JPH101499A - Amyloid precursor protein - Google Patents

Abstract

(57) [Abstract] [Solution] Amino acid sequence: KKKQYTSIHHGVVEVDAAVTPE
A polypeptide that is specified by EXHLSKMQQNGYENPTYKFFEQMQN (X is an amino acid residue other than arginine) and is an intracellular domain of amyloid precursor protein; and (a) a polypeptide that is an extracellular domain of wild-type amyloid precursor protein or an amino acid variant thereof (Excluding the part contained in the amyloid β domain), (b) amyloid β domain, (c)
A polypeptide that binds to the C-terminus of the amyloid β domain to constitute a cell membrane domain, and (d) a mutant amyloid precursor protein containing the polypeptide identified by the amino acid sequence and being an intracellular domain. [Effect] The productivity of amyloid β is enhanced, and by expressing this precursor protein in cells, amyloid β can be produced in a large amount.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a protein which acts as a precursor of amyloid β which is thought to be involved in Alzheimer's disease.

[0002]

2. Description of the Related Art Alzheimer's disease is a disease that presents with progressive dementia symptoms. Histopathologically, it is a neurodegenerative disease characterized by the appearance of remarkably large numbers of senile plaques and neurofibrillary tangles in the brain. is there. Alzheimer's disease often occurs in elderly people, and it is known that the proportion of affected individuals increases with aging. At present, it is impossible to cure Alzheimer's disease, and in response to the rapid increase of the elderly population in the future, methods for treatment and prevention of Alzheimer's disease and development of effective preventive and therapeutic agents against Alzheimer's disease will be developed. Coveted.

[0003] Senile plaques are extracellular deposits containing various components, the main components of which are called amyloid β.
It is a strongly cohesive peptide consisting of 43 amino acids (Aβ: this peptide is sometimes called β-protein, β-peptide, β-amyloid, A4 peptide, β / A4, etc.). In the senile plaques, amyloid β is deposited as a solid structure having a β sheet structure. The senile plaque begins with a “stain” -like deposit called the chronic senile plaque,
At this stage, no neurodegeneration has occurred, and it is thought that the chronic senile plaques become firm deposits, and that neurodegeneration and neurological degeneration cause symptoms of Alzheimer's disease such as dementia.

[0004] Amyloid β is a one-time transmembrane protein, amyloid precursor protein (APP, amyloid precu).
rsor protein; sometimes called βPP or βAPP)
Is biosynthesized by being cut out from (Nature, 32
5, p. 733, 1987). Amyloid precursor protein is a protein transcribed from a single amyloid precursor protein gene.
It is a generic term for proteins produced by translating various mRNAs generated by various splicing (alternating splicing) of mRNAs, and it is known that there are mainly three types of subclasses. These three types of amyloid precursor proteins are distinguished by the number of their constituent amino acids,
APP770 (770 amino acids), APP751 (751 amino acids), and APP695 (69 amino acids)
5) is called. Although these differ in the number of amino acids constituting the extracellular region, all of them are amyloid β
The region and the intracellular region are common, and have a property of functioning as a precursor protein of amyloid β.

[0005] The amino terminus of amyloid β is in the extracellular region of the amyloid precursor protein, and the enzyme that cleaves it is tentatively called β-secretase. In addition, the carboxyl terminus of amyloid β is present in the intracellular region of the amyloid precursor protein, and the enzyme that cleaves this is provisionally called γ-secretase. As an example of the amyloid precursor protein, the structure of APP695 (region within the molecule) and the cleavage site of APP695 are shown in Fig. 1 (Kitaguchi et al., Neuropsychopharmacology, 16 (1)
2), pp. 747-764, 1994). Furthermore, it has been shown that the processing of amyloid precursor protein is affected by calcium ion influx into cells, activation of protein kinase C, and the like (Kitaguchi et al., Supra).

[0006] It is also clear that the deposition of amyloid β is a lesion that occurs prior to neurofibrillary tangles, and that amyloid β, particularly aggregated amyloid β, has toxicity to cultured neurons. In addition, the existence of a family with familial Alzheimer's disease having mutations in and around amyloid β of the amyloid precursor protein gene has been demonstrated (Hardy, J., et a.
l., Nature, 349, p.704, 1991), and amyloid β is considered to have an important role in the development and progression of Alzheimer's disease (Tamaoka, Internal Medicine, 77 (5),
pp.843-851, 1996). In addition, substances that inhibit the production of amyloid β have been considered to be useful for preventing Alzheimer's disease and for preventing the progression of symptoms.
Action of β and γ secretases, which are excision enzymes, activation of protein kinase C, pH of organelles
The search for inhibitors of amyloid β production is being advanced using physiological actions such as the regulation action of amyloid as an indicator of the assay.

On the other hand, for the development of a prophylactic / therapeutic agent for human diseases, the construction of an experimental system using model animals is indispensable.
In particular, it is extremely important to develop a model animal that accurately reproduces the pathological features and pathological conditions of the target disease. As a model animal used for the development of a prophylactic / therapeutic agent for Alzheimer's disease, for example, a transgenic mouse into which a gene for the amyloid precursor protein (717V → F) having a mutation at the 717th amino acid has been introduced is known. (Games,
D., Nature, 373, pp. 523-527, 1995). This mouse
It has some pathological features of Alzheimer's disease, such as amyloid deposition and degenerated neurites in the brain, and is considered to be the best model animal at present (Hirai, senile dementia, 9 (2), pp.203-204, 199
Five) .

[0008]

An object of the present invention is to provide a means for increasing the productivity of amyloid β in a living body. More specifically, it is an object of the present invention to provide a polypeptide capable of increasing amyloid β productivity in a living body and a modified amyloid precursor protein containing the polypeptide. It is also an object of the present invention to provide a nucleotide sequence encoding a polypeptide having the above characteristics and a modified amyloid precursor protein. Further, a vector into which a DNA sequence as a preferred embodiment of the above nucleotide sequence has been introduced, a transformant transformed with the vector, and
It is also an object of the present invention to provide a transgenic animal into which the DNA has been introduced.

[0009]

Means for Solving the Problems The present inventors have found that a phosphorylation site exists in the intracellular region of the amyloid precursor protein, and when this site is phosphorylated by cdc2 kinase, the phosphorylation site of the amyloid precursor protein is reduced. Processing changes,
It was found that a qualitative change in the immature amyloid precursor protein, a decrease in the secretory amyloid precursor protein, and an increase in the carboxyl-terminal fragment of the intracellular amyloid precursor protein were caused (Suzuki, T. et al. , EMBO Jo
urnal, 13 (5), pp.1114-1122, 1994).

The present inventors have further studied the changes in processing of the amyloid precursor protein due to phosphorylation. As a result, the amyloid precursor protein APP695
The arginine residue is replaced with another amino acid residue, the 728th arginine residue of the amyloid precursor protein APP751 is replaced with an amino acid residue other than arginine, or the 747th arginine of the amyloid precursor protein APP770 is replaced. When the DNA encoding each mutant amyloid precursor protein in which an arginine residue is substituted with an amino acid residue other than arginine is expressed, the production of the carboxyl-terminal fragment of the mutant protein is qualitatively altered to include amyloid β. It has been found that polypeptides are accumulated in large amounts in cells. The present invention has been completed based on the above findings.

That is, the present invention provides the following amino acid sequence:
H ₂ N-Lys-Lys-Lys-Gln-Tyr-Thr-Ser-Ile-His-His-Gly-Va
l-Val-Glu-Val-Asp-Ala-Ala-Val-Thr-Pro-Glu-Glu-X-Hi
s-Leu-Ser-Lys-Met-Gln-Gln-Asn-Gly-Tyr-Glu-Asn-Pro-
The present invention provides a polypeptide specified by Thr-Tyr-Lys-Phe-Phe-Glu-Gln-Met-Gln-Asn-OH (in the sequence, X represents an amino acid residue other than arginine). Also,
The present invention provides the above polypeptide, which is an intracellular domain of amyloid precursor protein.

[0012] Further, one or more amino acid residues constituting the above amino acid sequence are substituted with other amino acid residues (however, X is not arginine), and one or more amino acid residues constituting the amino acid sequence are substituted. A polymorphic domain of the amyloid precursor protein, wherein two or more amino acid residues have been deleted and / or the amino acid sequence having one or more amino acid residues inserted therein is specified. Peptides are also provided. According to another aspect of the present invention, there is provided a nucleotide sequence encoding each of the above polypeptides.

According to still another aspect of the present invention, there is provided a mutant amyloid precursor protein comprising the above polypeptide as an intracellular domain, and in a preferred embodiment, the following polypeptide: (a) wild-type A polypeptide that is the extracellular domain of the amyloid precursor protein (excluding the portion contained in the amyloid β domain),
(b) a polypeptide that is an amyloid β domain, (c) a polypeptide that binds to the C-terminus of the amyloid β domain, and constitutes a part of a cell membrane domain, and (d) the polypeptide that is an intracellular domain. A mutant amyloid precursor protein is provided.

In another preferred embodiment, one or more of the following polypeptides: (e) one or more amino acid sequences constituting the extracellular domain of a wild-type amyloid precursor protein (excluding the portion contained in the amyloid β domain) Has been replaced with another amino acid residue, one or more amino acid residues constituting the amino acid sequence have been deleted, and / or one or more amino acid residues have been added to the amino acid sequence. A polypeptide which is identified by the amino acid sequence in which the residue has been inserted and which is located on the N-terminal side of the extracellular domain of the amyloid precursor protein; (f) a polypeptide which is an amyloid β domain; and (g) a polypeptide which is amyloid β domain. A mutant amylo comprising a polypeptide bound to the terminal side and constituting a part of a cell membrane domain, and (h) the above-mentioned polypeptide which is an intracellular domain. An id precursor protein is provided.

In still another preferred embodiment, the following polypeptides: (i) a polypeptide that is an amyloid β domain; (j) a polypeptide that binds to the C-terminus of the amyloid β domain and forms a part of a cell membrane domain; And (k)
A mutant amyloid precursor protein comprising the above polypeptide which is an intracellular domain is also provided. In these preferred embodiments, a mutant amyloid precursor protein in which the amyloid β domain is a polypeptide specified by the amino acid sequence of wild-type human amyloid β is a particularly preferred embodiment.

Further, according to the present invention, a nucleotide sequence encoding the above-mentioned mutant amyloid precursor protein, a DNA sequence as a preferred embodiment thereof, a recombinant vector containing the above-mentioned DNA sequence, and a plasmid transformed by the recombinant vector A transformant is provided. In addition, the above DN
A transgenic animal in which the A sequence has been introduced into the chromosome and amyloid β production in nerve tissue has been enhanced; a method for evaluating a substance having an action of inhibiting amyloid β production, comprising: Culturing the transformant according to claim 13 in the presence thereof, and measuring the amount of amyloid β produced in the culture solution; and co-culturing the nerve cell and the transformant in the method. A method is provided that includes:

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The polypeptide of the present invention has the following amino acid sequence: H ₂ N-Lys-Lys-Lys-Gln-Tyr-Thr-Ser-Ile-
His-His-Gly-Val-Val-Glu-Val-Asp-Ala-Ala-Val-Thr-Pr
o-Glu-Glu-X-His-Leu-Ser-Lys-Met-Gln-Gln-Asn-Gly-Ty
r-Glu-Asn-Pro-Thr-Tyr-Lys-Phe-Phe-Glu-Gln-Met-Gln-
Asn-OH (in the sequence, X represents an amino acid residue other than arginine), which is represented by KKKQYTSIHHGVVEVDAAVTPEE
XHLSKMQQNGYENPTYKFFFEQMQN (Similarly, X represents an amino acid residue other than arginine).

The above-mentioned polypeptide can be an intracellular domain of amyloid precursor protein. The amino acid residue represented by X is not particularly limited as long as it is an amino acid residue other than arginine. For example, an α-amino acid residue such as an alanine residue is preferable. As used herein, the term "amino acid" refers to L-α-amino acid,
Amino acids and non-α-amino acids (eg, β-alanine and ε
-Aminocaproic acid, etc.), but preferably refers to α-L-amino acids. In addition, the term `` amino acid residue '' refers to the amino acid of the amino acid which is generated by removing one hydroxyl group and two hydrogen atoms from a carboxyl group and an amino group involved in peptide bond among the functional groups of the amino acid constituting the polypeptide. The rest of the partial structure is meant.

In the polypeptide of the present invention, one or more amino acid residues constituting the above amino acid sequence, preferably one amino acid residue is substituted with another amino acid residue (where X is Arginine), 1 or 2 or more, preferably 1 amino acid residue constituting the above amino acid sequence is deleted, and / or 1 or 2 or more, preferably A polypeptide which is specified by an amino acid sequence having one amino acid residue inserted therein and which is an intracellular domain of amyloid precursor protein, similar to the above-mentioned polypeptides (may be referred to herein as "modified polypeptide" ) Are also included.

As used herein, the term “amyloid precursor protein” includes various m-splicing (alternating splicing) of pre-mRNA transcribed from a single amyloid precursor protein gene.
Various proteins produced by translation of RNA are included. In addition, one or more amino acid residues constituting the amino acid sequence of those proteins have been substituted with other amino acid residues, one or more amino acid residues have been deleted, and / or It is used as a concept including a variant in which one or more amino acid residues are inserted into the sequence. The origin of the amyloid precursor protein is not particularly limited, and may be derived from mammals such as monkeys, dogs and cats in addition to humans. However, the amyloid β domain present in the amyloid precursor protein preferably has the amino acid sequence of wild-type human amyloid β.

The amyloid precursor protein is composed of an extracellular domain (comprising a partial sequence on the N-terminal side of the amyloid β domain, or a peptide further containing one or more amino acids at the N-terminal of the partial sequence). A cell membrane domain containing an amyloid β domain other than the amyloid β domain contained in the extracellular domain and a polypeptide sequence bound to the C-terminal side of the domain; and an intracellular domain. . Amyloid β is generated by the N-terminal and C-terminal of the amyloid β domain contained in the amyloid precursor protein being cleaved by β-secretase and γ-secretase, respectively, or the extracellular domain is formed by a partial sequence of the amyloid β domain. It is produced by cleaving the C-terminus of the amyloid β domain of the constituted amyloid precursor protein by γ secretase.

Therefore, it has the above-mentioned structural characteristics, and has the action of β secretase and γ secretase,
It is to be understood that any protein that gives amyloid β by the action of only γ-secretase is included in the concept of amyloid precursor protein. In addition, as a typical amyloid precursor protein, APP770
(Number of constituent amino acids 770: Kitaguchi, N. et al., Nuture,
331, pp.530-532, 1988), APP751 (constituting amino acids 75
1: Ponte, P. et al., Nature, 331, pp. 525-527,198
8), and APP695 (constituent amino acids 695: Kang, J. et a
l., Nature, 325, pp. 733-736, 1987), and their amino acid sequences are all known. For reference, the structure of APP695 and the enzyme cleavage site are shown in FIG.

As used herein, the term "mutated amyloid precursor protein" refers to the intracellular domain of each of the above-mentioned amyloid precursor proteins (for example, from APP6495 to 649th lysine residue to 695th C-terminal asparagine). Polypeptide composed of 47 amino acids)
Is a protein that has been substituted by the above-mentioned polypeptides (including modified polypeptides) of the present invention. Without wishing to be bound by any particular theory, intracellularly expressed native amyloid precursor protein such as APP695 can be converted to amyloid β by the action of β-secretase and γ-secretase, or γ-secretase.
In the process of the formation of amyloid, a part of the amyloid β is cleaved by the action of α-secretase to give a fragment (see FIG. 1). On the other hand, the mutant amyloid precursor protein of the present invention is characterized by being less susceptible to degradation by α-secretase and producing a large amount of a polypeptide containing the full length of amyloid β.

The mutant amyloid precursor protein of the present invention includes, for example, amyloid precursor protein APP695
A mutant amyloid precursor protein in which the second arginine residue is substituted with an amino acid residue other than arginine; a mutant amyloid precursor protein in which the 728th arginine residue of the amyloid precursor protein APP751 is substituted with an amino acid residue other than arginine; And amyloid precursor protein
A mutant amyloid precursor protein in which the arginine residue at position 747 of APP770 is substituted with an amino acid residue other than arginine; and a mutant amyloid precursor protein in which the amino acid residue other than arginine is an alanine residue in these mutant amyloid precursor proteins Although a protein may be mentioned, the mutant amyloid precursor protein of the present invention is not limited to these.

In particular, regarding the extracellular domain of the mutant amyloid precursor protein, a considerable amount of amino acid residue substitution with respect to the amino acid sequence of the extracellular domain of the wild-type protein (excluding the portion contained in the amyloid β domain) is performed. , Insertions and deletions are tolerated. Therefore, according to a preferred embodiment of the present invention, the following mutant amyloid precursor protein is provided. The following polypeptides: (a) Extracellular domain of wild-type amyloid precursor protein (excluding the portion contained in the amyloid β domain)
(B) a polypeptide that is an amyloid β domain, (c) a polypeptide that binds to the C-terminus of an amyloid β domain, and constitutes a part of a cell membrane domain,
And (d) a mutant amyloid precursor protein comprising the polypeptide (including a variant polypeptide) that is an intracellular domain;

The following polypeptides: (e) one or more amino acid residues constituting the amino acid sequence that is the extracellular domain of the wild-type amyloid precursor protein (excluding the portion contained in the amyloid β domain) Has been substituted with an amino acid residue of
Or N-terminal of the extracellular domain of the amyloid precursor protein, which is specified by an amino acid sequence in which two or more amino acid residues have been deleted and / or one or more amino acid residues have been inserted into the amino acid sequence. (H) a polypeptide that binds to the C-terminal side of the amyloid β domain and constitutes a part of the cell membrane domain, and (h)
Mutant amyloid precursor protein comprising the polypeptide (including a variant polypeptide) that is an intracellular domain;
And the following polypeptides: (i) a polypeptide that is an amyloid β domain, (j) a polypeptide that binds to the C-terminus of the amyloid β domain and constitutes a part of a cell membrane domain, and (k) an intracellular domain. Mutant amyloid precursor protein comprising a polypeptide (including a variant polypeptide).

According to another aspect of the present invention, there is provided a nucleotide sequence encoding the above-mentioned polypeptide of the present invention (including a variant polypeptide). As used herein, the term "nucleotide sequence" includes RNA and DNA sequences. In the nucleotide sequence of the present invention, one or more nucleotides constituting the above nucleotide sequence are substituted with other nucleotides, one or more nucleotides are deleted, and / or A nucleotide sequence in which one or more nucleotides have been inserted into the nucleotide sequence, preferably DN
Also encompassed are nucleotide sequences that encode variant polypeptides that are A sequences and that are intracellular domains of amyloid precursor protein.

As a preferred example of the above nucleotide sequence of the present invention, the following DNA sequence (where the codon represented by X ₁ X ₂ X ₃ is CGT, CGC, CGA, CGG, AGA, AGG, TAA, TAG, Or TGA). AAGAAGAAAC AGTACACATC CATTCATCAT 30 GGTGTGGTGG AGGTTGACGC CGCTGTCACC 60 CCAGAGGAGX ₁ X ₂ X ₃ CACCTGTC CAAGATGCAG 90 CAGAACGGCT ACGAAAATCC AACCTACAAG 120 TTCTTTGAGC AGATGCAGAA C 141

According to still another aspect of the present invention, a nucleotide sequence encoding each of the above mutant amyloid precursor proteins, and 1
Or a nucleotide sequence in which two or more nucleotides have been replaced with other nucleotides, one or more nucleotides have been deleted, and / or one or more nucleotides have been inserted into the above nucleotide sequence, preferably Is a DNA sequence, and a nucleotide sequence encoding a protein substantially biologically equivalent to the mutant amyloid precursor protein described above is also provided.

Of these nucleotide sequences, a DNA sequence encoding a mutant amyloid precursor protein in which the 672th arginine residue of the amyloid precursor protein APP695 is substituted with an amino acid residue other than arginine; 728 of the amyloid precursor protein APP751 A DNA sequence encoding a mutant amyloid precursor protein in which the arginine residue is substituted with an amino acid residue other than arginine; and the 747th arginine residue of the amyloid precursor protein APP770 was substituted with an amino acid residue other than arginine A DNA sequence encoding a mutant amyloid precursor protein is preferred. In addition, in these mutant amyloid precursor proteins, a DNA encoding a mutant amyloid precursor protein in which amino acid residues other than arginine are alanine residues.
Sequences are also particularly preferred nucleotide sequences. It goes without saying that nucleotide sequences containing these nucleotide sequences as partial sequences are included in the scope of the present invention.

The above-mentioned polypeptide, mutant amyloid precursor protein and nucleotide sequence of the present invention may be produced by either a chemical synthesis method or a genetic engineering method, and such methods are well known to those skilled in the art. . Nucleotide variants include, for example, N-nitroso-N'-nitro-N-nitrosoguanidine
(NTG) to induce mutation, and the gene can be recovered from the cells. Alternatively, the DNA sequence to be modified may be directly treated with a drug such as sodium nitrite. In addition, the DNA sequence to be modified
Site-directed mutagenesis (Kramer, W., et al., Methods in E
nzymology, 154, p.350, 1987) and recombinant PCR
(PCR Technology, Stockton press, 1989) or the like, it is possible to insert, delete, or substitute a desired amino acid residue by directly introducing nucleotide insertion, deletion, or substitution.

According to another aspect of the present invention, there is provided a recombinant vector containing each of the above nucleotide sequences, preferably a DNA sequence, and a transformant transformed with the recombinant vector. Here, the term “transformant” is used to include cells, bacteria, fungi, or animals into which the transformed cells have been introduced by the recombinant vector. The type of vector is not particularly limited, and can be appropriately selected by those skilled in the art depending on conditions such as the combination with the host cell to be transformed. For example, a plasmid or the like can be used as a vector. As the host cell, for example, any of prokaryotic cells such as Escherichia coli, eukaryotic cells such as yeast, animal cells, and plant cells may be used. As a method for introducing a DNA sequence into a vector and a method for introducing a recombinant vector into a host cell, methods well known to those skilled in the art can be employed. Examples of them are described in detail in the embodiments of the present specification.

By using the transformant capable of expressing the mutant amyloid precursor protein of the present invention, it is possible to efficiently evaluate a substance having an action of inhibiting amyloid β production. Such an evaluation method provided by the present invention includes a screening method and the like. In a preferred embodiment, a step of culturing the transformant in the presence of a test substance and measuring the amount of amyloid β produced is performed. Contains. The measurement of the amount of amyloid β production includes any quantitative and qualitative measurement, and methods for performing the quantification include, for example, anti-amyloid β
RIA (radioimunoassay) or EIA (enzym
e. immunoassay) can be used. In the absence of the test substance or when the test substance is ineffective, there is no change in the amount of amyloid β secreted from the transformant. On the other hand, if the test substance has the action of suppressing the enzyme activity of β or γ secretase, amyloid β will not be produced in large quantities,
The amount of amyloid β secreted from the transformant decreases. In addition, a method of co-culturing the transformant and nerve cells may be adopted.

Furthermore, a transgenic animal can be prepared using a DNA sequence which is a preferred embodiment of the nucleotide sequence of the present invention. The type of animal is not particularly limited, for example, mice, rats, rabbits, cats, dogs,
Any mammal, such as a cow, horse, or monkey, can be used. Of these, mice are preferably used. Methods for producing transgenic animals by introducing DNA into embryos such as mice are well known to those skilled in the art, and those skilled in the art can select and employ an appropriate method.

As an example, Asano et al. (Cell Engineering, 13 (1
2), pp. 1138-1146, 1994), and a transgenic mouse can be produced. For example, a fertilized egg of a mouse is removed from the oviduct, and a small amount of a recombinant vector such as a plasmid incorporating the DNA of the present invention is injected into the male pronucleus of the fertilized egg using a glass injection pipette. Implant into the fallopian tubes of pregnant mice. A transgenic mouse can be produced by analyzing the chromosomal DNA of the offspring by an appropriate method and selecting individuals in which the DNA of the present invention has been inserted into the chromosomal DNA.

[0036]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to the following Examples. Example 1 Preparation of APP770 cDNA Deleting Part of Extracellular Region APP770 cDNA (Kitaguchi, N. et al., Nature, 331, pp.
530-532, 1988) immediately before the coding region.
Site, 250 bases downstream after completion of the coding region
Has an XmnI site. APP770 cDNA was transferred to NruI (Toyobo) and XmnI (New England Biolabs).
After insertion, insert into the HincII site in the polylinker of plasmid pUC19 (Toyobo Co., Ltd.) so that the upstream is located on the HindIII site side in the polylinker of pUC19, and insert a HindIII site and a 3 ′ end at the 5 ′ end. APP7 with XbaI site at
A plasmid carrying 70 cDNA was prepared (having XbaI downstream of the HincII site in the polylinker).

The HindIII site and XbaI
The DNA fragment obtained by cutting each site is
It was introduced into pcDNA1 / Neo (Invitrogen) to obtain plasmid pcDNA1 / Neo-APP770. This plasmid pcDNA1 /
Neo-APP770 was cut with XhoI and BglII to obtain DNA fragments. Next, the DNA fragment containing the upstream portion of the XhoI site and the downstream portion of the BglII site of the APP770 cDNA was
After blunt-ending with the Klenow fragment of E. coli DNA polymerase I in the presence of the species dNTPs,
The plasmid was prepared by ligation with ligase.

Further, using this plasmid, E. coli Top
The 10F ′ strain was transformed to prepare a large amount of a plasmid having an APP770 (APP770del) cDNA lacking from the XhoI site to the BglII site. This plasmid is called HindIII and X
After cutting each with baI, 1% agarose electrophoresis was performed, and a DNA fragment containing APP770del was recovered from the agarose gel. This DNA fragment was combined with plasmid pcDNA3 (Invitrogen) digested with HindIII and XbaI to T4D
Plasmid pcDNA3-AP obtained by binding with NA ligase
E. coli Top10F 'strain was transformed using P770del. In this plasmid, at the 5 'end of the APP770del cDNA
The T4 promoter is bound in the same direction as the cDNA, and the SP6 promoter is bound to the 3 'end in the opposite direction to the cDNA. In addition, restriction enzyme, Klenow fragment, and T4
The reaction conditions of the DNA ligase were in accordance with the conditions recommended by each reagent sales company. FIG. 2 shows an outline of the above steps.

Example 2: Preparation of cDNA of mutation 672A-APP770del in which arginine at position 672 is replaced with alanine (amino acid numbers are based on the amino acid sequence of APP695) (1) Base sequence of primer and mutation by synthetic PCR In order to carry out the introduction, four kinds of primers shown in the table were synthesized using a DNA automatic synthesizer (ABI). Primer 1 indicates that T, which is the base of the original APP770 cDNA, was replaced with G in order to introduce the EcoO65 recognition site, and indicates that the original APP770 cDNA was changed to change the arginine codon to an alanine codon. GC base CG
Indicates that substitution was performed. The underlined part in primer 3 is E
Base of the original APP770 cDNA to create the coO65I recognition site
Indicates that A was replaced with C. Primer 2 has a nucleotide sequence complementary to the SP6 promoter DNA, and primer 4 has a nucleotide sequence complementary to the T4 promoter DNA.

[0040]

[Table 1]

(2) Experimental method of PCR The amplification reaction of the DNA fragment by PCR was carried out in 100 μl of buffer. Buffer composition is 10 mM Tris-HCl (pH 8.3)
, 50 mM KCl, 1.5 mM MgCl ₂ , Pwo DNA polymerase (Boehringer Mannheim) 2.5 U, dNTPs mix
300 μm, 1 ng of plasmid pcDNA3-APP770del, and 100 pmol of each primer. The reaction was carried out using a DNA thermal cycler (Perkin-Elmer), and after preincubation at 94 ° C for 4 minutes, one cycle was repeated 30 times at 94 ° C for 1 minute, 50 ° C for 2 minutes, and 72 ° C for 3 minutes. Was.

(3) Preparation of cDNA of Mutation 672A-APP770del: PCR was performed with primer 1 and primer 2, electrophoresed on a 1% agarose gel, and a DNA fragment (fragment B) as a reaction product was recovered from the gel. . This fragment B contains the EcoO65I site and the introduced mutation. In addition, PCR and agarose gel electrophoresis were performed in the same manner using primer 3 and primer 4 to have an EcoO65I site on the downstream side and APP7
A DNA fragment (fragment A) containing the upstream side of the 70del cDNA was obtained.

After fragment A and fragment B were digested with Eco065I, both fragments were ligated with T4 DNA ligase, the reaction product was subjected to 1% agarose gel electrophoresis, and the mutation 672A-APP770del c
A DNA fragment containing the entire DNA was recovered from the gel. This DNA
Using the fragment and primer 1 and primer 2,
DNA amplification by PCR was performed according to the method of (2). However, the plasmid pcDNA3-APP770del was excluded. This PCR
The DNA fragment obtained in Step 3 was digested with HindIII and XbaI, and the obtained fragment was ligated with plasmid pcDNA3 previously digested with HindIII and XbaI with T4 DNA ligase, and Top10F ′
The strain was transformed to obtain plasmid pcDNA3-672A-APP770del. The nucleotide sequence of 672A-APP770delcDNA on this plasmid was confirmed by the dye terminator method. An outline of the above method is shown in FIG.

Example 3: Obtaining an expression cell line (1) Preparation of transformed cells 4 ml of medium (DMEM containing 10% fetal calf serum, high gl
293 cells (transformed cells derived from human fetal kidney, Dainippon Pharmaceutical, ATCC: CRL-1573) were inoculated on a 6 cm plate containing ucose gibco 12100 (Gibco) and cultured until they became semi-confluent. After washing twice with DMEM, 2 ml of DME
M was added. In this cell, plasmid pcDNA3-APP770de
l or plasmid pcDNA3-672A-APP770del DNA (10μ
g) and 30% (V / V) Lipofectin (Gibco) solution 100
After incubating for 12 hours, add 2 ml of DMEM containing 20% fetal calf serum, and in a CO2 incubator at 37 ° C.
Cultured for hours. After the culture, the cells were collected by pipetting and inoculated on a 10 cm plate containing 10 ml of a medium containing the antibiotic G418 (800 μg / ml). After culturing for 2 weeks while changing the medium every 3 to 4 days, several colonies of the transformed cells were isolated, and each cell was cultured on a 10 cm plate supplemented with 10 ml of medium until confluent, and the cells were cultured. It has become.

(2) APP770del and mutation 672A-APP770del
Obtaining expression cell lines Plasmid pcDNA3-APP770del and Plasmid pcDNA3-
For multiple transformed cell lines obtained using 672A-APP770del, the transgene product was detected by the method of (3) below,
By comparing the band intensities of the obtained full-length products, the respective high-producing strains, 293-APP770del-A strain and 293-6
72A-APP770del-B strain was acquired.

(3) Transgene products (APP770del and 672
A-APP770del) Detection method: The transformed 293 cell line was 10 cm in 10 ml medium.
Inoculate the plate at a rate of 1 × 10 ⁷ cells / plate, 37 ° C
After culturing in a carbon dioxide incubator for 4 days, the cells were collected by trypsinization. Transfer cells to DPBS (Dulbecco-
Dulbecco's phosphate-buffered sali
ne), and centrifuged at 800 × g for 5 minutes to collect the cells as a precipitate fraction. The cells were disrupted by adding 15 μl SDS-sample buffer and 15 μl 8 M urea and sonicating.

After boiling the obtained cell extract for 5 minutes,
SDS-polyacrylamide gel electrophoresis was performed using 15% polyacrylamide gel. After the electrophoresis, the protein in the gel was transferred to a nitrocellulose membrane (0.2 μm; S & S) using a semi-dry blotting apparatus. APP695
Antiserum (UT-421) obtained from a rabbit immunized with a synthetic peptide corresponding to positions 676 to 695 of
APP770de on nitrocellulose membrane using the ECL Western Blotting Analysis System (Amersham), as recommended by the Amersham protocol.
l and their degradation products, including the full-length form of 672A-APP770del and the C-terminal part, were detected.

Example 4: 293-APP770del-A strain and 293-672A-
APP770del and 672A-APP7 expressed in APP770del-B strain
Analysis of degradation product of 70del 293-APP770del-A and 293-672A- obtained in Example 3 (2)
Gene products produced by each of the APP770del-B strains were analyzed by the following method. 293-APP770del-A strain and 293-672A-APP7
Add 70del-B strain to a 10 cm plate containing 10 ml of medium.
The cells were inoculated at a rate of × 10 ⁷ cells / plate, cultured in a carbon dioxide incubator at 37 ° C. for 4 days, and then the cells were collected by trypsinization. Transfer cells to DPBS (Dulbecco's phosphate-buff
ered saline), and centrifuged at 800 × g for 5 minutes to collect cells as a precipitate fraction.

In this fraction, 120 μl of cell lysis buffer was added.
(50 mM Tris-HCl (pH 8.0), 0.27 M sucrose,
1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 1% Triton X-100, 0.1% 2-mercaptoethanol, 200 μg / ml pepstatin A, 200 μg / ml chymostatin, 200 μg / ml leupeptin) Then, the cells were lysed by holding them for 30 minutes in ice with occasional shaking. The lysate was centrifuged at 10,000 xg for 5 minutes, and the supernatant was
μl was collected. 100 μl of 0.1 M Tris-HCl solution (pH 7.4, containing 0.2% SDS and 5.44 M urea) was added to the supernatant, and after mixing, the mixture was boiled and the reaction product was centrifuged at 10,000 × g for 5 minutes. In 200 μl of the obtained reaction supernatant, add 60 μl of 5 M
NaCl, 40 μl 0.5 M EDTA, 150 μl 1 M Tris
(pH 7.4), 500 µl of NP-40 and 350 µl of double-distilled water were added, and the mixture was stirred well.
Allowed to react for hours.

The antigen-antibody complex was recovered by adding protein A-Sepharose (Pharmacia), and after washing, 20 μl of × 5 SDS-sample buffer and 20 μl of the resin were added to the resin.
8 M urea was added and mixed. Boil this mixture for 10,0
After centrifugation at 00 × g for 5 minutes, the resulting supernatant was subjected to SDS-polyacrylamide gel electrophoresis using 15% polyacrylamide gel. After the electrophoresis, the proteins in the gel were transferred to a nitrocellulose membrane using a semi-dry blotting apparatus. Western blotting was performed using antisera UT-421 and ¹²⁵ I-protein A (Amersham), and the full-length forms of APP770del and 672A-APP770del or their carboxyl-terminal degradation products were analyzed by autoradiography. FIG. 4 shows the results. 293-APP770d
In the el strain, one band was detected at around 15 kD, but 293
In the -672A-APP770del strain, a band having almost the same mobility as the band observed in the 293-APP770del strain and a band having a lower mobility than the band were observed.

To examine the amino terminal side of these gene products, a monoclonal antibody A-11 (1 to 1 of amyloid β) obtained by immunizing mice with a synthetic peptide corresponding to amino acids 1 to 16 of amyloid β was used. Binds to a synthetic peptide corresponding to the 16th amino acid, but amyloid β
(It does not bind to the synthetic peptide corresponding to the 3rd to 16th amino acids) and the binding to these bands was examined. As a result, the 15 kD band detected from the 293-APP770del strain and the higher mobility band detected from the 293-672A-APP770del strain showed no binding. On the other hand, 293-672A-A
The band with the lower mobility detected from the PP770del strain showed binding to monoclonal antibody A-11. These results indicate that the low-mobility gene product obtained from the 293-672A-APP770del strain is a so-called amyloidogenic fragment containing the full length of amyloid β.

[0052]

EFFECT OF THE INVENTION The mutant amyloid precursor protein containing the polypeptide of the present invention has enhanced amyloid β productivity, and is capable of producing a large amount of amyloid β by expressing this precursor protein in cells. Can be. Therefore, the DNA encoding the precursor protein of the present invention
By using a transformant transformed by the above method, an agent that suppresses the production of amyloid β can be efficiently screened. In addition, transgenic animals in which the DNA of the present invention has been introduced into the chromosome can be expected to be useful as a model animal for Alzheimer's disease because amyloid β can be expressed in large amounts in neural tissues.

[Brief description of the drawings]

Fig. 1 APP, an example of amyloid precursor protein
FIG. 7 shows the extracellular domain, cell membrane domain, and intracellular domain in the molecule of No. 695, and the amyloid β domain and enzyme cleavage site spanning the extracellular domain and cell membrane domain.

FIG. 2 is a diagram schematically showing a method for preparing APP770 cDNA in which a part of an extracellular region has been deleted according to Example 1 in Examples.

FIG. 3. Mutation of APP770del (672
FIG. 1 is a diagram showing an outline of a method for preparing cDNA of A).

FIG. 4. 293-APP770del obtained according to example 3 of the example
Gene product obtained from the strain and the 293-672A-APP770del strain.
FIG. 2 is a photograph showing an electrophoresis pattern, which was subjected to SDS-polyacrylamide gel electrophoresis using a 15% polyacrylamide gel and detected with UT-421 and ¹²⁵ I-protein A reacting on the carboxy terminal side of amyloid β.

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[Procedure amendment]

[Submission date] July 22, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 1

[Correction method] Change

[Correction contents]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction contents]

That is, the present invention provides the following amino acid sequence:
H -Lys-Lys-Lys-Gln-Tyr-Thr-Ser-Ile-His-His-Gly-Val
-Val-Glu-Val-Asp-Ala-Ala-Val-Thr-Pro-Glu-Glu-X-His
-Leu-Ser-Lys-Met-Gln-Gln-Asn-Gly-Tyr-Glu-Asn-Pro-T
The present invention provides a polypeptide specified by hr-Tyr-Lys-Phe-Phe-Glu-Gln-Met-Gln-Asn-OH (in the sequence, X represents an amino acid residue other than arginine). Also,
The present invention provides the above polypeptide, which is an intracellular domain of amyloid precursor protein.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0017

[Correction method] Change

[Correction contents]

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The polypeptide of the present invention has the following amino acid sequence: H -Lys-Lys-Lys-Gln-Tyr-Thr-Ser-Ile-H
is-His-Gly-Val-Val-Glu-Val-Asp-Ala-Ala-Val-Thr-Pro
-Glu-Glu-X-His-Leu-Ser-Lys-Met-Gln-Gln-Asn-Gly-Tyr
-Glu-Asn-Pro-Thr-Tyr-Lys-Phe-Phe-Glu-Gln-Met-Gln-A
(in the sequence, X is a amino acid residues <br/> shown other than arginine, the left end N-terminus is) sn-OH is a polypeptide that is specified by, in letter codes are, KKKQYTSIHHGV
VEVDAAVTPEEXHLSKKMQQNGYENP
TYKFFEQMQN (similarly, X represents an amino acid residue other than arginine).

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication location C12N 5/10 G01N 33/53 D 15/09 ZNA A61K 37/02 AAM C12P 21/02 C12N 5 / 00 B G01N 33/53 9282-4B 15/00 ZNAA // (C12N 1/21 C12R 1:19) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1:91)

Claims (16)

[Claims] 1. The following amino acid sequence: H ₂ N-Lys-Lys-Lys-Gln-Tyr-Thr-Ser-Ile-His-His- -Gly-Val-Val-Glu-Val-Asp-Ala-Ala -Val-Thr- -Pro-Glu-Glu-X-His-Leu-Ser-Lys-Met-Gln- -Gln-Asn-Gly-Tyr-Glu-Asn-Pro-Thr-Tyr-Lys- -Phe- Phe-Glu-Gln-Met-Gln-Asn-OH (X represents an amino acid residue other than arginine in the sequence)
A polypeptide identified by 2. The polypeptide according to claim 1, which is an intracellular domain of amyloid precursor protein. 3. The amino acid sequence according to claim 1, wherein one or more amino acid residues constituting the amino acid sequence according to claim 1 have been substituted with another amino acid residue (provided that X is not arginine). And / or one or more amino acid residues constituting
Or a polypeptide which is an intracellular domain of amyloid precursor protein, which is specified by an amino acid sequence having two or more amino acid residues inserted. 4. A nucleotide sequence encoding the polypeptide according to any one of claims 1 to 3. 5. A mutant amyloid precursor protein comprising the polypeptide according to any one of claims 1 to 3 as an intracellular domain. 6. The following polypeptides: (a) Extracellular domain of wild-type amyloid precursor protein (excluding a portion contained in amyloid β domain)
(B) a polypeptide that is an amyloid β domain; (c) a polypeptide that binds to the C-terminus of the amyloid β domain and forms part of a cell membrane domain; and (d) a polypeptide that is a part of a cell membrane domain. The mutant amyloid precursor protein according to claim 5, comprising: a polypeptide that is an intracellular domain according to any one of claims 1 to 6. 7. The following polypeptides: (e) Extracellular domain of wild-type amyloid precursor protein (excluding a portion contained in amyloid β domain)
Wherein one or more amino acid residues constituting the amino acid sequence are substituted with another amino acid residue, and one or more amino acid residues constituting the amino acid sequence are deleted, and / or Or a polypeptide specified by an amino acid sequence having one or more amino acid residues inserted into the amino acid sequence and arranged on the N-terminal side of the extracellular domain of amyloid precursor protein; (f) an amyloid β domain (G) a polypeptide which binds to the C-terminal side of the amyloid β domain and constitutes a part of a cell membrane domain; and (h) a polypeptide which is the intracellular domain according to any one of claims 1 to 3. The mutated amyloid precursor protein according to claim 5, comprising a polypeptide. 8. The following polypeptides: (i) a polypeptide that is an amyloid β domain; (j) a polypeptide that binds to the C-terminus of the amyloid β domain and forms a part of a cell membrane domain; and (k) a claim The mutant amyloid precursor protein according to claim 5, comprising: a polypeptide that is an intracellular domain according to any one of items 1 to 3. 9. The mutant amyloid precursor protein according to any one of claims 6 to 8, wherein the amyloid β domain is a polypeptide specified by the amino acid sequence of wild-type human amyloid β. 10. A nucleotide sequence encoding a mutant amyloid precursor protein according to any one of claims 5 to 9. 11. The nucleotide sequence according to claim 10, which is a DNA sequence. 12. A recombinant vector comprising the DNA sequence according to claim 11. A transformant transformed by the recombinant vector according to claim 12. 14. A transgenic animal, wherein the DNA sequence according to claim 11 has been introduced into a chromosome, and production of amyloid β in nerve tissue has been enhanced. 15. A method for evaluating a substance having an action of inhibiting amyloid β production, comprising culturing the transformant according to claim 13 in the presence of a test substance, and producing amyloid β in a culture solution. A method comprising measuring 16. The method according to claim 15, comprising a step of co-culturing the nerve cell and the transformant.