WO2002004625A1 - α-SYNUCLEIN BINDING PROTEIN - Google Patents

Abstract

A novel α-synuclein binding protein; a DNA encoding this protein; a method of screening a substance inhibiting the association or fibrosis of α-synuclein by using this a-synuclein binding protein; and preventives or retardants containing the above substance for neurodegenerative diseases in association with the fibrosis of a-synuclein such as Parkinson's disease.

Description

 Specification

 One-synuclein binding protein

The present invention relates to a novel protein that binds to a precursor of a non-amyloid component in amyloid protein [hi-synuclein (also referred to as NACP)], an antibody recognizing the protein, and a protein that binds to thynuclein (his-nuclein binding protein) The present invention relates to a method for screening a substance that inhibits the action of synuclein association or fibril formation promotion by the above method, an agent for preventing the onset of neurodegenerative disease obtained by the method, or an agent for delaying the progress.

The following abbreviations are used in this specification.

 B S A: bovine serum albumin

 DMSO: dimethyl sulfoxide

 EDTA: ethylene diamine tetraacetic acid

EGTA: Ethylene Glyco-lubis (? -Aminoethyl ether)-N, N, N ,, N,-

4-acetic acid (ethylene glycol bis (? -Aminoethyl ether) -Ν, Ν, Ν ', Ν' -tetraacetic acid

]

 FITC: fluorescein isothiocyanate HPLC: high performance liquid chromatography

 IPTG: isopropyl thiogalactoside

K LH: keyhole limpet hemocyanin MALD I / T0F: matrix-assisted laser desorption and ionization / time-of-flight ) MAP 5: Microtubule-associated protein 5

 MEM: Minimum essential medium)

 N A C: non-amyloid β component

 NACP: NAC precursor

 PBS: phosphate buffered saline

 PCR: Polymerase chain reaction

 PEG—1000: 1000 polyethylene glycol

-1000) P I PES: piperazine-Ν, Ν'-bis (2-ethanesulpf onic acid)

 PMSF: phenylmethylsulfonyl fluoride

 P VDF: Polyvinylidene difluoride

 RT-P CR: reverse transcription-PCR

 SD S: sodium dodecyl sulfate

 SD S-PAGE: SDS polyacrylamide gel electrophoresis

 TBS: Tris buffered saline

 T F A: trifluoroacetic acid Tris: Tris (hydroxymethyl) aminoniethane

Old style

 Synuclein, Maroteaux L. etal.,

J. Neurosci., S, 2804 (1988)], a protein named human human synuclein [Jakes. Et al., FEBS Lett., S, 27 (1994)] Amyloid proteins other than amyloid ^ previously accumulated in Alzheimer's disease The cDNA was cloned as a precursor protein of the non-amyloid beta component (NAC), a peptide found as a constituent [Ueda K. et al., Proc. Natl. Acad. Sci., USA,, 11282 (1993)], so it is also called NACP (NAC precursor).

 Human synuclein is abundant in presynaptic terminals of the central nervous system [Iwai A. etal.,

Neuron, 14, 467 (1995)], but its physiological function is unknown. However, in some families with familial Parkinson's disease, missense mutations in the human synuclein gene were found [Polymeropoulos MH et al., Science, 2045 (1997), ruger R. et al., Nature Genet. , 106 (1998)], attention has been paid to the relationship between human synuclein and the onset of Parkinson's disease. In addition, human synuclein is a major fibrous form of inclusions called Lewy bodies found in the nerves of Parkinson's disease and certain types of dementia, and intracellular inclusions found in multiple system atrophy. It was reported to be a constituent [Spillantini MG et al., Nature,, 839 (1997)]. Human sph-synuclein also associates in vitro to form fibers.

[Wood SJ et al., J. Biol. Chem., 214, 19509 (1999), Conway KA et al., Nature

Med., 4, 1318 (1998)]. Among the missense mutants, synuclein, found in families with familial Parkinson's disease, are more likely to associate or fibrillate in vitro than wild-type human synuclein [ Conway KA et al., Nat.

Med., I, 1318 (1998), Narhi L. et al., J. Biol. Chem., M, 9843 (1999), Conway.

KA et al., Proc. Natl. Acad. Sci., USA, 21, 571 (2000)]. from these things

The association or fibril formation of spike synuclein is thought to be involved in the development or progression of these diseases, but the mechanism has not been elucidated yet. In addition, most neurodegenerative diseases in which α-synuclein fibril formation is observed, such as Parkinson's disease, Lewy body dementia, multiple system atrophy, and Alzheimer's disease, are mostly familial rather than familial. Because of their onset, the brains of patients with these diseases have factors that promote the initiation and progression of sph-synuclein association and fiber formation, or degrade the sph-synuclein fibers formed. It is considered that the mechanism for performing the operation is reduced.

 Amyloid /? [Yoshimoto M. et al., Proc. Natl. Acad. Sci., USA, 92, 9141 (1995), Jensen PH et al., Biochem. J., 539 (1997)], 14-3-3 protein [Ostrerova N. et al., J. Neurosci., 12., 5782 (1999)], tau protein [Jensen PH et al., J. Biol. Chem., 21A, 25481 (1999)] and microtubule-associated protein 1B, Jensen PH et al., J. Biol. Chem., 2R, 21500 (2000)] (alias: microtubule-associated protein 5, MAP5) However, no effect on the association of these synuclein and fibril formation has been reported. It has been reported that fibril formation is promoted in the mouth of NBIT in the presence of amyloid ^ peptide in NAC, which has a different aggregation property from that of human synuclein [Han H. et al., Chem. Biol., A , 163 (1995)]. In addition, it has been reported that partial peptides of amyloid promote the aggregation of spike synuclein [Paik S et al, FEBS Lett., 421, 73 (1998)].

 Tubulin is a major constituent protein of microtubules, and there are two types, tubulin and tubulin. Tubulin is present in Lewy bodies [Galloway PG et al., J. Neuropathol. Exp. Neurol., 41, 654 (1988)], and inhibits the binding of protein to human synuclein. [Jensen PH et al., J. Biol. Chem., M, 25481 (1999)], but reports that it binds to hy-synuclein and reports on its association with association and fibril formation. There is no.

On the other hand, to find prophylactic or therapeutic agents for neurodegenerative diseases associated with fibril formation of sph-synuclein such as Parkinson's disease, proteins that bind to sph-synuclein in brain cells and are involved in its association or fibril formation have been sought. Have been. 昍

 The present inventors isolated a 42 kDa and 5 OkDa protein that specifically binds to human synuclein from human brain extract by human synuclein-immobilized affinity chromatography. By analyzing the partial amino acid sequences of these proteins, it was found that the 42 kDa protein is a human synuclein binding protein having a novel amino acid sequence, and the 50 kDa protein is a single tubulin. Identified. In addition, it was found that human synuclein binds not only to single tubulin but also to 5-tubulin (in the present invention, “tubulin” refers to single tubulin or /?-Thubulin).

 Furthermore, it was found that tubulin greatly promotes the association and fibril formation of spike-synuclein in the mouth of the intestine, and that the fiber morphology is very similar to the spike-synuclein fiber in the Lewy bodies of pathological tissues. Completed the invention.

 That is, the present application provides the following inventions (1) to (27).

 (1) It is derived from human brain, has a molecular weight in the range of 41600 to 42000, has an amino acid sequence represented by SEQ ID NO: 1 at the N-terminus, and has an amino acid sequence represented by SEQ ID NO: 2 therein, A protein that binds to katsuhi-synuclein.

 (2) The protein according to (1) above, which has a molecular weight of 418477.1 (M + H) + determined by MALD I / T OF mass spectrometry.

 (3) An antibody against the protein of (1) or (2).

 (4) A method for detecting or quantifying the protein of (1) or (2) using the antibody of (3).

 (5) [i] contacting human synuclein with human synuclein binding protein;

[ii] Observe and compare the association or fibril formation of human synuclein when human synuclein is contacted with human synuclein binding protein in the presence of the test substance. A method for screening a substance that inhibits the association or fibril-forming action of human synuclein possessed by a human synuclein-binding protein, comprising selecting a substance that inhibits the association or fibril formation of human synuclein from test substances.

 (6) The difference between the case where [i] human synuclein is brought into contact with the human synuclein binding protein and the case where [ii] the human synuclein is brought into contact with the human synuclein binding protein in the presence of the test substance. Comparing the amount of conjugates of synuclein and human synuclein-binding protein and selecting a substance that inhibits the binding of human synuclein to human-synuclein-binding protein from the test substance; A method for screening a substance that inhibits binding to a protein.

 (7) [i] The case where human synuclein is brought into contact with human synuclein binding protein and the case where [ii] human synuclein is brought into contact with human synuclein binding protein in the presence of a test substance. Compare the function of synuclein and select from the test substance a substance that inhibits the function of sph-synuclein.The function of sph-synuclein caused by the binding of sph-synuclein binding protein to α-synuclein is characterized by Screening methods for inhibitors.

 (8) a protein selected from the group consisting of the protein of (1) or (2) above, tubulin, amyloid / ?, 14_3-3 protein, tau protein and microtubule-associated protein (8). The method according to any one of the above (5) to (7).

 (9) A compound obtained by the method according to any one of the above (5) to (8), or a pharmacologically acceptable salt thereof.

(10) an antibody that inhibits the association or fibrogenesis promoting activity of the human synuclein binding protein possessed by the human synuclein binding protein from the antibody that recognizes the human synuclein binding protein; Of neutralizing antibodies against Method.

 (11) A neutralizing antibody obtained by the method of the above (10).

 (12) A neutralizing antibody against a human synuclein-binding protein, which inhibits the activity of human synuclein-binding protein to promote association or fibril formation of human synuclein.

 (13) The human synuclein binding protein is selected from the group consisting of the protein of (1) or (2) above, tubulin, amyloid 5, 14-3-3 protein, tau protein, and microtubule-associated protein 1B The method according to (10) above.

 (14) The group consisting of the protein of the above (1) or (2), tubulin, amyloid / ?, 14-3-3 protein, tau protein, and microtubule-associated protein 1B, wherein the human synuclein binding protein is (1) or (2). The neutralizing antibody according to (11) or (12), which is selected from the group consisting of:

 (15) A medicament comprising the compound of (9) or a pharmaceutically acceptable salt thereof, or the neutralizing antibody of (11), (12) or (14) as an active ingredient.

(16) The medicament according to the above (15), which is a preventive agent for the onset of neurodegenerative disease or an agent for delaying the progress.

 (17) The medicament according to the above (16), wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Lewy body dementia, multiple system atrophy and Alzheimer's disease.

(18) A polynucleotide encoding the protein of (1) or (2).

 (19) A recombinant DNA comprising the polynucleotide of the above (18).

 (20) A transformant obtained by introducing the recombinant DNA of (19) into a host cell

(21) The transformant according to (20), wherein the host cell is a cell selected from the group consisting of bacteria, yeast, insect cells, plant cells, and animal cells.

(22) Culture the transformant of (20) or (21) above in a culture medium and place it in the culture. A method for producing the protein according to the above (1) or (2), comprising producing and accumulating the protein according to the above (1) or (2), and collecting the protein from the culture.

 (23) an oligonucleotide having a sequence consisting of 5 to 60 consecutive nucleotides in the nucleotide sequence of DNA of the above (18), an oligonucleotide having a sequence complementary to the oligonucleotide, or Derivatives. '

 (24) A medicament comprising the DNA of (18) or the oligonucleotide or oligonucleotide derivative of (23) as an active ingredient.

 (25) The drug according to the above (24), which is a preventive agent for the onset of a neurodegenerative disease or an agent for delaying the progress thereof.

 (26) An agent for preventing the onset of neurodegenerative disease or an agent for delaying the progress, comprising as an active ingredient DNA encoding a synuclein-binding protein or a partial DNA thereof. (27) The medicament according to the above (25) or (26), wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Lewy body dementia, multiple system atrophy and Alzheimer's disease. Hereinafter, the present invention will be described in detail.

 1. Synuclein-binding protein

 (1) Isolation and analysis

The human synuclein binding protein can be isolated from a biological tissue or cell extract by affinity chromatography using a carrier on which human synuclein is immobilized. The human-synuclein-immobilized carrier is brought into contact with the cell extract to bind the human-synuclein-binding protein to the human-synuclein-immobilized carrier, and then the carrier is washed to remove unbound proteins. By treating this carrier with immobilized glycine, the protein can be eluted and isolated from the carrier. it can.

 Since human synuclein-binding protein is considered to bind to and coexist with human synuclein in cells, the source of the isolation is preferably a living tissue or cells rich in human synuclein, such as the brain. Tissues and cells are disrupted in a buffer containing an appropriate protease inhibitor, and the supernatant obtained by centrifugation can be used as an extract.

 Examples of the carrier for immobilizing hy-synuclein include agarose-based carriers such as Sepharose-14B. By activating the sugar such as agarose contained in the carrier or the group of the spacer molecule for immobilization, and reacting with the human synuclein, the human synuclein can be immobilized. For example, Sepharose-14B (manufactured by Amersham 'Pharmacia' Biotech) activated by cyanogen bromide is reacted with para-synuclein based on the manufacturer's manual to immobilize para-synuclein on sepharose. One can get 4 B.

 Hi-synuclein to be bound can be easily obtained with high purity by using a gene recombination method. Specifically, first, a cDNA clone pHBS6-1 of spike-synuclein

Natl. Acad. Sci., USA, M, 11282 (1993)]. CDNA was cut out using restriction enzymes, and the base sequence of c-DNA of human synuclein.

Natl. Acad. Sci., USA, M, 11282 (1993)] from human brain RNA using RT-PCR [McPherson MJ et al.,

PCR: A practical Approach, Oxford University Press (1991)], isolate a DNA fragment containing the cDNA for human synuclein, and use it as an expression vector in E. coli as a host.

For example, pET-15b (manufactured by Novazidin) and pET-3d (manufactured by Novazidin) are inserted into the cloning site to prepare a vector for expression of human synuclein. The transformed E. coli was prepared by introducing this human synuclein expression vector into an appropriate host for E. coli. After culturing, the cells are recovered, and the synuclein is isolated from the solution obtained by disrupting the cells by a conventional protein purification method such as ammonium sulfate precipitation ion exchange chromatography, hydrophobic chromatography, or reverse phase chromatography. It can be purified.

 The spun-synuclein-binding protein eluted from the spun-synuclein-immobilized carrier can be separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional gel electrophoresis. It can also be isolated and purified by chromatography such as ion exchange chromatography, hydrophobic chromatography, and reverse phase chromatography, or by excising the gel after SDS-PAGE and eluting it from the gel.

 The molecular weight of the isolated protein can be analyzed by SDS-PAGE or MALD I / TOF mass spectrometry.

 This isolated protein is used as it is, or after cleavage into peptides by a protease such as lysylendopeptidase or trypsin or chemical cleavage with cyanogen bromide, and the peptide separated by reverse-phase HP LC as a sample. By analyzing by a decomposition method, the N-terminal or internal partial amino acid sequence can be determined. Analysis by the Edman degradation method can be performed automatically by the amino acid sequencer.

 An isolated amino acid sequence was analyzed by analyzing a known amino acid sequence that matches the obtained amino acid sequence by using a homology analysis program against an amino acid sequence database such as SwissProt or PIR. The novelty of the synuclein binding protein can be determined and identified. Examples of the homology analysis program include BLASTX developed based on the algorithm BLAST [J. Mol.

Biol., 403 (1990)]. When analyzing an amino acid sequence by BLASTX based on BLAST, each parameter is, for example, score = 50 and wordlength = 3. BLAST and When using the Gapped BLAST program, use the default parameters of each program. Specific procedures for this are et al method of analysis is Ru Oh known (http://www.ncbi.nlm.nih.gov. ₀

 In other words, when there is no amino acid sequence that matches the known amino acid sequence, it can be determined that the protein has a new amino acid sequence, and when there is a matching amino acid sequence, the isolated human synuclein bond The protein is considered to be the protein in the database containing its amino acid sequence. In addition, the molecular weight of the protein in the database whose amino acid sequence was consistent was the same as that of the isolated human synuclein binding protein, and the N-terminal amino acid sequence of the isolated human synuclein binding protein was If the amino acid sequence at the N-terminus of the protein in the protein matches, the isolated human synuclein binding protein is almost identified as a protein in the database. The thus isolated human synuclein-binding protein had a molecular weight of 4188.1 (M + H) + by MALD I / T OF mass spectrometry and an amino acid sequence represented by SEQ ID NO: 1. A protein or a tubulin containing an amino acid sequence represented by SEQ ID NO: 2 at the N-terminus can be mentioned.

 Examples of the human synuclein binding protein that can be used in the present invention include, in addition to the above, —-tubulin, amyloid / ?, 14-13-3 protein, tau protein, microtubule-associated protein 1B, and the like.

 (2) Binding of human synuclein binding protein to human synuclein

 Whether or not the sph-synuclein-binding protein isolated in (1) is bound to sph-synuclein in vivo can be examined by immunoprecipitation. That is, using a brain extract obtained by the method described in (1) as a sample, an anti-synuclein antibody [EQ

VI, P QE 3: Arima K. et al., Brain Res., Spirit, 93 (1998), Arima K. et al.,

Acta Neuropathol., 2 £, 439 (1998)]. The immunoprecipitate containing crane is subjected to immunoblotting using, as a primary antibody, an antibody against the human synuclein binding protein described later in Section 2. When the human-synuclein binding protein is human tubulin or /?-Tubulin, a commercially available anti-human tubulin antibody or anti-tubulin antibody [DM1A etc .: Sigma-Aldrich (Sigma) -Aldrich) can also be used. If, as a result of immunobroth, spike-synuclein-binding protein can be detected in the immunoprecipitate, it can be confirmed that spike-synuclein-binding protein is bound to spike-synuclein even under physiological conditions in vivo. Conversely, immunoprecipitation was performed using an antibody against the human synuclein binding protein, and the immunoprecipitate containing the obtained human synuclein binding protein was subjected to immunoblotting using an anti-human synuclein antibody as the primary antibody. However, by detecting sph-synuclein in the immunoprecipitate, it can be confirmed that the sph-synuclein binding protein binds to the sph-synuclein even under physiological conditions in vivo. By such a method, 5-tubulin, single tubulin and the like can be mentioned as examples of proteins which have been confirmed to bind to sphine-synuclein under physiological conditions in vivo.

 (3) Promotion of association or fibrillation of synuclein

Prepare a synuclein solution using a buffer such as PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, 7.65 g of sodium chloride, 1 liter of distilled water, pH 7.2). By keeping the temperature at 37 ° C. for 2 to 30 days, sph-synuclein can self-associate in vitro to form fibers. An appropriate concentration of hy-synuclein is 50-700 jumo 1/1. The degree of fibrillation of human synuclein can be quantified as the degree of association of human synuclein by measuring light scattering at 400 nm. Alternatively, ultracentrifugation at 100,000 X g for 10 minutes to precipitate aggregated human synuclein and quantification of the human synuclein concentration in the supernatant by absorbance at 28 O nm [Wood SJ et al., J. Biol. Chem., 2ΊΑ, 9843 (1999)]. Also By observing with an electron microscope, the shape of the fiber can be compared with the shape of the human synuclein fiber in pathological tissues such as Parkinson's disease and Lewy body dementia.

 In this system for the formation of fibro-synuclein fibroin mouth, fibro-formation is not observed unless fi-synuclein alone is kept for at least 15 days. It can be confirmed that, when coexisting with the human synuclein binding protein, the human synuclein binding protein promotes association and fiber formation. This association and fiber formation can be further promoted by magnesium chloride, GTP or ATP.

2. Preparation of Antibodies to Hepato-Synuclein Binding Protein

 (1) Preparation of polyclonal antibody

 The administration of a peptide consisting of five or more contiguous sequences in the amino acid sequence of the human synuclein binding protein or its fragment or the human synuclein binding protein to an animal as an antigen allows a polyclonal protein to bind to the human synuclein binding protein. One null antibody can be made. Examples of the human synuclein-binding protein include the protein obtained above, tubulin, amyloid, 14-3-3 protein, tau protein, and microtubule-associated protein 1B.

 The peptide serving as the antigen can be chemically synthesized using a peptide synthesizer. Non-human mammals such as rabbits, goats, rats, mice, and hamsters can be used as animals to which the antigen is administered. The dose of the antigen is preferably 50 to 100 // g per animal. When a partial peptide is used as an antigen, it is desirable that the partial peptide be covalently bound to a carrier protein such as KLH or bovine thyroglobulin as the antigen.

 The administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. After each dose

Blood was collected from the fundus venous plexus on days 3-7, and the serum reacted with the antigen used for immunization. Enzyme-linked immunosorbent assay [Eiji Ishikawa, Enzyme-linked immunosorbent assay Medical Shoin (1978), Harlow E. & Lane D Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].

 A polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum has a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.

 Methods for separation and purification include centrifugation, salting out with ammonium sulfate 50% saturated, and prillic acid precipitation [Harlow E. & LaneD. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)], or DEAE- There is a method in which chromatography using a Sepharose column, an anion exchange column, a protein A or protein G column, a gel filtration column, or the like is performed alone or in combination.

 (2) Preparation of monoclonal antibody

 (2-1) Preparation of antibody-producing cells

 In (1) above, an animal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody-producing cells. As an animal, it is preferable to use a rat.

 On the 37th day after the final administration of the antigenic substance to the rat showing the antibody titer, the spleen is removed.

 The spleen is minced in MEM, loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.

 The spleen cells of the obtained precipitate fraction are treated with Tris-ammonium chloride buffer (PH7.65) for 12 minutes to remove red blood cells, washed three times with MEM, and the obtained spleen cells are used as antibody-producing cells. Used.

(2-2) Preparation of myeloma cells As myeloma cells, cell lines obtained from mice or rats are used. For example, 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line P3—X63Ag8-Ul [Yelton DE et al., Curr. Topics Microbiol. Immunol., SI, 1 (1978), Kohler G. & Milstein C., Eur. J. Immunol., 6, 511 (1976)], SP2 / OA14 [Shulman. Et al., Nature, 269 (1978)], P3-X63-8653 [Seeger RC J. Immunol., M, 1548 (1979)], P3-X63 Ag

[Kohler G. & Milstein C., Nature, 495 (1975)] and the like can be used. These cell lines, 8-Azaguanin medium [RPMI 1640 medium 1. 5 mm o 1/1 glutamine, ⁵ x 10- 5 mmo 1/1 2- mercaptoethanol, 10 〃G / ml Jen evening clarithromycin and 10% © Medium supplemented with fetal serum (CSL)

(Hereinafter referred to as a normal medium) and a medium supplemented with 15-g / ml 8-azaguanine], and cultured 3 to 4 days before cell fusion with a normal medium. Use 2 X 10 ⁷ or more.

 (2-3) Preparation of Hypri-Doma

 The antibody-producing cells obtained in (2-1) and the myeloma cells obtained in (2-2) were combined with MEM or PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, sodium chloride 7). After washing well with 65 g, 1 liter of distilled water, pH 7.2), mix the cells so that the number of live cells: myeloma cells = 5-10: 1, and centrifuge at 1200 rpm for 5 minutes. Discard the supernatant.

 The cell group of the obtained precipitate fraction is loosened well, and the cell group is stirred at 37 ° C while stirring.

, 10 ⁸ antibody-producing cells per, PEG- 1000 2 g, MEM2ml Oyobi DMSO

Add 0.2 ml of the mixed solution of 0.7 ml; add Lml, and further add MEM 1 every 1-2 minutes.

Add ~ 2 ml several times.

After the addition, add MEM to adjust the total volume to 50 ml. After centrifuging the preparation at 900 rpm for 5 minutes, discard the supernatant.

Gently loosen the cells in the obtained precipitate fraction, then gently aspirate with a pipette and gently blow out the HAT medium [1 O ^ mrno 1/1 hypoxanthine, 1.5 x 10 " ⁵ mmo 1 in normal medium. / 1 thymidine and ^{4 x 10- 7 mm o 1/} 1 Aminobuteri down the added medium] is suspended in 100 ml.

The suspension plate for 96 well culture 100〃1 / well portions dispensed, 5% C0 ₂ ink Yube - evening - in, at 37 ° C? Incubate for ~ 14 days.

 After culturing, a portion of the culture supernatant is removed, and antibody-producing cells are isolated by the enzyme immunoassay described in Harlow E. & Lane D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) Chapter14. To obtain them, select hybridomas that specifically react with the antigen used for immunization.

 The following method can be given as a specific example of the enzyme immunoassay.

 At the time of immunization, the full-length or partial fragment of the human synuclein-binding protein used as the antigen was coated on a suitable plate, and the hybridoma culture supernatant or the purified antibody obtained in (2-4) described later was used as the primary antibody. The antibody is reacted with an anti-rat immunoglobulin antibody labeled with piotin, an enzyme, a chemiluminescent substance, a radioactive compound, or the like as a secondary antibody, followed by a reaction according to the labeling substance. Those that specifically react with the combined protein are selected as hybridomas that produce monoclonal antibodies against the sph-synuclein binding protein.

Cloning is repeated twice by the limiting dilution method using the hybridoma [the first time uses HT medium (medium in which aminopterin is removed from HA Έ medium), and the second time uses normal medium], Those with a stable and strong antibody titer are selected as hybridoma strains producing a monoclonal antibody against the sph-synuclein binding protein. (2-4) Preparation of monoclonal antibody

0.5 ml of Pristane (Pristane; 2, 6, 10, 14-tetramethylpen-decane) was intraperitoneally administered to 8-10-week-old or nude mice bred for 2 weeks, using (2-3) Hyprideoma cells producing the monoclonal antibody to the obtained human synuclein binding protein are injected intraperitoneally with 5 to 20 × 10 ⁶ cells / animal. In 10 to 21 days, Hypridoma becomes ascites cancer.

 Ascites is collected from the mouse with ascites tumor and centrifuged at 3,000 rpm for 5 minutes to remove solids.

 From the obtained supernatant, a monoclonal antibody can be purified and obtained in the same manner as the method used for the polyclonal antibody.

 The antibody subclass is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The protein content is calculated by the Lowry method or from the absorbance at 280 nm.

3. Use of Antibodies to the Synapsein-Binding Protein

 Antibodies to the human synuclein binding protein recognize and specifically bind to the human synuclein binding protein, and thus can be used for detection, quantification, and purification of the human synuclein binding protein.

 Methods for immunologically detecting human synuclein-binding protein using an antibody against human synuclein-binding protein include immunohistochemical staining such as immunohistochemistry and immunocytostaining, immunoblot, and sandwich ELISA. Enzyme immunoassay (EIA) and radioimmunoassay (Radioimmunoassay; RIA)

And the literature [Sakuni Toyama, Tamoe Ando, Monoclonal Antibody Experiment Manual (Kodansha Scientific 1987), Seismic Chemistry Laboratory Course 5 Immunochemical Chemistry Research Method (Tokyo Chemical Doujinshi) 1986), Goding JW, Monoclonal Antibodies: Principles and Practice, Third edition (Academic Press 1996), Harlow E. & Lane D., Antibodies: A laboratory Manual (Cold Spring Harbor Laboratory 1988)] © ad.

 Immunohistochemical staining involves fixing tissue sections and cells, reacting an antibody against human synuclein-binding protein as a primary antibody, and labeling the antibody with fluorescent substances, enzymes, biotin, gold colloid, radioactive substances, etc. After reacting the immunoglobulin antibody or antibody fragment as a secondary antibody, if necessary, the labeled antibody is visualized and observed using a microscope to detect human synuclein-binding protein in tissues and cells. It is a way to do it.

Fluorescein'isothiocyanate (FITC), tetramethylrhodamine / isothiocyanate, etc., are used for fluorescent substance labeling, and are detected by observation with a fluorescence microscope. In the case of enzyme labeling, peroxidase, alkaline phosphatase or the like is used, and a color-developing reaction is performed by adding a substrate that develops a color by the enzyme, and the enzyme can be detected by observing with an optical microscope. In the case of biotin labeling, after reacting avidin labeled with an enzyme such as peroxidase, the same operation as for the enzyme-labeled antibody is performed. In the case of a gold colloid label, it is detected by observation with an electron microscope. The radioactive materials labeled used is ¹²⁵ 1 or the like, can be detected by observing the silver particles precipitated by radiation by coating the emulsion in an optical microscope.

 Imnobroth is obtained by fractionating tissues or cells or their crushed solutions by SDS-PAGE, and then printing the gel on a PVDF membrane or a nitrocellulose membrane, and coating the membrane with an antibody against a human synuclein binding protein or an antibody fragment thereof. The reaction is performed as a primary antibody, and enzymes such as peroxidase and alkaline phosphatase

After the anti Imunoguropurin antibody or antibody fragment labeled with a radioactive substance ¹²⁵ 1 such as by reacting a secondary antibody, shed - in a way of detecting the synuclein binding protein bands is there. In the case of enzyme labeling, an enzyme-colored substrate is added and reacted for visualization of the band of -synuclein-binding protein, or an enzyme-emitting substrate is added and X-ray film autoradiography is performed. Performed by In the case of radioactive substances, it is detected by X-ray film autoradiography.

 In a sandwich ELISA, a type of enzyme immunoassay, monoclonal antibodies to two types of human synuclein binding proteins having different antigen recognition sites are prepared, and one of the monoclonal antibodies or antibody fragments is adsorbed on a plate in advance. The other monoclonal antibody or antibody fragment should be labeled with an enzyme such as peroxidase or alkaline phosphatase. Prepare a cell lysate from tissues and cells and use it as a test sample. The test sample is allowed to react with the antibody adsorption plate, and then the monoclonal antibody or its antibody fragment against the enzyme-labeled human-synuclein-binding protein is reacted. By measuring the intensity of color development with a meter, detection or quantification of sph-synuclein binding protein in the sample is performed.

Radioimmunoassay, using antibodies labeled with radioactive material ¹²⁵ 1 or the like instead of the enzyme, the same operation as enzyme immunoassay, in a sample by measuring radiation at scintillation counter evening one This is a method for detecting or quantifying a synuclein-binding protein. '

In the enzyme immunoassay and the radioimmunoassay, in addition to the above-described sandwich method, instead of using an antibody, a sample of a gamma-synuclein-binding protein is labeled, and a certain amount of labeled α-synuclein-binding protein is used. The test sample is allowed to react with an antibody against the human synuclein-binding protein immobilized on the plate, and the enzymatic activity and radiation on the plate are measured to detect or quantify the human synuclein-binding protein in the sample. There are competition laws. In the immunoprecipitation method, an antibody against the human synuclein-binding protein and a test sample are mixed in a solution to bind to each other to form a complex of the human synuclein-binding protein and the antibody, and then immobilized on the carrier. This is a method of isolating the complex from a solution using a munoglobulin antibody, the antibody fragment, protein A, or the like. The complex was dissolved by treating the carrier with a sample buffer for SDS-PAGE, and the SDS-PAGE was associated with the sph-synuclein binding protein and the sph-synuclein binding protein in the test sample. Protein can be detected.

 An antibody against the human synuclein binding protein is immobilized on a carrier as in the case of human synuclein described in 1., and the solution containing the human synuclein binding protein is subjected to affinity mouth chromatography to obtain the human synuclein binding protein. Can be purified. Unlike affinity chromatography immobilized with human-synuclein, it is possible to specifically purify only the human-synuclein-binding protein recognized by the antibody.

Among the antibodies against the human synuclein binding protein, those that inhibit the activity of the human synuclein binding protein to promote the fiber formation of the human synuclein are called neutralizing antibodies against the human synuclein binding protein. Examples of the neutralizing antibody against the human synuclein binding protein include an antibody against the human synuclein binding protein that inhibits the binding of the human synuclein binding protein to the human synuclein. Whether an antibody against the human synuclein-binding protein inhibits the activity of promoting human synuclein association or fiber formation depends on the fiber formation system at the mouth of human synuclein described in 1. (3). When the human synuclein-binding protein and the antibody against the human synuclein-binding protein are added, compared with the case where only the human synuclein-binding protein is added and the antibody against the human synuclein-binding protein is not added, the human synuclein-binding protein is used. Inhibits the action of sperm-synuclein association or fibril formation by cysteine? You can find out if.

 4. Screening of sph-synuclein association or fiber formation inhibitors using sph-synuclein binding protein

 Since human synuclein-binding protein promotes human synuclein association or fiber formation, substances that inhibit the binding of human synuclein to human synuclein binding protein are considered to inhibit human synuclein association or fiber formation. Can be In neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies, in which association or fibril formation of hi-synuclein is involved in the onset or progression of the disease state, human synuclein-binding protein is involved in the association or promotion of fibrosis. Therefore, substances that inhibit the association or fibril formation of human synuclein can be used as therapeutic agents to prevent the onset or delay the progress of such diseases.

After labeling human synuclein with a radioactive substance or a fluorescent dye, contacting it with a certain amount of human synuclein binding protein and binding it, immunoprecipitation with an antibody against human synuclein binding protein is used. Separate free human synuclein binding protein from the reaction system. After washing, the amount of labeled substance can be measured to determine the amount of spun-synuclein bound to the spun-synuclein binding protein. Conversely, after labeling the human synuclein-binding protein, contacting it with a certain amount of human synuclein and binding it, free α-synuclein is reacted with the reaction system by immunoprecipitation with an anti-human synuclein antibody, etc. Can be separated, washed, and the amount of the sph-synuclein binding protein bound to the sph-synuclein can be measured. In such a system, the amount of binding of human synuclein or human synuclein-binding protein when the test substance is added is measured. Can be screened as a substance that inhibits the binding of tohi-synuclein binding protein. Labeling of human synuclein or human synuclein binding protein is described in [Harlow E. & Lane D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].

 Whether a substance that inhibits the binding of human synuclein to human synuclein binding protein inhibits human synuclein association or fibril formation depends on the in vitro fibril formation of human synuclein described in 1. (3). It is possible to determine whether or not spike-synuclein promotes fibril formation compared to when no test substance is added (α-synuclein-binding protein is added) when spike-synuclein binding protein and a test substance are added to the system. it can. Also, even if the test substance does not inhibit the binding of the human synuclein to the human synuclein-binding protein, α-synuclein may be added to the fibril formation system at the mouth of the human synuclein described in 1. (3). By adding the binding protein and the test substance, and examining whether the association of the synuclein or the promotion of fiber formation is inhibited as compared with the absence of the test substance (the addition of the human synuclein binding protein), It can screen substances that inhibit the association of sph-synuclein or the action of promoting fiber formation by the binding protein.

 If the synuclein-binding protein alters the physiological function or properties of the synuclein, such as the degree of phosphorylation or interaction with other proteins, the synuclein-binding protein and the synuclein-binding protein By inhibiting the action of this human synuclein-binding protein using a substance that inhibits the binding of human synuclein, neurons involved in the onset and progression of the disease state of human synuclein such as Parkinson's disease and Lewy body dementia. Treatment can prevent the onset of degenerative disease or delay its progress.

5. A substance containing a substance that inhibits the association or fibril formation promoting action of human synuclein binding protein possessed by human synuclein binding protein or a drug containing a neutralizing antibody of human synuclein binding protein

Spontaneous synuclein binding and fibrillogenesis promoted by spontaneous synuclein binding protein A drug containing a substance that inhibits the stimulatory action or a neutralizing antibody for human synuclein-binding protein can be administered as a drug alone or as a drug, but is usually pharmacologically acceptable. It is desirable to mix it with one or more carriers as described above and provide it as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics.

 It is desirable to use the most effective route for treatment, and it is preferable to use oral administration or parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, and intravenous administration. it can. Administration forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. For example, liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, soybean oil, p- It can be produced using preservatives such as hydroxybenzoic acid esters and flavors such as strawberry flavor and peppermint as additives. Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl alcohol, It can be produced using a binder such as hydroxypropylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives. Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. For example, an injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats and carboxylic acids. The propellant does not irritate the substance or the neutralizing antibody itself or the oral and respiratory mucosa of the recipient, and separates the substance or the neutralizing antibody as fine particles. It is prepared using a carrier or the like which is dispersed to facilitate absorption. Specific examples of the carrier include lactose and glycerin. Formulations such as aerosols and dry powders are possible depending on the properties of the compound and the carrier used. Also, in these parenteral preparations, the components exemplified as additives for oral preparations can be added.

 The dosage or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 mg / kg to 100 mg / kg per day for an adult.

6, Polynucleotide that encodes human synuclein binding protein

 If the sph-synuclein binding protein isolated in 1. has a novel amino acid sequence, the cDNA of the sph-synuclein binding protein, which is a polynucleotide encoding the novel sph-synuclein binding protein, is used. It can be isolated as follows.

 Because of the degeneracy of codons (there are multiple codons even if the amino acid sequence is the same), a mixture of multiple nucleotide sequences encoding the partial amino acid sequence obtained in 1. (1) (hereinafter referred to as “degenerate nucleotide sequence”) Can be designed. Since the base sequence of the portion encoding the partial amino acid sequence in the base sequence of the gene for the human synuclein binding protein is one of the above degenerate base sequences, Oligonucleotides are synthesized, and using this as a probe, a cDNA library of the tissue from which the human synuclein-binding protein is isolated is screened by colony hybridization or plaque hybridization. A cDNA clone of the human synuclein binding protein can be isolated. Oligonucleotides having a degenerated base sequence can be chemically synthesized using a DNA synthesizer.

c Preparation of DNA library, preparation of oligonucleotide probe, screening by colony hybridization or plaque hybridization Can be performed by the method described in an experimental book [J. Sambrook et al .; Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory (1989)].

 When two consecutive partial amino acid sequences of 5 amino acids or more are known for the human synuclein-binding protein, for example, the N-terminal amino acid sequence and the internal amino acid sequence are each determined at 5 amino acids or more consecutively. In some cases, or when 20 consecutive amino acids are determined (the first 5 amino acids and the last 5 amino acids yield 2), the degeneracy of the amino acid sequence closer to the N-terminus of both is An oligonucleotide having a base sequence and an oligonucleotide having a base sequence complementary to the degenerate base sequence for the amino acid closer to the C-terminus were synthesized, and the foresight was considered as a first primer and the latter as a reverse primer. RT-PCR [McPherson MJ et al., PCR: A Practical Approach, Oxford Unive] for RNA of the tissue from which the human synuclein-binding protein was isolated. rsity Press (1991)], it is possible to amplify and isolate a partial cDNA fragment of the human synuclein binding protein. By screening the cDNA library in the same manner as described above using the cDNA partial fragment as a probe, a cDNA clone encoding the human synuclein binding protein can be isolated.

In the present invention, a novel human-synuclein binding protein having an amino acid sequence represented by SEQ ID NO: 1 at the N-terminus and an amino acid sequence represented by SEQ ID NO: 2 therein was isolated. A human brain cDNA library is screened using an oligonucleotide probe having a degenerate nucleotide sequence represented or a nucleotide sequence of 14 or more consecutive nucleotides of these nucleotide sequences, thereby obtaining the human synuclein. The cDNA encoding the binding protein can be isolated. Furthermore, a degenerate nucleotide sequence represented by SEQ ID NO: 5 or a salt of 14 or more consecutive nucleotides of this nucleotide sequence Using a forward primer having a base sequence and a degenerate base sequence represented by SEQ ID NO: 7 or a reverse primer having a continuous base sequence of 14 bases or more of the base sequence, RT-P By performing the CR, it is possible to amplify and isolate a partial fragment of the cDNA of the human synuclein binding protein. The cDNA clone of the human synuclein binding protein can be isolated by screening the human brain cDNA library using the cDNA partial fragment as a probe.

 In addition, when a homology search program was used to search for a base sequence contained in the degenerate base sequence based on the base sequence data, a match was found with the base sequence of a known gene. No, but partial nucleotide sequence of random cDNA clone ES

T (expressed sequnece tag) 場合 If the nucleotide sequence of the human genomic gene matches the inferred cDNA sequence inferred from a cDNA inference program such as GENSCAN (http // genes.mit.edu / GENSCAN.html), , These sequences, EST and

An EST having a nucleotide sequence identical to the EST nucleotide sequence, and ESTs derived from the same clone as the ES * are collected as ESTs derived from the same gene. The nucleotide sequence of the cDNA of the human synuclein-binding protein can be revealed by connecting the nucleotide sequences considered to be derived from the cDNA of the human synuclein-binding protein. If the nucleotide sequence of cDNA cannot be determined solely from these nucleotide sequences, a sense primer having the most 5 or 5 nucleotides of the nucleotide sequence obtained by splicing or a nucleotide complementary to the 3 or 3 nucleotide sequence is obtained. Using an antisense primer having a sequence, RT—

By performing PCR, cDNA of the human synuclein binding protein can be obtained. When the obtained cDNA is not a full-length cDNA, screening of a cDNA library using this clone as a probe or RACE method [rapid amplification of cDNA ends; Frohman MA et al., Proc. Natl. Acad. Sci. . USA, 8998 (1988)] to obtain a full-length cDNA.

 The nucleotide sequence of the obtained cDNA clone was determined using a DNA sequencer or the like, and the nucleotide sequence was translated into an amino acid sequence in each frame. Then, the amino acid sequence of the human synuclein binding protein obtained in 1. (1) was determined. Search for an amino acid sequence that matches the partial amino acid sequence. The amino acid sequence of the open reading frame containing the amino acid sequence that matches the partial amino acid sequence can be determined as the amino acid sequence of the whole synuclein-binding protein.

 Examples of the polynucleotide encoding the human synuclein binding protein include a nucleotide sequence corresponding to the open reading frame and DNAs and RNAs including a nucleotide sequence encoding the amino acid sequence of the entire human synuclein binding protein. I can give it. .

 The c-DNA or its partial fragment of the c-synuclein binding protein, or the c-DNA base sequence or a nucleotide sequence complementary to the c-DNA sequence of at least 10 consecutive bases is used to form the c-synuclein binding protein. The expression level of the protein at the mRNA level can be measured by a method such as Northern plotting, RT-PCR, or dot hybridization.

 In addition, the above-mentioned cDNA, a partial fragment of the cDNA, an oligonucleotide, or a derivative thereof is used to suppress the expression of the human synuclein-binding protein gene, thereby inhibiting the association of human synuclein or the promotion of fibrosis. It can prevent the onset of neurological diseases related to the onset and progression of the disease state, such as dementia and Lewy body dementia, and treat the disease to delay the progression.

 Methods for suppressing the expression of the above genes include, for example, antisense RNA / DNA technology [Bioscience and Industry, 322 (1992), Chemistry, 681 (1991).

, Biotechnology, fl, 358 (1992), Trends in Biotechnology, 10., 87 (1992), Trends in Biotechnology, iQ, 152 (1992), Cell Engineering, J, 1463 (1997)], Tribble Helix Technology CTrends in Biotechnology, 10, 132 (1992)], Ribozyme Technology Current Opinion in Chemical Biology, 2, 274 (1999), FEMS Microbiology Reviews, 23, 257 (1999), Frontiers in Bioscience, i, D497 (1999), Chemistry & Biology, 6, R33 (1999), Nucleic Acids Research, 2 £, 5237 (1998), Trends In Biotechnology, H, 438 (1998)) or decoy DNA method (Nippon Rinsho-Japanese Journal of Clinical Medicine, 5 £, 563 (1998), Circulation Research, S2, 1023 (1998), Experimental Nephrology, 429 (1997) Nippon Rinsho-Japanese Journal of Clinical Medicine, 54, 2583 (1996)].

 Examples of the oligonucleotide derivative include an oligonucleotide derivative in which a phosphoric ester bond in an oligonucleotide is converted into a phosphorothioate bond, and a phosphoric diester bond in an oligonucleotide in the form of N3,1-P5, phosphoamido. Oligonucleotide derivatives converted to date bonds, ribose and phosphodiester bonds in oligonucleotides are converted to peptide nucleic acid bonds, fe oligonucleotide derivatives and peracyl in oligonucleotides are replaced by C-15 propynyl peracyl Oligonucleotide derivatives, oligonucleotide derivatives in which peracyl in the oligonucleotide is substituted with C-5 thiazole peracyl, oligonucleotide derivatives in which cytosine in the oligonucleotide is substituted with C-5 propynylcytosine, oligonucleotides An oligonucleotide derivative in which cytosine in tide is substituted with Fuenokisajin modified cytosine (phenoxazine- modified cytosine), ribonucleic in the oligonucleotide

Oligonucleotide derivatives in which the sugars are substituted with 2'-0-propyl ribose, or oligonucleotide derivatives in which the ribose in the oligonucleotides is replaced with 2'-methoxyethoxy ribose. £, 1463 (1997)]. The medicament containing the cDNA, a partial fragment of the cDNA, the oligonucleotide, or the derivative of the oligonucleotide as an active ingredient can be provided in the form and use of the pharmaceutical preparation described in 5. above.

 The human synuclein binding protein is described in J. Sambrook et al .; Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory (1989), DM Glover and BDHames jDNA Clonin 1: Core Techniques, A Practical Approach, Second. Edition, Oxford University Press (1995) and the like, the polynucleotide encoding the human synuclein binding protein can be expressed in host cells in large quantities and easily.

 That is, a recombinant vector in which DNA encoding the human synuclein binding protein is inserted downstream of the promoter of an appropriate expression vector is constructed, and the vector is introduced into a host cell to obtain the human synuclein. By obtaining a transformant expressing the binding protein and culturing the transformant, the human synuclein binding protein can be produced.

 The expression vector contains a promoter capable of autonomous replication in a host cell or integration into a chromosome and capable of transcribing mRNA from DNA encoding the synuclein binding protein in the host cell. Things are used.

 As the host cell, any cells that can express the target gene, such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.

 The method of culturing the above-obtained transformant in a medium can be performed according to a usual method used for culturing a host.

The method for producing the human synuclein binding protein includes a method of producing the protein in a host cell, a method of secreting the protein out of the host cell, and a method of producing the protein on the host cell outer membrane. By changing the structure of the protein to be produced, You can choose the method.

 In order to isolate and purify the hishinuclein-binding protein produced by the above transformant, a conventional method for isolating and purifying an enzyme can be used. For example, in the case where the synuclein-binding protein is expressed in a lysed state in the cells, the cells are collected by centrifugation after completion of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, etc. The cells are disrupted with a Manton-Gaurin homogenizer, Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal enzyme isolation / purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Tylaminoethyl (DEAE)-anion exchange chromatography using a resin such as Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), or a resin such as S-Sepharose FF (manufactured by Pharmacia) Cation exchange chromatography, hydrophobic chromatography using resins such as petyl sepharose and phenylsepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, A purified sample can be obtained by using techniques such as electrophoresis such as isoelectric focusing alone or in combination.

 In addition, when the synuclein-binding protein is expressed by forming an insoluble form in the cells, the cells are similarly collected, crushed, and centrifuged to remove the insoluble form of the protein as a precipitate fraction. to recover. The recovered insoluble form of the protein is solubilized with a protein denaturant. The protein is returned to a normal conformation by diluting or dialyzing the solubilized solution and reducing the concentration of the polypeptide denaturing agent in the solubilized solution. After this operation, a purified sample of the protein can be obtained by the same isolation and purification method as described above.

When a synuclein-binding protein or a derivative such as a protein having a sugar chain added to the protein is secreted extracellularly, the protein or a derivative of the protein can be recovered in the culture supernatant. That is, the culture is centrifuged in the same manner as described above. The culture supernatant is obtained by further treatment, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.

 The protein thus obtained, for example, binds to a single nuclein having an amino acid sequence represented by SEQ ID NO: 1 at the N-terminus and having an amino acid sequence represented by SEQ ID NO: 2 therein. Protein, tubulin, amyloid, 14-3-3 protein, tau protein and microtubule-associated protein 1B.

 The synuclein-binding protein can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). . Chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Perkin-Elma, Pharmaci, Protein Technology Instrument, Synthecell-Vega, PerSeptive, and Shimadzu. In the drawing

FIG. 1 is a view showing the action of tubulin for promoting the fiber formation of spike synuclein. “a” shows the aggregation degree of light-synuclein (light scattering degree of 40 Onm) after 15 days when the concentration of light-synuclein was changed. The horizontal axis is the concentration of spike-synuclein (^ mo 1 /

1) The vertical axis is the light scattering at 440 nm. If the mouth is only synuclein and nothing is added, ▲ indicates 1〃mo 1/1 tubulin is added, Hata is l は mol

1/1 tubulin and 1 / mo 1/1 magnesium chloride, GTP and AT

The case where P was added is shown.

b shows the time-course change in the cohesion degree of human synuclein up to 96 hours after addition of 1 mo 1/1 tubulin to 300 / mo 1/1 human synuclein. The horizontal axis is time (unit: time), and the vertical axis is light scattering at 400 nm. Mouth is only for synuclein When nothing was added, ▲ indicates that 1〃mo 1/1 tubulin was added, and 參 indicates that 1 mo 1/1 tubulin and 1 / mo 1/1 magnesium chloride, GTP and ATP were added. Show the case.

 c is a bar graph showing the same result as a, showing the specific light scattering at 400 nm for each concentration of sph-synuclein. From the left, C 1: 300 zmo1 / If nothing is added, only C 2: 1 jumo 1/1 tubulin (no spike-synuquine), C 3: 300 μ〃mo 1/1 spike-synuclein When mo 1/1 BSA is added, C4: 300〃mol / l BSA, l mo 1/1 tubulin is added (no synuclein is present), 100, 3 The figures show the case where 1 zmo 1/1 of tubulin was added to each of 0, 500, and 700-mo 1/1 of human synuclein. + (Black bar graph) indicates the case where l mol / 1 of magnesium chloride, GTP and ATP were further added, and-(gray bar graph) indicates the case where no addition was made.昍 昍 暴 暴

 The present invention will be described in more detail with reference to the following Examples, which are merely illustrative of the present invention and do not limit the scope of the present invention. Unless otherwise indicated, methods for handling DNA in the examples are described in J. Sambrook et al .; Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory (1989), DM Glover and BD Hames; DNA Cloning 1: The method described in Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995) and the like was used. Example 1 Isolation and analysis of a protein that binds to human synuclein

 (1) Expression and purification of spike synuclein by genetic recombination

Human synuclein cDNA clone pHB S 6-1 [UedaK. Etal., Proc. Natl. Acad. Sci., USA, SO, 11282 (1993)] A fragment containing human human synuclein cDNA obtained by digestion with NoooT ^ Rg1II was used as an expression vector pET—

Plasmid pET-hi-synuclein for human sph-synuclein expression was prepared by inserting it between NcoT / RamHT sites of 15b [Novagen]. ρΕ

Escherichia coli strain transformed with Τ—hi-synuclein K s che ri oh i a. Co 1 丄 BL21 (DE 3) pLy s S (manufactured by Novagen) in a medium containing ampicillin LB (0% .5% yeast extract, 1% sodium chloride

After culturing at pH 7.2) and adding isopropylthiogalactoside (IPTG) to induce the expression of the human synuclein gene, the culture was incubated at 4,000 xg at 4 ° C.

The supernatant was removed by centrifugation for 20 minutes, and the cells were collected. Buffer [20mmo l / l Tris-hydrochloric acid (PH7.5), 20 Ommo 1/1 sodium chloride, Z Ommo lZ

1 Ethylenediamine tetraacetic acid (EDTA)]

. After centrifugation at 18,000 rpm for 60 minutes, the supernatant was recovered.

 The precipitated insoluble fraction was suspended again in the above buffer, centrifuged at 4 ° C. and 18,000 rpm for 60 minutes, and the supernatant was combined with the first supernatant. Add 40 to this combined supernatant.

The protein is salted out by adding ammonium sulphate to a saturation of 4%,

The mixture was centrifuged at 000 rpm for 60 minutes, and the precipitated protein was recovered. This precipitate is

Ommo 1/1 Tris-dissolved in hydrochloric acid (pH 7.5), dialyzed in 2 Ommo 1/1 Tris-hydrochloric acid (PH 7.5), and purified by anion exchange HPLC

. That is, the recombinant dialysate was adsorbed through a solution after dialysis through a DEAE-5PW column (manufactured by Tosoichi Co., Ltd.), washed, and eluted sequentially by increasing the sodium chloride concentration stepwise. Further, the fraction containing the eluted human spleen synuclein was again subjected to DE.

After passing through an AE-5 PW column to adsorb human spike synuclein, the column was washed and eluted with an eluent having a linear concentration gradient of sodium chloride. Eluted human sperm The fractions containing the crane were purified by hydrophobic HP LC using Phenyl Toyopearl (manufactured by Tosoichi Co.), and further purified by reversed-phase HP LC. The purity of the purified human sperm-synuclein was estimated to be 98% or more based on SDS-PAGE, Immunob mouth, MALD I / TOF mass spectrometry, and amino acid sequencing.

 (2) Preparation of human brain extract

 -After crushing 75 g of neonatal cortex of Alzheimer's disease patient frozen at 80 ° C into small pieces of 4 mm square, extraction buffer [2 Ommo 1/1 Tris-HCl (pH 8.0), 15 Ommo 1 / 1 Sodium chloride, lmmo l / 1 EDTA, 1 mmo 1/1

PMSF]. A 9-fold amount of extraction buffer was added, and the mixture was ultracentrifuged at 140,000 g at 4 ° C for 60 minutes, and the supernatant was recovered. The precipitate was suspended in an extraction buffer, homogenized again with a polytron homogenizer, and similarly centrifuged to collect the supernatant. This precipitate was subjected to the same homogenization and separation of the supernatant by ultracentrifugation, and the collected supernatants were all combined to obtain a human brain extract.

 (3) Preparation of affinity column

 After washing the sintered glass filter in a 5 ml column with distilled water and then with 1 mmo 1/1 hydrochloric acid, Sepharose-4B activated with cyanogen bromide

(Amersham Pharmacia's Biotech) was suspended in lmmo 1/1 hydrochloric acid, swelled, and poured onto a sintered glass filter of the column. After continuously washing the Sepharose-14B column with lmmo 1/1 hydrochloric acid, the buffer solution for ligand immobilization [0

lmmo 1/1 sodium bicarbonate (pH 8, 3), 50 Ommo 1/1 sodium chloride]. The equilibrated Sepharose-14B was quickly transferred from the column to the ligand immobilization buffer containing 27 mg of the purified recombinant human human synuclein prepared in (1) using an aspirator and mixed. The immobilization reaction was performed by gentle stirring at 4 ° C overnight. Cephalo immobilized with human human synuclein -Synuclein-Sepharose-1B was separated from the solution of human synuclein by centrifugation at 3,000 rpm for 20 minutes, washed with a buffer for immobilizing ligand, and then treated with 50 Ommo The cells were washed with 10 Ommo 1/1 acetate buffer (pH 4.0) containing 1/1 sodium chloride, and then washed again with a ligand immobilization buffer. The washed human synuclein-sepharose-14B was transferred to a ligand immobilization buffer containing 20 Ommo 1/1 glycine (pH 8.0) and reacted at 4 ° C for an additional 16 hours. After unreacted active groups were blocked with glycine, they were washed with a buffer solution for immobilizing a ligand to obtain a poly-synuclein-Sepharose-14B column. For control, a glycine-Sepharose-14B column in which only glycine was immobilized without using synuclein was also prepared.

 (4) Isolation and purification of human synuclein binding protein

 After equilibrating the H-synuclein-Sepharose-14B column prepared in (3) with PBS [50mmo 1/1 sodium phosphate (pH 7.0), 150mm o 1/1 sodium chloride] The extract was passed through the human brain extract prepared in (2). After washing proteins that do not bind to H-synuclein-Sepharose-14B with excess PBS, pass through a 50 Ommo 1/1 sodium citrate buffer (pH 3.0) containing 50 Ommo 1/1 sodium chloride. The protein bound to human synuclein-Sepharose-14B was eluted. All the above operations were performed at 4 ° C. As a control, the same operation was performed using the human brain extract on the glycine-Sepharose-14B column prepared in (3).

 About the eluate from the obtained spike-synuclein-sepharose-1 4B,

0 'Farrell 2D polyacrylamide gel electrophoresis [1st dimension is isoelectric focusing

(Carrier-ampholite pH gradient), the second dimension was SDS-PAGE]. As a result, molecular weight 15-20 kDa (PI 7.5-9.0), 40-45 kDa ( Several protein spots such as pI 6.5 to 8.5) and 50 to 55 kDa (pI 6.5 to 8.5) were observed.

 Further, SDS-PAGE (gel concentration: 13.5%) of the eluate from human-synuclein-Sepharose-14B was performed, and a band was detected by silver staining. As a control, a human brain extract before being adsorbed to H-synuclein-Sepharose-14B, an eluate from glycine-Sepharose-14B, a fraction that passed without being adsorbed to H-synuclein-Sepharose-14B, Recombinant human synuclein prepared in (1) was simultaneously subjected to SDS-PAGE. As a result, two bands, 42 kDa and 5 OkDa, were found in the eluate from sph-synuclein-sepharose-14B. These bands were not found in the eluate from glycine-sepharose-14B, and were considered to be specific proteins for binding to synuclein.

 The 42-kDa and 50-kDa human-synuclein-binding proteins eluted from human synuclein-sepharose-14B were further purified by anion exchange HP LC. The eluate containing the protein synthesizing human-synuclein binding from Chichi-Synuclein-Sepharose-14B is passed through a column of £ 5 £ -5 (4.6 x 75 mm, manufactured by Tosoh Corporation). After the human synuclein-binding protein was adsorbed, it was washed with 2 Ommo 1/1 Tris-monohydrochloric acid (pH 9.0). Elution was carried out sequentially with 2 Ommo 1/1 tris-hydrochloric acid (pH 9.0) in which the concentration of sodium chloride was increased stepwise by 10 Ommo 1/1 from 10 Ommo 1/1 to 50 Ommo 1/1.

Further, purification by gel excision was performed. That is, the eluate of anion-exchange HP LC containing the 42-kDa and 50-kDa spike-synuclein-binding proteins was subjected to gel electrophoresis, quickly immersed in distilled water, and stained with 30 Ommo 1/1 copper chloride solution. The band of the detected human synuclein-binding protein was cut out with a microtome knife, transferred to an eppendorf tube, and used for 10 times the amount of gel. mmo 1/1 Tris-glycine (pH 8.3) buffer was added. After washing the gel with 12.5 mMol / 1 Tris-glycine (pH 8.3) buffer, a solution containing 0.01% SDS [l O Ommol / 1 Tris (pH 8.8), 100 mmo1 / 1 ED TA] was added to elute the protein from the gel. The eluate was centrifuged to remove insoluble matters such as gel, and further purified by reversed-phase HP LC using a Phenyl-1 5PW RP column (4.6 x 75 mm). After adsorption to the column, elution was performed with a linear gradient of 10% -80% acetonitrile-0.1% trifluoroacetic acid (TFA) at a flow rate of lml / min for 15 minutes. The eluted protein was monitored by the absorbance at 280 nm. (5) Analysis of molecular weight and amino acid sequence of sph-synuclein binding protein

 MALD I / TOF mass spectrometry was performed on each of the two purified human synuclein-binding proteins obtained in (4). As a result, it was found that the protein of 42 kDa had a molecular weight of 41847. 1 (+ Μ) +, and the protein of 50 kDa had a molecular weight of 501611.1 (M + H) +.

 To determine the amino acid sequence of each, the purified human synuclein-binding protein was digested with lysyl endopeptidase at an enzyme: substrate ratio of 1:50 (w / v). The produced peptide was separated by reversed-phase HPLC using Super Sphere RP-100-100 C18 column [4.0 x 250 mm, Merck], ie, 5% acetonitrile. After adsorption of the peptide on the column equilibrated with 0.1% TFA, elution was performed at room temperature, at a flow rate of lml / min for 30 minutes, with 5% -60% linear concentration gradient of acetonitrile—0.1% Made in TF A. The eluted peptide was monitored by absorbance at 210 nm. The amino acid sequences of the obtained peptide and the purified non-enzyme-treated purified human-synuclein binding protein were determined by the Edman degradation method.

As a result, the 42 kDa protein has the amino acid sequence represented by SEQ ID NO: 1 at the N-terminal. And found to have the amino acid sequence represented by SEQ ID NO: 2 inside. The presence or absence of an amino acid sequence having homology to these amino acid sequences is checked by using the homology search software Frame Search (manufactured by Compugen) with respect to the amino acid sequence databases SwissProt, PIR, and Genpept. As a result, there was no matching sequence, and it was presumed to be a novel protein.

 Similarly, the 5 OkDa protein was found to have the amino acid sequence represented by SEQ ID NO: 3 at the N-terminal and the amino acid sequence represented by SEQ ID NO: 4 internally. When these amino acid sequences were searched against the amino acid sequence database Swiss PRot using the above-described homology search software, it was found that both of them were included in the amino acid sequence of human human tubulin. The molecular weight of the 50 kDa protein determined by MALD I / T OF mass spectrometry, 50161.1, is in good agreement with the molecular weight, 50158, deduced from the published amino acid sequence of h-tubulin, and the 5-okDa h-synuclein The binding protein was thought to be spike-tubulin.

Example 2 Binding of tubulin and spike-synuclein in the brain

 (1) Binding of Hi-tubulin to Hi-synuclein

 The physiological binding of sph-tubulin and sph-synuclein in vivo was examined by immunoprecipitation and immunoblot.

 A brain extract was prepared from rat brain in the same manner as in Example 1 (2), and RIPA buffer [

5 Ommo 1/1 Tris-hydrochloric acid (pH 7.4), 150 mmo 1/1 sodium chloride, lmmol / 1 EGTA, 1% nonidetate P-40, 0.5% sodium deoxycholate] Rat brain extracts were prepared using two types of human-synuclein-specific antibodies EQV1 and PQE3 [Arima K. et al., Brain Res.

(1998), Arima K. et al., Acta Neuropathol., 439 (1998)], anti-tupurin mouse monoclonal antibody DM1 A [Sigma-Aldrich (Sigma-Aldrich) ), And reacted at 4 ° C for 2 hours.

 The antigen-antibody conjugate was bound to protein A Sepharose (Sigma-Aldrich). After the reaction, wash the protein A sepharose in PBS containing 1% Triton X-100 and repeat the centrifugation washing operation 5 times, and then in SDS-sample buffer at 100 ° C for 5 minutes. Upon heating, the bound antigen-antibody complex was dissolved in SDS-one sample buffer. The antigen-antibody complex dissolved in the SDS-sample buffer is subjected to SDS-PAGE (gel concentration: 12%), and then electrically transferred to nitrocellulose membrane (manufactured by S & S Portran) and fixed. And 3% BSA in TBST buffer [2 Ommo 1/1 Tris-HCl (H7.6), 150 mmo 1/1 sodium chloride, 0.05% Tween 20] at 25 ° C for 30 minutes. Non-specific binding sites were blocked for minutes. After washing for 30 minutes while exchanging the TBST buffer five times, each primary antibody diluted with the TBST buffer was reacted for 60 minutes at 25 ° C. After washing the primary antibody, it is reacted with a secondary antibody (peroxidase-labeled anti-immunoglobulin antibody) at 25 ° C for 60 minutes, washed, and then a chemical luminescence reagent [NEN Life Science Products (NEN Life Science Products)] Inc.)] and exposed to hyperfilm ECL (Hyperfilm ECL; Amersham 'Falmacia' Biotech) to detect bands [ArimaK. Et al., Brain Res., Thigh, 93 (1998), Arima. Et al., Acta Neuropathol.,, 439 (1998)] First, the antigen-antibody complex immunoprecipitated with anti-synuclein antibody PQE3 or EQV1 was used as the primary antibody. 1 A (1: 500

(Dilution 0), immuno-blot was detected in both immunoprecipitates, and immuno-precipitation of human-tubulin together with human-synuclein was detected by the anti-human-synuclein antibody. all right. Also anti-tubing Of the antigen-antibody conjugate immunoprecipitated with the immunoprecipitated antibody DM 1 A using the anti-synuclein antibody PQE 3 or EQV 1 (1: 1000 dilution) as the primary antibody Further, it was found that the band of spike-synuclein was detected by both anti-spike-synuclein antibodies, and that the anti-spike-tubulin antibody immunoprecipitated spike-synuclein together with spike-tubulin. From the above, it was confirmed that spike-tubulin and spike-synuclein were physiologically bound in the brain.

(2) Binding of? -Tubulin and spike-synuclein

 For the antigen-antibody complex immunoprecipitated with the anti-synuclein antibody of Example 2 (1), the anti-tubulin antibody TUB 2.1 (Sigma-Aldrich) was used as the primary antibody. When immunoblotting was performed, a band of /?-Tubulin was detected from the immunoprecipitate, and the anti-synuclein antibody immunoprecipitated not only with human -synuclein but also with human -tubulin. I understood. Thus, in the brain, it was thought that physiologically sph-synuclein was bound not only to sph-tubulin but also to ??-tubulin.

 (3) Binding of microtubules to spike synuclein

 Since tubulin and / or tubulin are the major constituent proteins of microtubules, to confirm the binding of microtubules to human synuclein, microtubules were purified from rat brain and examined for the presence of human synuclein. Was.

63 brains of rats were treated with PIPES buffer (PH6) containing lmmol / 1 EGTA, 1 mo1 / magnesium chloride, 1 g / ml bushtin, and 10 zg / ml leptin. 8) Crushed in to obtain a homogenate. Utilizing the property that tubulin associates to form microtubules at 37 ° C and in the absence of GTP, and dissociates back to tubulin in the absence of GTP at 4 ° C and Shelanski According to these methods [Shelanski ML et al., Proc. Natl. Acad. Sci., USA, 10, 765 (1973)], microtubules were isolated and purified by repeating association-dissociation.

 For purified microtubules, anti-tubulin antibody (1 ': 5000 dilution), anti-tubulin antibody (1: 5000 dilution), anti-tau protein antibody Tau-2 (Neomakers) (1: 500 dilution), anti-MAP5 antibody [Chemicon] (1: 500 dilution) was used as a primary antibody, subjected to the same Imnob analysis as in (1), and purified from brain. It was confirmed that tubulin, tubulin, tau protein, and MAP5 were present in the microtubules. The same purified microtubules were subjected to the same immunoblot analysis using two types of sph-synuclein-specific antibodies EQVI and PQE3 as primary antibodies, and the amount of purified microtubules used was 15, 30, 60. When g was increased, gamma-synuclein was detected by both gamma-synuclein-specific antibodies, and the amount of gamma-synuclein was detected in the purified microtubules. confirmed. In addition, three kinds of human synuclein-specific antibodies MDV 2 [Arima K. et al., Brain Res., Ling, 93 (1998)] (recognizing the N-terminal region), EQ VI (recognizing the NAC domain), As a result of immunoblot analysis using PQE 3 (recognizing the C-terminal region) as the primary antibody, it was confirmed that all three types of antibodies contained human synuclein in purified microtubules. It was also found that the whole tube was associated with the whole synuclein.

 (4) Coexistence of spike-synuclein and tubulin in the pathological tissues of neurodegenerative diseases Dorsal nucleus of the vagus nerve in patients with Parkinson's disease, cervical sympathetic ganglion in patients with dementia with Lewy bodies

Then, for each of the tissues at the bottom of the pons of a patient with multiple system atrophy, formalin-fixed and paraffin-embedded sections, anti-synuclein antibody PQE 3 (1: 400 dilution) or anti-hybrid ubulin antibody (1: (200 dilution) was used as the primary antibody for fluorescent antibody staining.

. For anti-synuclein antibody, Rhodamine-labeled anti-magpie Ig G antibody [1:80 dilution "*; made by Biosource International (Biosource International)"] For anti-tubulin antibody, FITC-labeled anti-mouse IgG antibody (1:30 dilution, Biosource International) Company).

 As a result, anti-synuclein antibody stained very strongly in all of these tissues for Levi bodies, Pale bodies, which are considered to be the early state of Lewy bodies, and Levi body-associated neurites. Although the anti-tubulin antibody was weaker than the anti-synuclein antibody, it stained Lewy bodies, Veil bodies, and Lewy body-related neurites in all tissues.

 When the images stained with both antibodies (rhodamine label: red, FITC label: green) were superimposed, yellow fluorescence appeared. The stained sites with both antibodies completely overlapped, and the Levy small part of the histopathological tissue was observed. It was confirmed that splenic synuclein and tubulin coexist in the body. When fluorescent antibody staining was performed using the / 5-tubulin antibody as the primary antibody, the same site was stained in each tissue.

Example 3 Tubulin Stimulates Fibril Formation of S-synuclein

 The effect of tubulin, a human synuclein binding protein, on the fiber formation of human synuclein was examined.

 The microtubules isolated from the rat brain in Example 2 (3) were dissociated into tubulin, and subjected to phosphocellulose column chromatography using Whatman P11 [Weingarten M et al., Proc. Natl. Acad. Sci., USA, 11, 1858 (1975)] to purify tubulin.

 PBS containing 100, 300, 500, 700 PBSmo 1/1 spike synuclein

25 Add l〃mo 1/1 tubulin to 1 and incubate at 37 ° C for 15 days.

The fibril formation of -synuclein was examined by immuno-microscopy and the aggregation of -synuclein. The cohesion of human synuclein is 400 nm light scattering [MaJ. Etal., Nature, , 92 (1994)] was measured with a spectrophotometer [Gene Spec III manufactured by Hitachi, Ltd.] using the reaction solution 1-1. In the immunoelectron microscopy, the formed fibers are isolated, placed on a grid of an electron microscope, and the primary antibody, a sph-synuclein-specific antibody EQV1 (1:40 dilution) or an anti-sph-tubulin antibody (1:40) After the reaction, a secondary antibody bound to gold colloid particles with a diameter of 1 O nm [1:20 dilution, manufactured by British Biocell (British BioCell)] was detected as a probe. As controls, a system containing only human synuclein without tubulin, a system containing 1 / mo 11/1 BSA instead of tubulin, a system containing only tubulin, and a system containing 300 g instead of human synuclein were used. The same measurement was performed in a system using / mo 1/1 BSA and tubulin.

 The results are shown in a and c of FIG. When tubulin was not added, aggregation of spike-synuclein hardly occurred, whereas with the addition of tubulin, the degree of aggregation was increased in proportion to the concentration of spike-synuclein. BSA had no effect. In addition, when 300-mo 1/1 of human synuclein was used, the aggregation of human synuclein was measured over time up to 96 hours from the start of the reaction (Fig. 1! 3). At the beginning, aggregation was found to reach near saturation in 72 hours.

 In addition, in order to examine the effect of tubulin on the formation of microtubules when very small microtubules were formed, magnesium chloride, GTP and ATP, which are microtubule formation promoting factors, were added to 1〃mo 1/1 (normal (Approximately 1/1000 of the concentration used for microtubule formation) was further added, and the same measurement was carried out. As shown in a and b in Fig. 1, compared to the case where only tubulin was added, It was found that aggregation of spike synuclein was promoted.

Observation of spun-synuclein in the presence of tubulin after the reaction with an electron microscope showed that It was confirmed that it had a fibrous structure with a diameter of 10 nm and a length of about 1 m. In addition, by immunoelectron microscopy using three types of human synuclein-specific antibodies MDV2, EQV1, PQE3 or anti-human tubulin antibodies, anti-human tubulin antibodies, it was confirmed that human It was confirmed that tubulin and 5_ tubulin were present. In addition, ultrathin sections prepared by fixing the dorsal nucleus tissue of the vagus nerve of a patient with Lewy body dementia to formalin and embedding in LR white resin (manufactured by London Resin) were used for the human synuclein specific antibody PQE 3 ( (1:40 dilution) as the primary antibody. As a detection probe, a gold colloid particle-bound secondary antibody having a diameter of 5 nm was used. As a result, it was found that the diameter and morphology of the fiber of sph-synuclein formed in the presence of tubulin in vitro were very similar to the fiber of the sph-synuclein in the pathological tissue of patients with dementia with Lewy bodies. The fiber morphology of spiky-synuclein formed at the mouth was well consistent with that of spike-synuclein reported in the brains of patients with Parkinson's disease and dementia with Lewy bodies.卜 の i ilfflwr calf

 According to the present invention, there is provided a novel human synuclein-binding protein, a DNA encoding the protein, and a method for screening a substance that inhibits the association or fibril formation of human synuclein using the human synuclein-binding protein, A prophylactic or retardant drug for neurodegenerative diseases associated with spike synuclein fibril formation such as Parkinson's disease can be developed.

"Sequence List Free Text"

SEQ ID NO: 5: synthetic DNA SEQ ID NO: 6: synthetic DNA SEQ ID NO: 7: synthetic DNA

Claims

The scope of the claims  1. Derived from human brain, has a molecular weight in the range of 41600 to 42000, has an amino acid sequence represented by SEQ ID NO: 1 at the N-terminus, has an amino acid sequence represented by SEQ ID NO: 2 therein, and has amyloid A protein that binds to the precursor of the non-amyloid component in the protein (hereinafter referred to as "hy-synuclein"). 2. The protein according to claim 1, which has a molecular weight of 4188.1 (M + H) ^{+ as} determined by MALD I / T OF mass spectrometry.  3. An antibody against the protein according to claim 1 or 2.  4. A method for detecting or quantifying the protein according to claim 1 or 2, using the antibody according to claim 3.  5. [i] Contact between human synuclein and a protein that binds to human synuclein (hereinafter referred to as human synuclein binding protein); and [ii] human synuclein and human protein in the presence of the test substance. -Observes and compares the degree of association of fibrin synuclein or the degree of fiber formation when brought into contact with synuclein-binding protein, and selects from the test substance a substance that inhibits association or fiber formation of human synuclein. A method for screening a substance that inhibits the association or fibril-forming action of sph-synuclein possessed by the sph-synuclein binding protein.  6. [i] The contact between human synuclein and the synuclein-binding protein, and [ii] the contact between human synuclein and the human synuclein-binding protein in the presence of the test substance. Comparing the amount of the bound substance of the human synuclein binding protein and selecting from the test substance a substance that inhibits the binding of the human synuclein to the human synuclein binding protein, wherein the human synuclein and the human synuclein binding protein are selected. A method for screening a substance that inhibits binding to ¾. 7. [i] contacting human synuclein with human synuclein binding protein; ii] Compare the function of human synuclein in the case where human synuclein is contacted with human synuclein binding protein in the presence of the test substance, and select a substance that inhibits the function of human synuclein from the test substance A method for screening for a substance that inhibits the function of human synuclein induced by binding of human synuclein-binding protein to human synuclein.  8. The human synuclein binding protein is selected from the group consisting of the protein according to claim 1 or 2, tubulin, amyloid ^, 1413-3-3 protein, tau protein, and microtubule-associated protein 1B. A method according to any one of claims 5 to 7. 9. A compound obtained by the method according to any one of claims 5 to 8, or a pharmacologically acceptable salt thereof.  10. An antibody against human synuclein binding protein, which selects an antibody that inhibits the activity of human synuclein binding protein to promote association or fibrogenesis of human synuclein binding protein from antibodies recognizing human synuclein binding protein. Screening method for sum antibody.  11. A neutralizing antibody obtained by the method according to claim 10.  12. A neutralizing antibody against a human synuclein binding protein, which inhibits the human synuclein binding protein's association or fiber formation promoting action of the human synuclein binding protein.  13. The human synuclein binding protein is selected from the group consisting of the protein according to claim 1 or 2, tubulin, amyloid ^, 14-3-3 protein, tau protein and microtubule-associated protein IB. The method of claim 10.  14. The human synuclein binding protein is selected from the group consisting of the protein according to claim 1 or 2, tubulin, amyloid / ?, 14-3-3 protein, tau protein, and microtubule-associated protein 1B. The neutralizing antibody according to claim 11 or 12, which is: 15. The compound according to claim 9, or a pharmacologically acceptable salt thereof, or the claim. 11. A medicament comprising the neutralizing antibody according to 1, 12, or 14 as an active ingredient.  16. The medicament according to claim 15, which is an agent for preventing the onset of neurodegenerative disease or an agent for delaying the progress thereof. 17. The medicament according to claim 16, wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Lewy body dementia, multiple system atrophy and Alzheimer's disease.  18. A polynucleotide encoding the protein according to claim 1 or 2.  19. A recombinant DNA comprising the polynucleotide of claim 18.  20. A transformant obtained by introducing the recombinant DNA of claim 19 into a host cell. 21. The transformant according to claim 20, wherein the host cell is a cell selected from the group consisting of bacteria, yeast, insect cells, plant cells, and animal cells.  22. A transformant according to claim 20 or 21 is cultured in a medium, the protein according to claim 1 or 2 is produced and accumulated in the culture, and the protein is collected from the culture. The method for producing a protein according to claim 1 or 2.  23. An oligonucleotide having a sequence consisting of consecutive 5 to 60 bases in the nucleotide sequence of DNA according to claim 18, an oligonucleotide having a sequence complementary to the oligonucleotide, or a derivative of these oligonucleotides.  24. A medicament comprising the DNA according to claim 18 or the oligonucleotide or oligonucleotide derivative according to claim 23 as an active ingredient.  25. The medicament according to claim 24, which is an agent for preventing the onset of neurodegenerative disease or an agent for delaying the progress. 26. An agent for preventing the onset of neurodegenerative disease or delaying the progress of the disease, comprising DNA encoding a human synuclein binding protein or a partial DNA thereof. 27. The method according to claim 25 or 26, wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Lewy body dementia, multiple system atrophy and Alzheimer's disease. 6 S0 / T0df / X3d card 0 OAV