A novel carboxypeptidase and the encoding gene thereof were successfully identified by screening a human hippocampus extract using brain-APP-cleaving activity as an index. Antibodies against the protein were also prepared. The antibodies are useful in, for example purifying the protein, or examining and diagnosing diseases, such as Alzheimer's disease, which cause accumulation of A.beta. in the brain.

Description of the Invention

An objective of the present invention is to provide a novel carboxypeptidase expressed in brain and comprising a peptidase activity for brain APP, and the gene, a method of production, and uses thereof.

The present inventors previously analyzed the processing of APP and A.beta.-containing peptides in lymphoblastoid cells derived from patients with familial AD (Matsumoto, A. and Fujiwara, Y., 1993, Eur. J. Biochem. 217: 21-27; Matsumoto, A. and Matsumoto, R., 1994, Eur. J. Biochem. 225: 1055-1062). In the culture media of familial AD cells, the inventors found a 68 kDa serine protease that shows .beta.-secretase-like activity for synthetic substrates (Matsumoto, A. and Fujiwara, Y., 1994, Biochemistry 33: 3941-3948). This protease also cleaves natural APP prepared from lymphoblastoid cells (LAPP) and its A.beta.-containing fragment at a site(s) in the vicinity of the A.beta.-N-terminus (Matsumoto, A. et al., 1995, Neurosci. Lett. 195:171-174). This serine protease, however, does not degrade natural brain APP as revealed by measuring this protease's activity using natural brain APP prepared from normal human brain as the substrate. Therefore, the inventors thought that natural APP as a substrate has tissue-specificity, and that it should be possible to identify a novel protease that uses natural brain APP as the substrate by screening proteases expressed in brains using natural brain APP-cleaving activity as an index.

The present inventors searched for protease activity towards natural brain APP in the homogenate fractions of the human hippocampus, and by analyzing the active fractions, succeeded in separating a 40-kDa protein that has the activity to produce A.beta.-containing peptide by cleaving brain APP at multiple sites. Analysis of this protein revealed that it was a novel protease belonging to the carboxypeptidase (CP) family. This protease (brain carboxypeptidase B; brain CPB) is not an alternatively spliced isoform of plasma CPB, rather it has such specific features as 14 C-terminal amino acids. Northern analysis using a human brain CPB-cDNA probe and Western analysis using a human brain CPB-specific antibody detected a brain-specific expression of this protease. An immunohistochemical study showed that the protease is expressed in neuronal perikarya, particularly in that of the pyramidal neurons of the hippocampus, ependymal-choroid plexus cells, and in a portion of the microglia of normal brains. This protease existed in spinal fluid and blood a little. In brains of patients with sporadic AD, decreased neuronal expression and a cluster of microglia with protease immunoreactivity associated with its extracellular deposition being detected. Furthermore, Western analysis using anti-A.beta. N terminus antibody proved that human brain CPB has exopeptidase activity in which the A.beta. peptide is digested from its C-terminal end.

These findings suggest that brain CPB isolated by the inventors has a physiological function in APP processing and may have significance in AD pathophysiology. Therefore, brain CPB itself, isolated by the present inventors, could be used as a drug for preventing and treating Alzheimer's disease, and for screening drugs for prevention and treatment. Furthermore, brain CPB isolated by the inventors may be used in tests for Alzheimer's disease.

In addition, brain CPB isolated by the present inventors, and compounds that may regulate its activity, may be applied not only for the Alzheimer's disease mentioned above, but also for the prevention, treatment, and diagnosis of various other diseases causing accumulation of A.beta..

Therefore, the present invention provides: (1) a protein comprising the amino acid sequence of any one of SEQ ID NOs: 2 to 4, (2) a protein comprising peptidase activity towards brain APP, wherein said protein is selected from, (a) a protein comprising an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and/or added to the amino acid sequence of any one of SEQ ID NOs: 2 to 4, (b) a protein encoded by a DNA that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1, (3) a DNA encoding the protein of (1) or (2), (4) the DNA of (3) comprising the coding region of the nucleotide sequence of SEQ ID NO: 1, (5) a vector into which the DNA of (3) or (4) is inserted, (6) a host cell carrying the vector of (5), (7) a method for producing the protein of (1) or (2), wherein said method comprises the steps of culturing the host cell of (6), and collecting from the cell or its culture supernatant, a recombinant protein expressed within the cell, (8) an antibody against the protein of (1) or (2), (9) a partial peptide of the protein of (1) or (2), (10) a polynucleotide comprising at least 15 nucleotides, which hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or its complementary strand, (11) a method for screening a compound that binds to the protein of (1) or (2), comprising the steps of: (a) contacting a test sample with the protein or a partial peptide thereof, (b) detecting the binding activity between the test sample and the protein or the partial peptide thereof, and (c) selecting a compound that has an activity to bind to the protein or the partial peptide thereof, (12) a compound that binds to the protein of (1) or (2), (13) the compound of (12), wherein said compound is isolated by the method of (11), (14) a method for screening a compound that promotes or inhibits the peptidase activity of the protein of (1) or (2), comprising the steps of: (a) contacting the protein of (1) or (2) with its substrate in the presence of a test sample, (b) detecting the cleavage of the substrate, and (c) selecting a compound comprising the activity to increase or decrease substrate cleavage caused by the protein of (1) or (2), in comparison to the cleavage in the absence of the test sample (control) (15) the method of (14), wherein said substrate is brain APP, (16) a compound comprising the activity to promote or inhibit peptidase activity of the protein of (1) or (2), (17) the compound of (16), wherein said compound is isolated by the method of (14) or (15), (18) an A.beta. production regulator, comprising the protein of (1) or (2) as an active ingredient, (19) a drug for treating a disease that causes accumulation of A.beta. in the brain, wherein said drug comprises the protein of (1) or (2) as an active ingredient, (20) the drug of (19), wherein said disease that causes accumulation of A.beta. in the brain is selected from the group consisting of senile dementia, Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhage, and cephalic contusion, (21) an A.beta. production regulator, comprising a compound of any one of (12), (13), (16), or (17) as an active ingredient, (22) a drug for treating a disease that causes accumulation of A.beta. in the brain, comprising a compound of any one of (12), (13), (16), or (17) as an active ingredient, (23) the drug of (22), wherein said disease that causes accumulation of A.beta. in the brain is selected from the group consisting of senile dementia, Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhage, and cephalic contusion, (24) a kit for screening a compound that promotes or inhibits peptidase activity of the protein of (1) or (2), wherein said kit comprises the protein of (1) or (2), (25) the kit of (24), further comprising a substrate of the protein of (1) or (2), (26) the method of (25), wherein said substrate is brain APP, (27) a method for testing a disease that causes accumulation of A.beta. in the brain, comprising the steps of: (a) preparing a sample from a subject, and (b) detecting the amount of the protein of (1) or (2) contained within the sample, using the antibody of (8), (28) the method of (27), wherein said sample is spinal fluid or serum, (29) the method of (27) or (28), wherein said disease that causes accumulation of A.beta. in the brain is selected from the group consisting of senile dementia, Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhage, and cephalic contusion, (30) a reagent for testing a disease that causes accumulation of A.beta. in the brain, comprising the antibody of (8), and (31) the reagent of (30), wherein said disease that causes accumulation of A.beta. in the brain is selected from the group consisting of senile dementia, Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhage, and cephalic contusion.

The present invention relates to a novel brain carboxypeptidase B (brain CPB) protein expressed in the brain, which has peptidase activity towards brain APP. Human brain CPB protein included in the protein of this invention was isolated from an extract of human hippocampus using decomposition activity towards brain APP as an index. In addition, the gene encoding human brain CPB protein was cloned by immunological screening using antibodies against human brain CPB protein and by RT-PCR and RACE using a primer that was synthesized based on the partial amino acid sequence of human brain CPB protein. SEQ ID NO: 1 shows the nucleotide sequence of a cDNA encoding the human brain CPB isolated by the present inventors, and SEQ ID NO: 2 shows the amino acid sequence of human brain CPB protein (prepro-form) encoded by this cDNA. The human brain CPB of this invention is thought to be initially synthesized as a preproprotein, then a signal sequence is removed by cleavage to produce the proprotein, furthermore the activation peptide is removed by cleavage to produce the mature protein. SEQ ID NO: 3 shows the amino acid sequence of the pro-protein of human brain CPB of this invention, and SEQ ID NO: 4 shows the amino acid sequence of the mature protein. In the present invention, "brain APP" refers to APP molecular species expressed in the brain. Examples of molecular species of human brain APP are APP695 (Kang, J. et al. (1987) Nature 325, 733-736) and Appican (Shioi, J. et al. (1992) J. Biol. Chem. 267, 13819-13822), but are not limited to these examples.

Human brain CPB has the activity to produce A.beta.-containing peptides by cleaving APP, prepared from the human brain, at multiple sites. It is thought that the human brain. CPB is synthesized as a 40-kDa preproprotein, and therefrom, a 30-kDa mature protein is produced. From the characteristics of the amino acid sequence, this protein was determined to be a novel protease belonging to the CPB family (brain carboxypeptidase B; brain CPB). This protease has high homology to a known human plasma CPB (HPCPB) belonging to the CPB family, but since it has specific characteristics, such as a unique set of 14 amino acids at its C terminus, this was presumed to be an independent gene, and not an isoform produced by alternative splicing of plasma CPB.

Among human organs examined by Northern analysis, human brain CPB (HBCPB) was found only in the brain. Antibody against C-terminal 14 amino acids of HBCPB (anti C14 antibody) showed signals in the neuronal perikaryon and a portion of the microglia (FIG. 7, see Original Patent), but not in the liver in which HPCPB is synthesized. Furthermore, in the RT-PCR analysis of brain mRNAs, only a 1077 bp band that is unique to a processed form of prepro-HBCPB was detected (FIG. 4C, see Original Patent). Moreover, immunological screening done with anti prepro-HBCPB antibody gave three cDNA clones that had almost identical restriction enzyme patterns, indicating that HBCPB is the only CPB isoform expressed in human brain.

Immunohistochemical analysis for normal brains indicates that HBCPB is expressed in various neuronal perikaria, particularly in that of the pyramidal neurons of the hippocampus, ependymal cells, choroid plexus cells, and in a portion of the microglia, but not in astrocytes. In AD brains, HBCPB seems to be expressed in a portion of the clustered microglia as parenchymal deposits with or without glial infiltration. Furthermore, in senile plaques from five AD brains, there was partial colocalization of the protease immunoreactivity, ranging from 10% to 60% of all the plaques in each brain. Moreover, in parenchyma of the hippocampus, round homogeneous deposits with HBCPB- and C14-immunoreactivities were common to all five AD brains, which differ markedly from amyloid bodies (FIG. 9G, see Original Patent). Enhanced infiltration of microglia is a common finding in aged and AD brains. The significance of the microglia in relation to senile plaque formation is not clear. Wisniewski et al. (Wisniewski, H. M. et al., 1989, Can. J. Neurol. Sci. 16: 535-542) reported A.beta. fibrils in the endoplasmic reticulum of activated microglia in the immediate vicinity of A.beta. deposits, suggesting that activated microglia may synthesize as well as process A.beta.-peptides. APP isoforms in the microglia, which are mesodermal in origin, have LAPP, and APP with the Kunitz protease inhibitor domain, neither of which are expressed neurons. Another hypothesis is that the microglia are not involved in initial plaque formation, rather they process A.beta. peptides through phagocytosis (Frackowiak, J. et al., 1992, Acta Neuropathol. 84: 225-233). Choroid plexus cells secrete cerebrospinal fluid (CSF) and ependymal cells are speculated to have secretion, absorption and transport functions and to provide a barrier between the brain and CSF (Del Bigio, M. R., 1995, Glia 14: 1-13). The expression of anti C14 immunoreactivity in these cells suggests that HBCPB is necessary for processing the peptides synthesized in brain cells, including the appropriate isoform of APP. Under physiological conditions, target substrates in the neurons appear to have at least two chances to come across the protease, in the perikaria and in ependymal cells before it is released into the CSF. In AD, the presence of HBCPB in the extracellular space associated with enhanced activation of the microglia may represent a pathophysiological condition.

In addition, experiments confirmed that A.beta. peptide is a substrate of the human brain CPB.

From the above-mentioned findings, the brain CPB protein of this invention is thought to be deeply involved with the onset of Alzheimer's disease and with the production of A.beta. formed by decomposition of brain APP.

That is, the loss of balance the functions of proteases including the brain CPB protein of this invention, appears to be causing the abnormal accumulation of A.beta..

The human brain CPB of this invention may have the function of reducing nerve cell death by decreasing and metabolizing the highly self-aggregating .beta.-amyloid peptides (especially .beta.-amyloid 1-42) accumulating in the brain.

Besides, its use as a subject for research to elucidate the molecular mechanism of A.beta. production, the brain CPB protein of this invention itself can be used as a drug. For example, it could be possible to improve A.beta. metabolism by continuously infusing the protein of this invention into the cerebrospinal fluid of an AD patient to elevate the concentration of human brain CPB in the cerebrospinal fluid, and thus promoting the uptake of the protein into nerve cells via ependymal cells to supplement the decreased or depleted human brain CPB.

In addition, the brain CPB of this invention can be used as a tool for screening promoters or inhibitors of the protein of this invention useful as a drug. Furthermore, the protein of this invention or a compound that regulates the activity of the protein of this invention is useful as a drug for preventing or treating various diseases that involve pathological conditions of accumulation of A.beta. peptide in the brain, and as an agent for testing and diagnosing such diseases. Examples of such diseases are senile dementia, Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhage (Holland type, etc.), and cephalic contusion (boxer's brain), and such.

As long as there is a peptidase activity towards brain APP, the present invention includes proteins that are structurally similar to the natural human brain CPB protein (SEQ ID NOs: 2, 3, and 4). Such structurally similar proteins include mutants of natural human brain CPB protein, and brain CPB proteins derived from other organisms.

One skilled in the art would readily prepare these proteins using, for example, standard mutagenesis methods. Known methods for altering amino acids in proteins include Kunkel's method (Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488; Kunkel, T. A. et al. (1987) Methods in Enzymology 154, 367), Gapped duplex method (Kramer, W. et al. (1984) Nucl. Acids. Res. 12, 9441; Kramer, W. et al. (1987) Methods in Enzymology 154, 350), Oligonucleotide-directed Dual Amber (ODA) method (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275; Zoller, M. J. and Smith, M. (1983) Methods in Enzymology 100, 468), etc. In artificial alteration of amino acids in proteins, the number of amino acid residues to be altered is usually 30 or less, preferably 10 or less, and more preferably 5 or less. Alteration of amino acids in proteins could occur spontaneously. Such proteins having amino acid sequences different from that of the natural human brain CPB protein (SEQ ID NOs: 2, 3, and 4) due to artificial or spontaneous substitution, deletion, addition and/or insertion of amino acid residues, are also included in this invention as long as they have a peptidase activity to brain APP.

An amino acid having properties similar to those of the amino acid to be substituted is preferably used for substitution. Since Ala, Val, Leu, Ile, Pro, Met, Phe and Trp are, for example, all classified into the non-polar amino acid, they are considered to have similar properties. Non-charged amino acids include Gly, Ser, Thr, Cys, Tyr, Asn, and Gln. Acidic amino acids include Asp and Glu, while basic amino acids include Lys, Arg and His.

Proteins structurally similar to the human brain CPB protein having a peptidase activity against brain APP can be prepared using the known hybridization technique (2.9 Southern Blotting and Hybridization 2.9.1-2.9.10 (Selden, R. F.), 4.9 Analysis of RNA by Northern Hybridization 4.9.1-4.9.8 (Selden, R. F.), 6.3 Hybridization with Radioactive Probes Using DNA Fragments 6.3.1-6.3.6 (Straus, W. M.), 6.4 Hybridization with Radioactive Probes Using Oligonucleotides 6.4.1-6.4.5 (Duby, A.), in `Current Protocols in Molecular Biology` (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. eds.) John Wiley & Sons, New York (1987)) and polymerase chain reaction (PCR) technique (Optimization of PCRs 3-12 (Innis, M. A. and Gelfand, D. H.), Amplification of Genomic DNA 13-20 (Saiki, R. K.), Amplification of RNA 21-27 (Kawasaki, E. S.), RACE: Rapid Amplification of cDNA Ends 28-38 (Frohman, M. A.), Degenerate Primers for DNA Amplification 39-45 (Compton, T), cDNA Cloning Using Degenerate Primers 46-53 (Lee, C. C. and Caskey, T.), in `PCR Protocols` (Innis, M. A., Gelfand, D. H., Sninsky, J. J., White, T. J. eds.) Academic Press, San Diego (1990)). It is routine for one skilled in the art to isolate DNA highly homologous to human brain CPB cDNA from various other organisms using the human brain CPB cDNA (SEQ ID NO: 1) or portions thereof as a probe and oligonucleotides specifically hybridizing with the human brain CPB cDNA (SEQ ID NO: 1) as a primer to obtain proteins structurally similar to the human brain CPB protein from the isolated DNA.

Proteins encoded by DNAs hybridizing with the human brain CPB cDNA are included in this invention as long as they have a peptidase activity against brain APP. Other organisms used for isolating such proteins include, for example, monkeys, mice, rats, rabbits, goats, cattle, sheep, pigs, dogs, and so on, but are not limited thereto. cDNAs encoding such proteins can be isolated from such sources as brains of these organisms, for example, hippocampus.

DNAs encoding the brain CPB protein derived from organisms other than human are usually highly homologous to human brain CPB. Being highly homologous means at least 40% or more, preferably 55% or more, and further preferably 70% or more (e.g. 85% or more, 90% or more, 95% or more) sequence identity at the amino acid level. Sequence homology can be determined by the homology search program of Data Bank of Japan: DDBJ (National Institute of Genetics), such as FASTA, BLAST, etc.

One skilled in the art can readily determine conditions for hybridization to isolate DNAs encoding proteins functionally equivalent to the human brain CPB protein. One example of hybridization conditions when the DNAs are isolated from a brain cDNA library using .sup.32P-labeled probe is as follows. After hybridization, washing is done under the low stringent conditions of 55.degree. C., 2.times.SSC, and 0.1% SDS, more preferably, moderately stringent conditions of 55.degree. C., 0.2.times.SSC, and 0.1% SDS, and still more preferably, highly stringent conditions of 68.degree. C., 0.2.times.SSC, and 0.1% SDS. In this case, although plural factors including the temperature, salt concentration, and such are thought to influence the stringency of hybridization, one skilled in the art would readily determine the stringent conditions similar to above by suitably selecting these factors. When the cDNAs are isolated from other tissues, washing is preferably done under high stringent conditions since similar CPBs may be expressed therein.

The protein of this invention can be prepared as either a natural protein or a recombinant protein utilizing gene recombination techniques. Natural proteins can be prepared by, for example, subjecting extracts from tissues that are supposed to contain the brain CPB protein (for example, hippocampus) to affinity chromatography using the antibody to the brain CPB protein as described below. In addition, as indicated in the Examples, a natural protein can be prepared by collecting the fraction that bound to a benzamidine-Sepharose column, and by appropriately combining preparative PAGE, etc. On the other hand, recombinant proteins may be prepared by culturing cells transformed with DNA encoding the brain CPB protein, allowing the transformants to express the protein, and recovering the protein as described below.

The invention also includes partial peptides of the proteins of this invention. An example of partial peptides of the proteins of this invention may be a peptide containing the boundary portion of the activation peptide and the mature protein, among the proteins of this invention. Such a peptide is thought to act antagonistically in the production of the mature form of the protein of this invention by acting on the protease mediating the reaction that cleaves the mature protein from the proprotein In addition, an example of the partial peptides of the proteins of this invention may be a peptide that corresponds to the substrate-binding site of the protein of this invention. This type of partial peptide may be utilized as inhibitors, and such, of the protein of this invention by biological administration. These partial peptides are useful as an inhibitor or activator of signal transduction that is mediated by the protein of this invention. Additionally, examples of the partial peptides of this invention may be a partial peptide of the C-terminal region of the protein of this invention (for example, SEQ ID NOs: 2, 3, and 4), and this peptide may be utilized to prepare an antibody. In addition, as the partial peptides of the proteins of this invention, an activation peptide may be cited. Partial polypeptides comprising the amino acid sequence specific to the protein of this invention have at least 7, preferably at least 8, more preferably at least 9 amino acid residues. Partial peptides of this invention can be produced by, for example, genetic engineering techniques, known peptide synthetic methods, or by digestion of the protein of this invention with appropriate peptidases.

This invention also relates to DNAs encoding the proteins of the invention. DNAs encoding the protein of this invention are not particularly limited as long as they can encode the proteins of this invention, including cDNAs, genomic DNAs, and synthetic DNAs. DNAs having any desired nucleotide sequences based on the degeneracy of genetic codes are also included in this invention as long as they can encode the proteins of this invention.

cDNAs encoding the proteins of this invention can be screened, for example, by labeling cDNA of SEQ ID NO: 1 or segments thereof, RNAs complementary to them, or synthetic oligonucleotides comprising partial sequences of said cDNA with .sup.32P, etc., and hybridizing them with a cDNA library derived from tissues (e.g., hippocampus, etc.) expressing the proteins of this invention. Such cDNAs can be cloned by synthesizing oligonucleotides corresponding to nucleotide sequences of these cDNAs, and amplifying them by PCR with cDNA derived from suitable tissues (e.g. hippocampus, etc.) as a template. The genomic DNA can be screened, for example, by labeling cDNA of SEQ ID NO: 1 or segments thereof, RNAs complementary to them, or synthetic oligonucleotides comprising partial sequences of said cDNA with .sup.32P, etc., and hybridizing them with a genomic DNA library. Alternatively, the genomic DNA can be cloned by synthesizing oligonucleotides corresponding to nucleotide sequences of these cDNAs, and amplifying them by PCR with genomic DNA as a template. Synthetic DNAs can be prepared, for example, by chemically synthesizing oligonucleotides comprising partial sequences of cDNA of SEQ ID NO: 1, annealing them to form double strand, and ligating them with DNA ligase.

These DNAs are useful for the production of recombinant proteins. The proteins of this invention can be prepared as recombinant proteins by inserting DNAs encoding the proteins of this invention (e.g. DNA of SEQ ID NO: 1) into an appropriate expression vector, transforming suitable cells with the vector, culturing the transformants, and purifying expressed proteins from the transformants or culture supernatant of them.

For example, when expressing a recombinant protein using E. coli as the host, examples of systems that can be used are: the pET System (Novagen) that allows induction of expression by T7 RNA polymerase and attachment of various tags; the pET CBD Fusion System 34b-38b (Novagen) that allows expression of the protein of this invention as a fusion protein with the cellulose binding domain followed by purification by affinity chromatography using a cellulose carrier; and, the GST Gene Fusion System (Pharmacia) that allows expression of the protein of this invention as a fusion protein with glutathione S-transferase followed by purification by glutathione Sepharose 4B. In addition, there is no limitation on the host used to express the proteins of this invention, and it is possible to use a known vector system for expression in yeast, insect cells, mammalian cells, and such. For the purpose of expression in neurons, a system using Semliki Forest Virus (SFV) vector (Tienari, P. J. et al., EMBO J. 15: 5218-5229 (1996), etc.) may be used, for example.

Recombinant proteins expressed in host cells can be purified by known methods. The protein of this invention expressed in the form of a fusion protein, for example, with a histidine residue tag or glutathione-S-transferase (GST) attached at the N-terminus can be purified by a nickel column or a glutathione sepharose column, etc. In addition, when the protein of the present invention is expressed as a fusion protein, the portion comprising the protein of this invention can be recovered by inserting cleavage sites, such as Thrombin and Factor Xa, into the boundary area.

The DNA encoding the protein of this invention or its antisense DNA, and such, may also be utilized for gene therapy of diseases caused by abnormalities in the protein of this invention (abnormalities in expression and function). There is no limitation on the vector to be used for gene therapy, as long as a therapeutically effective expression can be achieved by the vector, and examples of vectors are adenovirus vectors, Semliki Forest virus vectors, and such (Chen, J. et al. (1998) Cancer Res. 58, 3504-3507; Barkats, M. et al. (1998) Progress in Neurobiology 55, 333-341).

For example, gene-therapy can be carried out after producing a vector in which prepro-human brain CPB-cDNA has been inserted by matching the frame with a Semliki Forest virus vector (pSFV-1), completing the genetically recombined virus using a helper virus, then selecting a patient appropriate for treatment by analyzing the general condition of the patient and virus antibody value, and conducting gene therapy on this patient.

The present invention also relates to polynucleotides hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence, and containing at least 15 nucleotides. Preferably, the polynucleotides specifically hybridize with the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence. "Specifically hybridize" means that a polynucleotide does not significantly cross-hybridize with DNAs encoding other proteins under usual hybridization conditions, preferably under stringent hybridization conditions as described above. Such polynucleotides include probes, primers, nucleotides or nucleotide derivatives (e.g. antisense oligonucleotides and ribozymes, etc.), which can specifically hybridize with DNAs encoding the proteins of this invention, or DNAs complementary to said DNAs.

cDNAs encoding the proteins of this invention or oligonucleotides comprising partial sequences thereof can be used for cloning genes and cDNAs encoding the proteins of this invention, or amplifying them by PCR. The cDNAs and oligonucleotides can also be utilized for detecting polymorphism or abnormality (gene diagnosis, etc.) of the gene or cDNA by the restriction fragment length polymorphism (RFLP) method, single strand DNA conformation polymorphism (SSCP) method, etc.

This invention also relates to antibodies binding to the proteins of the invention. The antibodies against the proteins of this invention can be prepared by known method (Scheidtmann, K. H., Immunological detection of known sequence, in "Protein structure", T. E. Creighton ed., IRL Press, Oxford University Press, pp. 93-115). To prepare polyclonal antibodies, for example, the naturally derived protein of this invention prepared from the hippocampus tissue or the recombinant protein in combination with an adjuvant are used to immunize animals such as rabbit, guinea pig, goat, and such. By performing the immunization several times, the antibody titer can be elevated. After the final immunization, antiserum can be obtained by taking a blood sample from the immunized animal. This antiserum, for example, can be fractionated by ammonium sulfate precipitation or anion-exchange chromatography, and purified by affinity chromatography using protein A or immobilized antigens, to prepare polyclonal antibodies. It is also possible to use the partial peptide of the protein of this invention as the antigen. On the other hand, to prepare a monoclonal antibody, for example, the protein of this invention or its partial peptide is used to immunize the immunization animal in the same manner as described above, and after the final immunization, the spleen or the lymph nodes are obtained from the immunized animal. Antibody producing cells contained within the spleen or lymph nodes are fused with myeloma cells using polyethylene glycol, and such, to prepare hybridomas. The hybridomas of interest are screened, then cultivated, and from this culture supernatant, monoclonal antibodies can be prepared. Monoclonal antibodies can be purified, for example, through fractionation by ammonium sulfate precipitation and anion-exchange chromatography, and by affinity chromatography purification using protein A or immobilized antigens.

Antibodies thus prepared are used for the affinity purification of the proteins of this invention. They can also be used for the test and diagnosis of disorders caused by abnormal expression and structural abnormality of the proteins of this invention and for detection of the expression level of the protein, etc.

As indicated in the Examples, a decrease in the expression of the protein of this invention and deposits to the interstitium thereof are observed in Alzheimer's disease. Since the proteins of this invention are detected in the spinal fluid and sera, it is possible to test and diagnose diseases that cause accumulation of A.beta. in the brain by detecting the proteins of this invention contained within these samples. The antibodies against the proteins of this invention are useful for this type of disease diagnostics.

The test for diseases that cause accumulation of A.beta. in the brain, which uses the antibodies of this invention, can be carried out by a method comprising the steps of, (a) preparing samples from the subject, and (b) detecting the amount of protein of the present invention contained within the sample using antibodies against the protein. Diseases targeted by test of this invention are not limited as long as they are diseases that cause accumulation of A.beta. in the brain, and may be, for example, senile dementia, Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhage, and cephalic contusion. As a sample to be prepared from the subject for testing, spinal fluid or serum is preferable.

The protein of this invention included in the sample can be detected, for example, by Western blotting, immunoprecipitation, ELISA, and such. The antibody used for detection may be either monoclonal antibody or polyclonal antibody. When the antibody is used as a testing reagent, pH buffer (for example, phosphate buffer, HEPES buffer, etc.) is used, and carriers (for example bovine serum albumin or gelatin, etc.), preservatives (sodium azide), and such may be mixed as necessary.

As a result of this detection, if an increase or decrease in the amount of antibody-reactive protein, abnormal molecular weight of the antibody-reactive protein, and/or abnormal molecular weight ratio of the respective proteins, are confirmed when comparing the sample of the subject to that of a healthy person, the subject is determined to have the above-mentioned disease, or is suspected of having the above-mentioned disease. Since this test is simple and places a small burden on the subject, it is an excellent method to test and diagnose diseases of the CNS.

Antibodies of this invention can be applied to the antibody treatment. For antibody treatment, they are preferably humanized or human antibodies. Such antibodies can be prepared by known methods (Parren, P. W. (1992) Hum. Antibodies Hybridomas 3, 137-145).

The present invention also relates to a method for screening a compound binding to the protein of this invention. The screening method of this invention comprises: (a) contacting a test sample with the protein of this invention or partial peptide thereof, (b) detecting the binding activity between the test sample and the protein of this invention or the partial peptide thereof, and (c) selecting a compound that has an activity to bind to the protein of this invention or the partial peptide thereof.

Test samples used for screening include, for example, cell extracts, expression products of a gene library, synthetic low molecular weight compounds, synthetic peptides, modified peptides, natural compounds, etc., but are not limited thereto. Those test samples used for screening may be labeled prior to use as the occasion demands. Labels include, for example, radioactive and fluorescent ones, etc., but are not limited to them.

Screening of proteins that bind to the protein of this invention can be carried out, for example, by applying the culture supernatant or cell extract of cells that are thought to express proteins that bind to the protein of this invention, to an affinity column that has an oligopeptide, and such, consisting of the 14 C-terminal amino acids (C14) of the protein of this invention, and by purifying the protein that specifically binds to this column. The screening of proteins that bind to the protein of this invention, or genes thereof, can be accomplished by utilizing, for example, West-western blot technique (Tarassishin, L. A. and Russell, W. C. (1997) Biochemistry (Mosc.) 62, 38-40; Matthews, D. A. and Russell, W. C. (1998) J. Gen. Viol. 79, 1671-1675) and two-hybrid system (Vidal, M. (1997) The Reverse Two-Hybrid System in "The Yeast Two-Hybrid System" (Bartel, P. and Fields, S. eds.) Oxford University Press, New York; Fields, S. and Song, O. K. (1995) Microbiology Rev. 59, 94; Vidal, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 10321). Methods to isolate compounds that bind to the protein of this invention by high-throughput screening (Cerretani, M. et al. (1999) Anal. Biochem. 266, 192-197; Kenny, B. A. et al. (1998) Prog. Drug. Res. 51, 245-269; Gonzalez, J. E. and Negulescu, P. A. (1998) Curr. Opin. Biotechnol. 9, 624-631) using combinatorial chemistry techniques (Myers, P. L. (1997) Curr. Opin. Biotechnol. 8, 701-707; Campbell, D. B. (1997) Q. J. Nucl. Med. 41, 163-169), and such, are also well known to those skilled in the art.

In addition, the present invention relates to a method to screen compounds that promote or inhibit peptidase activity of the protein of this invention. This method uses as an index, the substrate cleaving activity of the protein of this invention, and specifically comprises the steps of, (a) contacting the protein of this invention with its substrate in the presence of a test sample, (b) detecting the cleavage of the substrate, and (c) selecting a compound comprising the activity to increase or decrease substrate cleavage caused by the protein of this invention, in comparison to the cleavage in the absence of the test sample (control).

Test samples used for this screening method include, for example, cell extracts, expression products of a gene library, synthetic low molecular weight compounds, proteins, natural or synthetic peptides, natural products, sera, etc., but are not limited to them. The test samples can be compounds isolated by the above-described screening method monitoring the binding activity of the compounds to the protein of this invention. The protein of this invention to be used for screening may be a natural protein or a recombinant protein.

Brain APP is preferred as a substrate. Natural brain APP can be prepared by the method described in the Examples. In addition, as long as the protein of this invention can show a cleaving activity, partial peptides of brain APP, other APP isoforms and APP-like polypeptides, and oligopeptides synthesized to contain the cleavage site of the substrate may be used.

Cleavage of the substrate can be detected, for example, by incubating the protein of this invention with a substrate, then performing SDS-PAGE, and finally performing Western blotting using an antibody against the substrate. Alternatively, when using a synthetic oligopeptide as a substrate, detection can be carried out by Tris-tricine-type PAGE, or by thin-layer chromatography (TLC), and such, by labeling the N terminus, the C terminus, and such in advance. Otherwise, if one end of the substrate peptide is immobilized onto a support such as a microplate, and the other end is labeled, substrate fragments could be easily detected If cleavage of the substrate by the protein of this invention is promoted when a test sample is present, compared to when the test sample is absent, that compound is determined to be the compound promoting substrate-cleaving activity of the protein of this invention. Conversely, if substrate cleavage by the protein of this invention is inhibited, when the test sample is present, that compound is determined to be the compound inhibiting substrate-cleaving activity of the protein of this invention. In the present invention, "inhibiting substrate-cleaving activity" includes cases where the cleaving activity is completely inhibited and cases where the inhibition is partial.

To determine the substrate cleavage site by the protein of this invention, for example, the cleavage site is estimated by first incubating brain APP and the protein of this invention, then upon separating the cleaved brain APP by SDS-PAGE, Western blotting is carried out using domain-specific antibodies for APP (for example, for A.beta., A.beta. 1-17 antibody, A.beta. 17-24 antibody, A.beta. 1-40 C-terminal antibody, A.beta. 1-42 C terminal antibody, etc.). Next, as an advanced system, the substrate fragments cleaved by the protein of this invention are purified, and by performing N-terminal or C-terminal amino acid sequencing, the cleavage site can be determined. Alternatively, the cleavage site can be estimated using a synthetic oligopeptide (for example, A.beta. 1-42, etc.) as a substrate, and upon incubation with the protein of this invention, free amino acids that are digested from the C terminus can be measured qualitatively and quantitatively by an amino acid analyzer.

The protein of this invention and the compounds isolated by the screening method of this invention may be applied, for example, as agents for regulating A.beta. production, and as drugs for preventing and treating cerebral diseases in which the protein of the present invention is involved (for example, diseases causing A.beta. accumulation, such as Alzheimer's disease).

When the proteins of this invention or compounds isolated by the screening methods of this invention are used as drugs, they may be administered to patients as they are or as pharmaceutical preparations produced by known methods. They can be formulated together with, for example, pharmaceutically acceptable carriers or media such as sterilized water, physiological saline, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, etc. They may be administered to patients by methods well known in the art, for example, by intra-arterial, intravenous, subcutaneous injection. They can also be administered intranasally, intrabronchially, intramuscularly, or orally. Doses may vary depending on the body weight and age of patients as well as administration method, and such, and can be suitably selected by those skilled in the art. When the compound is encoded by DNA, gene therapy may be performed by inserting the DNA into a vector for gene therapy. Doses and method for its administration may vary depending on the body weight, age, symptoms, and so on of patients, but can be suitably selected by those skilled in-the art.

In addition, the present invention relates to a kit for screening a compound that promotes or inhibits peptidase activity of the protein of this invention, wherein said kit comprises the protein. Examples of the protein of this invention in the kit of this invention may be a purified or crudely purified protein, may be in a form expressed outside or inside a desired cell (including a transformant made to express the protein), or in a form bound to a support. The kit of this invention preferably comprises a substrate as another element in addition to the protein preparation mentioned above. As substrate., brain APP is preferred, although there is no limitation as long as the protein of this invention shows cleaving activity. Natural brain APP can be prepared by the method described in the Examples. In addition, partial peptides of brain APP, other APP isoforms and APP-like polypeptides, and oligopeptides synthesized to contain a cleavage site of a substrate may be included as substrates. The substrate may be labeled. Furthermore, buffer for the reaction between the protein of this invention and the substrate, washing solution, and a reagent for detecting cleavage of the substrate may be included.
 

Claim 1 of 6 Claims

1. An isolated antibody that binds to human brain carboxypeptidase B consisting of SEQ ID NO:9.

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