領域、３’端パリンドローム領域または３’端ヘアピンループなどは、好ましい対象領域として選択しうるが、ポリペプチドをコードする遺伝子内の如何なる領域も対象として選択しうる。
　目的核酸と、対象領域の少なくとも一部に相補的なポリヌクレオチドとの関係については、目的核酸が対象領域とハイブリダイズすることができる場合は、その目的核酸は、当該対象領域のポリヌクレオチドに対して「アンチセンス」であるということができる。アンチセンスポリヌクレオチドは、２－デオキシ－Ｄ－リボースを含有しているポリヌクレオチド、Ｄ－リボースを含有しているポリヌクレオチド、プリンまたはピリミジン塩基のＮ－グリコシドであるその他のタイプのポリヌクレオチド、非ヌクレオチド骨格を有するその他のポリマー（例えば、市販のポリペプチド核酸および合成配列特異的な核酸ポリマー）または特殊な結合を含有するその他のポリマー（但し、該ポリマーはＤＮＡやＲＮＡ中に見出されるような塩基のペアリングや塩基の付着を許容する配置をもつヌクレオチドを含有する）などが挙げられる。それらは、２本鎖ＤＮＡ、１本鎖ＤＮＡ、２本鎖ＲＮＡ、１本鎖ＲＮＡ、ＤＮＡ：ＲＮＡハイブリッドであってもよく、さらに非修飾ポリヌクレオチド（または非修飾オリゴヌクレオチド）、公知の修飾の付加されたもの、例えば当該分野で知られた標識のあるもの、キャップの付いたもの、メチル化されたもの、１個以上の天然のヌクレオチドを類縁物で置換したもの、分子内ヌクレオチド修飾のされたもの、例えば非荷電結合（例えば、メチルホスホネート、ホスホトリエステル、ホスホルアミデート、カルバメートなど）を持つもの、電荷を有する結合または硫黄含有結合（例、ホスホロチオエート、ホスホロジチオエートなど）を持つもの、例えばポリペプチド（例、ヌクレアーゼ、ヌクレアーゼ・インヒビター、トキシン、抗体、シグナルペプチド、ポリ－Ｌ－リジンなど）や糖（例、モノサッカライドなど）などの側鎖基を有しているもの、インターカレント化合物（例、アクリジン、ソラレンなど）を持つもの、キレート化合物（例えば、金属、放射活性をもつ金属、ホウ素、酸化性の金属など）を含有するもの、アルキル化剤を含有するもの、修飾された結合を持つもの（例えば、αアノマー型の核酸など）であってもよい。ここで「ヌクレオシド」、「ヌクレオチド」および「核酸」とは、プリンおよびピリミジン塩基を含有するのみでなく、修飾されたその他の複素環型塩基をもつようなものを含んでいて良い。このような修飾物は、メチル化されたプリンおよびピリミジン、アシル化されたプリンおよびピリミジン、あるいはその他の複素環を含むものであってよい。修飾されたヌクレオチドおよび修飾されたヌクレオチドはまた糖部分が修飾されていてよく、例えば、１個以上の水酸基がハロゲンや脂肪族基などで置換されていたり、またはエーテル、アミンなどの官能基に変換されていてよい。 An antisense polynucleotide (nucleic acid) corresponding to the gene capable of inhibiting replication or expression of the gene encoding the polypeptide of the present invention is a cloned or determined DNA sequence encoding the polypeptide Design and synthesize based on information. Such an antisense polynucleotide can hybridize with RNA of a gene encoding the polypeptide of the present invention, and can inhibit the synthesis or function of the RNA.
The above-described antisense polynucleotide is useful for regulating / controlling the expression of a gene encoding the polypeptide of the present invention in vivo and in vitro, and also useful for treatment or diagnosis of diseases. The term “corresponding” means homologous to or complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. "Corresponding" between a nucleotide, base sequence or nucleic acid and polypeptide usually refers to the amino acid of the polypeptide (instruction) derived from the nucleotide (nucleic acid) sequence or its complement. 5 'end hairpin loop of gene encoding polypeptide, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation initiation codon, polypeptide coding region, ORF translation stop codon, 3' end non A translation region, 3′-end palindromic region, 3′-end hairpin loop, or the like can be selected as a preferred target region, but any region in a gene encoding a polypeptide can be selected as a target.
Regarding the relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region, if the target nucleic acid can hybridize to the target region, the target nucleic acid It can be said that it is “antisense”. Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, non-polynucleotides, Other polymers with a nucleotide backbone (eg, commercially available polypeptide nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a base as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a base). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Such as polypeptides (eg, nucleases, nuclease inhibitors, toxins, antibodies, One having a side chain group such as a guanal peptide, poly-L-lysine, etc., sugar (eg, monosaccharide, etc.), one having an intercurrent compound (eg, acridine, psoralen, etc.), chelate compound (eg, , Metals, radioactive metals, boron, oxidizing metals, etc.), alkylating agents, and modified bonds (eg, alpha anomeric nucleic acids) Also good. Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens or aliphatic groups, or converted to functional groups such as ethers, amines, etc. May have been.

　本発明のアンチセンスポリヌクレオチドは、ＲＮＡ、ＤＮＡまたは修飾された核酸（ＲＮＡ、ＤＮＡ）である。修飾された核酸の具体例としては、核酸の硫黄誘導体、チオホスフェート誘導体、ポリヌクレオシドアミドやオリゴヌクレオシドアミドの分解に抵抗性のものなどが挙げられる。本発明のアンチセンスポリヌクレオチドは、例えば、以下のように設計されうる。すなわち、細胞内でのアンチセンスポリヌクレオチドをより安定なものにする、アンチセンスポリヌクレオチドの細胞透過性をより高める、目標とするセンス鎖に対する親和性をより大きなものにする、また、もし毒性があるような場合はアンチセンスポリヌクレオチドの毒性をより小さなものにする。このような修飾は、例えばPharm Tech Japan, 8巻, 247頁または395頁, 1992年、Antisense Research and Applications, CRC Press, 1993年などで数多く報告されている。
　本発明のアンチセンスポリヌクレオチドは、変化せしめられて良く、また、修飾された糖、塩基、結合を含有していて良く、リポゾーム、ミクロスフェアのような特殊な形態で供与されたり、遺伝子治療により適用されたり、付加された形態で与えられたりすることができうる。こうして付加形態で用いられるものとしては、リン酸基骨格の電荷を中和するように働くポリリジンのようなポリカチオン体、細胞膜との相互作用を高め、あるいは核酸の取込みを増大せしめるような脂質（例、ホスホリピド、コレステロールなど）などの疎水性のものが挙げられる。付加するに好ましい脂質としては、コレステロールやその誘導体（例、コレステリルクロロホルメート、コール酸など）が挙げられる。こうしたものは、核酸の３’端または５’端に付着させることができ、塩基、糖、分子内ヌクレオシド結合を介して付着させることができうる。その他の基としては、核酸の３’端または５’端に特異的に配置されたキャップ用の基で、エキソヌクレアーゼ、ＲＮａｓｅなどのヌクレアーゼによる分解を阻止するためのものが挙げられる。こうしたキャップ用の基としては、ポリエチレングリコール、テトラエチレングリコールなどのグリコールをはじめとした当該分野で知られた水酸基の保護基が挙げられるが、それに限定されるものではない。 The antisense polynucleotide of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acids include nucleic acid sulfur derivatives, thiophosphate derivatives, polynucleoside amides and oligonucleoside amides that are resistant to degradation. The antisense polynucleotide of the present invention can be designed, for example, as follows. That is, to make the antisense polynucleotide in the cell more stable, to increase the cell permeability of the antisense polynucleotide, to increase the affinity for the target sense strand, and to reduce the toxicity In some cases, the antisense polynucleotide is less toxic. Many such modifications have been reported in, for example, Pharm Tech Japan, 8, 247 or 395, 1992, Antisense Research and Applications, CRC Press, 1993, and the like.
The antisense polynucleotide of the present invention may be altered and may contain modified sugars, bases and bonds, and may be provided in special forms such as liposomes, microspheres, or by gene therapy. It can be applied or given in an added form. Thus, the additional form can be used as a polycationic substance such as polylysine that works to neutralize the charge of the phosphate group skeleton, a lipid that enhances the interaction with the cell membrane or increases the uptake of nucleic acid ( Examples include hydrophobic ones such as phospholipid and cholesterol. Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 ′ end or 5 ′ end of the nucleic acid, and can be attached via a base, sugar, intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid and for preventing degradation by a nuclease such as exonuclease or RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

（ｅ）本発明のポリペプチドをコードするポリヌクレオチドに対するsiRNAまたはshRNA
　本発明のポリペプチドをコードするＲＮＡの一部を含有する二重鎖ＲＮＡ（本発明のポリヌクレオチドに対するｓｉＲＮＡ（small (short) interfering RNA）、ｓｈＲＮＡ（small (short) hairpin RNA）など）は、公知の方法（例、Nature, 411巻, 494頁, 2001年）に準じて、本発明のポリヌクレオチドの配列を基に設計して製造することができる。　 (E) siRNA or shRNA against a polynucleotide encoding the polypeptide of the present invention
Double-stranded RNA containing a part of the RNA encoding the polypeptide of the present invention (such as siRNA (small (short) interfering RNA) and shRNA (small (short) hairpin RNA) for the polynucleotide of the present invention) is publicly known. In accordance with the method (eg, Nature, 411, 494, 2001), it can be designed and produced based on the sequence of the polynucleotide of the present invention.

　ＩＭＰＡ２阻害薬は、安全で低毒性であるので、ＩＭＰＡ２関連末梢性疾患の予防・治療を目的として、哺乳動物（例えば、ヒト、ラット、マウス、モルモット、ウサギ、ヒツジ、ブタ、ウシ、ウマ、ネコ、イヌ、サルなど；ヒトが好ましい）に対して経口的または非経口的に投与することができる。
　ＩＭＰＡ２阻害薬は、肥満症などのＩＭＰＡ２関連末梢性疾患の予防および／または治療に有用である。また、ＩＭＰＡ２関連中枢性疾患（例、神経変性疾患、統合失調症）の予防および／または治療にも有用である。
　ＩＭＰＡ２阻害薬の投与量は、その作用、対象疾患、投与対象、投与ルートなどにより異なるが、肥満症などの治療の目的でＩＭＰＡ２阻害薬を経口投与する場合、一般的に成人（体重６０ｋｇとして）においては、一日につきＩＭＰＡ２阻害薬を約０.１～１００ｍｇ、好ましくは約１．０～５０ｍｇ、より好ましくは約１．０～２０ｍｇ投与する。非経口的に投与する場合は、ＩＭＰＡ２阻害薬の１回投与量は投与対象、対象疾患などによっても異なるが、例えば、肥満症などの治療の目的でＩＭＰＡ２阻害薬を注射剤の形で通常成人（体重６０ｋｇとして）に投与する場合、一日につきＩＭＰＡ２阻害薬を約０．０１～３０ｍｇ程度、好ましくは約０．１～２０ｍｇ程度、より好ましくは約０．１～１０ｍｇ程度を注射により投与するのが好都合である。他の動物の場合も、体重６０ｋｇ当たりに換算した量を投与することができる。 Since IMPA2 inhibitors are safe and have low toxicity, mammals (eg, humans, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats) are used for the prevention and treatment of IMPA2-related peripheral diseases. , Dogs, monkeys, etc .; humans are preferred) orally or parenterally.
IMPA2 inhibitors are useful for the prevention and / or treatment of IMPA2-related peripheral diseases such as obesity. It is also useful for the prevention and / or treatment of IMPA2-related central diseases (eg, neurodegenerative diseases, schizophrenia).
The dose of the IMPA2 inhibitor varies depending on its action, target disease, administration subject, administration route, etc., but when an IMPA2 inhibitor is orally administered for the purpose of treating obesity or the like, generally an adult (with a body weight of 60 kg) In this case, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of IMPA2 inhibitor is administered per day. When administered parenterally, the single dose of the IMPA2 inhibitor varies depending on the administration subject, the target disease, etc. For example, the IMPA2 inhibitor is usually administered in the form of an injection for the purpose of treating obesity or the like. When administered (with a body weight of 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of IMPA2 inhibitor is administered by injection per day. Is convenient. In the case of other animals, an amount converted per 60 kg body weight can be administered.

　ＩＭＰＡ２関連末梢性疾患の予防・治療剤は、薬学的に許容し得る担体などと共に、有効成分としてＩＭＰＡ２阻害薬を含有する医薬組成物として、自体公知の方法により製造することができる。
　例えば、経口投与のための組成物としては、固体または液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤などが挙げられる。かかる組成物は自体公知の方法によって製造され、製剤分野において通常用いられる担体、希釈剤もしくは賦形剤を含有するものである。例えば、錠剤用の担体、賦形剤としては、乳糖、でんぷん、蔗糖、ステアリン酸マグネシウムなどが用いられる。
　非経口投与のための組成物としては、例えば、注射剤、坐剤などが用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤、関節内注射剤などの剤形を包含する。かかる注射剤は、自体公知の方法に従って、例えば、ＩＭＰＡ２阻害薬を通常注射剤に用いられる無菌の水性もしくは油性液に溶解、懸濁または乳化することによって調製する。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液などが用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン界面活性剤〔例、ポリソルベート８０、ＨＣＯ－５０（polyoxyethylene(50mol)adduct of hydrogenated castor oil）〕などと併用してもよい。油性液としては、例えば、ゴマ油、大豆油などが用いられ、溶解補助剤として安息香酸ベンジル、ベンジルアルコールなどを併用してもよい。調製された注射液は、通常、適当なアンプルに充填される。直腸投与に用いられる坐剤は、ＩＭＰＡ２阻害薬を通常の坐薬用基剤に混合することによって調製される。 The prophylactic / therapeutic agent for IMPA2-related peripheral diseases can be produced by a method known per se as a pharmaceutical composition containing an IMPA2 inhibitor as an active ingredient together with a pharmaceutically acceptable carrier and the like.
For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As a composition for parenteral administration, for example, injections, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections. Includes dosage forms such as agents. Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the IMPA2 inhibitor in a sterile aqueous or oily liquid usually used for injections. As an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. Suppositories used for rectal administration are prepared by mixing an IMPA2 inhibitor with a normal suppository base.

　ＩＭＰＡ２阻害薬がアンチセンスポリヌクレオチド、siRNAまたはshRNAである場合、これらをレトロウイルスベクター、アデノウイルスベクター、アデノウイルスアソシエーテッドウイルスベクターなどの適当なベクターに挿入した後、常套手段に従って、哺乳動物（例、ヒト、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど；ヒトが好ましい）に対して経口的または非経口的に投与することができる。前記アンチセンスポリヌクレオチド等は、そのままで、あるいは摂取促進のために補助剤などの生理学的に認められる担体とともに製剤化し、遺伝子銃やハイドロゲルカテーテルのようなカテーテルによって投与できる。あるいは、エアロゾル化して吸入剤として気管内に局所投与することもできる。
　さらに、体内動態の改良、半減期の長期化、細胞内取り込み効率の改善を目的に、前記のアンチセンスポリヌクレオチド等を単独またはリポソームなどの担体とともに製剤（注射剤）化し、静脈、皮下等に投与してもよい。 When the IMPA2 inhibitor is an antisense polynucleotide, siRNA or shRNA, it is inserted into a suitable vector such as a retroviral vector, adenoviral vector, adenoviral associated viral vector, etc., and then a mammal (eg, , Human, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc .; human is preferred) orally or parenterally. The antisense polynucleotide or the like can be formulated as it is or with a physiologically recognized carrier such as an auxiliary agent for promoting intake, and can be administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the above-mentioned antisense polynucleotides or the like are formulated (injection) alone or together with a carrier such as liposomes, and are administered intravenously, subcutaneously, etc. It may be administered.

・スクリーニングキット
　本発明は、本発明のスクリーニングに使用されるキット（本明細書中、本発明のスクリーニングキットと略記する場合がある）も提供する。本発明のスクリーニングキットは、本発明で用いられるポリペプチド、または本発明のポリペプチドを産生する能力を有する細胞、あるいは必要に応じてさらにバッファーなどを含有する。
　また、本発明のスクリーニングキットは、本発明のポリペプチドのホスファターゼの基質化合物を含んでもよい。そのような化合物としては、例えば、６－ホスホグルコン酸およびその類縁化合物としてミオイノシトール－１－リン酸、ミオイノシトール－４－リン酸、ミオイノシトール－１，３－二リン酸、グルコース－１－リン酸、β－グリセロリン酸、ミオイノシトール－１，４－二リン酸、ミオイノシトール－３－リン酸、６－ホスホグルコノ－ｄ－ラクトン、リブロース５－リン酸、キシロース５－リン酸、エリトロース４－リン酸、または２，３－ジホスホ－Ｄ－グリセリン酸、およびグリセロール－３－リン酸などが挙げられるが、これに限定されない。より好ましくは、本発明のスクリーニングキットは、６－ホスホグルコン酸を含む。また、本発明のスクリーニングキットは、上記のスクリーニング方法において用いられる試薬、対照となる試験化合物を含むことができる。
　本発明のスクリーニング方法または本発明のスクリーニングキットを用いて得られる化合物またはその塩は、上記した試験化合物、例えば、ペプチド、ポリペプチド、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液、血漿などから選ばれた化合物またはその塩であり、本発明のポリペプチドの活性（例、ホスファターゼ活性、細胞内トリグリセリド蓄積促進活性など）を阻害、該ポリペプチドの遺伝子の発現を阻害、または本発明のポリペプチドの発現を阻害する化合物またはその塩である。
　該化合物の塩としては、前記した本発明のポリペプチドの塩と同様のものが用いられる。
　本発明のポリペプチドの活性を阻害する化合物またはその塩、本発明のポリペプチドをコードする遺伝子の発現を阻害する化合物またはその塩、本発明のポリペプチドの発現を阻害する化合物またはその塩は、低毒性であり、ＩＭＰＡ２関連末梢性疾患（例えば、上記の細胞内トリグリセリド蓄積が関与する疾患、または細胞内ミトコンドリア活性が関与する疾患など：特に肥満症）の予防・治療剤として有用である。
　本発明のスクリーニング方法またはスクリーニングキットを用いて得られる化合物またはその塩を上述の予防・治療剤として使用する場合、常法に従って製剤化することができる（後述参照）。 -Screening Kit The present invention also provides a kit used in the screening of the present invention (sometimes abbreviated as the screening kit of the present invention). The screening kit of the present invention contains a polypeptide used in the present invention, a cell having the ability to produce the polypeptide of the present invention, or a buffer as necessary.
The screening kit of the present invention may also contain a phosphatase substrate compound of the polypeptide of the present invention. Examples of such compounds include 6-phosphogluconic acid and its related compounds such as myo-inositol-1-phosphate, myo-inositol-4-phosphate, myo-inositol-1,3-diphosphate, glucose-1- Phosphate, β-glycerophosphate, myo-inositol-1,4-diphosphate, myo-inositol-3-phosphate, 6-phosphoglucono-d-lactone, ribulose 5-phosphate, xylose 5-phosphate, erythrose 4- Examples thereof include, but are not limited to, phosphoric acid, 2,3-diphospho-D-glyceric acid, and glycerol-3-phosphate. More preferably, the screening kit of the present invention contains 6-phosphogluconic acid. In addition, the screening kit of the present invention can contain a reagent used in the above screening method and a test compound as a control.
The compound obtained by using the screening method of the present invention or the screening kit of the present invention or a salt thereof is a test compound as described above, for example, a peptide, a polypeptide, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, A compound selected from plant extracts, animal tissue extracts, plasma and the like, or a salt thereof, which inhibits the activity of the polypeptide of the present invention (eg, phosphatase activity, intracellular triglyceride accumulation promoting activity, etc.), A compound or a salt thereof that inhibits gene expression or inhibits expression of the polypeptide of the present invention.
As the salt of the compound, those similar to the aforementioned salt of the polypeptide of the present invention can be used.
A compound or salt thereof that inhibits the activity of the polypeptide of the present invention, a compound or salt thereof that inhibits the expression of the gene encoding the polypeptide of the present invention, a compound or salt thereof that inhibits the expression of the polypeptide of the present invention, It has low toxicity and is useful as a prophylactic / therapeutic agent for IMPA2-related peripheral diseases (for example, diseases involving intracellular triglyceride accumulation or diseases involving intracellular mitochondrial activity: especially obesity).
When the compound or salt thereof obtained using the screening method or screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method (see below).

・診断方法
　本発明のポリペプチドは、ホスファターゼ活性を介して細胞内トリグリセリド蓄積作用または細胞内β酸化活性に関与するので、ＩＭＰＡ２関連末梢性疾患の早期診断、症状の重症度の判定、疾患進行の予測のためのマーカーとして有用である。したがって、本発明のポリペプチドの活性を阻害する化合物またはその塩、本発明のポリペプチドに対する抗体、本発明のポリペプチドをコードする遺伝子のアンチセンスポリヌクレオチドなどは、例えば、上記疾患の診断薬などとして使用して、これら疾患を診断・予測することができる。
　本発明のポリペプチドはまた、細胞内ミトコンドリア活性、またはミトコンドリア量に関与するので、ＩＭＰＡ２関連末梢性疾患の早期診断、症状の重症度の判定、疾患進行の予測のためのマーカーとして有用である。したがって、本発明のポリペプチドの活性を阻害する化合物またはその塩、本発明の抗体、本発明のアンチセンスポリヌクレオチドなどは、例えば上記疾患の診断薬などとして使用して、これら疾患を診断・予測することができる。
　本発明はまた、哺乳動物から採取した細胞含有試料における、本発明のポリペプチドまたはそれをコードする遺伝子の発現を測定することを特徴とする、肥満度の測定法、またはＩＭＰＡ２関連末梢性疾患の診断方法も提供する。例えば、本発明のポリペプチドをコードする遺伝子が過剰に発現している場合、細胞内トリグリセリド蓄積量が高いことが予想されるので、肥満度が高い、またはＩＭＰＡ２関連末梢性疾患に罹患しているもしくは罹患する可能性が高いと予測することができる。したがって、該細胞含有試料において本発明のポリペプチドまたはそれをコードする遺伝子の発現を測定することは、肥満度の予測またはＩＭＰＡ２関連末梢性疾患の診断・予測などに有益であるといえる。
　本発明はまた、哺乳動物から採取した細胞含有試料における、本発明のポリペプチドまたはそれをコードする遺伝子の発現を測定することを特徴とする、ＩＭＰＡ２関連末梢性疾患などの診断方法も提供する。例えば、本発明のポリペプチドをコードする遺伝子が過剰に発現している場合、細胞内ミトコンドリア活性が高いことが予想されるので、ＩＭＰＡ２関連末梢性疾患に罹患しているもしくは罹患する可能性が高いと予測することができる。したがって、該細胞含有試料において本発明のポリペプチドまたはそれをコードする遺伝子の発現を測定することは、ＩＭＰＡ２関連末梢性疾患の診断・予測などに有益であるといえる。
　上述の診断方法においては、例えば、上記の本発明のポリペプチドなどの定量方法において説明されるように、本発明の抗体を用いて、試料（例えば、患者から採取した血液、細胞、組織など）中の本発明のポリペプチドの発現量を測定（定量）する。 Diagnosis method Since the polypeptide of the present invention is involved in intracellular triglyceride accumulation action or intracellular β-oxidation activity through phosphatase activity, early diagnosis of IMPA2-related peripheral diseases, determination of symptom severity, disease progression Useful as a marker for prediction. Accordingly, a compound or a salt thereof that inhibits the activity of the polypeptide of the present invention, an antibody against the polypeptide of the present invention, an antisense polynucleotide of a gene encoding the polypeptide of the present invention, etc. Can be used to diagnose and predict these diseases.
Since the polypeptide of the present invention is also involved in intracellular mitochondrial activity or mitochondrial mass, it is useful as a marker for early diagnosis of IMPA2-related peripheral diseases, determination of the severity of symptoms, and prediction of disease progression. Therefore, the compounds or salts thereof that inhibit the activity of the polypeptide of the present invention, the antibody of the present invention, the antisense polynucleotide of the present invention, etc. are used as diagnostic agents for the above diseases, for example, to diagnose and predict these diseases. can do.
The present invention also relates to a method for measuring obesity or an IMPA2-related peripheral disease characterized by measuring the expression of the polypeptide of the present invention or a gene encoding the same in a cell-containing sample collected from a mammal. A diagnostic method is also provided. For example, when the gene encoding the polypeptide of the present invention is overexpressed, it is expected that the amount of intracellular triglyceride accumulation is high, so that the degree of obesity is high or the patient is suffering from an IMPA2-related peripheral disease Or it can be predicted that there is a high possibility of being affected. Therefore, it can be said that measuring the expression of the polypeptide of the present invention or a gene encoding the same in the cell-containing sample is useful for predicting obesity or diagnosing / predicting IMPA2-related peripheral diseases.
The present invention also provides a method for diagnosing an IMPA2-related peripheral disease, which comprises measuring the expression of the polypeptide of the present invention or a gene encoding the same in a cell-containing sample collected from a mammal. For example, when the gene encoding the polypeptide of the present invention is overexpressed, intracellular mitochondrial activity is expected to be high, so that the patient is or is likely to suffer from an IMPA2-related peripheral disease. Can be predicted. Therefore, it can be said that measuring the expression of the polypeptide of the present invention or a gene encoding the same in the cell-containing sample is useful for the diagnosis / prediction of IMPA2-related peripheral diseases.
In the above-described diagnostic method, for example, as described in the above-described quantification method for the polypeptide of the present invention, a sample (for example, blood, cells, tissue, etc. collected from a patient) using the antibody of the present invention. The expression level of the polypeptide of the present invention is measured (quantified).

　さらには、本発明の抗体を用いて本発明のポリペプチドの濃度を定量することによって、本発明のポリペプチドの濃度の増加が検出された場合、ＩＭＰＡ２関連末梢性疾患に罹患しているか、またはそれら疾患に罹患するリスクが高いと診断することができる。
　本発明は、上記の診断方法・予測方法も提供する。
　また、本発明の抗体は、体液や組織などの被検体中に存在する本発明のポリペプチドを検出するために使用することができる。また、本発明の抗体は、本発明のポリペプチドを精製するために使用する抗体カラムの作製、精製時の各分画中の本発明のポリペプチドの検出、被検細胞内における本発明のポリペプチドの挙動の分析などのために使用することができる。 Further, if an increase in the concentration of the polypeptide of the present invention is detected by quantifying the concentration of the polypeptide of the present invention using the antibody of the present invention, or the patient is suffering from an IMPA2-related peripheral disease, or It can be diagnosed that the risk of suffering from these diseases is high.
The present invention also provides the above diagnostic method and prediction method.
Moreover, the antibody of the present invention can be used for detecting the polypeptide of the present invention present in a subject such as a body fluid or tissue. In addition, the antibody of the present invention can be prepared by preparing an antibody column used for purifying the polypeptide of the present invention, detecting the polypeptide of the present invention in each fraction during purification, and detecting the polypeptide of the present invention in a test cell. It can be used for analysis of peptide behavior.

・遺伝子診断薬
　本発明のＤＮＡは、例えば、プローブとして使用することにより、哺乳動物（例えば、ヒト、ラット、マウス、モルモット、ウサギ、トリ、ヒツジ、ブタ、ウシ、ウマ、ネコ、イヌ、サル、チンパンジーなど；ヒトが好ましい）における本発明のポリペプチドまたはその部分ペプチドをコードするＤＮＡまたはｍＲＮＡの異常（遺伝子異常）を検出することができるので、例えば、該ＤＮＡまたはｍＲＮＡの損傷、突然変異あるいは発現低下や、該ＤＮＡまたはｍＲＮＡの増加あるいは発現過多などの遺伝子診断薬として有用である。このような遺伝子診断薬は、本発明に含まれる。
　本発明のＤＮＡを用いる上記の遺伝子診断は、例えば、自体公知のノーザンハイブリダイゼーションやPCR-SSCP法（Genomics，第5巻，874～879頁, 1989年、Proceedings of the National Academy of Sciences of the United States of America，第86巻，2766～2770頁，1989年）などにより実施することができる。
　例えば、ノーザンハイブリダイゼーションにより発現過多が検出された場合やPCR-SSCP法によりＤＮＡの突然変異が検出された場合は、その被検体は、ＩＭＰＡ２関連末梢性に罹患しているか、またはそれら疾患に罹患するリスクが高いと診断することができる。 -Gene diagnostic agent The DNA of the present invention can be used as, for example, a mammal, such as a mammal (eg, human, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey, Since abnormality (gene abnormality) of DNA or mRNA encoding the polypeptide of the present invention or a partial peptide thereof (genetic abnormality) in chimpanzees and the like (preferably human) can be detected, for example, damage, mutation or expression of the DNA or mRNA It is useful as a genetic diagnostic agent for reduction, increase or overexpression of the DNA or mRNA. Such a gene diagnostic agent is included in the present invention.
The above gene diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879, 1989, Proceedings of the National Academy of Sciences of the United States). States of America, 86, 2766-2770, 1989).
For example, if an overexpression is detected by Northern hybridization, or if a DNA mutation is detected by the PCR-SSCP method, the subject is affected by the IMPA2-related peripherality or affected by these diseases. Can be diagnosed as being at high risk.

　本明細書および配列表において、塩基やアミノ酸などを略号で表示する場合、IUPAC-IUB Commission on Biochemical Nomenclature による略号あるいは当該分野における慣用略号に基づくものであり、その例を下記する。またアミノ酸に関し光学異性体があり得る場合は、特に明示しなければＬ体を示すものとする。
　ＤＮＡ　　　　　：デオキシリボ核酸
　ｃＤＮＡ　　　　：相補的デオキシリボ核酸
　　Ａ　　　　　　：アデニン
　　Ｔ　　　　　　：チミン
　　Ｇ　　　　　　：グアニン
　　Ｃ　　　　　　：シトシン
　ＲＮＡ　　　　　：リボ核酸
　ｍＲＮＡ　　　　：メッセンジャーリボ核酸
　ｄＡＴＰ　　　　：デオキシアデノシン三リン酸
　ｄＴＴＰ　　　　：デオキシチミジン三リン酸
　ｄＧＴＰ　　　　：デオキシグアノシン三リン酸
　ｄＣＴＰ　　　　：デオキシシチジン三リン酸
　ＡＴＰ　　　　　：アデノシン三リン酸
　ＥＤＴＡ　　　　：エチレンジアミン四酢酸
　ＳＤＳ　　　　　：ドデシル硫酸ナトリウム
　Ｇｌｙ　　　　　：グリシン
　Ａｌａ　　　　　：アラニン
　Ｖａｌ　　　　　：バリン
　Ｌｅｕ　　　　　：ロイシン
　Ｉｌｅ　　　　　：イソロイシン
　Ｓｅｒ　　　　　：セリン
　Ｔｈｒ　　　　　：スレオニン
　Ｃｙｓ　　　　　：システイン
　Ｍｅｔ　　　　　：メチオニン
　Ｇｌｕ　　　　　：グルタミン酸
　Ａｓｐ　　　　　：アスパラギン酸
　Ｌｙｓ　　　　　：リジン
　Ａｒｇ　　　　　：アルギニン
　Ｈｉｓ　　　　　：ヒスチジン
　Ｐｈｅ　　　　　：フェニルアラニン
　Ｔｙｒ　　　　　：チロシン
　Ｔｒｐ　　　　　：トリプトファン
　Ｐｒｏ　　　　　：プロリン
　Ａｓｎ　　　　　：アスパラギン
　Ｇｌｎ　　　　　：グルタミン
　ｐＧｌｕ　　　　：ピログルタミン酸
　Ｓｅｃ　　　　　：セレノシステイン（selenocysteine） In the present specification and sequence listing, bases, amino acids and the like are indicated by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when there are optical isomers with respect to amino acids, L form is shown unless otherwise specified.
DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphate dCTP: deoxycytidine triphosphate ATP: adenosine triphosphate EDTA: ethylenediaminetetraacetic acid SDS: sodium dodecyl sulfate Gly: glycine Ala: alanine Val: valine Leu: leucine Ile: isoleucine Ser: threonine CyrM: threonine : Methionine Glu: Glutamic acid Asp: Aspa Gin acid Lys: lysine Arg: arginine His: histidine Phe: phenylalanine Tyr: tyrosine Trp: tryptophan Pro: proline Asn: asparagine Gln: glutamine pGlu: pyroglutamic acid Sec: selenocysteine (selenocysteine)

　また、本明細書中で繁用される置換基、保護基および試薬を下記の記号で表記する。
　Ｍｅ　　　　　　：メチル基
　Ｅｔ　　　　　　：エチル基
　Ｂｕ　　　　　　：ブチル基
　Ｐｈ　　　　　　：フェニル基
　ＴＣ　　　　　　：チアゾリジン－４（Ｒ）－カルボキサミド基
　Ｔｏｓ　　　　　：ｐ－トルエンスルフォニル
　ＣＨＯ　　　　　：ホルミル
　Ｂｚｌ　　　　　：ベンジル
　Cl2-Bzl　　　　 ：２，６－ジクロロベンジル
　Ｂｏｍ　　　　　：ベンジルオキシメチル
　Ｚ　　　　　　　：ベンジルオキシカルボニル
　Ｃｌ－Ｚ　　　　：２－クロロベンジルオキシカルボニル
　Ｂｒ－Ｚ　　　　：２－ブロモベンジルオキシカルボニル
　Ｂｏｃ　　　　　：ｔ－ブトキシカルボニル
　ＤＮＰ　　　　　：ジニトロフェニル
　Ｔｒｔ　　　　　：トリチル
　Ｂｕｍ　　　　　：ｔ－ブトキシメチル
　Ｆｍｏｃ　　　　：Ｎ－９－フルオレニルメトキシカルボニル
　ＨＯＢｔ　　　　：１－ヒドロキシベンズトリアゾール
　ＨＯＯＢｔ　　　：３,４－ジヒドロ－３－ヒドロキシ－４－オキソ－
　　　　　　　　　　１,２,３－ベンゾトリアジン
　ＨＯＮＢ　　　　：1-ヒドロキシ-5-ノルボルネン-2,3-ジカルボキシイミド
　ＤＣＣ　　　　　：Ｎ，Ｎ'－ジシクロヘキシルカルボジイミド Further, substituents, protecting groups, and reagents frequently used in the present specification are represented by the following symbols.
Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine -4 (R) - carboxamide group Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl 2 -Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenyl Trt: trityl Bum: t-butoxymethyl Fmoc : N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOBt: 3,4-dihydro-3-hydroxy-4-oxo-
1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N′-dicyclohexylcarbodiimide

　本明細書の配列表の配列番号は、以下の配列を示す。
〔配列番号：１〕ヒトＩＭＰＡ２のアミノ酸配列
〔配列番号：２〕ヒトＩＭＰＡ２をコードするDNAの塩基配列
〔配列番号：３〕マウスＩＭＰＡ２のアミノ酸配列
〔配列番号：４〕マウスＩＭＰＡ２をコードするDNAの塩基配列
〔配列番号：５〕ラットＩＭＰＡ２のアミノ酸配列
〔配列番号：６〕ラットＩＭＰＡ２をコードするDNAの塩基配列
〔配列番号：７〕ウシＩＭＰＡ２のアミノ酸配列
〔配列番号：８〕ウシＩＭＰＡ２をコードするDNAの塩基配列
〔配列番号：９〕イヌＩＭＰＡ２のアミノ酸配列
〔配列番号：１０〕イヌＩＭＰＡ２をコードするDNAの塩基配列
〔配列番号：１１〕サルＩＭＰＡ２のアミノ酸配列
〔配列番号：１２〕サルＩＭＰＡ２をコードするDNAの塩基配列
〔配列番号：１３〕３６Ｂ４順方向プライマーの塩基配列
〔配列番号：１４〕３６Ｂ４逆方向プライマーの塩基配列
〔配列番号：１５〕３６Ｂ４プローブ配列
〔配列番号：１６〕ｓｉＲＮＡ１　センス鎖の塩基配列
〔配列番号：１７〕ｓｉＲＮＡ１　アンチセンス鎖の塩基配列
〔配列番号：１８〕ｓｉＲＮＡ２　センス鎖の塩基配列
〔配列番号：１９〕ｓｉＲＮＡ２　アンチセンス鎖の塩基配列
〔配列番号：２０〕マウスＩＭＰＡ２の増幅及び検出用プライマー１
〔配列番号：２１〕マウスＩＭＰＡ２の増幅及び検出用プライマー２
〔配列番号：２２〕マウスＩＭＰＡ２の検出用プライマー
〔配列番号：２３〕ラットＩＭＰＡ２の増幅及び検出用プライマー１
〔配列番号：２４〕ラットＩＭＰＡ２の増幅及び検出用プライマー２
〔配列番号：２５〕ラットＩＭＰＡ２の検出用プライマー
〔配列番号：２６〕ＦＬＡＧ配列導入用オリゴヌクレオチド１
〔配列番号：２７〕ＦＬＡＧ配列導入用オリゴヌクレオチド２ The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1] Amino acid sequence of human IMPA2 [SEQ ID NO: 2] DNA nucleotide sequence encoding human IMPA2 [SEQ ID NO: 3] Amino acid sequence of mouse IMPA2 [SEQ ID NO: 4] DNA encoding mouse IMPA2 Base sequence [SEQ ID NO: 5] Amino acid sequence of rat IMPA2 [SEQ ID NO: 6] Base sequence of DNA encoding rat IMPA2 [SEQ ID NO: 7] Amino acid sequence of bovine IMPA2 [SEQ ID NO: 8] Codes bovine IMPA2 DNA base sequence [SEQ ID NO: 9] amino acid sequence of canine IMPA2 [SEQ ID NO: 10] DNA base sequence of DNA encoding canine IMPA2 [SEQ ID NO: 11] amino acid sequence of monkey IMPA2 [SEQ ID NO: 12] monkey IMPA2 Base sequence of DNA to be encoded [SEQ ID NO: 13] Base sequence of 36B4 forward primer [SEQ ID NO: 4] Base sequence of 36B4 reverse primer [SEQ ID NO: 15] 36B4 probe sequence [SEQ ID NO: 16] siRNA1 base sequence of sense strand [SEQ ID NO: 17] siRNA1 base sequence of antisense strand [SEQ ID NO: 18] siRNA2 Base sequence of sense strand [SEQ ID NO: 19] siRNA2 Base sequence of antisense strand [SEQ ID NO: 20] Primer 1 for amplification and detection of mouse IMPA2
[SEQ ID NO: 21] Primer 2 for amplification and detection of mouse IMPA2
[SEQ ID NO: 22] Primer for detection of mouse IMPA2 [SEQ ID NO: 23] Primer 1 for amplification and detection of rat IMPA2
[SEQ ID NO: 24] Primer 2 for amplification and detection of rat IMPA2
[SEQ ID NO: 25] Primer for detection of rat IMPA2 [SEQ ID NO: 26] Oligonucleotide 1 for introducing FLAG sequence
[SEQ ID NO: 27] Oligonucleotide 2 for introducing FLAG sequence

　以下に実施例を示して、本発明をより詳細に説明するが、これらは本発明の範囲を限定するものではない。なお、大腸菌を用いての遺伝子は、モレキュラー・クローニング（Molecular cloning）に記載されている方法に従った。 Hereinafter, the present invention will be described in more detail with reference to examples, but these do not limit the scope of the present invention. In addition, the gene using Escherichia coli followed the method described in the molecular cloning (Molecular cloning).

実施例１：C3H10T1/2分化脂肪細胞での分化に伴うIMPA2発現上昇
　マウスC3H10T1/2細胞を、10％CS含有D-MEM培地で培養した。脂肪細胞への分化誘導は以下の方法で行った。C3H10T1/2細胞を96 well Collagen type 1コートプレート（IWAKI）に10,000 cells/wellで播種し、0.5％CO2雰囲気下、37℃で培養した。一晩培養した後、10μM ピオグリタゾンを含む10％FBS含有D-MEM培地に交換して脂肪細胞への分化を誘導した。
　脂肪細胞への分化過程におけるIMPA2のmRNA発現量の変化を調べるため、脂肪細胞への分化過程の各段階で細胞を回収し、RNeasy mini kit（Qiagen）を用い、添付のプロトコルに従ってトータルRNAを調製した。調製したトータルRNAから、High-Capacity cDNA Archive（ABI）を用い、添付のプロトコルに従ってcDNAを調製した。次に、調製したcDNAサンプルから、TaqMan PCR法により、添付のプロトコルに従ってIMPA2のmRNA量を定量した。TaqMan PCR法のプローブとプライマーセットに関して、IMPA2のmRNAに対してはGene expression assayのAssay ID: Mm00475141_m1（TaqMan(R) Gene Expression Assays（Part Number: 4331182）、Assay ID:Mm00475141_m1、Applied Biosystems）、対照となる36B4のmRNAに対しては配列番号:13と14のプライマーと、配列番号:15のプローブ（VIC-TAMRA）を用いた。その結果、脂肪細胞への分化に伴ってIMPA2のmRNA発現が上昇することを明らかにした（図１）。 Example 1: Increase in IMPA2 expression accompanying differentiation in C3H10T1 / 2 differentiated adipocytes Mouse C3H10T1 / 2 cells were cultured in D-MEM medium containing 10% CS. Differentiation into adipocytes was induced by the following method. C3H10T1 / 2 cells were seeded in a 96-well Collagen type 1 coated plate (IWAKI) at 10,000 cells / well and cultured at 37 ° C. in a 0.5% CO 2 atmosphere. After overnight culture, the cells were replaced with 10% FBS-containing D-MEM medium containing 10 μM pioglitazone to induce differentiation into adipocytes.
To examine changes in the expression level of IMPA2 mRNA during differentiation into adipocytes, cells were collected at each stage of differentiation into adipocytes, and total RNA was prepared using the RNeasy mini kit (Qiagen) according to the attached protocol. did. From the prepared total RNA, cDNA was prepared using the High-Capacity cDNA Archive (ABI) according to the attached protocol. Next, the mRNA amount of IMPA2 was quantified from the prepared cDNA sample by TaqMan PCR according to the attached protocol. Regarding the probe and primer set of TaqMan PCR method, for the mRNA of IMPA2, Gene ID assay Assay ID: Mm00475141_m1 (TaqMan (R) Gene Expression Assays (Part Number: 4331182), Assay ID: Mm00475141_m1, Applied Biosystems), control For the 36B4 mRNA, the primers of SEQ ID NOs: 13 and 14 and the probe of SEQ ID NO: 15 (VIC-TAMRA) were used. As a result, it was clarified that the mRNA expression of IMPA2 increases with differentiation into adipocytes (FIG. 1).

実施例２：siRNAを用いたIMPA2発現抑制による脂肪細胞内トリグリセリド(TG)蓄積の抑制
　実施例1と同様の方法で分化誘導させたC3H10T1/2分化脂肪細胞に、以下の方法でIMPA2に対するsiRNAを導入した。トランスフェクション試薬HiperFect（Qiagen）を用い、脂肪分化誘導後2日目に添付のプロトコルに従って配列番号:16と17、あるいは配列番号:18と19のsiRNAを分化脂肪細胞に導入した。siRNA導入後4日目に、以下の方法を用いて細胞内のトリグリセリド蓄積量を定量した。細胞を2.5％グルタルアルデヒドで15分間処理した後、PBSで100倍希釈したAdipoRed溶液（CAMBREX）で20分間処理し、細胞内トリグリセリドを染色した。染色したトリグリセリドはEnVision（485nm/ 535nm, PerkinElmer）で検出した。さらに同条件でsiRNAを導入した細胞を用い、実施例1と同様にしてIMPA2のmRNA量を定量し、siRNAによる遺伝子発現抑制効果を評価した。その結果、siRNAを用いてIMPA2の遺伝子発現を抑制すると、脂肪細胞のトリグリセリド蓄積が抑制されることを明らかにした（図２）。 Example 2: Inhibition of adipocyte triglyceride (TG) accumulation by suppression of IMPA2 expression using siRNA siRNA against IMPA2 was applied to C3H10T1 / 2 differentiated adipocytes induced by the same method as in Example 1 by the following method. Introduced. Using the transfection reagent HiperFect (Qiagen), siRNAs of SEQ ID NOs: 16 and 17, or SEQ ID NOs: 18 and 19 were introduced into differentiated adipocytes on the second day after induction of fat differentiation according to the attached protocol. On the fourth day after siRNA introduction, intracellular triglyceride accumulation was quantified using the following method. The cells were treated with 2.5% glutaraldehyde for 15 minutes and then treated with AdipoRed solution (CAMBREX) diluted 100-fold with PBS for 20 minutes to stain intracellular triglycerides. Stained triglycerides were detected with EnVision (485 nm / 535 nm, PerkinElmer). Furthermore, using the cells into which siRNA was introduced under the same conditions, the amount of mRNA of IMPA2 was quantified in the same manner as in Example 1, and the gene expression suppression effect by siRNA was evaluated. As a result, it was clarified that the suppression of IMPA2 gene expression using siRNA suppresses triglyceride accumulation in adipocytes (FIG. 2).

実施例３：ヒトIMPA2とヒトIMPA1タンパク質（ポリペプチド）の大量調製
　ヒトIMPA2遺伝子の発現ベクターpcDNA3.1F-hIMPA2は以下のように作製した。まず、pcDNA3.1(+)（Invitrogen）にFLAG配列を導入したpcDNA3.1FFベクターを、FLAG配列を有する合成 DNA を用いて構築した。すなわち、2種類のPrimer、FLAGNHENOT-F 5’-CTAGCGCCACCATGGACTACAAGGACGACGATGACAAGGGATCCGACTACAAGGACGACGATGACAAGTGAGC-3’（配列番号：26）及びFLAGNHENOT-R 5’-GGCCGCTCACTTGTCATCGTCGTCCTTGTAGTCGGATCCCTTGTCATCGTCGTCCTTGTAGTCCATGGTGGCG-3’（配列番号：27）を98℃10分反応後、室温で徐冷してアニールさせた。これをNhe I及びNot I（ともにタカラバイオ）で切断したpcDNA3.1(+)と連結し、大腸菌JM109（ニッポンジーン）にクローニングしてpcDNA3.1FFを作製した。次に以下の方法で、ヒトIMPA2のN末端側にFLAGを融合させたタンパク質（ポリペプチド）を発現させるためのベクターpcDNA3.1F-hIMPA2を構築した。すなわちInvitrogenより購入したエントリーベクターpENTRTM-IMPA2（Invitrogen）からIMPA2の5’端にBamHIを、3’端にXhoI認識配列を付加したprimerを用いたPCRによりIMPA2フラグメントを増幅し、増幅フラグメントをBamHI/XhoI制限部位を利用してpcDNA3.1FFに挿入し、得られたプラスミドでDH5α株（Invitrogen）を形質転換した。得られたクローンからプラスミドDNAをQIAprep spin Miniprep kit(50)（QIAGEN）によって抽出し、配列確認（ABI PRISM 3130xl Genetic Analyzer、ABI）を行った。
　次に以下の方法で、ヒトIMPA1のN末端側にFLAGを融合させたタンパク質（ポリペプチド）を発現させるためのベクターpcDNA3.1F-hIMPA1を構築した。HEK293細胞から、RNeasy mini kit（QIAGEN）を用いてtotal RNAを抽出し、抽出したトータルRNAをHigh Capacity cDNA Reverse Transcription Kit（ABI PRISM）により逆転写し、cDNAを得た。同cDNAからIMPA1の5'端にBglII、3'端にXhoＩ認識配列を付加したプライマーを用いてPCR反応によりIMPA1フラグメントを増幅し、増幅フラグメントをBamHＩ/XhoＩ制限部位を利用してpcDNA3.1FFに挿入し、得られたプラスミドでDH5α株（Invitrogen）を形質転換した。得られたクローンをT7 forward primer、BGH reverse primer、Insert check Ready（TOYOBO）を用いたコロニーPCRにより陽性クローンを選択し、プラスミドDNAをQIAprep spin Miniprep kit(50)（QIAGEN）によって抽出し、配列確認（ABI PRISM 3130xl Genetic Analyzer、ABI）を行った。
　pcDNA3.1F-hIMPA2及びpcDNA3.1F-hIMPA1をそれぞれFreeStyle293細胞に導入し、抗FLAG抗体を用いたアフィニティー精製法でヒトIMPA2タンパク質（ポリペプチド）及びヒトIMPA1タンパク質（ポリペプチド）を大量調製した。すなわちFreeStyle293を0.6×106cells/mLの細胞濃度で90 mLのFreeStyle293 expression medium（Invitrogen）に播種し、これを一晩培養した。翌日細胞数を計測し、1.1×106 cell/mLとなるように細胞を培地で希釈した。pcDNA3.1F-hIMPA2またはpcDNA3.1F-hIMPA190μgを50 mLチューブ（Falcon）へ添加し、そこへOpti-MEM（Invitrogen）を3 ml加え混合した。また別の50 mLファルコンチューブに293fectin 120μlおよびOpti-MEM 3 mLを添加し、混合した。DNA希釈液と293fectin希釈液を混合し、室温で20分間反応させた。20分経過後、DNA-293fectin反応液を培養液に添加し、その後細胞を72時間培養した。72時間経過後、細胞を回収し、-80℃で保存した。保存した細胞液50 mL分をLysis buffer（20 mM TrisHCl(pH 7.5), 0.25 M スクロース, 1.2 mM EGTA, 1 mM オルトバナジン酸ナトリウム, 1 mM ピロリン酸ナトリウム, 1 mM NaF, 1％Triton X-100, 0.5％Nonidet P-40, 150 mM NaCl）で溶解した。Wash buffer（20 mM Tris-HCl (pH7.5), 0.1 mM EDTA, 100 mM NaCl, 0.05％Tween20）で平衡化したAnti-FLAG M2 Affinity resin 200μl（50％スラリー）（Sigma）に溶解物を加え、4℃で1時間、ローテーターを用いて混合し、目的のFLAG融合タンパク質（ポリペプチド）をレジンに結合させた。溶解物とレジンとの混合物をカラムに移し、Wash buffer をレジンの10倍量（2 mL）用いてレジンを洗浄した。洗浄後のフロースルーについての波長280 nmにおける吸光度を測定し、吸光度0.05以下になるまで洗浄操作を4回行った。その後、レジンと結合した目的のFLAG融合タンパク質（ポリペプチド）を、150 ng/μLの3×FLAGペプチド（1×Wash Bufferに溶解）（Sigma）を含む溶液をレジン容量の5倍量（1 mL）用いて競合溶出させ、ヒトIMPA2タンパク質（ポリペプチド）およびヒトIMPA1タンパク質（ポリペプチド）をそれぞれ得た。 Example 3: Large-scale preparation of human IMPA2 and human IMPA1 protein (polypeptide) The human IMPA2 gene expression vector pcDNA3.1F-hIMPA2 was prepared as follows. First, a pcDNA3.1FF vector in which a FLAG sequence was introduced into pcDNA3.1 (+) (Invitrogen) was constructed using a synthetic DNA having a FLAG sequence. That is, two types of Primer, FLAGNHENOT-F 5'-CTAGCGCCACCATGGACTACAAGGACGACGATGACAAGGGATCCGACTACAAGGACGACGATGACAAGTGAGC-3 '(SEQ ID NO: 26) and FLAGNHENOT-R 5'-GGCCGCTCACTTGTCATCGTCGTCCTTGTAGTCTCGATCTGTTTGCATCGTCTC Slow cooling and annealing. This was ligated with pcDNA3.1 (+) cleaved with Nhe I and Not I (both Takara Bio) and cloned into E. coli JM109 (Nippon Gene) to produce pcDNA3.1FF. Next, a vector pcDNA3.1F-hIMPA2 for expressing a protein (polypeptide) in which FLAG was fused to the N-terminal side of human IMPA2 was constructed by the following method. That is, the IMPA2 fragment was amplified by PCR using a primer with the BAMHI at the 5 'end of IMPA2 and the XhoI recognition sequence added at the 3' end from the entry vector pENTR TM -IMPA2 (Invitrogen) purchased from Invitrogen. Using the / XhoI restriction site, it was inserted into pcDNA3.1FF, and the resulting plasmid was transformed into DH5α strain (Invitrogen). Plasmid DNA was extracted from the obtained clone with QIAprep spin Miniprep kit (50) (QIAGEN), and sequence confirmation (ABI PRISM 3130xl Genetic Analyzer, ABI) was performed.
Next, a vector pcDNA3.1F-hIMPA1 for expressing a protein (polypeptide) in which FLAG was fused to the N-terminal side of human IMPA1 was constructed by the following method. Total RNA was extracted from HEK293 cells using RNeasy mini kit (QIAGEN), and the extracted total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (ABI PRISM) to obtain cDNA. From the same cDNA, the IMPA1 fragment was amplified by PCR using primers with BglII at the 5 'end and XhoI recognition sequence added at the 3' end, and the amplified fragment was converted into pcDNA3.1FF using BamHI / XhoI restriction sites. The DH5α strain (Invitrogen) was transformed with the resulting plasmid. Positive clones were selected by colony PCR using T7 forward primer, BGH reverse primer, Insert check Ready (TOYOBO), and plasmid DNA was extracted with QIAprep spin Miniprep kit (50) (QIAGEN) for sequence confirmation. (ABI PRISM 3130xl Genetic Analyzer, ABI) was performed.
pcDNA3.1F-hIMPA2 and pcDNA3.1F-hIMPA1 were introduced into FreeStyle293 cells, respectively, and human IMPA2 protein (polypeptide) and human IMPA1 protein (polypeptide) were prepared in large quantities by affinity purification using anti-FLAG antibody. That is, FreeStyle293 was seeded in 90 mL of FreeStyle293 expression medium (Invitrogen) at a cell concentration of 0.6 × 10 6 cells / mL and cultured overnight. On the next day, the number of cells was counted, and the cells were diluted with a medium so as to be 1.1 × 10 6 cells / mL. pcDNA3.1F-hIMPA2 or pcDNA3.1F-hIMPA190 μg was added to a 50 mL tube (Falcon), and 3 ml of Opti-MEM (Invitrogen) was added thereto and mixed. In another 50 mL falcon tube, 120 μl of 293fectin and 3 mL of Opti-MEM were added and mixed. The DNA diluted solution and the 293fectin diluted solution were mixed and reacted at room temperature for 20 minutes. After 20 minutes, the DNA-293fectin reaction solution was added to the culture solution, and then the cells were cultured for 72 hours. After 72 hours, cells were collected and stored at -80 ° C. 50 mL of the stored cell solution was added to Lysis buffer (20 mM TrisHCl (pH 7.5), 0.25 M sucrose, 1.2 mM EGTA, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM NaF, 1% Triton X-100 , 0.5% Nonidet P-40, 150 mM NaCl). Add the lysate to 200 μl of Anti-FLAG M2 Affinity resin (50% slurry) (Sigma) equilibrated with Wash buffer (20 mM Tris-HCl (pH7.5), 0.1 mM EDTA, 100 mM NaCl, 0.05% Tween20) The mixture was mixed with a rotator at 4 ° C. for 1 hour to bind the desired FLAG fusion protein (polypeptide) to the resin. The mixture of the lysate and the resin was transferred to a column, and the resin was washed with a wash buffer 10 times as much as the resin (2 mL). The absorbance at a wavelength of 280 nm for the flow-through after washing was measured, and washing operation was performed four times until the absorbance was 0.05 or less. Then, a solution containing the desired FLAG fusion protein (polypeptide) bound to the resin and 150 ng / μL of 3 × FLAG peptide (dissolved in 1 × Wash Buffer) (Sigma) is 5 times the resin volume (1 mL) ) And competitive elution to obtain human IMPA2 protein (polypeptide) and human IMPA1 protein (polypeptide), respectively.

実施例４：ヒトIMPA2のホスファターゼ活性
　標準測定系は、50 mM Tris-HCl(pH 8.0)、0.1 mM EGTA, 1 mM 基質（下記表１に記載の化合物）、上記実施例３において取得したIMPA1またはIMPA2 70 ng（40μl）を用いて37℃にて24時間行った。それぞれの反応液20μlに、30μLの50 mM Tris-HCl(pH 8.0) 0.1 mM EGTAのbuffer、100μLのBiomol Green reagent（BIOMOL Research Lab., Plymouth Meeting, PA）を添加し、600 nmの吸光度を測定し、IMPA2のIMPA1に対する相対活性を表１に示した。

Figure JPOXMLDOC01-appb-T000001
　上記の表に記載の化合物のうち、IMPA1とは反応性の異なる、IMPA2に相対活性の高い基質化合物として６－ホスホグルコン酸を特定した。この６－ホスホグルコン酸は、IMPA2の基質としては新規であり、かつIMPA1にも弱いながら基質となりうるが、明らかにＩＭＰＡ２に対する基質特異性が高い。
　したがって、この６－ホスホグルコン酸の類縁化合物は、IMPA2特異的な基質である可能性が非常に高い。この６－ホスホグルコン酸に基づき類縁化合物や誘導体などを調製し、本発明のスクリーニング方法を行うことにより、ＩＭＰＡ２により高い特異性、および／またはより高い阻害活性を有する化合物の単離が期待できる。 Example 4: Phosphatase activity of human IMPA2 The standard measurement system was 50 mM Tris-HCl (pH 8.0), 0.1 mM EGTA, 1 mM substrate (compound described in Table 1 below), IMPA1 obtained in the above Example 3 or IMPA2 70 ng (40 μl) was used at 37 ° C. for 24 hours. To 20 μl of each reaction solution, add 30 μL of 50 mM Tris-HCl (pH 8.0) 0.1 mM EGTA buffer and 100 μL of Biomol Green reagent (BIOMOL Research Lab., Plymouth Meeting, PA), and measure the absorbance at 600 nm The relative activity of IMPA2 to IMPA1 is shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
 Among the compounds listed in the above table, 6-phosphogluconic acid was identified as a substrate compound having a different reactivity from IMPA1 and having a high relative activity to IMPA2. This 6-phosphogluconic acid is a novel substrate for IMPA2 and can be a substrate although it is weak against IMPA1, but it clearly has a high substrate specificity for IMPA2.
Therefore, this 6-phosphogluconic acid related compound is very likely to be an IMPA2-specific substrate. By preparing similar compounds and derivatives based on this 6-phosphogluconic acid and performing the screening method of the present invention, it is possible to expect isolation of compounds having higher specificity and / or higher inhibitory activity by IMPA2.

実施例５：マウス筋組織でのIMPA2 mRNA発現
　正常（ｍ＋／ｍ＋）マウス（BKS.Cg-m +/m +/Jcl、９週齢、雄、５匹）より、ヒラメ筋並びに腓腹筋の赤色の濃い部分及び薄い部分をそれぞれ分けて採取した。それぞれの組織は、Isogen（ニッポンジーン社）およびRNeasy mini（キアゲン社）にて、マニュアルに従いトータルＲＮＡを調製した。このトータルRNA 1.35μgから、High Capacity cDNA Reverse Transcription Kit（ABI社）を用いてマニュアルに従ってcDNAを合成した。マウスのIMPA2 mRNA発現量測定には、Sequence Detection System Prism 7900（ABI社）を用いた。マウスＩＭＰＡ２の増幅と検出のためのプライマーとして5’-TCAGTTTGGCCTTCACTGCTG-3’（配列番号：20）、5’-GCACGACATGAGGTCAAGGG-3’（配列番号：21）、およびTaqMan probeとして5’-(FAM)-AGGTGTCAATCACAATGCCGCCTGCT-(TAMRA)-3’（配列番号：22）を使用した。RT-PCR反応液はExpress qPCR Supermix Universal（Invitrogen社）5μlに、それぞれ100μMのプライマー溶液を0.09μl、5μMのTaqMan probeを0.5μl、および上記で調製したcDNA溶液を10 ng加え、蒸留水で総反応液量を10μlとした。PCR反応は50℃　2分、95℃　10分の後、95℃　15秒、60℃　1分のサイクルを40回繰り返した。得られたマウス各種組織におけるIMPA2のmRNA発現量を、トータルRNA 10ngあたりのコピー数として算出した（図３）。
　ヒラメ筋、あるいは腓腹筋の赤い部分ではミトコンドリア量が多く、かつミトコンドリアの各種活性が高いことが知られているので、図３に示す結果は、IMPA2がミトコンドリアの機能あるいは活性に強く関わっていることを強く示唆する。 Example 5: IMPA2 mRNA expression in mouse muscle tissue From normal (m + / m +) mice (BKS.Cg-m + / m + / Jcl, 9 weeks old, male, 5 mice), the soleus and gastrocnemius red The dark part and the thin part were collected separately. For each tissue, total RNA was prepared with Isogen (Nippon Gene) and RNeasy mini (Qiagen) according to the manual. From 1.35 μg of this total RNA, cDNA was synthesized according to the manual using High Capacity cDNA Reverse Transcription Kit (ABI). Sequence Detection System Prism 7900 (ABI) was used to measure the expression level of mouse IMPA2 mRNA. 5'-TCAGTTTGGCCTTCACTGCTG-3 '(SEQ ID NO: 20), 5'-GCACGACATGAGGTCAAGGG-3' (SEQ ID NO: 21) as primers for amplification and detection of mouse IMPA2, and 5 '-(FAM)-as TaqMan probe AGGTGTCAATCACAATGCCGCCTGCT- (TAMRA) -3 ′ (SEQ ID NO: 22) was used. RT-PCR reaction solution was added to Express qPCR Supermix Universal (Invitrogen) 5 μl, 0.09 μl of 100 μM primer solution, 0.5 μl of 5 μM TaqMan probe, and 10 ng of the cDNA solution prepared above, respectively. The reaction volume was 10 μl. In the PCR reaction, a cycle of 50 ° C. for 2 minutes and 95 ° C. for 10 minutes was followed by 40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute. The mRNA expression level of IMPA2 in the obtained various mouse tissues was calculated as the copy number per 10 ng of total RNA (FIG. 3).
Since the red part of the soleus or gastrocnemius muscle is known to have a large amount of mitochondria and high mitochondrial activity, the results shown in Fig. 3 indicate that IMPA2 is strongly involved in mitochondrial function or activity. Strongly suggest.

実施例６：ラット脂肪組織でのIMPA2 mRNA発現
　Wistarラット（９週齢、雄）から褐色脂肪組織、皮下脂肪、腸間膜脂肪及び精巣上体脂肪部分をそれぞれ分けて採取した。それぞれの組織は、ISOGEN（ニッポンジーン社）にて、マニュアルに従いトータルＲＮＡを調製した。このトータルRNA 750ngから、High Capacity cDNA Reverse Transcription キットを用いてマニュアルに従ってｃＤＮＡを合成した。ラットのIMPA2 mRNA発現量は、Sequence Detection System Prism 7900（ABI社）を用いた。ラットIMPA2の増幅と検出のためのプライマーとして5’-GTGGCATTGTGATTGACACCTC-3’（配列番号：23）、5’-GAGCTATGAGCACTGCCATCTC-3’（配列番号：24）、およびTaqMan probeとして5’-(FAM)-AGCCACCACTCTGCACGACATGAGGT-(TAMRA)-3’（配列番号：25）を使用した。Express qPCR Supermix Universal（Invitrogen社）5.0μlに、それぞれ100μMのプライマー溶液を0.18μl、5μMのTaqMan probeを1μl、および上記で調製したcDNA溶液を2μl加え、蒸留水で総反応液量を10μlとした。PCR反応は50℃　2分、95℃　10分の後、95℃　15秒、60℃　1分のサイクルを40回繰り返した。得られたラット各種組織におけるIMPA2のmRNA発現量を、トータルRNA 10ngあたりのコピー数として算出した（図４）。
　褐色脂肪ではミトコンドリア量が多く、かつミトコンドリアの各種活性が高いことが知られているので、図４に示す結果は、IMPA2がミトコンドリアの機能あるいは活性に強く関わっていることを強く示唆する。 Example 6: IMPA2 mRNA expression in rat adipose tissue Brown adipose tissue, subcutaneous fat, mesenteric fat and epididymal fat were separately collected from Wistar rats (9 weeks old, male). For each tissue, total RNA was prepared by ISOGEN (Nippon Gene) according to the manual. CDNA was synthesized from 750 ng of this total RNA using the High Capacity cDNA Reverse Transcription kit according to the manual. For the expression level of rat IMPA2 mRNA, Sequence Detection System Prism 7900 (ABI) was used. 5'-GTGGCATTGTGATTGACACCTC-3 '(SEQ ID NO: 23), 5'-GAGCTATGAGCACTGCCATCTC-3' (SEQ ID NO: 24) as primers for amplification and detection of rat IMPA2, and 5 '-(FAM)-as TaqMan probe AGCCACCACTCTGCACGACATGAGGT- (TAMRA) -3 ′ (SEQ ID NO: 25) was used. Express qPCR Supermix Universal (Invitrogen) 5.0 μl, 0.1 μl of 100 μM primer solution, 1 μl of 5 μM TaqMan probe, and 2 μl of the cDNA solution prepared above were added, and the total reaction volume was adjusted to 10 μl with distilled water. . In the PCR reaction, a cycle of 50 ° C. for 2 minutes and 95 ° C. for 10 minutes was followed by 40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute. The mRNA expression level of IMPA2 in the obtained rat tissues was calculated as the copy number per 10 ng of total RNA (FIG. 4).
Since brown fat is known to have a large amount of mitochondria and high mitochondrial activity, the results shown in FIG. 4 strongly suggest that IMPA2 is strongly involved in mitochondrial function or activity.

実施例７：siRNAを用いたIMPA2発現抑制による脂肪酸β酸化活性の上昇
　10％CS含有D-MEM培地で培養したC3H10T1/2細胞に、IMPA2に対するsiRNAを実施例2と同様の方法で導入した。用いたsiRNAも実施例２と同じものを使用した。導入後5日目に反応培地（5 nCi/ml [3H]-オレイン酸（GE Healthcare, TRK140）、0.1 mM オレイン酸（Wako）、1.1％エタノール、0.12％BSA（Sigma）含有DMEM（Invitrogen））に置換し、6時間培養した。Norit（Wako）を充填したMultiScreen DV 0.65 mm プレート（Millipore)に培養上清液を通し、脂肪酸酸化により生成したトリチウム水を含む水溶性画分の放射活性をTopCount（PerkinElmer）で測定した（図５、p<0.05）。
　本実施例により、IMPA2のsiRNA1およびsiRNA2を用いた細胞内IMPA2発現の抑制によって、細胞内脂肪酸β酸化活性の上昇が生じた。これによって細胞内トリグリセリド量は低下するので、IMPA2阻害薬がＩＭＰＡ２関連末梢性疾患、特に肥満症などのトリグリセリド蓄積量に関する疾患の予防・治療剤として有用であることが示唆される。 Example 7: Increase in fatty acid β oxidation activity by suppression of IMPA2 expression using siRNA siRNA against IMPA2 was introduced into C3H10T1 / 2 cells cultured in D-MEM medium containing 10% CS in the same manner as in Example 2. The same siRNA as in Example 2 was used. 5 days after introduction, reaction medium (5 nCi / ml [ 3 H] -oleic acid (GE Healthcare, TRK140), 0.1 mM oleic acid (Wako), 1.1% ethanol, 0.12% BSA (Sigma) -containing DMEM (Invitrogen) ) And cultured for 6 hours. The culture supernatant was passed through a MultiScreen DV 0.65 mm plate (Millipore) filled with Norit (Wako), and the radioactivity of the water-soluble fraction containing tritium water produced by fatty acid oxidation was measured with TopCount (PerkinElmer) (FIG. 5). , P <0.05).
According to this example, suppression of intracellular IMPA2 expression using siRNA1 and siRNA2 of IMPA2 resulted in an increase in intracellular fatty acid β-oxidation activity. This reduces the amount of intracellular triglycerides, suggesting that IMPA2 inhibitors are useful as prophylactic / therapeutic agents for IMPA2-related peripheral diseases, particularly diseases related to triglyceride accumulation such as obesity.

実施例８：ヒトIMPA2とヒトIMPA1タンパク質（ポリペプチド）の大量調製
　pcDNA3.1F-hIMPA2及びpcDNA3.1F-hIMPA1をそれぞれFreeStyle293細胞に導入し、抗FLAG抗体を用いたアフィニティー精製法でヒトIMPA2タンパク質（ポリペプチド）及びヒトIMPA1タンパク質（ポリペプチド）を大量調製した。すなわち、FreeStyle293を0.6×106 cells/mLの細胞濃度で90 mLのFreeStyle293 expression medium（Invitrogen）に播種し、これを一晩培養した。翌日、細胞数を計測し、1.1×106 cell/mLとなるように細胞を培地で希釈した。pcDNA3.1F-hIMPA2（またはpcDNA3.1F-hIMPA1）90μgを50 mLチューブ（Falcon）へ添加し、そこへOpti-MEM（Invitrogen）を3 ml加え、混合した。次いで、別の50 mLファルコンチューブに293fectin 120μlおよびOpti-MEM 3 mLを添加し、混合した。得られたDNA希釈液と293fectin希釈液を混合し、さらに室温で20分間反応させた。得られたDNA-293fectin反応液を培養液に添加し、細胞を72時間培養し、細胞を回収後、-80℃で保存した。保存した細胞液50 mL分をLysis buffer（TBS、10％グリセロール、1 mM DTT）で溶解し、ポリトロンで20000 rpm、30 秒間を2回、破砕した後、3000 rpm、10 分間、遠心分離した。上清を14000 rpm、20分間でさらに遠心分離をした後、上清を回収し、Anti-FLAG M2 アガロースレジン 3 mLを加え、4℃で1時間、ローテーターを用いて混合し、目的のFLAG融合タンパク質（ポリペプチド）をレジンに結合させた。上清とレジンの混合物をカラムに移し、Lysis bufferでレジンを洗浄した。フロースルーについての波長280 nmにおける吸光度を測定し、吸光度0.05以下になるまで洗浄操作を4回行った。その後、レジンと結合した目的のFLAG融合タンパク質（ポリペプチド）を、Elution buffer（TBS、10％Glycerol、1 mM DTT、200 ng/μL 3×FLAGペプチド(Sigma)）を用いて競合溶出させた。取得したヒトIMPA2タンパク質（ポリペプチド）およびヒトIMPA1タンパク質（ポリペプチド）をElution bufferで2 mg/mlのタンパク濃度に調製し、-80℃で保存した。 Example 8: Large-scale preparation of human IMPA2 and human IMPA1 protein (polypeptide) pcDNA3.1F-hIMPA2 and pcDNA3.1F-hIMPA1 were introduced into FreeStyle293 cells, respectively, and human IMPA2 protein ( Polypeptide) and human IMPA1 protein (polypeptide) were prepared in large quantities. That is, FreeStyle293 was seeded in 90 mL of FreeStyle293 expression medium (Invitrogen) at a cell concentration of 0.6 × 10 6 cells / mL and cultured overnight. The next day, the number of cells was counted, and the cells were diluted with a medium so as to be 1.1 × 10 6 cells / mL. 90 μg of pcDNA3.1F-hIMPA2 (or pcDNA3.1F-hIMPA1) was added to a 50 mL tube (Falcon), and 3 ml of Opti-MEM (Invitrogen) was added thereto and mixed. Then, 120 μl of 293fectin and 3 mL of Opti-MEM were added to another 50 mL falcon tube and mixed. The obtained DNA diluted solution and 293fectin diluted solution were mixed and further reacted at room temperature for 20 minutes. The obtained DNA-293fectin reaction solution was added to the culture solution, the cells were cultured for 72 hours, and the cells were collected and stored at −80 ° C. 50 mL of the stored cell solution was dissolved in Lysis buffer (TBS, 10% glycerol, 1 mM DTT), crushed twice with Polytron at 20000 rpm for 30 seconds, and then centrifuged at 3000 rpm for 10 minutes. After further centrifuging the supernatant at 14000 rpm for 20 minutes, collect the supernatant, add 3 mL of Anti-FLAG M2 agarose resin, mix at 4 ° C for 1 hour using a rotator, and target FLAG fusion Protein (polypeptide) was conjugated to the resin. The mixture of the supernatant and the resin was transferred to a column, and the resin was washed with Lysis buffer. The absorbance at a wavelength of 280 nm for the flow-through was measured, and washing was performed four times until the absorbance was 0.05 or less. Thereafter, the target FLAG fusion protein (polypeptide) bound to the resin was competitively eluted using an Elution buffer (TBS, 10% Glycerol, 1 mM DTT, 200 ng / μL 3 × FLAG peptide (Sigma)). The obtained human IMPA2 protein (polypeptide) and human IMPA1 protein (polypeptide) were prepared with Elution buffer to a protein concentration of 2 mg / ml and stored at -80 ° C.

実施例９：スクリーニング及び選択性試験に用いたヒトIMPA2ホスファターゼ活性測定系
　50 mM Tris-HCl(pH 7.5)、0.1 mM EGTA、2 mM MgCl2、0.01％BSA、1 mM DTT、0.01％CHAPS、1 mM 6-phosphogluconic acid、上記実施例８において取得したIMPA2の27 ng（9μl）及び3μM（もしくは、0.3～100μMの選択性試験の各濃度）の試験化合物を用いて、室温にて50分間反応を行った。それぞれの反応液に20μLのBiomol Green reagent（BIOMOL Research Lab., Plymouth Meeting, PA）を添加し、620 nmの吸光度を測定し、ヒトIMPA2ホスファターゼ活性を確認した。
　また、50 mM MES（pH 6.5）、0.1 mM EGTA、10 mM MgCl2、0.01％BSA、1 mM DTT、0.01％CHAPS、0.5 mM D-myo-inositol 1-monophosphate、上記実施例８において取得したIMPA1の27 ng（9 μl）及び0.3～100μMの選択性試験の各濃度の試験化合物を用いて室温にて50分間反応を行った。それぞれの反応液に20μLのBiomol Green reagent（BIOMOL Research Lab., Plymouth Meeting, PA）を添加し、620 nmの吸光度を測定し、ヒトIMPA1ホスファターゼ活性を確認した。 Example 9: Human IMPA2 phosphatase activity measurement system used for screening and selectivity test 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 2 mM MgCl 2 , 0.01% BSA, 1 mM DTT, 0.01% CHAPS, 1 Reaction was performed at room temperature for 50 minutes using mM 6-phosphogluconic acid, 27 ng (9 μl) of IMPA2 obtained in Example 8 above, and 3 μM (or each concentration of 0.3-100 μM selectivity test). went. 20 μL of Biomol Green reagent (BIOMOL Research Lab., Plymouth Meeting, PA) was added to each reaction solution, and the absorbance at 620 nm was measured to confirm human IMPA2 phosphatase activity.
In addition, 50 mM MES (pH 6.5), 0.1 mM EGTA, 10 mM MgCl 2 , 0.01% BSA, 1 mM DTT, 0.01% CHAPS, 0.5 mM D-myo-inositol 1-monophosphate, IMPA1 obtained in Example 8 above The reaction was carried out at room temperature for 50 minutes using 27 ng (9 μl) of the test compound at each concentration in the selectivity test of 0.3-100 μM. 20 μL of Biomol Green reagent (BIOMOL Research Lab., Plymouth Meeting, PA) was added to each reaction solution, and the absorbance at 620 nm was measured to confirm human IMPA1 phosphatase activity.

　本発明のスクリーニング方法によれば、ＩＭＰＡ２関連末梢性疾患（特に肥満症）の予防・治療剤を効率良く探索することができる。
　また、ＩＭＰＡ２阻害薬を含有してなる、ＩＭＰＡ２関連末梢性疾患の予防・治療剤は、上述のＩＭＰＡ２関連末梢性疾患（特に肥満症）の予防・治療剤として有用であるとともに、低毒性且つ安全である。 According to the screening method of the present invention, a prophylactic / therapeutic agent for IMPA2-related peripheral diseases (particularly obesity) can be efficiently searched.
In addition, the preventive / therapeutic agent for IMPA2-related peripheral diseases comprising an IMPA2 inhibitor is useful as a preventive / therapeutic agent for the above-mentioned IMPA2-related peripheral diseases (particularly obesity), and has low toxicity and safety. It is.

　配列番号１３：36B4フォワードプライマー
　配列番号１４：36B4リバースプライマー
　配列番号１５：36B4プローブ
　配列番号１６：siRNA1ss
　配列番号１７：siRNA1as
　配列番号１８：siRNA2ss
　配列番号１９：siRNA2as
　配列番号２０：マウスIMPA2プライマー
　配列番号２１：マウスIMPA2プライマー
　配列番号２２：マウスIMPA2 mRNA定量用に設計されたプローブであり、5'-末端がFAMで、3'-末端がTAMRAで標識されたプライマー
　配列番号２３：ラットIMPA2プライマー
　配列番号２４：ラットIMPA2プライマー
　配列番号２５：ラットIMPA2 mRNA定量用に設計されたプローブであり、5'-末端がFAMで、3'-末端がTAMRAで標識されたプライマー
　配列番号２６：Flagをコードするプライマー
　配列番号２７：Flagをコードするプライマー
  SEQ ID NO: 13: 36B4 forward primer SEQ ID NO: 14: 36B4 reverse primer SEQ ID NO: 15: 36B4 probe SEQ ID NO: 16: siRNA1ss
Sequence number 17: siRNA1as
SEQ ID NO: 18: siRNA2ss
SEQ ID NO: 19: siRNA2as
SEQ ID NO: 20: Mouse IMPA2 primer SEQ ID NO: 21: Mouse IMPA2 primer SEQ ID NO: 22: Probe designed for quantification of mouse IMPA2 mRNA, labeled with FAM at the 5′-end and TAMRA at the 3′-end SEQ ID NO: 23: Rat IMPA2 primer SEQ ID NO: 24: Rat IMPA2 primer SEQ ID NO: 25: A probe designed for rat IMPA2 mRNA quantification, labeled with FAM at the 5′-end and TAMRA at the 3′-end SEQ ID NO: 26: Primer encoding Flag SEQ ID NO: 27: Primer encoding Flag

Claims (13)

　配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むポリペプチドもしくはその部分ペプチドまたはその塩、あるいは該ポリペプチドまたはその部分ペプチドを産生する細胞を用いることを特徴とする、ＩＭＰＡ２関連末梢性疾患の予防・治療剤のスクリーニング方法。 A polypeptide comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, or a partial peptide thereof or a salt thereof, or the polypeptide or a partial peptide thereof A screening method for a prophylactic / therapeutic agent for IMPA2-related peripheral diseases, characterized by using cells that produce the cells. 　ＩＭＰＡ２関連末梢性疾患が肥満症である請求項１記載の方法。 The method according to claim 1, wherein the IMPA2-related peripheral disease is obesity. 　さらに６－ホスホグルコン酸またはその類縁化合物を用いる、請求項１または２に記載の方法。 The method according to claim 1 or 2, further using 6-phosphogluconic acid or an analogous compound thereof. 　配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むポリペプチドをコードするポリヌクレオチドまたはその部分ポリヌクレオチド、あるいは該ポリペプチドまたはその部分ペプチドに対する抗体をさらに用いることを特徴とする、請求項１または２に記載の方法。 A polynucleotide encoding a polypeptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11, or a partial polynucleotide thereof, or the polypeptide or the The method according to claim 1 or 2, further comprising using an antibody against the partial peptide. 　配列番号：４、６、８、１０または１２で示される塩基配列と同一または実質的に同一な塩基配列を含むポリヌクレオチドが不活性化された、該ポリヌクレオチド発現不全非ヒト哺乳動物を用いることを特徴とする、脂肪分化促進剤のスクリーニング方法。 Use of the polynucleotide expression-deficient non-human mammal in which a polynucleotide comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12 is inactivated A screening method for a fat differentiation promoter. 　外来性の、配列番号：２、４、６、８、１０または１２で示される塩基配列と同一または実質的に同一な塩基配列を含むポリヌクレオチドを有する非ヒトトランスジェニック動物を用いることを特徴とする、ＩＭＰＡ２関連末梢性疾患の予防・治療剤のスクリーニング方法。 Use of a non-human transgenic animal having an exogenous polynucleotide comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 A screening method for a prophylactic / therapeutic agent for an IMPA2-related peripheral disease. 　ＩＭＰＡ２関連末梢性疾患の予防・治療剤が、配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含有するポリペプチドの活性を促進または阻害する化合物またはその塩である、請求項１～４および６のいずれか１項に記載の方法。 An agent for the prophylaxis or treatment of IMPA2-related peripheral diseases promotes the activity of a polypeptide containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11 Alternatively, the method according to any one of claims 1 to 4 and 6, wherein the compound is a inhibiting compound or a salt thereof. 　配列番号：１、３、５、７、９または１１で示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むポリペプチドもしくはその部分ペプチドまたはその塩、あるいは該ポリペプチドまたはその部分ペプチドを産生する細胞、および６－ホスホグルコン酸またはその類縁化合物を用いることを特徴とする、ＩＭＰＡ２阻害薬のスクリーニング方法。 A polypeptide comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, or a partial peptide thereof or a salt thereof, or the polypeptide or a partial peptide thereof A method for screening an IMPA2 inhibitor, which comprises using a producing cell and 6-phosphogluconic acid or a similar compound thereof. 　ＩＭＰＡ２阻害薬を含有してなるＩＭＰＡ２関連末梢性疾患の予防・治療剤。 An agent for preventing / treating IMPA2-related peripheral diseases comprising an IMPA2 inhibitor. 　ＩＭＰＡ２関連末梢性疾患が肥満症である、請求項９記載の剤。 The agent according to claim 9, wherein the IMPA2-related peripheral disease is obesity. 　ＩＭＰＡ２阻害薬がsiRNAである、請求項９または１０に記載の剤。 The agent according to claim 9 or 10, wherein the IMPA2 inhibitor is siRNA. 　哺乳動物に対して、ＩＭＰＡ２阻害薬の有効量を投与することを特徴とするＩＭＰＡ２関連末梢性疾患の予防・治療方法。 A method for preventing and treating an IMPA2-related peripheral disease, comprising administering an effective amount of an IMPA2 inhibitor to a mammal. 　ＩＭＰＡ２関連末梢性疾患の予防・治療のための、ＩＭＰＡ２阻害薬。
  An IMPA2 inhibitor for the prevention and treatment of IMPA2-related peripheral diseases.
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