Patent Application
20060160146
The present invention relates to a method for measuring β-amyloid, which is considered to be one of the causes of Alzheimer's disease. The invention also relates to a method for screening a substance which affects the production of β-amyloid.
Alzheimer's-type dementia is a progressive neurodegenerative disease, and is characterized by cognitive dysfunction typified by recent-memory disturbance and pathological changes (a senile plaque, neurofibrillary degeneration, and neurocyte death).
β-amyloid is a polypeptide composed of about 40-amino. acid residues, and is accepted as a principal component of the senile plaque that is a pathological feature of Alzheimer's-type dementia. As currently known, accumulation of β-amyloid in the brain is observed as a pathological change in the early stages of this disease.
β-amyloid is considered to be one of the causes for the onset of this disorder for the following reasons: β-amyloid itself is toxic to neurocytes, there exists a family with genetic mutation in a precursor of a β-amyloid, i.e., a β-amyloid precursor protein (APP) and the family develops Alzheimer's-type dementia with high frequency, and, in a transgenic mouse to which the mutant gene was introduced, a large amount of β-amyloid was accumulated in the brain.
APP is classified into a type I membrane protein by structure. In the living body, there are various kinds of molecular species distinguished by the existence or absence of the Kunitz-Type protease inhibitor domain. Although APP695, which is composed of 695 amino acid residues, is a principal molecule in the nervous system, the physiological function of APP 695 is almost unknown.
As shown in
As a minor processing system for APP, there exists an amyloidgenic system which produces β-amyloid. This processing is a system in which β-secretase acts on APP 695 at the C terminal side of a methionine residue at position 596 of APP695 to generate sAPPβ and a C-terminal fragment of 99 residues (C99). C99 is further cleaved by γ-secretase to generate β-amyloid, which is considered to be a cause of Alzheimer's disease. This pathway may be 10% or less of the entire processing of APP (β-amyloid production system).
The α-secretase used herein denotes an enzyme which acts on the α-secretase cleavage site in APP to cleave a peptide bond.
In the early stage of the above-described APP processing, α- and β-secretases act in a competitive manner (Journal of Molecular Neuroscience 17, 157-180, 2001).
At present, some attempts are being made to prevent and/or treat Alzheimer's disease by inhibiting β-amyloid production, and β-secretase is being studied as one of the targeted candidate molecules. β-secretase has already been identified as BACE1. It is reported that mice deficient in BACE1 have a normal phenotype based on knowledge obtained from BACE1 knockout mice (Nature Neurosci., 4, 231, 2001 and Nature Neurosci., 4, 233, 2001). Therefore, the suppression of β-amyloid production by inhibiting β-secretase activity is expected to be effective in a prophylactic and/or therapeutic agent for Alzheimer's disease. A substance for improving β-secretase activity is useful for elucidating the onset mechanism of Alzheimer's disease, the production of a disease model, and proof of an amyloid hypothesis.
A method for screening a compound which has β-secretase inhibition activity is practically examined. The method comprises expressing in a cell a protein created by fusing the C terminus of a wild type APP into the C terminus of the secretory form of alkaline phosphatase (SEAP), and determining SEAP activity secreted in the culture supernatant (CIS Diagnostic Co., Ltd.).
However, since α- and β-secretases act in a competitive manner as described above, and α-secretase processing makes up 90% of all processing, β-amyloid production efficiency is extremely low. As is clear from the above, it is unlikely that the screening system using the fusion protein will be able to efficiently screen substances which affect the β-amyloid production.
A primary object of the invention is to provide a material and a process for efficiently measuring the production of β-Amyloid related to the onset of Alzheimer-type dementia. Another primary object of the invention is to provide a process for efficiently screening substances which affect the production of β-Amyloid.
The inventors conducted intensive research on a material and a process for efficiently measuring the production of β-amyloid and found that a specific amino acid substitution in APP eliminates the α-secretase cleavage without affecting β-secretase cleavage. The inventors also found that a substance which affects β-amyloid production can be efficiently screened by fusing the APP-modified protein into a detectable secretory protein and stably expressing the same in a cell. The inventors conducted further research and accomplished the present invention based on these findings.
The present invention includes the following items:
Item 1. A modified protein of β-amyloid precursor protein (APP), wherein (i) a β-secretase cleavage site is contained and (ii) one or more amino acids are mutated so that the β-secretase cleavage is not affected and α-secretase cleavage does not occur.
Item 2. A nucleic acid molecule which encodes an APP modified protein according to Item 1.
Item 3. A test cell which expresses an APP modified protein according to Item 1.
Item 4. A fusion protein, comprising an APP modified protein according to Item 1 and a detectable secretory protein.
Item 5. A nucleic acid molecule encoding a fusion protein, comprising an APP modified protein according to Item 1 and a detectable secretory protein.
Item 6. A test cell expressing a fusion protein comprising an APP modified protein according to Item 1 and a detectable secretory protein.
Item 7. A method for examining whether or not a subject substance affects β-secretase activity, the method comprising culturing in the presence of the subject substance a test cell expressing a fusion protein comprising an APP modified protein according to Item 1 and a detectable secretory protein, and measuring detectable secretory proteins which were cleaved by β-secretase and secreted outside the cell.
Item 8. A method for examining whether or not a subject substance affects β-amyloid production, the method comprising culturing in the presence of the subject substance a test cell expressing a fusion protein comprising an APP modified protein according to Item 1 and a detectable secretory protein, and measuring detectable secretory proteins which were cleaved by β-secretase and secreted outside the cell.
Item 9. A method for screening a substance useful as an active ingredient for a therapeutic agent or prophylactic agent for Alzheimer's-type dementia, the screening method comprising culturing in the presence of a subject substance a test cell expressing a fusion protein comprising an APP modified protein according to Item 1 and a detectable secretory protein, and measuring detectable secretory proteins which were cleaved by β-secretase and secreted outside the cell.
Item 10. A method for examining the cytotoxicity of a subject substance according to anyone of Items 7 to 9, the method comprising performing a cell viability test of the test cell cultured in the presence of the subject substance.
Item 11. An APP modified protein according to Item 1, wherein Swedish familial Alzheimer's disease mutations are introduced to the protein.
Item 12. A fusion protein comprising an APP modified protein according to Item 1 and a detectable secretory protein, wherein Swedish familial Alzheimer's disease mutations are introduced to the fusion protein.
Item 13. An APP modified protein according to Item 1, wherein the protein has an amino acid sequence of any one of SEQ ID Nos. 2 to 6.
Item 14. An APP modified protein according to Item 1, wherein the protein has an amino acid sequence of any one of SEQ ID Nos. 7 to 11.
Item 15. A fusion protein comprising an APP modified protein according to Item 1 and a detectable secretory protein, wherein the fusion protein has an amino acid sequence of any one of SEQ ID Nos. 12 to 16.
Item 16. A fusion protein comprising an APP modified protein according to Item 1 and a detectable secretory protein, wherein the fusion protein has an amino acid sequence of any one of SEQ ID Nos. 17 to 21.
Item 17. An APP modified protein according to Item 1, wherein the protein has the following amino acid sequence: Lys-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-X-X-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val, wherein X represents any amino acid residue.
Item 18. An APP modified protein according to Item 1, wherein the protein has the following amino acid sequence: Asn-Leu-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-X-X-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val, wherein X represents any amino acid residue.
Item 19. A fusion protein comprising an APP modified protein according to Item 17 or 18 and a detectable secretory protein.
Item 20. A nucleic acid molecule which encodes a protein according to any one of Items 11 to 19.
Item 21. A test cell which expresses an APP modified protein according to any one of Items 11, 13, 14, 17, and 18.
Item 22. A test cell which expresses a fusion protein according to any one of Items 12, 15, 16, and 19.
Item 23. A method for examining whether or not a subject substance affects β-secretase activity, the method comprising culturing in the presence of the subject substance a test cell according to Item 22, and measuring detectable secretory proteins which were cleaved by β-secretase and secreted outside the cell.
Item 24. A method for examining whether or not a subject substance affects β-amyloid production, the method comprising culturing in the presence of the subject substance a test cell according to Item 22, and measuring detectable secretory proteins which were cleaved by β-secretase and secreted outside the cell.
Item 25. A method for screening a substance useful as an active ingredient for a therapeutic agent or prophylactic agent for Alzheimer's-type dementia, the screening method comprising culturing in the presence of a subject substance a test cell according to Item 22, and measuring detectable secretory proteins which were cleaved by β-secretase and secreted outside the cell.
Item 26. A method for examining the cytotoxicity of a subject substance according to any one of Items 23 to 25, the method comprising performing a cell viability test of the test cell cultured in the presence of the subject substance.
Hereinafter, the present invention is described in more detail.
β-amyloid Precursor Protein (APP) Modified Protein
The β-amyloid precursor protein (APP) modified protein of the invention is a protein which is characterized by containing a β-secretase cleavage site in APP and which is modified by an amino acid mutation or mutations which do not affect β-secretase cleavage.
Here, using APP695 as an example, the β-secretase cleavage site is a site between a methionine residue at the 596 position and an aspartic acid residue at position 597 (see
The amino acid sequence of APP695 is shown in SEQ ID No. 1 of
A number of molecular species of APP in addition to APP 695, such as APP751 and APP770, are known to exist. They are identical to each other in the α-secretase and β-secretase cleavage sites, and thus APP695 is used to describe the invention. The APP used for the APP modified protein of the invention, however, is not limited to APP695.
In the APP modified protein of the invention, there is no limitation to the length of the amino acid sequence at the N-terminus to the extent that a region required for exhibiting the above characteristics is contained and the β-secretase cleavage is not affected. All of the amino acid sequences at the C terminus are not necessarily required to the extent that the β-amyloid production caused by γ-secretase generated after the β-secretase region is cleaved is not affected.
Taking APP695 as an example, the region of positions 593 to 648 of the APP695 is required to demonstrate the above characteristics.
596 and 597 positions as β-secretase recognition site need to be contained in the region. Since it has been reported that about 4 residues at the N terminus are necessary for the β-secretase to recognize APP695 as a substrate (Neuron, 14, 661-670 (1995)), the N terminus should start with at least position 593. 612 and 613 positions as the α-secretase site are also necessary. When the β-amyloid region (from 597 to 638) for confirming the production amount of the β-Amyloid is included, positions 593 to 638 are required. Since the APP is a type I membrane protein, positions 625 to 648 of a transmembrane region are required so that the APP may be normally fixed to a membrane and maybe undergo processing in the fixed state. This is why positions 593 to 648, which include these positions, are mentioned as an example.
Specific examples of proteins containing such a region include a protein having the C terminal region containing positions 581 to 695 of the amino acid sequence of APP695.
In the modified protein of the invention, an amino acid mutation or mutations which do not affect β-secretase cleavage and do not cause α-secretase cleavage is referred to as an amino acid mutation or mutations which do not cause α-secretase cleavage, and such an amino acid mutation or mutations do not eliminate nor reduce β-secretase cleavage activity.
Such an amino acid mutation or mutations are caused by deletion, substitution, addition, etc., of the amino acid at, for example, the α-secretase cleavage site.
There is no limitation to such an amino acid mutation or mutations to the extent that the mutation or mutations do not affect β-secretase cleavage nor cause α-secretase cleavage.
Particularly preferable examples of amino acid mutations include an amino acid mutation in which the leucine residue at position 17 counting from the amino acid at the N terminal β-amyloid is substituted by glutamic acid, proline, alanine, or lysine, or an amino acid mutation in which the lysine residue at position 16 is substituted by tryptophan.
Specific examples of proteins having the above amino acid mutation include an APP modified protein which has an amino acid sequence of any one of SEQ ID Nos. 2 to 6.
Further, in order to make an attack of β-secretase easier, the generally-known APP amino acid mutation or mutations in the hereditary Alzheimer's disease may be introduced into the modified protein of the invention. Specific examples of the APP amino acid mutation or mutations in the hereditary Alzheimer's disease include a Swedish familial Alzheimer's disease APP mutations.
Specific examples of proteins in to which such a mutation or mutations are introduced include modified proteins having an amino acid sequence of any one of SEQ ID Nos. 7 to 11.
The APP modified protein of the invention may be obtained by modifying a gene encoding an APP protein to a DNA sequence that encodes the amino acid sequence of the modified protein, introducing the mutated gene into a suitable expression vector, to be expressed in a host cell. Subcloning, etc. may be appropriately performed to acquire the target gene in each step. For example, an insertion of the target gene into a suitable cloning vector, and a screening of the target gene by a routine procedure may be conducted to increase the amount of the gene itself.
The selection of codons in the DNA molecule corresponding to the amino acid of an APP modified region is not limited, and any combination can be accepted to the extent that the DNA sequence encodes the amino acid sequence. The codons can be selected in a routine manner. For example, codons can be selected with reference to the codon usage frequencies in the host to be employed.
The target DNA molecular arrangement may be acquired by the DNA amplification method using PCR, for example. The primers used when PCR is employed can be suitably determined based on the sequence information of the target amino acid mutation or mutations, and can be synthesized according to a routine method.
The amplified DNA fragments can be isolated and purified by a routine method, such as gel electrophoresis, molecular sieving, etc.
The base sequence of the nucleic acid molecule of the invention can be determined according to the dideoxy method, the Maxam-Gilbert method, etc., or determined simply by using a commercially-available sequencing kit, etc.
The protein is produced based on the sequence information of the DNA molecule provided by the above DNA molecule according to a common gene recombination technique (see Sambrook, J., Frisch, E. F., and Maniatis, T., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989; Current Protocols in Molecular Biology, John Wiley Sons, Inc. 1998, etc.)
More specifically, the protein is produced by preparing a recombinant DNA (expression vector) in which a DNA molecule coding the desired protein can be expressed in a host cell, introducing the recombinant DNA into the host cell to be transformed, culturing the transformant, and subsequently collecting such proteins from the obtained culture.
Specific examples of host cells include cells derived from prokaryotes, cells derived from eukaryotes, etc.
Specific examples of those derived from prokaryotes include those derived from colibacillus, Bacillus subtilis, etc. Preferable examples of colibacillus are those belonging to the genus Escherichia, such as Escherichia coli K12 strain. Preferable examples of Bacillus subtilis are Bacillus strains, such as Bacillus subtilis Marburg 168.
When prokaryotic cells are used as a host cell, a vector which is replicable in the host cell is used, and a promoter, SD (Shine and Dalgarno) base sequence, and an initiation codon (e.g., ATG), which is necessary for initiation of the protein synthesis, are provided upstream of the gene in the vector, so that the DNA molecule may express.
Various known vectors can be used as the vector mentioned above, and plasmids derived from Escherichia coli, such as pBR322, pBR325, pUC12, pUC13, etc., are generally used. Examples of the vectors for use in expression systems using E. coli include pGEX-4T (Amersham Pharmacia Biotech), pMAL-C2, pMAL-P2 (New England Biolabs), pET21, pET21/lacq (Invitrogen) and pBAD/His (Invitrogen).
There is no limitation to usable promoters. Any promoter can be used. When a strain of the genus Escherichia is used as the host, tryptophan (trp) promoters, lpp promoters, lac promoters, recA promoters, PL/PR promoters, etc., are preferably used. When the host is a strain of the genus Bacillus, SP01 promoters, penP promoters, etc., are preferably used.
Usable cells derived from eukaryotes are cells derived from mammal, for example, and specific examples of such cells include the monkey cell line COS [Cell, 23: 175 (1981)], Chinese hamster ovary cells and a dihydrofolate reductase-defective cell line thereof [J. Exp. Med., 108: 945 (1958), Proc. Natl. Acad. Sci., 77:4216 (1980), NIH3T3 cells (J. Viol., 4:549 (1969)), Jurkat cells (J. Immunol., 133:123 (1984), etc. Cells of yeast belonging to the genus Saccharomyces, etc., are preferably used.
When cells derived from eukaryotes are used as the host cell, vectors having a promoter upstream of the DNA molecule to be expressed, an RNA splice site, a polyadenylation site and a transcription termination sequence are usually used as the expression vector. This vector may also have a replication origin as required. A specific example of the expression vector includes pSV2dhfr harboring an early promoter of SV40 [Mol. Cell. Biol., 1:854(1981)]. In addition to the above, various known commercially available vectors may be used. Examples of commercial vectors which are used in expression systems using animal cells include vectors for animal cells, such as pCI (Promega), pIND (Invitrogen), pcDNA3.1/His (Invitrogen), pEF1/V5-His (Invitrogen), etc., and vectors for insect cells, such as pFastBac HT (GibciBRL), pAcGHLT (PharMingen), pAc5/V5-His, pMT/V5-His and pMT/Bip/V5-his (all Invitrogen), etc.
Examples of preferable promoters for use when animal cells are used as the host cell include SV40-derived promoters, retrovirus promoters, metallothionein promoters, heat shock promoters, cytomegalovirus promoters, and SRα promoters, adenovirus promoters, the elongation factor promoters, etc. When a yeast is used as the host, pH05 promoters, PGK promoters, GAP promoters, ADH promoters, etc., are preferably used.
Introduction of expression vectors into the host cell and transformation caused by the introduction can be carried out without limitation in accordance with various general methods, such as an electroporation, liposome method, calcium phosphate method, DEAE dextran method, etc.
The protein of the invention can be obtained by culturing the host cells, and appropriately isolating and purifying the produced proteins.
The protein thus obtained can be used per se for screening a compound affecting β-secretase.
When the protein is produced using cells derived from eukaryotes, isolated proteins or the whole cell may be used.
In the coexistence of the test substance and the protein (or cells expressing the protein), β-secretase is acted and its influence is measured, thereby screening a substance which affects β-secretase or the production of β-amyloid.
Proteins can be detected by Western blotting, ELISA method, or the like, using an antibody which recognizes the amino acid sequence of APP, for example.
Fusion Protein
The fusion protein of the invention denotes a protein obtained by fusing proteins other than the APP modified protein, e.g., a detectable secretory protein, into the N-terminal protein of the above-described APP modified protein of the invention.
Here, the detectable secretory protein denotes a protein or a peptide which does not contain the localization sequence for each intracellular tissue in the amino acid sequence, and which is released or emitted outside the cell to demonstrate this function. More specifically, this refers to a protein which is cleaved at the C terminal region of the protein by β-secretase and secreted outside the cell.
Preferable examples of detectable secretory proteins include a protein which can be easily detected using a substrate like an enzyme; a protein which has physical properties and thus the amount secreted outside the cell or into the culture supernatant can be quantatively measured using an instrument, a reagent, etc.; or a protein which demonstrates a bioactivity outside the cell. In addition to the above, usable proteins include those in which the bioactivity resulting from the physiologically active protein in the culture supernatant can be determined, or in which the amount of the secretory protein in the culture supernatant can be generally determined using the antibody against the secretory protein with ELISA or Western blotting, for example.
More specifically, secretory alkaline phosphatase, β-galactosidase, peroxidase, luciferase, etc., can be mentioned. These can be quantified as the amount of enzyme activity by, for example, adding an enzyme substrate in the culture supernatant.
Other examples include a green fluorescence protein (GFP), aequorin, immunoglobulin, growth hormone, histidine tag, c-myc tag, Flag tag, hemagglutinin (HA) tag, glutathione S transferase (GSP) tag, T7 tag, V5 epitope tag, X-press tag, BE11 tag, etc.
Furthermore, a physiologically activating protein group belonging to cytokine, for example, interleukin 2, can be mentioned. The protein released outside the cell can be detected by measuring the bioactivity in a culture medium or by measuring with a commercially-available ELISA kit.
Although these secretory proteins are desirably produced and secreted as a secretory-type protein having an original signal peptide, they can be secreted using a signal peptide of the secretory protein of APP and other secretory proteins.
These secretory proteins can be partially modified, deleted, and inserted to the extent that they do not significantly affect the detected enzyme activities, immunoreactivity, bioactivity, etc.
The APP modified protein encompassed in the fusion protein of the invention is a protein subjected to the above-described amino acid mutation or mutations such that the β-secretase cleavage site is contained, the β-secretase cleavage is not affected, and the α-secretase cleavage does not arise.
There is no limitation to the amino acid mutation or mutations to the extent that the amino acid mutation or mutations do not affect the β-secretase cleavage nor cause the α-secretase cleavage. Particularly preferable is an amino acid mutation in which the leucine residue at position 17 counting from the N terminal amino acid of β-amyloid is substituted by glutamic acid, proline, alanine, or lysine, or the lysine residue at position 16 is substituted by tryptophan.
Specific examples of fusion proteins of the invention include those having an amino acid sequence of any one of SEQ ID Nos. 12 to 16.
Further, in order to make the β-secretase attack easier, a generally-known APP amino acid mutation or mutations in hereditary Alzheimer's disease, such as a Swedish familiar Alzheimer's disease APP mutations, may be introduced into the modified protein of the invention.
Specific examples of fusion proteins include those having an amino acid sequence of any one of SEQ ID Nos. 17 to 21.
In the fusion protein, the N terminal region of the APP modified protein may be extended, or a spacer may be suitably inserted between a secretory protein and an APP modified protein, as required, so that the β-secretase cleavage site in the APP does not have a steric hindrance that affects the attack of the β-secretase.
The fusion protein can be produced from a gene which encodes the above-described APP modified protein of the invention and a gene which encodes secretable protein according to the genetic recombination procedure in the same manner as in the above-described production process for the APP modified protein of the invention.
Test Cell
Hereinafter, the test cell of the invention which stably expresses the above described APP modified protein or fusion protein of the invention is described.
The test cell used in the invention denotes a cell used for a test to measure the effect, toxicity, etc., of a compound and to evaluate the measurement results.
The test cell can be produced using the same genetic recombination procedure and materials (expression vector) as previously described.
In the cell used as the test cell, it is preferable that the gene expression mechanism, protein expression mechanism, and secretion mechanism are similar to human components or human mechanisms. As the cell expressing a fusion protein, it is preferable to use, for example, a cell with a mechanism such that secretory proteins are released outside the cell after cleavage at the APP site. The cell which has a rapid growth rate and maintains the stability even after subculturing is preferable. These include, for example, human-derived cells or mammalian-derived cells such as CHO-K1 cell (J. Exp. Med., 108: 945 (1958), a Hela cell (Cancer Res., 12: 264 (1952)), a NIH3T3 cell (J. Viol., 108: (1958)), a Jurkat cell (J. Immunol., 133: 123 (1984)), a 293 cell (J. Gen. Viol., 36: 59 (1977)), etc.
The following kind of procedure may be applied to obtain a cell strain with stable expression as a test cell. For example, usable is a method comprising integrating a drug resistance gene, such as aminoglycoside phosphotransferase (Th5neo), hygromycin B phosphotransferase (hygr), xanthin guanine phosphoribosyl transferase (Eco-gpt), dihydrofolate reductase (dhfr), etc., into an expression vector, introducing the target gene-containing expression vector into a cell, and cultivating it in a medium containing the selective drug, such as G418, hygromycin, a HAT, and a methotrexate. For example, pSV2neo, pRSVneo, pHyg, pSV2gpt, or pSV2dhfr is used as an expression vector.
In order to screen a highly expressive cell strain, secreted proteins in the culture supernatant or fusion proteins may be measured. For example, the enzyme activity in the culture supernatant may be measured. Detection using the antibody recognizing the C terminal region of APP is performed according to Western blotting or ELISA. Measurement by the Northern blotting method or RT-PCR method is performed at the gene expression level.
Measurement Method
The measurement method of the invention comprises processing the above-described test cell with a subject substance, and measuring detectable secretory proteins which are cleaved at the β-secretase cleavage site and are secreted outside the cell.
The following processes can be adopted. The test cells are initially suspended in a culture medium, and are then seeded on a suitable culture plate. The seeded cells are incubated, as required, for a given period of time until they are stabilized. Any plate can be used by considering the number of seeded cells and the measurement sensitivity of the secretory proteins. For example, a 96-well plate is suitable.
The subject substance is then added to an appropriate solvent and dissolved, and the solution is added to a plate as a fixed concentration. In this case, amphiphilic solvents, such as DMSO, may be used when the subject substance is a fat-soluble compound or an insoluble compound. Water-soluble solvents, such as physiological saline, can be used when the subject substance is a hydrophilic compound. The concentration of the solvent used is preferably adjusted to a degree such that the cells are not adversely affected. After the subject substance is added, incubation is conducted for a given-period-of-time. The incubation period can be determined without limitation according to a test system to the extent that the subject substance is incorporated into a cell and sufficiently reacted with a target molecule.
After the incubation is complete, a given quantity of the culture supernatant is taken, and the quantity of the secretory proteins in the supernatant is measured according to the properties. When the secretory protein is, for example, an enzyme, a substrate is added to measure the enzyme activity. When the secretory proteins have characteristic physical properties, such as a specific absorption spectrum, an absorbance meter is used for measurement. The target proteins may be quantified by monitoring them through a separation operation using HPLC, etc., as required. When a secretory protein is a physiologically active protein, such as cytokine, the bioactivity resulting from the proteins in the culture supernatant may be measured. The measurement and quantification may be made using the commercially-available ELISA kit, etc.
The existence form of a fusion protein expressing in a cell may be monitored. More specifically, it can be directly confirmed whether or not a fusion protein is cleaved at the β-secretase cleavage site by measuring the amount of 99-amino acid C-terminal fragment (C99) which is produced by the β-secretase cleavage. For example, after lysis of cell, C99 can be detected and quantified using an antibody which recognizes C99 according to Western blotting or ELISA. The above-described measurement method of C99 is applicable not only to the expression cell of a fusion protein but to the expression cell of an APP modified protein. Therefore, the cell can be used for screening a compound affecting β-secretase activity, a compound affecting β-amyloid production, or a substance usable as the active ingredient for a therapeutic or prophylactic agent of Alzheimer's-type dementia.
The effect of the subject substance on the β-secretase activity or the β-amyloid production can be determined based on a comparison between the measurement value obtained when the subject substance is added according to the above-described measurement method using the modified protein of the invention and the base value obtained when the subject substance is not added (solvent only). More specifically, when the measurement value is lower than the control value, the β-amyloid production is judged to be suppressed. In contrast thereto, when the measurement value is higher than the control value, the β-amyloid production is judged to be promoted.
Whether or not the subject substance affects the β-amyloid production is judged by performing statistical analysis, i.e., a significant difference test.
Whether or not the subject substance affects the β-secretase activity can be examined by the above-described procedure. Further, it impossible to screen the substance as a candidate for the active ingredient for a therapeutic or prophylactic agent of Alzheimer's-type dementia.
The measurement method of the invention may further comprise a step of examining whether or not the subject substance shows toxicity to a cell.
Whether or not the subject substance has toxicity to a cell can be judged by culturing the test cell with the subject substance, measuring the detectable secretory protein, and then measuring the survival degree of the cultured test cells. The number of survival cells can be determined by measuring the number of cells dyed by, for example, calcein AM. The number of dead cells can be determined by measuring the number of cells dyed by, for example, propidium iodide. Alternatively, the number of dead cells can be measured by counting the number of cells dyed by trypan blue. Alternatively, the number of survival cells may be determined by colorimetry measuring the formazan dye of the tetrazolium salt (MTT, MTS, WST-1, WST-8, etc.) produced by the survival cells. The number of dead cells may be determined by measuring the lactate dehydrogenase (LDH) which has flowed into the culture supernatant due to cell death, such as apotosis. Of course, these determination methods are not limited to the above.
Hereinafter, the present invention is described with reference to Examples, however it is not limited thereto.
BE11-hAPP695 was used as a starting material which was produced by adding a BE11 epitope tag immediately after a signal peptide of the N-terminus of a human-type APP gene (
In Table 1, “↓” indicates a reduced amount of secretase cleavage product, “→” indicates an unchanged amount of each secretase cleavage product, “↑” indicates an increased amount of secretase cleavage product, and “0” indicates no detection of each secretase cleavage product.
In
As can be seen from the result shown in
The β-secretase cleavage was not affected by these mutations.
When position 17 was replaced with lysine, the amount of α-secretase cleavage product decreased and the amount of β-secretase cleavage product slightly increased.
It was confirmed based on the above results that the protein introducing these mutations of the invention was effectively used for efficiently screening substances which affect β-Amyloid production.
(1) Construction of a Fusion Protein Gene
A fusion protein was constructed using an APP modified protein which was mutated by replacing position 17 of β-amyloid with glutamic acid.
cDNA (pSEAP2-Basic (Clontech, Cat. No. 6049-1)), which encodes secretory alkaline phosphatase (SEAP), was treated with EcoRI and XbaI to produce an EcoRI-XbaI fragment. However, the region corresponding to the last ten amino acids of the SEAP coding region was excluded.
Subsequently, EcoRI-XbaI fragment was isolated by PCR using a human APP modified protein cDNA fragment, i.e., a C terminal region (581 to 695 amino acids from the N-terminus of APP). The following primers were used:
A gene encoding the APP modified protein of the invention which was mutated by replacing position 17 of β-amyloid with glutamic acid was used as a template DNA. As a reaction solution, 25 μl of a solution of the following composition was used (20 mM Tris-HCl with pH 8.0 containing 10 mM KCl, 10 mM(NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 400 μM NTP, 0.5 μM Primer (each sense and antisense), 10 ng template DNA, 0.01% BSA, and 0.25 unit Ventr'q DNA polymerase (NEB, Cat. No. 254L)). Amplification was conducted under the reaction condition at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 2 minutes, and 25 cycles were repeated. The amplified PCR fragments were cloned using pT7 blue-3 (Novagen Inc., Cat. No. 70029-3). E. coli DH5a (Toyobo, Cat. No. DNA-901) was used for the transformation. The above-described process produces pT7 blue-3/cAPPE.
Subsequently, the following processes were conducted to insert Swedish type mutations. More specifically, a DNA adapter containing BglII and an EcoRI site was initially produced. The following sequences were used as oligonucleotide sequences:
The DNA adapter was inserted into pT7 blue-3/cAPPE, which was previously digested by BglII and EcoRI, and subcloning was performed according to the routine procedure, producing pT7 blue-3/APPE.
Subsequently, pT7 blue-3/APPE was processed by XbaI and NotI according to the routine procedure, and the XbaI-NotI fragment obtained was used to produce a fusion protein.
With respect to SEAP, pSEAP2-Basic was processed by EcoRI and XbaI, producing an SEAP EcoRI-XbaI DNA fragment.
The two types of DNA fragments, i.e., the pT7 blue-3/APPE XbaI-NotI DNA fragment and the SEAP EcoRI-XbaI DNA fragment were ligated into expression vector pEF1/V5-His which was previously processed by EcoRI and NotI, and the construction of a fusion protein expression gene was completed.
(2) Formation of a Test Cell Line for a Fusion Protein Stable Expression
The expression vector was transfected using a transfection reagent of Polyfect™ (Qiagen, Cat. No. 301105) to Chinese hamster ovary cells (CHO-K1, ATCC CCL61), which was cultivated on a 60 mm-culture dish. The culture was carried out using a culture medium which was obtained by adding cow fetus blood serum (FBS) (JRH Bioscience, Cat. No. 12103-789, Lot. No. 9K2087) to a DMEM/F12 culture medium (Sigma Chemical Co., Cat. No. D8062) to bring the final concentration to 10%. Forty-eight hours after culture initiation, the processed cells were suspended in the above-described culture medium containing Geneticin (500 μg/ml), and thereafter the resultant cells were seeded to a 96-well microplate at ratios of 1, 10, and 100 cells/well. Two weeks later, the wells containing a positive clone were screened. The alkaline phosphatase activity of the culture supernatant was determined using the alkaline phosphatase substrate (Blue Phos™, KPL, Cat. No. 50-88-00). Clones showing stable expression were screened based on the determined alkaline phosphatase activity.
It was confirmed in the obtained clone (CHO/sw) that cells proliferated with time (
The fusion protein expression cell (CHO/sw) established thus secreted alkaline phosphatase as a β-secretase cleavage product in the culture supernatant as the cell proliferation and culture period proceeded. It was clarified that the activity of the secreted alkaline phosphatase correlated with the production amount of β-amyloid secreted. It was confirmed based on the above result that a target cell line which allows detection of the β-amyloid production amount by determining the activity of secreted alkaline phosphatase was successfully produced.
Further, it was confirmed that the production amount of β-amyloid correlated with the alkaline phosphatase activity by measuring the β-amyloid in the culture supernatant by ELISA (
(3) Confirmation of a Gene Introduced into a Fusion Protein
Whether or not a gene was actually transferred was further confirmed. The gene-introduced CHO-K1 cell, which was cultured to be confluent state on a 100-mm dish was suspended in 500 μl of a digestion buffer (10 mM Tris-HCl with pH8.0 containing 100 mM NaCl, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml protein-kinase K (proteinase K)) and was incubated at 50° C. overnight. The cell lysate was then extracted with phenol chloroform (1:1), isopropanol was added in equal proportions to an aqueous layer, and DNA was collected by centrifugation at 1,700×g for 2 minutes. Subsequently, DNA was suspended in 0.5 mg/ml of TE buffer solution (Tris-HCl with pH8.0 containing 1 mM EDTA), and was completely dissolved at 60° C. PCR was conducted using the prepared DNA (2.5 μg) as a template using Takara Ex Taq (Takara, Cat. No. RR001A) and 0.5 μM of each primer described later. The PCR was conducted at 94° C. for 30 seconds, 60° C. for 1 minute, and 72° C. for 2 minutes, and 30 cycles were repeated. Primers for the fusion protein are as follows:
Primers for β-Actin are as follows:
The β-Actin gene was used as a control gene.
The results are shown in
As can be seen from the result shown in
Next, it was confirmed by Western blotting whether or not the obtained clone expressed the target fusion protein.
A CHO-K1 cell cultured to be a confluent state on a 100-mm dish was suspended in 1 ml of a cytolysis buffer (50 mM Tris-HCl with pH8.0 containing 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% Chaps, 10 mg/ml aprotinin, 10 mg/ml leupeptine), and the suspension was intermittently vortexed for 30 minutes on ice. Cytolysis was centrifuged at 4° C. for 10 minutes at 14,000 rpm, and the supernatant was used as a sample for Western blotting. Subsequently, a cell extract and 16 μl of a culture supernatant were mixed into a sample preparation buffer (0.624 M Tris-HCl with pH6.8 containing 10 mM EDTA, 10% SDS, 25% glycerol, 0.1% BPB, and 1% 2-mercaptoethanol), and the mixture was electrophoresed with 13% polyacrylamide gel electrophoresis (10×10 cm slab gel). After the electrophoresis, the protein was transferred to a nitrocellulose membrane (Hybond™-ECL, Amersham Pharmacia Biotech, Cat. No. RPN3032D) using a semidry blotter (Trans-Blot SD, Bio Rad, Cat. No. 170-3940). Subsequently, the nitrocellulose membrane was blocked at room temperature for 1 hour in a Blotto-B buffer (PBS containing 1% BSA, 1% skim milk, and 0.05% tween-20), and was treated with the APP recognition antibody solution (at room temperature for 2 hours). The nitrocellulose membrane was sufficiently washed with a washing solution (PBS containing 0.05% Tween20), and treated with the secondary antibody (Peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology Inc. Cat. No. SC-2030) and Peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology Inc. Cat. No. SC-2005)) at room temperature for 1 hour. A chemiluminescence detection kit (Super Signal'q, Pierce Co., Cat. No. 34080) was used for the detection.
The anti-human APP antibody (C terminal portion recognition antibody, Sigma Chemical Co., Cat. No. A-8717), the anti-sAPPα antibody (Otsuka Pharmaceutical Co., Ltd.), and the anti-sAPPβ antibody (Immuno-Biological laboratories Co., Ltd.) were used as primary antibodies.
It was confirmed by the result of Western blotting for cell lysate (
The result of Western blotting for the culture supernatant shows that no α-cleavage product was detected in any culture supernatant (
In contrast, a β-secretase cleavage product was detected only in the fusion protein expression cell line (S) (
It was confirmed by the above result that the fusion protein expression cell line expressed the desired fusion protein at a protein level, and the fusion protein was subjected to preferential β-cleavage to be secreted outside the cell, as intended.
The C terminal region produced by the cleavage of APP was also confirmed. The Western-blotting sample (supernatant of a cell lysate) prepared as described above was concentrated using acetone. Specifically, to 167 μl of the Western-blotting sample was added cold acetone in the amount of 8 times the sample, the mixture was stirred, allowed to stand overnight at −20° C., centrifuged at 4° C. at 14,000 rpm for 10 minutes, and an anti-human APP C terminus antibody (Sigma Chemical Co., Cat. N.A-8717) was detected as a primary antibody.
The fusion protein expression cell line (S) was subjected to preferential β-cleavage, and it was observed, as shown in
As described above, it was confirmed that the test cell of the invention contained a β-secretase cleavage site; expressed a fusion protein containing a β-amyloid precursor protein (APP) modified protein which was subjected to an amino acid mutation without affecting the β-secretase cleavage and without causing α-secretase cleavage at the α-secretase cleavage site; and was subjected to the target processing (β-secretase cleavage) to secrete a detectable secretory protein. Further, it was clarified that the APP modified protein into which Swedish type mutations were introduced exhibited a high substrate specificity of β-secretase, and produced 5 to 10 times as much β-amyloid.
(1) Determination of Effects on β-Amyloid Production
The cell obtained in Example 2, i.e., a stable cell line with a gene of fusion protein (SEAP-APPsw), was seeded to a 96-well microplate at 5,000 cells/well, a subject substance previously dissolved in DMSO was added thereto so that the final concentration became 5 μl/ml, and incubated under 5% CO2 overnight (18 to 24 hours). Thereafter, 50 μl of the culture supernatant was transferred to a microplate for measuring the alkaline phosphatase activity, 50 μl of an alkaline phosphatase substrate (Blue Phos™, KPL, Cat. No. 50-88-00) was added and reacted for 1 hour, a reaction stop solution (Blue Phos™, KPL, Cat. No. 50-89-01) 100 μl was added, and 650-nm absorption was measured using a microplate reader (Labsystems Multiskan'q Multisoft) (
A subject substance which shows a significant difference in absorption between when it was added and when it was not added was regarded as a candidate substance for affecting β-amyloid production.
(2) Determination of Toxicity of a Subject Substance to Cells
50 μl (1 mg/ml culture medium) of an MTT solution was added to each well of the microplate onto which the above-described test cells were seeded, and the reaction was conducted in a 5% CO2 incubator for 4 hours. After the reaction, the supernatant was discarded, 100 μl of DMSO was added to each well and stirred, and 575-nm absorption was measured using a microplate reader (Labsystems Multiskan'q Multisoft) (
The measurement results of Example 3 are shown in
The values in
Based on the results, the decrease in alkaline phosphatase activity was observed in 6D, 10G, and 11B. Cytotoxicity was observed in 5G, 8C, 8D, 10G, and 11B. Considering these results overall, the 6D compound was screened as a compound which affects β-amyloid production, and has little cytotoxicity.
The present invention provides a substance which can efficiently monitor β-amyloid production. Specifically, the present invention provides a β-amyloid precursor protein (APP) modified protein, a fusion protein containing the modified protein, a test cell expressing those proteins, etc. The use of such material allows the efficient screening of substances affecting β-amyloid production. Substances obtained by the screening serve as active ingredients for therapeutic or prophylactic agents of Alzheimer's disease.
As described above, the present invention particularly provides an excellent means for use in techniques for the prevention or treatment of Alzheimer's disease.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2002-159472 | May 2002 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP03/06319 | 5/21/2003 | WO | 11/29/2004 |
