JP2006204150A - Method for screening preventive or therapeutic agent for alzheimer's disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening a preventive or therapeutic agent for diseases caused by the deposition of amyloid β protein and further provide a splice variant isoform of CLAC (collagen-like Alzheimer amyloid plaque component) that can be used in the screening method according to the invention and the like. <P>SOLUTION: The screening of agents that are effective for prevention and treatment of the diseases caused by the deposition of amyloid β protein, for example, Alzheimer's disease is carried out by selecting the specimens showing the action to reduce the resistance of amyloid β fiber decomposition of the CLAC/amyloid β fiber complex. In this screening, a new splice variant isoform of CLAC is employed. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for screening an agent useful for the treatment or prevention of diseases caused by amyloid β protein deposition such as Alzheimer's disease. The present invention also relates to a splice variant of CLAC that can be used in the screening method of the present invention.

Amyloid β is considered to be a causative substance that is deposited on nerve cells or blood vessels in the brain of patients with certain types of neurological diseases such as Alzheimer's disease and causes symptoms such as dementia. Amyloid β is a physiological peptide that is constantly produced in the normal brain, but is prone to aggregation and deposition, and is also a major component of senile plaques found in Alzheimer's disease, Down's syndrome, and the elderly's brain. Amyloid β is composed of 39 to 43 amino acids derived from a transmembrane precursor protein (βAPP), and is said to occur in the normal metabolic process of βAPP in the cytoplasm. The accumulation of amyloid β in the brains of Alzheimer's disease patients is thought to result from deposition due to overexpression due to defects in the amyloid β degradation system or abnormal production mechanisms. Therefore, substances involved in amyloid β fiber degradation are considered useful for the treatment and prevention of diseases caused by amyloid deposition such as Alzheimer's disease.
Conventionally, a method for removing and decomposing amyloid β or a method for screening a substance that prevents accumulation and fibrosis of amyloid β is composed of only amyloid β and a test compound. However, no pharmaceuticals that can solve this problem have been obtained with conventional techniques.
Although the main component of senile plaque amyloid is amyloid β, several other proteinaceous components have been identified and are considered to be involved in the formation of amyloid β fibers and the onset of Alzheimer's disease. Hashimoto et al. Reported a 50/100 kDa protein CLAC (collagen-like Alzheimer amyloid plaque component) constituting senile plaque amyloid (Patent Document 1, Non-Patent Document 1). CLAC is a novel single-transmembrane protein with a collagen-like sequence that repeats in the extracellular part, and accumulates in the brain of Alzheimer's disease patients and Down syndrome patients with known amyloid β overexpression along with amyloid β Is known. It is also known that CLAC is specifically expressed in the brain and specifically binds to fibrotic amyloid β rather than solubilized amyloid.
In 2003, it was proved that CLAC is identical to AMY (Alzheimer disease amyloid-associated protein) which has been studied conventionally (Non-patent Document 2). Therefore, although it is estimated that CLAC is deeply involved in the pathological mechanism of Alzheimer's disease, which still has many unknown parts, there are still many unknown parts regarding its functions and roles.
International Publication Number WO 01/58343 Hashimoto, T., et.al.EMBO J 2002 Apr 2; 21 (7): 1524-34 Soderberg, Let., Al. J Neuropathol Exp Neurol 2003 Nov; 62 (11): 1108-17

An object of the present invention is to provide a screening method for substances useful for the treatment and prevention of diseases caused by amyloid β protein deposition.
Another object of the present invention is to provide a CLAC splice variant that can be used in the screening method of the present invention.

The present inventors have determined that it is necessary to develop a new method in order to solve the above-mentioned problems, and as a result of intensive research on amyloid β, focusing on components deposited in the brain of Alzheimer's disease patients, amyloid For the first time, the protein CLAC (collagenous Alzheimer amyloid plaque component or collagen-like Alzheimer amyloid plaque component), which is deposited together with β in the brain of Alzheimer's disease, binds to amyloid β fiber, thereby providing in vivo enzymatic degradation resistance. Revealed. Furthermore, six new types of CLAC splice variant isoforms were identified.
Based on these findings, the present inventors screen for a substance that reduces the amyloid β degradation resistance of a complex of CLAC and amyloid β fiber (hereinafter referred to as “CLAC / amyloid β fiber complex”). Thus, it was confirmed that an agent useful for the treatment or prevention of diseases caused by deposition of amyloid β protein such as Alzheimer, which could not be found by conventional screening methods, could be found. In addition, the newly discovered CLAC splice variant can be used not only for the implementation of the screening method of the present invention, but also has been convinced that it is useful for elucidating and researching new functions of CLAC, and completed the present invention. It came to do.

That is, the present invention is as follows:
[1] A screening method for a substance that decreases the resistance to amyloid β fibril degradation of a CLAC / amyloid β fibril complex, comprising the following steps (a) and (b):
(A) contacting the CLAC / amyloid β-fiber complex with a proteolytic enzyme in the presence of a test substance;
(B) measuring the amyloid β fiber degradation resistance of the CLAC / amyloid β fiber complex, and selecting a test substance that decreases the amyloid β fiber degradation resistance of the complex;
[2] A screening method for a substance that decreases amyloid β fibril degradation resistance of a CLAC / amyloid β fibril complex, comprising the following steps (a), (b) and (c):
(A) a step of contacting CLAC with amyloid β-fiber in the presence of a test substance to form a CLAC / amyloid β-fiber complex;
(B) contacting the formed CLAC / amyloid β fibril complex with a proteolytic enzyme;
(C) measuring the amyloid β fiber degradation resistance of the CLAC / amyloid β fiber complex, and selecting a test substance that decreases the amyloid β fiber degradation resistance of the complex;
[3] The screening method according to [1] or [2] above, wherein the proteolytic enzyme is an in vivo proteolytic enzyme,
[4] The screening method according to any one of [1] to [3], wherein the proteolytic enzyme is neprilysin or an insulin-degrading enzyme,
[5] The screening method according to any one of the above [1] to [4], wherein the method for measuring amyloid β fiber degradation resistance is a Coomassie brilliant blue staining method, a Western blot method or a membrane permeation method,
[6] The screening method according to any one of [1] to [5] above, for screening an agent effective for treating or preventing a disease caused by amyloid β protein deposition,
[7] The screening method according to the above [6], for screening an agent effective for treatment or prevention of Alzheimer's disease,
[8] A gene comprising the following DNA (a), (b) or (c):
(A) CLAC DNA consisting of the base sequence represented by any of SEQ ID NOs: 3, 5, 7, 9, 11 or 13;
(B) DNA encoding CLAC comprising the amino acid sequence represented by any of SEQ ID NOs: 4, 6, 8, 10, 12 or 14;
(C) a protein having the characteristics of hybridizing with the base sequence represented by any of SEQ ID NOs: 3, 5, 7, 9, 11 or 13 under a stringent condition and forming a complex with amyloid β fiber DNA encoding,
[9] CLAC comprising the following protein (a) or (b):
(A) a protein comprising an amino acid sequence represented by any of SEQ ID NOs: 4, 6, 8, 10 or 12,
(B) a protein encoded by the gene of [8] above.

The present invention is based on a completely new finding that amyloid β fibers are not easily degraded by in vivo proteolytic enzymes by forming a complex with CLAC, that is, they have amyloid β fiber degradation resistance.
According to the present invention, a new screening method based on a new mechanism of action different from previously known inhibitors is provided, and an agent effective for a disease caused by amyloid β protein deposition such as Alzheimer's disease is screened. can do. In conventional screening, screening systems have been constructed targeting only amyloid β, but there have been few reports of clinical application so far. This is because it is presumed that amyloid-binding proteins such as CLAC are important for the progression of the disease state in addition to amyloid β in the actual disease state of Alzheimer's disease patients. Therefore, in the screening method of the present invention, compounds that cannot be obtained by conventional screening systems can be selected, and substances that can be more clinically applied can be found. The substance obtained by the screening method of the present invention can be effectively used as a therapeutic or prophylactic agent for Alzheimer's disease and the like.

  The present invention also provides new CLAC splice variants. The splice variant can be used not only for carrying out the screening method of the present invention, but also for screening methods using the known CLAC, research for elucidating the function of CLAC, and the like.

  Hereinafter, in the present specification, indications by abbreviations such as amino acids, (poly) peptides, (poly) nucleotides, etc., are defined by IUPAC-IUB (IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 ( 1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office) and conventional symbols in the field.

  In the present specification, “gene” or “DNA” is used to include not only double-stranded DNA but also each single-stranded DNA such as a sense strand and an antisense strand constituting the DNA. The length is not particularly limited. Therefore, in this specification, unless otherwise specified, a gene (DNA) is a double-stranded DNA containing human genomic DNA and a single-stranded DNA containing DNA (positive strand) and a sequence having a complementary sequence to the positive strand. Both double-stranded DNA (complementary strand) and fragments thereof are included. In addition, the “gene” or “DNA” includes not only “gene” or “DNA” represented by a specific base sequence (SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15), Included are “genes” or “DNAs” that encode proteins (eg, variants) that are equivalent in biological function to the proteins encoded by these. The “gene” or “DNA” encoding such a mutant is specifically represented by the aforementioned SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15 under stringent conditions. And a “gene” or “DNA” having a base sequence that hybridizes with a complementary sequence of the specific base sequence.

  In the present specification, the “CLAC / amyloid β-fiber complex” refers to a protein complex formed by bringing amyloid β fiber and CLAC into contact with each other so as to bind to each other. The CLAC / amyloid β fiber complex can be prepared according to a known method, for example, by contacting CLAC with amyloid β fiber (Methods Enzymol 1999; 309: 386-402).

  In the present specification, “CLAC” means not only a protein containing the known amino acid sequence represented by SEQ ID NO: 2, but also an amino acid sequence similar to SEQ ID NO: 2 as long as it has a feature of binding to amyloid β fiber, A protein (polypeptide) containing an amino acid sequence consisting of a part thereof is also included.

  In addition, “CLAC” in the present specification includes a spliced variant isoform of CLAC newly found by the present inventors. Examples of CLAC splice variant isoforms include CLAC-P1, CLAC-P3 / P8, CLAC-TA7, CLAC-TA9, CLAC-TA10 and AMY (CLAC-P).

  In the present specification, CLAC-P1 is a portion lacking a portion encoded by a base sequence corresponding to exons located at positions 1 to 531 and positions 952 to 969 of the base sequence shown in SEQ ID NO: 1. It is a splice variant of CLAC characterized by CLAC-P1 is not only a protein containing the amino acid sequence shown by SEQ ID NO: 4, but also has an amino acid sequence similar to SEQ ID NO: 4 or an amino acid comprising a part thereof as long as it has a feature of binding to amyloid β fiber Also included are proteins (polypeptides) containing the sequence.

  In this specification, CLAC-P3 / P8 is a portion encoded by a base sequence corresponding to an exon located at positions 1 to 531 and positions 2296 to 2607 of the base sequence shown in SEQ ID NO: 1. Is a splice variant of CLAC characterized by the lack of CLAC-P3 / P8 is not only a protein containing the amino acid sequence shown in SEQ ID NO: 6, but also an amino acid sequence similar to SEQ ID NO: 6 or a part thereof as long as it has a feature of binding to amyloid β fiber. A protein (polypeptide) containing the amino acid sequence is also included.

  In this specification, CLAC-TA7 is a base corresponding to an exon located at positions 1 to 531, 952 to 969, and 2296 to 2607 of the nucleotide sequence shown in SEQ ID NO: 1. It is a CLAC splice variant characterized by the lack of the portion encoded by the sequence. CLAC-TA7 is not only a protein containing the amino acid sequence shown by SEQ ID NO: 8, but also has an amino acid sequence similar to SEQ ID NO: 8 or an amino acid comprising a part thereof as long as it has a feature of binding to amyloid β fiber Also included are proteins (polypeptides) containing the sequence.

  In the present specification, CLAC-TA9 means positions 1 to 531, positions 952 to 969, positions 1507 to 1551 and positions 2296 to 2607 of the nucleotide sequence shown in SEQ ID NO: 1. This is a CLAC splice variant characterized by lacking a portion encoded by a base sequence corresponding to an exon located at. CLAC-TA9 is not only a protein containing the amino acid sequence shown in SEQ ID NO: 10, but also has an amino acid sequence similar to SEQ ID NO: 10 or an amino acid comprising a part thereof as long as it has the feature of binding to amyloid β fiber. Also included are proteins (polypeptides) containing the sequence.

  In this specification, CLAC-TA10 refers to positions 1 to 531, positions 952 to 969, positions 1507 to 1551, positions 2161 to 2187 of the nucleotide sequence shown in SEQ ID NO: 1. A CLAC splice variant characterized by lacking a portion encoded by a base sequence corresponding to an exon located at positions 2188 to 2241 and 2296 to 2607. CLAC-TA10 is not only a protein containing the amino acid sequence shown by SEQ ID NO: 12, but also has an amino acid sequence similar to SEQ ID NO: 12 or an amino acid comprising a part thereof as long as it has a feature of binding to amyloid β fiber Also included are proteins (polypeptides) containing the sequence.

  In this specification, AMY (CLAC-P) refers to the 1st to 531st positions, the 952th to 969th positions, the 1507th position to the 1551st position, the 2296th position to the base sequence shown in SEQ ID NO: 1. A CLAC splice variant characterized by lacking a portion encoded by a base sequence corresponding to an exon located at positions 2607 and 2494 to 2607. AMY (CLAC-P) is not only a protein containing the amino acid sequence shown by SEQ ID NO: 14, but also has an amino acid sequence similar to SEQ ID NO: 14 or a part thereof as long as it has a feature of binding to amyloid β fiber A protein (polypeptide) containing an amino acid sequence consisting of

Here, the term “similar amino acid sequence” refers to, for example, one or more (preferably about 1 to 9, more preferably about 1 to 5, more preferably 1 to 3) amino acids in the amino acid sequence. In which the amino acid sequence is substituted.
The “partial amino acid sequence” is, for example, a CLAC having the sequence of SEQ ID NO: 2 that is known to exhibit an activity of binding to amyloid β fiber, and may be any sequence having this activity. In addition, the activity of binding to amyloid β fiber was confirmed by the CLAC binding test in which amyloid β was immobilized on an assay plate (Soderberg L, Kakuyama. H., Moller A, Ito A, Winblad B, Tjernberg L, Naslund J. J Biol Chem 2005; 280: 1007-1015).

  Therefore, when a term such as “CLAC gene” is used, it is a gene encoding the above “CLAC”. Therefore, unless otherwise specified, not only the gene containing the known base sequence shown in SEQ ID NO: 1, but also the sequence And a base sequence encoding the above-mentioned “protein (peptide) containing a partial amino acid sequence”, which is a gene containing a similar base sequence, or a part thereof.

  Furthermore, the “CLAC gene” includes a gene encoding a splicing variant isoform of CLAC newly found by the present inventors. Therefore, “CLAC gene” in the present specification includes CLAC-P1 gene, CLAC-P3 / P8 gene, CLAC-TA7 gene, CLAC-TA9 gene, CLAC-TA10 gene, and AMY (CLAC-P) gene. .

  In the present specification, the CLAC-P1 gene is a gene encoding the above-mentioned CLAC-P1, and is therefore located at positions 1 to 531 and positions 952 to 969 of the base sequence described in SEQ ID NO: 1. The base sequence corresponding to the exon is lacking. Unless otherwise stated, the CLAC-P1 gene is not only a gene containing the nucleotide sequence shown in SEQ ID NO: 3, but also a gene containing a nucleotide sequence similar to the nucleotide sequence or a part thereof, A base sequence encoding a “protein (peptide) containing a partial amino acid sequence” is also included.

  In the present specification, the CLAC-P3 / P8 gene is a gene encoding the above-mentioned CLAC-P3 / P8. The nucleotide sequence corresponding to the exon located at position 2607 is lacking. Unless otherwise stated, the CLAC-P3 / P8 gene is not only a gene containing the nucleotide sequence shown in SEQ ID NO: 5, but also a gene containing a nucleotide sequence similar to the nucleotide sequence, or a part thereof. A base sequence encoding the above-mentioned “protein (peptide) containing a partial amino acid sequence” is also included.

  In the present specification, the CLAC-TA7 gene is a gene encoding the above-mentioned CLAC-TA7. Therefore, the 1st to 531st positions, the 952th to 969th positions, and the A base sequence corresponding to an exon located at positions 2296 to 2607 is lacking. Unless otherwise stated, the CLAC-TA7 gene is not only a gene containing the nucleotide sequence shown in SEQ ID NO: 7, but also a gene containing a nucleotide sequence similar to the nucleotide sequence or a part thereof, A base sequence encoding a “protein (peptide) containing a partial amino acid sequence” is also included.

  In the present specification, the CLAC-TA9 gene is a gene that encodes the above-mentioned CLAC-TA9, so that the first position to the 531st position, the 952nd position to the 969th position, A base sequence corresponding to exons located at positions 1507 to 1551 and positions 2296 to 2607 is lacking. Unless otherwise stated, the CLAC-TA9 gene is not only a gene containing the base sequence represented by SEQ ID NO: 9, but also a gene containing a base sequence similar to the base sequence or a part thereof, A base sequence encoding a “protein (peptide) containing a partial amino acid sequence” is also included.

  In the present specification, the CLAC-TA10 gene is a gene encoding the above-mentioned CLAC-TA10, and therefore, the first to 531st positions, the 952th to 969th positions, A base sequence corresponding to exons located at positions 1507 to 1551, 2161 to 2187, 2188 to 2241 and 2296 to 2607 is lacking. Unless otherwise stated, the CLAC-TA10 gene is not only a gene containing the base sequence represented by SEQ ID NO: 11, but also a gene containing a base sequence similar to the base sequence or a part thereof, A base sequence encoding a “protein (peptide) containing a partial amino acid sequence” is also included.

  In the present specification, the AMY (CLAC-P) gene is a gene encoding the above-mentioned AMY (CLAC-P). Therefore, the first to 531st positions and the 952nd position of the base sequence described in SEQ ID NO: The base sequences corresponding to the exons located at positions 969, 1507 to 1551, 2296 to 2607, and 2494 to 2607 are lacking. Unless otherwise specified, the AMY (CLAC-P) gene is not only a gene containing the nucleotide sequence shown in SEQ ID NO: 13, but also a gene containing a nucleotide sequence similar to the nucleotide sequence or a part thereof. Also included are base sequences encoding the aforementioned “protein (peptide) containing a partial amino acid sequence”.

  The “gene containing a similar base sequence” means that one or a plurality of bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13. And a gene containing a base sequence having high homology with the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13. The “gene containing a base sequence having high homology” means a gene that hybridizes under stringent conditions. For example, in the case of a known CLAC gene, a gene containing a base sequence having a homology of 75% or more, preferably 80% or more, more preferably 90% or more with the base sequence shown in SEQ ID NO: 1. Is mentioned. Examples of the CLAC-P1 gene include a gene containing a base sequence having 75% or more, preferably 90% or more, more preferably 95% or more homology with the base sequence represented by SEQ ID NO: 3. In the case of the CLAC-P3 / P8 gene, a nucleotide sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more with the nucleotide sequence shown in SEQ ID NO: 5. The gene to contain is mentioned. The CLAC-TA7 gene contains a nucleotide sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more with the nucleotide sequence shown in SEQ ID NO: 7. Gene. The CLAC-TA9 gene contains a nucleotide sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more with the nucleotide sequence shown in SEQ ID NO: 9. Gene. The CLAC-TA10 gene contains a nucleotide sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more with the nucleotide sequence represented by SEQ ID NO: 11. Gene. In the case of the AMY (CLAC-P) gene, a nucleotide sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more with the nucleotide sequence represented by SEQ ID NO: 13 A gene containing

  The stringent conditions can be adjusted by appropriately changing the temperature, salt concentration, etc. during the hybridization reaction and washing, and are set according to the desired homology. For example, the salt concentration: 6XSSC, Temperature: 65 degreeC conditions are mentioned.

CLAC is based on the sequence information (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13) of the gene provided by the present invention, based on DNA cloning, construction of each expression vector, transfection into a host, It can be obtained by culturing the transformant and recovering the protein from the culture.
Specific methods for transfection of expression vectors into the host include calcium phosphate method, DEAE-dextran method, electroporation method, a method using a lipid for gene transfer (Lipofectamine, Lipofectin; Gibco-BRL), and microinjection. Known methods such as the method can be mentioned. The expression vector may be designed so that a fusion protein with a commonly used marker protein such as GST or His-tag is expressed.
These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.

As used herein, “amyloid β fibril” refers to a complex in which amyloid β protein is polymerized. The amyloid β fiber can be prepared according to a known method for fibrosis of amyloid β protein, such as the method described in Myszka, DG, Wood, SJ, and Biere, AL Methods Enzymol 1999; 309: 386-402.
Here, the amyloid β protein may be any commonly used amyloid β protein and synthesized by a known method (Reference: Organic Synthesis <4> Acid, Amino Acid, Peptide Laboratory Chemistry Course, Chemical Society of Japan (edit) (1999/09-ISBN: 4621045822-Maruzen), peptide synthesis basics and experiment Nobuo Izumiya (1985/01 -ISBN: 4621029622-Maruzen)). For amyloid β1-40 and the like, commercially available freeze-dried products (American peptide, Peptide Institute, Bachem) can also be obtained.

  In this specification, “amyloid β fibril degradation resistance” means that the amyloid β fiber of the CLAC / amyloid β fiber complex is more resistant to proteolytic enzymes than fibrillated amyloid β fiber. Say.

  Therefore, “a substance that reduces amyloid β fibril degradation resistance” means that the amyloid β degradation resistance of the CLAC / amyloid β fibril complex is reduced when the CLAC / amyloid β fibril complex is brought into contact with a proteolytic enzyme. Any substance can be used without any limitation. Specifically, for example, a substance that promotes the degradation of the complex by directly acting on the CLAC / amyloid β complex, or the protease resistance of the complex by participating in the formation of the CLAC / amyloid β complex. Examples include substances that weaken sex. If a test substance is added when forming a CLAC / amyloid β fiber complex with CLAC and amyloid β fiber, a substance that weakens the resistance to proteolytic enzymes can be found by participating in the formation of the complex. . On the other hand, if a test substance is added after formation of the CLAC / amyloid β complex, a compound capable of decomposing a stronger complex can be selected.

  In the present specification, the “proteolytic enzyme” may be a proteolytic enzyme having an activity of degrading amyloid β fiber, and is not limited at all. The proteolytic enzyme is preferably an in vivo proteolytic enzyme, more preferably a human-derived in vivo proteolytic enzyme. The in vivo proteolytic enzyme is a proteolytic enzyme existing in vivo among proteolytic enzymes. Specific examples of in vivo proteolytic enzymes include neprilysin, insulin-degrading enzyme, proteinase K, trypsin, and the like, and more preferably neprilysin and insulin-degrading enzyme.

  When using a term such as neprilysin in the present specification, it includes not only a known enzyme (protein) represented by International Enzyme Commission classification number EC 3.4.24.11, but also its homologues and mutants, unless otherwise specified. Used for purposes. Neprilysin-like neutral endopeptidase described in JP-A-2002-34596 can also be used as neprilysin in the present specification. A method for obtaining neprilysin is known as described in Neuroscience Letters, Volume 350, Issue 2, 23 October 2003, Pages 113-116 and JP-A-2002-34596. Neprilysin can be obtained by, for example, DNA cloning, construction of each plasmid, transfection into a host, culture of a transformant, and recovery of a protein from the culture based on the base sequence information of the neprilysin gene. These operations are performed according to methods known to those skilled in the art or methods described in the literature (Molecular Cloning T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). It can be carried out. Commercially available neprilysin (Alpha Diagnostic International, Inc) can also be used.

When using a term such as insulin-degrading enzyme in this specification, unless otherwise specified, not only a known enzyme (protein) represented by International Enzyme Commission classification number EC3.4.22.11, but also homologues and variants thereof Used for the purpose of inclusion. A method for obtaining an insulin-degrading enzyme is known as described in J. Biol. Chem. 278, 4978949794 (2003). Insulin-degrading enzyme is purified from rat liver, human erythrocytes, human placenta, etc., or the relevant cDNA (Science, 242, 1415 (1988)) is expressed in microorganisms, plant cells or animal cells and then isolated and purified. Can be obtained. Specifically, for example, based on the nucleotide sequence information of the insulin degrading enzyme gene (for example, accession No: BX648462, M21188, SEQ ID NO: 15), DNA cloning, construction of each plasmid, transfection into a host, transformation It can be obtained by manipulation of body culture and protein recovery from the culture. These operations are performed according to methods known to those skilled in the art or methods described in the literature (Molecular Cloning T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). It can be carried out.
As the proteolytic enzyme, proteinase K (Sigma), which is a commercially available proteolytic enzyme, can also be used.

  The present invention provides a method of screening for a substance that decreases the resistance to amyloid β fiber degradation of a CLAC / amyloid β fiber complex.

  In the present invention, as shown in the Examples, a complex of amyloid β fiber and CLAC (CLAC / amyloid β fiber complex) has a property that amyloid β fiber is less likely to be degraded by proteolytic enzyme than amyloid β fiber alone. That is, it is based on a completely new finding that it has amyloid β fiber degradation resistance.

The screening method of the present invention can be carried out by carrying out the step of contacting the CLAC / amyloid β fibril complex with a proteolytic enzyme under a test substance.
In that case, examples of the screening method of the present invention include those comprising the following steps (a) and (b):
(A) contacting the CLAC / amyloid β-fiber complex with a proteolytic enzyme in the presence of a test substance;
(B) A step of measuring the amyloid β-fiber degradation resistance of the CLAC / amyloid β-fiber complex and selecting a test substance that decreases the amyloid β-fiber degradation resistance of the complex.
Specifically, the test substance is selected by adding the test substance (candidate substance) to the amyloid β fiber degradation resistance by the proteolytic enzyme of the CLAC / amyloid β fiber complex to which the test substance (candidate substance) is added. This can be achieved by lowering the amyloid β fiber degradation resistance of the CLAC / amyloid β fiber complex.

Alternatively, the screening method of the present invention can also be performed by carrying out the step of forming a CLAC / amyloid β fibril complex in the presence of a test substance.
In that case, examples of the screening method of the present invention include those comprising the following steps (a), (b) and (c):
(A) a step of contacting CLAC with amyloid β-fiber in the presence of a test substance to form a CLAC / amyloid β-fiber complex;
(B) contacting the formed CLAC / amyloid β fibril complex with a proteolytic enzyme;
(C) A step of measuring the amyloid β fiber degradation resistance of the CLAC / amyloid β fiber complex and selecting a test substance that decreases the resistance to amyloid β fiber degradation of the complex.
Here, the selection of the test substance is specifically performed by the CLAC / amyloid formed in the absence of the test substance when the amyloid β fiber degradation resistance of the CLAC / amyloid β fiber complex formed in the presence of the test substance is This can be achieved by lowering the amyloid β-fiber degradation resistance of the β-fiber complex.
The “step of forming a CLAC / amyloid β-fiber complex” is sufficient if CLAC / amyloid β-fiber complex is formed by contacting CLAC and amyloid β-fiber, and the conditions such as reaction temperature and reaction time are not limited. Not. For example, it can be performed according to known conditions (Methods Enzymol 1999; 309: 386-402) or the conditions described in the Examples.

  Examples of substances that can be used as test substances include, but are not limited to, nucleotides, peptides, proteins, organic compounds, inorganic compounds, and the like. Specifically, screening is performed using these test substances or samples (test samples) containing these test substances. / Can be performed in contact with amyloid β-fiber complex. Such test samples include, but are not limited to, cell extracts containing test substances, gene library expression products, synthetic low-molecular compounds, synthetic peptides, natural compounds, and the like.

The step of “measuring amyloid β fibril degradation resistance” may be any measurement method that can detect the degradation of amyloid β fibrils of the CLAC / amyloid β fibril complex, and can take various embodiments. For example, it can be measured by visualization of amyloid β molecular weight characteristics.
Hereinafter, a specific example of a method for measuring amyloid β fibril degradation resistance is illustrated.

(1) Measurement of amyloid β fibril degradation resistance by staining method The measurement of amyloid β fibril degradation resistance can be carried out by a method using a staining reagent such as Coomassie Brilliant Blue R250. It can be carried out by using a method such as silver staining Coomassie blue staining, silver staining or spirolby staining, such as treatment with aldehyde followed by treatment with ammoniacal silver / formaldehyde.
Specifically, for example, after performing a degradation reaction of the CLAC / amyloid β fiber complex with a proteolytic enzyme, SDS-PAGE is performed based on a conventional method. After SDS-PAGE, CLAC remaining after the degradation reaction can be obtained by performing Coomassie Brilliant Blue (Gel Code Blue stain reagent: PIRCE) staining, silver staining Coomassie blue staining, silver staining, or spirolby staining according to known methods. The molecular weight of the amyloid β fibril of the amyloid β fibril complex can be visualized, and the amyloid β fibril degradation resistance of the CLAC / amyloid β fibril complex can be measured.
When SDS-PAGE is performed, for example, the remaining amyloid β fiber is solubilized using formic acid, etc., formic acid is removed, and then dissolved again using urea or the like and then SDS-PAGE is performed. Preferably it is done.

(2) Measurement of amyloid β fibril degradation resistance by Western blotting The measurement of amyloid β fibril degradation resistance can be carried out using Western blotting.
Specifically, for example, after performing a degradation reaction of the CLAC / amyloid β fiber complex with a proteolytic enzyme, SDS-PAGE is performed based on a conventional method. After completion of SDS-PAGE, amyloid β-fiber degradation resistance of the CLAC / amyloid β-fiber complex can be measured by detecting undegraded amyloid β-fiber by Western blotting.
When performing SDS-PAGE, for example, the remaining amyloid β fiber is solubilized using formic acid, etc., and after removing formic acid, it is dissolved again using urea or the like and then SDS-PAGE is performed. Is preferred.
The antibody used for detection of amyloid β fiber may be any antibody that can specifically recognize amyloid β, and examples thereof include 6E10 and 4G8 (Signet Laboratories), which are amyloid β-specific antibodies.

(3) Measurement of amyloid β fibril degradation resistance by membrane permeation method Measurement of amyloid β fibril degradation resistance can be carried out using a membrane permeation method.
Specifically, for example, after the CLAC / amyloid β-fiber complex is decomposed with a proteolytic enzyme, the amyloid β fiber remaining in the reaction solution is adsorbed on the membrane to be separated from the enzyme degradation product. Next, if the amount of residual amyloid β fibers adsorbed on the membrane is visualized by a general immunochemical measurement method using an antibody that specifically recognizes amyloid β, the amyloid β of the CLAC / amyloid β fiber complex Fibrosis resistance can be evaluated.
The membrane used here only needs to be capable of adsorbing the CLAC / amyloid β fiber complex and separating the complex from the enzymatic reaction degradation product, and examples thereof include nitrocellulose and cellulose acetate. .

  It can also be evaluated using amyloid β fibers in which labeled amyloid β (such as radioisotope label) is mixed at a certain ratio. In that case, after the degradation reaction of the CLAC / amyloid β fiber complex with a proteolytic enzyme, the amyloid β fiber remaining in the reaction solution is adsorbed on the membrane and separated from the enzyme degradation product. Next, if the amount of residual amyloid β fiber adsorbed on the membrane is measured by detecting the radioactivity of the labeled amyloid fiber, the resistance to amyloid β fiber degradation of the CLAC / amyloid β fiber complex can be evaluated. it can.

  EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

The amyloid β fiber degradation reaction by proteinase K on amyloid β fiber and CLAC complex was examined.
(1) Preparation of amyloid β fiber The step of fibrillating amyloid β to prepare amyloid β fiber was performed according to the method of Myszka, DG, Wood, SJ, and Biere, AL Methods Enzymol 1999; 309: 386-402. . 1 mg of lyophilized amyloid β1-40 trifluoroacetate (manufactured by Bachem) was dissolved in a phosphate buffer, and then fiberized while stirring with a stirrer at room temperature for 2 days. Fibrotic amyloid β was prepared to a final concentration of 100 μM and stored at −70 ° C. until used for experiments.

(2) Preparation of recombinant CLAC A cell culture containing a cell line expressing recombinant human CLAC was prepared according to the method of Soderberg L. et, al (2004) J Biol Chem in press.

(3) Reaction of amyloid β fiber and CLAC In order to contact CLAC purified from cell culture medium with amyloid β fiber, amyloid β fiber prepared in (1) (final concentration 50 μM, weight 20 μg), and (2) The prepared recombinant CLAC (final concentration 200 nM, weight 5 μg) was contacted in phosphate buffer at room temperature for 2 hours to form a complex of amyloid β fiber and CLAC.

(4) Degradation reaction of amyloid β fiber by in vivo proteolytic enzyme Following (3), the amyloid β fiber CLAC complex was contacted with the in vivo degrading enzyme. The amyloid β fiber CLAC complex was mixed with proteinase K (manufactured by Sigma) in a weight ratio of 1:10 (enzyme: amyloid β fiber) and reacted at 37 ° C. overnight. The reaction was stopped by adding 70% formic acid, and after adding formic acid, it was left overnight to solubilize amyloid β fibers. Next, after removing formic acid using a speed bag (centrifugal concentrator), the formic acid was dissolved again in an aqueous solution of 9M urea (100 mM Tris, pH 10.5) and subjected to the analysis analysis by SDS-PAGE of the next (5).

(5) Analysis of amyloid β fiber degradation activity Analysis of amyloid β fiber degradation activity was evaluated by a molecular weight characterization method using SDS-PAGE. For SDS-PAGE, 10-20% tricine gel (Invitrogen) and Tricin SDS buffer (Invitrogen) were used. After completion of SDS-PAGE, amyloid β was visualized using Coomassie Brilliant Blue (Gel Code Blue stain reagent: PIRCE) staining and Western blotting. In Western blotting, amyloid β separated by SDS-PAGE is transferred to a PVDF membrane (0.2 μm BIO-RAD), then the primary antibody is amyloid β-specific antibody 6E10, 4G8 (Signet Laboratories), and the secondary antibody Horseradish peroxidase binding Anti-mouse sheep IgG (Amersham) was used for visualization using ECL plus western blotting regents (Amersham).

As shown in Fig. 1, as a result of the Coomassie Brilliant Blue staining method, a band specific to amyloid β (amyloid β fiber content) was detected in the CLAC addition group even when reacted with proteinase K for 2 hours and overnight. It was done. On the other hand, in the CLAC non-added group, the band specific to amyloid β (amyloid β fiber amount) was already attenuated after the reaction for 2 hours, and the amyloid β fiber was completely decomposed and not detected in the specimen after the overnight reaction. In other words, it was found that the CLAC / amyloid β fiber complex is given resistance to the amyloid β fiber degradation action of proteinase K, an in vivo proteolytic enzyme, by binding CLAC to amyloid β fiber.
The result by Western blotting showed the same result as that by Coomassie Brilliant Blue. 6E10 is an antibody that recognizes the vicinity of the amyloid βN terminal, and 4G8 is an antibody that recognizes the vicinity of the center of the amino acid sequence of amyloid β. Even when antibodies that recognize two different regions were used, it was confirmed that CLAC imparted proteolytic enzyme resistance to amyloid β fibers. The molecular weight size characteristic of the remaining amount of amyloid β fiber decreased by the reaction of (4) could also be determined by SDS-PAGE.
From these results, it is possible to find compounds that weaken amyloid β fiber degradation resistance, such as compounds that promote amyloid β fiber degradation of the CLAC-amyloid β fiber complex, by screening using this principle. found.

(1) Reaction of amyloid β fiber and CLAC After preparing amyloid β fiber and CLAC according to the procedures of (1) and (2) of Example 1, CLAC purified from the cell culture solution and amyloid β fiber were contacted. The amyloid β fiber prepared in Example 1 (1) (final concentration 0.1 μM) and the recombinant CLAC prepared in Example 1 (2) (final concentration 100 nM) were contacted in phosphate buffer at room temperature for 2 hours. A complex of amyloid β fiber and CLAC was formed.

(2) Degradation reaction of amyloid β fiber by in vivo proteolytic enzyme Amyloid β fiber CLAC complex is mixed with proteolytic enzyme proteinase K (manufactured by Sigma) at a weight ratio of 58: 1 (enzyme: amyloid β fiber), The reaction was allowed to proceed overnight at 37 ° C. in the presence of 0.4% final concentration of SDS (sodium dodeclsulfate). The reaction was stopped by returning the temperature of the reaction solution to room temperature and further diluting the reaction solution by adding 10% SDS (final concentration 5 SDS%).

(3) Analysis of amyloid β fiber degradation activity Analysis of amyloid β fiber degradation activity was performed by a membrane permeation method. A membrane permeation method using a cellulose acetate membrane (pore size 0.2 μm, Schleicher & Schuell) and a dot blot apparatus (Slotblot, Amersham) was used for separation of residual amyloid β fibers and enzyme degradation products. To visualize the amount of residual amyloid β fiber, amyloid β-specific antibody 6E10 (Signet Laboratories) was used as the primary antibody, horseradish peroxidase-conjugated anti-mouse sheep IgG (Amersham) was used as the secondary antibody, and ECL plus western blotting regents (Amersham) ) Was used.
As a result of evaluation by the membrane permeation method, as shown in FIG. 2, a decrease in the amount of amyloid β fiber was recognized in the enzyme added group (lanes 5-12) than in the enzyme non-added group (lanes 1-4). Among the enzyme added groups, the amount of residual amyloid β in the CLAC added group (lanes 9-12) was higher than that in the CLAC non-added group (lanes 5-8). Therefore, the addition of CLAC suppressed the decrease of amyloid β fiber due to enzymatic degradation. That is, in the membrane permeation method, it was confirmed that the proteolytic enzyme resistance was imparted to the amyloid β fiber of CLAC.

(1) Screening method 1
Amyloid β fiber and CLAC were prepared according to the procedure of Example 1 (1) (2) above, and the prepared amyloid β fiber and recombinant CLAC were contacted in a phosphate buffer solution at room temperature for 2 hours to obtain CLAC / amyloid β. A fiber composite is formed.
Next, in the presence of the test compound, the amyloid β fiber CLAC complex and the in vivo proteolytic enzyme (neprilysin or insulin degrading enzyme) are mixed and reacted overnight at 37 ° C., according to the procedure of Example 2 (3), Analysis of amyloid β fiber degradation activity is performed by membrane permeation method. A product obtained by reacting the enzyme with the CLAC / amyloid β fiber complex in the absence of the test compound can be used as a control.

(2) Screening method 2
Amyloid β fiber and CLAC were prepared according to the procedure of Example 1 (1) and (2) above, and the prepared amyloid β fiber and recombinant CLAC were contacted in phosphate buffer at room temperature for 2 hours in the presence of the test compound. To form a CLAC / amyloid β fiber complex.
Next, amyloid β-fiber CLAC complex and in vivo proteolytic enzyme (neprilysin or insulin degrading enzyme) are mixed and reacted overnight at 37 ° C., and amyloid β is obtained by membrane permeation according to the procedure of Example 2 (3). Analysis of fiber degradation activity.

Six new splice variant isoforms of CLAC were cloned from a human brain cDNA library and the sequence was identified.
(1) Cloning of CLAC isoform splice variants
The CLAC precursor (CLAC-P) was cloned using a human brain cDNA library (QUICK-Clone cDNA, BD Bioscience Clontech, Palo Alto, Calif.). That is, 1 ng of the library was used as a template, and touch-down PCR was performed using forward 1 (F1) and reverse 1 (R1) primers (2) (Table 1). The sequence used as a template is Homo sapiens collagen-like Alzheimer amyloid plaque component precursor type I mRNA CLAC-P nucleotide sequence (accession no. AF293340).
Next, the CLAC sequence amplified by PCR was inserted into the pCR 2.1-TOPO TA cloning vector (Invitrogen, Life Technologies) for subcloning.

Table 1 shows the sequences of the primers contained in the CLAC cDNA.

(2) Gene sequence analysis
Sequence analysis of the inserted CLAC cDNA was performed using ABI PRISM 3100 Genetic Analyzer sequencer (Applied Biosystems Foster City CA), BigDye Terminator Cycle Sequencing kit and F1-F4, R1-R3 (Thermo-Hybaids, Thermo Electron Corporation, (Ulm, Gerrmay). The cDNA clone was sequenced using a labeled dye nucleotide according to a known instruction (Big-Dye, Perkin-Elmer Corp., Norwalk, CT).
The purified product was analyzed using an automatic sequence analyzer ABI PRISM (registered trademark) 3100 Genetic Analyzer (Applied Biosystems Foster City CA). Primers F1-F4 and R1-R3 used in the experiment were extended to the full length in both directions of the CLAC-P cDNA. Each primer was used at least 3 times for analysis of the inserted sequence.
Next, the sequence of each cDNA clone was analyzed using (Conting Assembly Program) Cap3 assembly software (available at http://bio.ifom-firc.it/ASSEMBLY/assemble.html).

As a result, new CLAC-P isoforms with a total of 6 different lengths were found. The newly found sequences were named P1, P3 / P8, TA7, TA9, TA10 and AMY / CLAC-P, respectively. AMY / CLAC-P has already published its amino acid sequence (Soderberg L, Kakuyama.H., Moller A, Ito A, Winblad B, Tjernberg L, Naslund J. J Biol Chem 2005; 280: 1007-1015). However, the encoded nucleotide sequence has not been published.
Table 2 shows the homology (%) of the results of comparison with a known clone having the longest reported base sequence (Homo sapiens collagen-like Alzheimer amyloid plaque component precursor type I mRNA sequence AF293340: NCBI sequence database). Shown together. In Table 2, the deleted base sequence position indicates the position of the defective base sequence in the base sequence described in SEQ ID NO: 1.

Table 2 shows the results of comparing the new CLAC isoform with AF293340.

According to the present invention, there is provided a screening method for an agent effective for a disease caused by amyloid β protein deposition such as Alzheimer's disease based on a new mechanism of action different from the known inhibitors. The substance obtained by the screening method of the present invention can be effectively used as a therapeutic or prophylactic agent for Alzheimer's disease and the like.
Furthermore, the present invention provides a novel splice variant isoform of CLAC that can be used in the screening method of the present invention.

CLAC imparts proteolytic enzyme resistance to the fibrillar amyloid β fiber. A shows the result by Western blotting, and B shows the result by Coomassie brilliant blue staining. CLAC +: CLAC added group, CLAC-: CLAC non-added group. 2h: Example of contact reaction with enzyme for 2 hours, on: Example of reaction with enzyme contact reaction overnight (approximately 12 hours).

The evaluation result by the membrane permeation method is shown. CLAC imparts proteolytic enzyme resistance to the fibrillar amyloid β fiber. Amyloid β fiber + CLAC: Lanes 1-4, Amyloid β fiber + Enzyme: Lane 5-8, Amyloid β fiber + CLAC + Enzyme: Lane 9-12

Claims (9)

A screening method for a substance that decreases the resistance to amyloid β fibril degradation of a CLAC / amyloid β fibril complex, comprising the following steps (a) and (b):
(A) contacting the CLAC / amyloid β-fiber complex with a proteolytic enzyme in the presence of a test substance;
(B) A step of measuring the amyloid β-fiber degradation resistance of the CLAC / amyloid β-fiber complex and selecting a test substance that decreases the amyloid β-fiber degradation resistance of the complex. A screening method for a substance that decreases the resistance to amyloid β fibril degradation of a CLAC / amyloid β fibril complex, comprising the following steps (a), (b) and (c):
(A) a step of contacting CLAC with amyloid β-fiber in the presence of a test substance to form a CLAC / amyloid β-fiber complex;
(B) contacting the formed CLAC / amyloid β fibril complex with a proteolytic enzyme;
(C) A step of measuring the amyloid β fiber degradation resistance of the CLAC / amyloid β fiber complex and selecting a test substance that decreases the resistance to amyloid β fiber degradation of the complex. The screening method according to claim 1 or 2, wherein the proteolytic enzyme is an in vivo proteolytic enzyme. The screening method according to claim 1, wherein the proteolytic enzyme is neprilysin or an insulin-degrading enzyme. The screening method according to any one of claims 1 to 4, wherein the method for measuring amyloid β fiber degradation resistance is a Coomassie brilliant blue staining method, a Western blot method, or a membrane permeation method. The screening method according to any one of claims 1 to 5, for screening an agent effective for treating or preventing a disease caused by deposition of amyloid β protein. The screening method of Claim 6 for screening the agent effective in the treatment or prevention of Alzheimer's disease. A gene comprising the following DNA (a), (b) or (c):
(A) CLAC DNA consisting of the base sequence represented by any of SEQ ID NOs: 3, 5, 7, 9, 11 or 13;
(B) DNA encoding CLAC comprising the amino acid sequence represented by any of SEQ ID NOs: 4, 6, 8, 10, 12 or 14;
(C) a protein having the characteristics of hybridizing with the base sequence represented by any of SEQ ID NOs: 3, 5, 7, 9, 11 or 13 under a stringent condition and forming a complex with amyloid β fiber DNA encoding CLAC comprising the following protein (a) or (b):
(A) a protein comprising an amino acid sequence represented by any of SEQ ID NOs: 4, 6, 8, 10 or 12,
(B) a protein encoded by the gene according to claim 8;

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