JP2000157263A - Novel human cathepsin y protein, and gene encoding the same, and their uses - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel protein that is a human-originating cathepsin Y protein or a protein that has an amino acid sequence homology with cysteine protease activity and antigenicity, thus is useful in the elucidation and therapy for Alzheimer's disease, neurodegenerative diseases, cancers and the like. SOLUTION: This novel protein is a human-originating cathepsin Y protein or a novel protein that has at least 49% homology with the human-originating cathepsin protein, and has cysteine protease activity or has the same level of antigenicity or their salt. This protein participates to the intracellular protein metabolism, antigen presentation, activation of hormone precursors, and bone modification and participates in Alzheimer's disease, pulmonary emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, infiltration and metastasis of cancers and is useful in the elucidation of pathogenic mechanism and for their treatment. This protein is obtained by incorporating the gene that is cloned from a human-originating cDNA library into the vector and expressing the gene in the host cells of a microorganism, for example, Escherichia coil.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a polynucleotide (or nucleic acid) and a polypeptide (or protein) (or a part thereof) or a salt thereof which have been newly identified; Mutants and derivatives of the polynucleotide (or nucleic acid) and polypeptide (or protein), and methods for producing the mutants and derivatives thereof; agonists and antagonists of the polypeptide (or protein); (Or proteins), in particular monoclonal antibodies; and the use of said polynucleotides (or nucleic acids), polypeptides (or proteins), variants, derivatives, agonists and antagonists. Above all, in these and other respects,
The present invention relates to cathepsin Y.

[0002]

BACKGROUND ART Cysteine proteases are a group of enzymes that catalyze various protein hydrolysis and are widely distributed in various cells and play a major role in the degradation and metabolism of intracellular proteins. These cysteine proteases are classified into more than 20 families such as calpain, streptopain, clostripain, viral cysteine protease and caspase, in addition to the papain family, which accounts for the most majority. In fact, the papain family of cysteine proteases occupy the majority of enzymes in both prokaryotes and eukaryotes, the major ones being also expressed in bacteria, molds, protozoa, plants and humans. In recent years, members of the human cysteine protease belonging to the papain family that have been analyzed to the amino acid sequence level have increased, reaching 10 species.

That is, cathepsin B (Chan, SJ, et.
al., Proc. Natl. Acad. Sci. USA, 83: 7721-7725,
1986), cathepsin L (Gal, S., et al., Biochem. J.,
253: 303-306, 1988), Cathepsin H (Ritonja, A., et.
al., FEBS Lett., 228: 341-345, 1988), cathepsin
S (Shi, GP, et al., J. Biol. Chem., 267: 7258-72
62, 1992), cathepsin C (Paris, A., et al., FEBS
Lett., 369: 326-330, 1995), cathepsin O (Velasco,
G., et al., J. Biol. Chem., 269: 27136-27142, 199
4), Cathepsin K (Inaoka, T., et al., Res.
n., 206: 89-96, 1995), Cathepsin W (Linnevers, C.,
et al., FEBS Lett., 405: 253-259, 1997), cathepsin L2 (Santamaria, I, et al., Cancer Res., 58: 16
24-1630, 1998) and cathepsin Z (Santamaria, I, et.
al., J. Biol. Chem., 273: 16816-16823, 1998)
Included in this human cysteine protease.

[0004] Structural analysis has revealed that these enzymes conserve a series of characteristic sequences containing essential cysteine residues in their active sites. In addition, all of these cysteine proteases are synthesized as preproenzymes, cleaved into proenzymes, and
-It is known that phosphoric acid is incorporated into lysosomes. However, these enzymes have different characteristics from each other in terms of tissue distribution, substrate specificity and pH stability. Functional analysis has revealed that these proteases are involved in many normal cellular processes, including intracellular protein metabolism, antigen presentation, bone modification, and activation of hormone precursors. On the other hand, cysteine protease has been suggested to be involved in various diseases such as emphysema, muscular dystrophy, osteoporosis, Alzheimer's disease, rheumatoid arthritis and cancer invasion / metastasis. Thus, inhibitors that block deviated cysteine protease activity in these diseases can be used as therapeutics for these diseases.

[0005]

Cysteine proteases are involved in many normal cellular processes, including intracellular protein metabolism, antigen presentation, activation of hormone precursors, and bone modification, while Alzheimer's disease, Emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis,
It has been suggested that it plays an important role in many diseases such as neurodegenerative diseases and cancer invasion and metastasis. Identifying and isolating novel cysteine proteases and elucidating the function of cysteine proteases associated with various diseases, especially cancer, will elucidate the pathogenesis of these diseases,
It is crucial for the development of therapeutics and therapeutics.

[0006]

SUMMARY OF THE INVENTION The present inventors considered that there is a novel member of the cysteine protease family that has not yet been reported, and screened a human prostate cDNA library. As a result, a novel cysteine protease is cloned and encoded
The entire nucleotide sequence of the cDNA was determined. An anticipated new cysteine protease was identified as "cathepsin Y"
". The present invention relates to a novel protein which is a human-derived factor having cathepsin Y activity, in particular, a polypeptide (or protein) referred to herein as cathepsin Y, a method for producing the same and its use, and a gene encoding the protein (or Polynucleotides or nucleic acids) and uses thereof. The present invention further provides an antibody that specifically reacts with cathepsin Y, a method for immunologically measuring cathepsin Y using the antibody, and a reagent for measuring the same. Further, the present invention provides a hybridization probe specific to the cathepsin Y gene.

The present invention relates to a novel human cathepsin Y gene, human-derived cathepsin Y, and analogs thereof. Furthermore, the present invention provides a novel human cathepsin Y
DNA sequence encoding all or part of a DN such as
It also relates to vectors having the A sequence and host cells transformed or transfected with such vectors. Furthermore, the present invention includes a method for producing recombinant human cathepsin Y and its use. It also relates to antibodies that bind to human cathepsin Y, especially monoclonal antibodies and hybridoma cells producing the antibodies. Nucleic acids that hybridize to the human cathepsin Y gene, such as DN
A, pertaining to RNA probes. Further, the present invention provides a reagent for detecting and measuring human cathepsin Y using the above product, and a method for detecting and measuring using the reagent.

Further, the present invention provides a human cathepsin Y agonist. Particularly preferred agonists are
Cathepsin Y that binds to a cathepsin Y binding molecule or a cathepsin Y receptor molecule and / or causes or enhances a cathepsin Y-induced reaction
It is a pseudo molecule. Also, preferred agonists interact with cathepsin Y or a cathepsin Y polypeptide, or other cathepsin Y activity regulator, to produce cathepsin Y.
It is a molecule that enables or enhances the action or effect of Y or more. The present invention provides a human cathepsin Y antagonist. Particularly preferred antagonists are cathepsin Y mimetic molecules that bind to a cathepsin Y binding molecule or a cathepsin Y receptor molecule but do not cause or inhibit one or more cathepsin Y-induced reactions. Also preferred antagonists are cathepsin Y or cathepsin Y polypeptide, or other cathepsin Y
It is a molecule that interacts with an activity controlling substance and inhibits or suppresses one or more actions and effects of cathepsin Y.

The present invention provides a method for detecting a substance that inhibits the cysteine protease activity of human cathepsin Y and a substance that inhibits the proteolytic activity, and provides a drug containing the substance or derivative as an active ingredient, such as cysteine protease activity. Alternatively, the present invention relates to a neutralizing antibody that inhibits proteolytic activity, in particular, a monoclonal antibody that binds to human cathepsin Y. Also human cathepsin Y
The present invention also relates to a partial peptide of human cathepsin Y which does not have human cathepsin Y activity and which binds to an antibody against the same, and a derivative thereof or a salt thereof. Also, human cathepsin Y
TECHNICAL FIELD The present invention relates to an antisense oligomer and a derivative thereof for inhibiting transcription or translation of a drug, and a drug containing the same as an active ingredient.

That is, the present invention relates to [1] a human-derived cathepsin Y protein or an amino acid sequence of the cathepsin Y protein, which
% Of a novel cathepsin protein or a salt thereof derived from human, characterized by having a homology of at least% and having cysteine protease activity or equivalent antigenicity; [2] a sequence represented by SEQ ID NO: 1 Amino acid sequences having amino acid sequences 271 to 484, those having amino acid sequences 20 to 484, and those having amino acid sequences 1 to 484 A protein having an amino acid sequence substantially equivalent to any one of them, and a protein having an amino acid sequence having a biological activity substantially equivalent to any one of them. [3] a partial peptide of the protein of the above-mentioned [1] or [2] or a salt thereof;

[4] a nucleic acid having a base sequence encoding the protein according to the above [1] or [2]; [5] a base sequence represented by SEQ ID NO: 2 in the sequence listing; The nucleic acid according to the above [4], which has an open reading frame portion or a nucleotide sequence substantially equivalent thereto; [6] The nucleic acid according to the above [4] or [5]. [7] a transformant comprising the vector of [6]; [8] a host cell comprising Escherichia coli, yeast, CHO cells and CO
The transformant according to the above [7], which is selected from the group consisting of S cells;

[9] the protein of the above-mentioned [1] or [2], which is obtained by being expressed by the transformant of the above-mentioned [7];

[10] An antibody against the following: (i) the protein of the above-mentioned [1] or a salt thereof, (ii)
The protein according to the above [2] or a salt thereof, or
(iii) a partial peptide or a salt thereof according to the above [3]; [11] a monoclonal antibody specifically immunoreactive with the protein or a salt thereof according to the above [1] or [2] is used as a measuring reagent. [12] a monoclonal antibody that specifically immunoreacts with the protein or salt thereof according to [1] or [2]; An immunoassay reagent for the protein or salt thereof according to the above [1] or [2]; [13] a nucleotide sequence represented by SEQ ID NO: 2 or a part thereof. A hybridization probe for a human-derived cathepsin Y gene, comprising:

[14] The protein according to any one of the above [1] to [3], a partial peptide thereof or a salt thereof; or the nucleic acid according to the above [4] or [5]; or the above [10] [15] A pharmaceutical comprising the antibody of [15], which promotes the biological activity of the protein of any one of [1] to [3], a partial peptide thereof, or a salt thereof. Or a medicament characterized by containing a compound or a salt thereof that inhibits; or [16] an organism of the protein according to any one of the above [1] to [3], a partial peptide thereof, or a salt thereof. Provided are a screening method and a screening kit for a compound that promotes or inhibits a biological activity.

According to still another aspect, the present invention relates to a method for producing a human cathepsin Y or a salt thereof, which has substantially the same cysteine protease activity or antigen activity, or [18] The amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, wherein the protein or the salt thereof or a part thereof has substantially the same primary structure conformation. A group having the 271st to 484th amino acid sequence, a group having the 20th to 484th amino acid sequence, and a group having the 1st to 484th amino acid sequence A homology of more than 50%, preferably 80% or more, and preferably 90% or more to an amino acid sequence selected from That the above-mentioned [1] protein or a salt thereof, or a portion thereof according;

[19] Among the amino acid sequences represented by SEQ ID NO: 1 in the sequence listing, those having the 271st to 484th amino acid sequences and those having the 20th to 484th amino acid sequences And a human cathepsin Y protein having an amino acid sequence selected from the group consisting of those having the first to 484th amino acid sequences, or one or more amino acid residues unique to the protein (for example, 100, preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20, especially 1 to 10
Deletion analog, one or more amino acid residues unique to the human cathepsin Y (eg, 1-100,
Substituted analogs in which preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20, especially 1 to 10, etc., are substituted with other residues, and one or more (e.g. For example, 1 to 100, preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20, especially 1 to 10
) Or a salt thereof or a part thereof, wherein the protein or salt thereof is selected from the group consisting of addition analogs to which amino acid residues are added.

[20] a nucleic acid having a base sequence encoding the protein according to any one of [17] to [19]; [21] a human cD of EMBL accession number: AJ007331
Sequence with at least 50% homology to NA; EMBL accessio
n number: sequence having at least 55% homology with human cDNA of AJ007331; EMBL accession number: sequence having at least 60% homology with human cDNA of AJ007331; EMBLa
ccession number: AJ007331 human cDNA and at least 65
% Homology sequence; EMBL accession number: AJ007
331 human cDNA sequences with at least 70% homology;
EMBL accession number: A sequence having at least 75% homology to the human cDNA of AJ007331; EMBL accession number:
A sequence having at least 80% homology with the human cDNA of AJ007331; EMBL accession number: a sequence having at least 85% homology with the human cDNA of AJ007331; EMBL accession n
umber: sequence having at least 90% homology to human cDNA of AJ007331; EMBLaccession number: human c of AJ007331
A sequence with at least 95% homology to DNA; and EMBL ac
cession number: AJ007331 human cDNA and at least 97%
A nucleic acid having a region selected from the group consisting of:

[22] a sequence having at least 50% homology with the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing; at least 55% homology with the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing A sequence having at least 60% homology with the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing; having at least 65% homology with the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing Sequence; a sequence having at least 70% homology with the nucleotide sequence represented by SEQ ID NO: 2 in the Sequence Listing; SEQ ID NO: 2 in the Sequence Listing
A sequence having at least 75% homology with the nucleotide sequence represented by SEQ ID NO: 2; a sequence having at least 80% homology with the nucleotide sequence represented by SEQ ID NO: 2 in the sequence list; A sequence having at least 85% homology with the base sequence to be obtained; at least 90% with the base sequence represented by SEQ ID NO: 2 in the sequence listing
A sequence having at least 95% homology with the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing; and a sequence having at least 97% homology with the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing A nucleic acid having a region selected from the group consisting of sequences having homology; [23] a nucleic acid comprising the nucleic acid according to any one of [20] to [22]. Vector to be;

[24] a transformant comprising the vector according to [23]; [25] a host cell selected from the group consisting of Escherichia coli, yeast, CHO cells and COS cells [24] The transformant according to [24]; [26] The transformant according to [17] to [17], which is obtained by expression by the transformant according to [25].
[19] the protein of any one of [19]; [27] an antibody of the above [10], which is a solid-phased monoclonal antibody; [28] a characteristic of a labeled monoclonal antibody [29] The antibody according to [10], which is labeled with an enzyme; [30] The monoclonal antibody labeled with Fab ′. The antibody according to the above [10], which is characterized in that:

[31] a base sequence containing at least a part of the base sequence represented by SEQ ID NO: 2 and capable of hybridizing with the human cathepsin Y gene under stringent conditions [32] A method for measuring a human-derived cathepsin Y-encoding nucleic acid in a sample, which comprises: (i) a human-derived cathepsin Y
Under a condition effective to specifically hybridize with the nucleic acid, and (ii) hybridization of the probe with respect to the nucleic acid in the sample. Measuring the hybridization, and the probe is represented by SEQ ID NO:
Has at least 49% homology to the nucleotide sequence represented by 2
[33] a method for measuring a biological activity such as cysteine protease activity or antigenicity of the protein according to [1] or [2], which has a sequence of 10 or more base pairs; A method of inhibiting; and

[34] The inhibitor is selected from the group consisting of peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and antibodies, for example, monoclonal antibodies. [33] The method according to the above [33], which is characterized in that According to another aspect, the present invention provides: [35] The above [1] to [3] and [17] to [19]
Characterized by using a protein selected from the group consisting of the protein according to any one of the above, a partial peptide thereof, and a salt thereof. [1] to [3] and [17] to [1]
9] A method for screening a compound that promotes or inhibits the biological activity of the protein, a partial peptide thereof, or a salt thereof according to any one of [9]; [36] The above [1] to [3] and [17] to [19]
A method for screening a compound that promotes or inhibits the biological activity of a protein, a partial peptide thereof, or a salt thereof according to any one of the above (1) to (1), wherein
(3) and the case where the substrate is brought into contact with the protein according to any one of [17] to [19], a partial peptide thereof or a salt thereof, and (ii) the above [1] to [3] and [ 1
7] a screening method, which comprises comparing a protein and a partial peptide thereof or a salt thereof according to any one of [19] with a substrate and a test compound;

[37] The above [1] to [3] and [1]
[7] to [19], comprising a protein selected from the group consisting of the protein according to any one of [19], a partial peptide thereof, and a salt thereof. A kit for screening a compound that promotes or inhibits the biological activity of the protein according to any one of [19] to [19], a partial peptide thereof, or a salt thereof; [38] (i) [16], [ 35] or [36]
The screening method described above or (ii) obtained by using the screening kit described in the above [16] or [37], wherein [1] to [3] and [1]
7] a compound or salt thereof that promotes or inhibits the biological activity of the protein according to any one of [19], some of the peptides or salts thereof; and [39] (i) the above [16], [35] or [36]
The protein according to any one of the above [1] to [3] and [17] to [19], which is obtained by using the screening method according to the above or (ii) the screening kit according to the above [16] or [37]. A compound which promotes or inhibits the biological activity of some peptides or salts thereof, or a salt thereof.

According to still another aspect, the present invention relates to the aforementioned [1], wherein the monoclonal antibody specifically immunoreacting with the protein of the above [1] or a salt thereof is used as a measuring reagent. [41] The protein or salt thereof according to [1], which comprises a monoclonal antibody that specifically immunoreacts with the protein or salt thereof according to [1]. [42] The antibody according to [10], wherein at least two of (i) a solid-phased antibody and (ii) a labeled antibody are used. [43] The method according to [42], which comprises quantitatively measuring the protein or salt thereof according to [1];

[44] The method as described above, wherein the measurement sample is selected from the group consisting of serum, plasma, synovial fluid, urine, saliva, tissue extract, cultured cell extract and cell culture supernatant. [42] The method according to [43]; [45] The method according to any one of [42] to [44], wherein (i) the antibody according to [10]; Reagent containing solid-phased
(ii) a reagent set for measuring human cathepsin Y having at least two kinds of reagents, including the antibody of the above-mentioned [10] and a reagent containing a labeled one; [46] (a) the above-mentioned [1] Producing a monoclonal antibody against a protein or a salt thereof and (b) a partial peptide or a salt thereof selected from the group consisting of:
And (a) the protein of the above-mentioned [1] or a salt thereof;
(b) Obtained by cell fusion between spleen cells of an animal such as a mouse immunized with a member selected from the group consisting of the partial peptide or a salt thereof and cells that can be cultured continuously such as myeloma cells of an animal such as a mouse. (47) (a) the spleen of an animal such as a mouse immunized with a protein selected from the group consisting of the protein of the above-mentioned [1] or a salt thereof, and (b) a partial peptide thereof or a salt thereof; The cells are fused with cells that can be permanently cultured, such as myeloma cells of an animal such as a mouse, and selected from the group consisting of (a) the protein or its salt described in [1] above and (b) its partial peptide or its salt. Providing a method for producing a hybridoma according to the above [46], wherein a cell capable of continuous culture that produces a monoclonal antibody against the selected one is selected. I do.

Further, according to another aspect, the present invention provides: [48] a nucleic acid having a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence represented by SEQ ID NO: 2; [49] a vector containing the nucleic acid of the above [48]; [50] a transformant containing the vector of the above [49]; [51] the above [50] [52] a protein or a salt thereof obtained by expressing a transformant of [52], wherein the nucleic acid is DNA; To the nucleic acid according to any one of [22] and [48]; [53] culturing the transformant according to [7], [8], [24], [25] or [50], ) The above [1]
A protein or a salt thereof, and (b) a method for producing the product, wherein a product selected from the group consisting of a partial peptide or a salt thereof is produced, accumulated, and collected;

[54] (i) a method obtained by using the screening method described in [16], [35] or [36] or (ii) a screening kit described in [16] or [37]. The above [1] to [3]
[55] (i) The compound according to any one of [16], [35] or [36] above, which promotes the biological activity of the protein, partial peptide or salt thereof according to any one of [1] to [3]. The screening method or (ii) the protein according to any one of the above [1] to [3], which is obtained by using the screening kit according to the above [16] or [37], or a part thereof. A compound or a salt thereof that inhibits the biological activity of the peptide or a salt thereof; [56] the screening method of [16], wherein the biological activity of [16] is a protease activity; [57] the screening kit of [16], wherein the biological activity of [16] is a protease activity; [58] the biological kit of [38]; The compound or salt thereof according to the above [38], wherein the physical activity is a protease activity; [59] The compound according to the above [15], wherein the biological activity according to the above [15] is a protease activity. 15] the medicament described above;

[60] The antibody of [10] is combined with a test sample and a labeled protein of any of [1] to [3], a partial peptide thereof, or a salt thereof. Reacting competitively and measuring the ratio of the labeled protein of any one of the above [1] to [3] bound to the antibody, a partial peptide thereof or a salt thereof. The above [1] to [3] in the test sample
[61] The method according to any one of [1] to [10], wherein the test sample and the carrier are insolubilized on a carrier.
Reacting the antibody of the above [10] with the labeled antibody of the above [10], and measuring the label on the insolubilized carrier or the label that does not bind to the insolubilized carrier; [3] A method for quantifying the protein according to any one of [3], a partial peptide thereof, or a salt thereof. As used herein, the term “and / or” means that both (1) merged connection and (2) selective connection exist, eg, “therapeutic and / or prophylactic”. In some cases, it is used to mean both (1) treatment and prevention and (2) treatment or prevention. Elsewhere, the term “and / or” is similarly used to encompass both (1) merged connectivity and (2) selective connectivity.

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, a human-derived cathepsin Y protein or a cathepsin Y
A protein or salt thereof having at least 49% homology with the amino acid sequence of the protein and having cysteine protease activity or equivalent antigenicity, a characteristic partial peptide of the protein or a salt thereof, and a gene encoding them A vector or plasmid containing the gene such that it can be manipulated by genetic recombination techniques, such as DNA, RNA, etc .; a host cell transformed with such a vector; and a transgene containing the gene. Transgenic animals such as transgenic mice, knockout animals such as knockout mice in which the gene is specifically deleted (deleted), and a method for producing the protein or a salt thereof by culturing the host cells thereof. Characteristic of protein or its salt or its protein Partial peptide or antibodies obtained using a salt thereof, in particular monoclonal antibodies, or using hybridoma cells producing the antibody, the isolated gene, for example DNA, RNA and the like as a probe,
Alternatively, a measurement / diagnosis means and a reagent using the antibody are provided. Furthermore, the present invention provides use of the active ingredient disclosed and described herein, for example, a drug or a reagent containing the active ingredient is provided, and a disease, disease or abnormal condition using such an active ingredient is provided. Treatment and / or
Alternatively, a prevention method, a screening method, and the like are provided.

More specifically, the human cathepsin of the present invention
As Y, among the amino acid sequences represented by SEQ ID NO: 1 in the sequence listing, those having the 271st to 484th amino acid sequences, those having the 20th to 484th amino acid sequences, Having the amino acid sequence of positions 1 to 484, and at least 49% of any one of them
Higher homology, preferably 50% or more homology, more preferably 55% or more homology, also preferably 60% or more homology, more preferably 65% or more homology, more preferably 70% or more Homology, even more preferably 75%
Or more homology, more preferably 80% or more homology,
Even more preferably, it has a homology of 85% or more, particularly preferably 90% or more, particularly preferably 95% or more, most preferably 97% or more, and cysteine protease activity or All those having an amino acid sequence having substantially the same biological activity such as the same antigenicity and the like can be mentioned. The human cathepsin Y of the present invention may be any one of a papain family having cysteine protease activity and having a novel amino acid sequence. More preferably, the present invention provides an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, one having the 271st to 484th amino acid sequence, and one having the 20th to 484th amino acid sequence. A protein selected from the group consisting of those having the first to 484th amino acid sequences and those having an amino acid sequence substantially equivalent to any one of them. A cathepsin Y protein or a salt thereof. Furthermore, human cathepsin Y of the present invention may have a part or all of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. Any having such an arrangement may be included.

As used herein, the term "homology" refers to each amino acid residue or two amino acid residues constituting a chain between two chains in a polypeptide sequence (or amino acid sequence) or a polynucleotide sequence (or base sequence). It refers to the amount of each base that can be determined to be identical in the mutual relationship with each other, and refers to the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences. "Homology" can be easily calculated. A preferred method of measuring "homology" is to use a computer with a computer program designed to obtain the largest match between the two sequences tested. Can be achieved by applying Suitable computer program methods for measuring homology between two sequences include:
For example, the GCG software package (Devereux, J. e.
t al., Nucleic Acids Research, 12 (1): 387 (1984))
And the like, but not limited thereto, and a method known in the art can be used.

The gene encoding human cathepsin Y of the present invention typically contains the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing and the gene represented by SEQ ID NO: 2 in the sequence listing. Starting from ATG at positions 46-48 of the sequence, from 1498
Contains a nucleotide sequence consisting of the TGA at position 1500 (the stop codon TGA from position 1498 to position 1500 is TAA or TAG
Or a sequence containing the base sequence from position 103 to position 1500 of the base sequence represented by SEQ ID NO: 2 in the sequence listing, and represented by SEQ ID NO: 2 in the sequence listing. Containing a nucleotide sequence from positions 856 to 1500 of the base sequence, those having an initiation codon added thereto, for example, a codon encoding Met, and at least 49% of the protein encoded by the nucleotide sequence. It has an amino acid sequence having the above homology and contains a nucleotide sequence equivalent to that encoding a protein having substantially the same biological activity as that of cysteine protease activity or equivalent antigenicity. Anything may be used. The gene encoding human cathepsin Y is a nucleic acid such as single-stranded DNA, double-stranded DNA, RNA, DNA: RNA hybrid, synthetic DNA, and human genomic DNA.
, A human genomic DNA library, a cDNA derived from a human tissue / cell, or a synthetic DNA. The nucleotide sequence of the gene encoding human cathepsin Y can be modified (eg, added, removed, substituted, etc.),
Such modifications may be included. Moreover,
As described below, the nucleic acid of the present invention may encode the protein of the present invention or a part thereof,
Preferred is DNA. The “equivalent nucleotide sequence” is, for example, one which hybridizes with the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing under stringent conditions, and encodes an amino acid sequence substantially equivalent to human cathepsin Y. And the like.

The DNA of the present invention containing the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing or a nucleotide sequence having the same effect as the nucleotide sequence is as follows:
For example, it can be obtained by the following method. The gene recombination technique is described in, for example, T. Maniatis et al., "Molecula
r Cloning ", 2nd ed., Cold Spring Harbor Laboratory,
Cold Spring Harbor, NT (1989); DM Glover et
al. ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (Th
ePractical Approach Series), IRL Press, Oxford Uni
versity Press (1995); edited by The Biochemical Society of Japan, “Seismological Chemistry Laboratory Course 1, Genetic Research Method II”, Tokyo Chemical Doujin (198
6); edited by The Japanese Biochemical Society, “Lecture on New Chemistry Experiment 2, Nucleic Acid III”
(Recombinant DNA technology) ", Tokyo Kagaku Doujin (1992); R. Wu
ed., "Methods in Enzymology", Vol. 68, Academic Pre
ss, New York (1980); R. Wu et al. ed., "Methods i
nEnzymology ", Vol. 100 & 101, Academic Press, New
York (1983); R. Wu et al. Ed., "Methods in Enzymolo
gy ", Vol. 153, 154 & 155, Academic Press, New York
(1987); JH Miller ed., "Methods in Enzymolog
y ", Vol. 204, Academic Press, New York (1991); R.
Wu et al. Ed., "Methods in Enzymology", Vol. 218, A
cademic Press, New York (1993), or the method described in the literature cited therein, or a method substantially similar to or a modification thereof.
(The statements therein are included in the disclosure herein by reference thereto).

First, a sequence showing significant similarity to the amino acid sequence of a known human cysteine protease family (more preferably papain family, particularly preferably human cathepsin) from which the entire sequence or a partial sequence has been cloned is obtained from a gene database. Identify by search. Examples of sequences showing significant similarity include, for example, cDNA sequences derived from animals, and more preferably cDNA sequences derived from ovarian cancer (H39591; registered by Hillier et al., Wash
U-Merck EST Project). Appropriate primers are designed and synthesized based on the sequence thus identified, and the target sequence is PCR-amplified, preferably using a cDNA library derived from an animal that is the origin of the identified sequence. The primer can be prepared by a method known in the art. For example, the primer can be synthesized using an automatic DNA synthesizer by a phosphodiester method, a phosphophotoester method, a phosphoramidite method, or the like.

Using the obtained DNA fragment as a probe, a human genomic DNA library or a human-derived cDNA library constructed from various human tissues or cultured cells is screened, and a clone that hybridizes to the probe is selected. The base sequence of the inserted sequence of DNA in the clone is determined, and a DNA fragment having a novel human cysteine protease gene sequence is determined and obtained. If necessary, the inserted sequence of the DNA in the clone can be subcloned. The nucleotide sequence is determined by the dideoxy method,
For example, M13 dideoxy method, etc., can be performed using the Maxam-Gilbert method, etc.
It can be performed using an automatic base sequencer, for example, a fluorescent DNA sequencer. Polymerases used in the dideoxy method include, for example, Klenow fragment of DNA polymerase I, AMV reverse transcriptase, Taq
DNA polymerase, T7 DNA polymerase, modified T7 DNA
Polymerase and the like.

Based on the DNA sequence which is considered to have the novel human cysteine protease gene thus analyzed, a gene encoding the human cysteine protease is obtained. The gene can be obtained by preparing a specific hybridization probe from the clone identified as described above, screening a human-derived DNA library, and selecting a clone that hybridizes to the probe. it can. In addition, based on the DNA sequence of the analyzed novel human cysteine protease gene, sense primers and antisense primers are synthesized, and these primers and various human-derived tissues or cultured cells (particularly, human Leukemia HL-60, leukemia K-562, leukemia, leukemia, colon, small intestine, ovary, testis, prostate, thymus, spleen, pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, heart, etc. Leukemia such as MOLT-4, Burkitt's lymphoma such as Raji, colorectal cancer such as SW480, lung cancer such as A549, melanoma cell line such as G361) cDNA library using polymerase chain reaction (polymerase) chain reac
tion: PCR) to amplify the cDNA.
The cDNA library used as a template can be prepared as described below, or a commercially available cDNA library derived from various tissues can be directly used.
A cDNA library commercially available from ne, Invitrogen, Clontech and the like can be used.

As used herein, the term "polymerase chain reaction" or "PCR" generally refers to a method as described in US Pat. No. 4,683,195. Refers to a method for enzymatic amplification. In general, the PCR method involves repeating a cycle of performing primer extension synthesis using two oligonucleotide primers that can preferentially hybridize with a template nucleic acid. Typically, as the primer used in the PCR method, a primer complementary to the nucleotide sequence to be amplified inside the template can be used, for example, complementary to the nucleotide sequence to be amplified at both ends. Those which are targeted or adjacent to the nucleotide sequence to be amplified can be preferably used.

The PCR reaction can be carried out by a method known in the art or a method substantially similar to or a modification thereof. For example, R. Saiki, et al., Science, 230: 13
50, 1985; R. Saiki, et al., Science, 239: 487, 198
8; HA Erliched., PCR Technology, Stockton Pres
s, 1989; DM Glover et al. ed., "DNA Cloning", 2
nd ed., Vol. 1, (The Practical Approach Series), I
RLPress, Oxford University Press (1995); MA In
nis et al. ed., "PCRProtocols: a guide to methods
and applications ", Academic Press, New York (199
0)); MJ McPherson, P. Quirke and GR Taylor
(Ed.), PCR: a practical approach, IRL Press, Oxfor
d (1991); MA Frohman et al., Proc. Natl. Acad.
Sci. USA, 85, 8998-9002 (1988), or a modification or modification thereof. In addition, the PCR method can be performed using a commercially available kit suitable for the PCR method, and can also be performed according to a protocol specified by a kit manufacturer or a kit distributor.

As used herein, the term "oligonucleotide" refers to a relatively short single-stranded or double-stranded polynucleotide, preferably a polydeoxynucleotide.
Agnew.Chem.Int.Ed.Engl., Vol.28, p.716-734 (19
89), and can be chemically synthesized by a known method such as a triester method, a phosphite method, a phosphoramidite method, or a phosphonate method. It is generally known that synthesis can be conveniently performed on a modified solid support, for example, using an automated synthesizer, which is commercially available. The oligonucleotide may contain one or more modified bases, for example,
It may contain an unnatural base such as inosine or a tritylated base. The obtained PCR product is cloned, the nucleotide sequence of the obtained PCR product is determined, and a DNA fragment having a novel human cysteine protease gene sequence can be obtained. Also, various cDNA libraries can be screened in the same manner using this DNA fragment as a probe to isolate a target DNA. The cloning of PCR products, for example, p-Direct (Clontech), pCR-Script TM SK (+) (Stra
tagene), pGEM-T (Promega) and pAmp ^™ (Gibco-BRL).

In order to obtain a full-length novel human cysteine protease gene sequence including a complete open reading frame, the novel human cysteine protease gene obtained as described above may be constructed, if necessary. Using the DNA fragment as a probe, a cDNA library constructed from various human tissues or cultured cells as described above is screened, a clone that hybridizes to the probe is selected, and the cDNA insertion sequence in the clone is The nucleotide sequence is determined, and a novel DNA fragment constituting the novel human cysteine protease gene is identified and obtained. Of course, if necessary, the cDNA in the clone
Can be subcloned. The target DNA can be isolated based on the determined base sequence. Preferably, a cDNA library derived from human brain or prostate is screened, and the nucleotide sequence is determined to isolate the target DNA. To label a probe or the like with a radioactive isotope, a commercially available labeling kit,
For example, random primed DNA labeling kit (Boeh
ringer Mannheim).

In order to construct a cDNA library, the cD
It is necessary to obtain NA, which can be obtained, for example, as follows. First, mRNA is isolated from various human tissues or cultured cells as described above. The isolation of mRNA can be performed by a method known in the art or a method substantially similar to or a modification thereof.
l., "Molecular Cloning", 2nd ed., Chapter 7, Cold S
pringHarbor Laboratory, Cold Spring Harbor, NT
(1989); DM Glover etal. Ed., "DNA Cloning", 2n
d ed., Vol. 1, (The Practical ApproachSeries), IRL
Press, Oxford University Press (1995); L. Grossm
an et al.ed., "Methods in Enzymology", Vol. 12, Pa.
rt A & B, Academic Press, NewYork (1968); SL Be
rger et al. ed., "Methods in Enzymology", Vol. 152,
p.33 & p.215, Academic Press, New York (1987); Bio
Chem., 18: 5294-5299, 1979, etc., for example, guanidine-cesium chloride method, guanidine thiocyanate method, phenol method and the like.
Kits used for mRNA isolation include, for example, Phar
Examples include those commercially available from macia, Stratagene, Gibco-BRL, and the like. If necessary, the total RNA obtained is an oligo (dT) -cellulose column, spin column, oligo (dT)
Purified using bound magnetic beads, etc. to poly (A) ⁺ mRNA
Can be obtained.

A cDNA is prepared using the mRNA and reverse transcriptase (RNA-dependent DNA polymerase). In the reverse transcription reaction, an oligo (dT) primer can be used. An oligo (dT) primer having preferably 12 to 18 T residues can be used. When performing directional cloning, it is also preferable to use a synthetic oligonucleotide primer in which a restriction enzyme site is linked to the 5 'side of 12 to 18 T residues. Examples of such primers include the Xba I oligo
(dT) primer adapter and the like. When a random hexamer primer is used, the possibility of obtaining the 5 'end of mRNA is increased. This random hexamer primer can be used alone or in combination with an oligo (dT) primer. In the reverse transcription reaction, RNas
e Inhibitors such as RNasin (Boehringer) can be added. cDNA synthesis using mRNA and reverse transcriptase can be carried out by a method known in the art or a method substantially similar thereto or a modified method, but H. Land et al.,
Nucleic Acids Res., 9: 2251, 1981; U. Gubler et a
l., Gene, 25: 263-269, 1983; SL Berger et al.e
d., "Methods in Enzymology", Vol. 152, p. 307, Acad
emic Press, New York (1987).

Based on the cDNA thus prepared, a cDNA is prepared by using a phage vector or a plasmid vector.
Can build libraries. In addition to using a phage vector, transformation of host cells such as E. coli can be performed by, for example, the calcium method, the rubidium / calcium method, the calcium / manganese method, the TFB high efficiency method, the FSB freezing competent cell method, the rapid colony method. Method, electroporation, or any other method known in the art or a method substantially similar thereto (D. Hanahan, J.
Mol. Biol., 166: 557, 1983). Further, using the cDNA thus prepared as a template, a novel DNA fragment constituting the novel human cysteine protease gene, a primer is designed based on the determined nucleotide sequence, and PCR is further performed. This also makes it possible to obtain the gene encoding human cathepsin Y of the present invention.
Typically, the amino acid sequence obtained from the papain family is a known cysteine protease enzyme, the entire sequence or a partial sequence of which is cloned.
More preferably, a highly conserved amino acid sequence selected from the sequenced or deduced amino acid sequence of human cathepsin Y (for example, a sequence near the cleavage site of the precursor (pro form) and the active form) or Amino acid sequence of a region specifically present in cathepsin (eg, an amino acid sequence of a C-terminal region, an amino acid sequence of a region existing between a propeptide domain and a catalytic domain,
A degenerated primer is prepared based on the amino acid sequence of the active site region of the catalytic domain. Using this primer and the cDNA prepared above, PCR
I do. PCR can be performed as described above. The resulting PCR product was cloned as above,
The nucleotide sequence of the obtained PCR product can be determined to obtain a DNA fragment having a novel human cathepsin Y gene sequence. Screening of various cDNA libraries in the same manner using this DNA fragment as a probe
DNA can also be isolated.

In order to isolate the desired DNA, reverse transcription PCR (polymerase chain reaction coupled reverse tr
anscription; RT-PCR), RACE (rapid amplification o
f cDNA ends) can be applied. RACE is described, for example, in MA Innis et al. Ed., "PCR Protocols" (MA
Frohman, "a guide to methods and applicarions"),
pp. 28-38, Academic Press, New York (1990). The RT-PCR product can be cloned into a plasmid vector, which can be introduced into highly efficient competent cells. Furthermore, a method capable of isolating and purifying RNA from a small amount of cells or tissues,
For example, REXkit, United States Biochmeical; Glass M
AX ^TM RNA spin cartridge system, using commercially available kits, such as Gibco-BRL, The obtained RNA was reverse transcribed using oligo (dT) primers were synthesized 1st strand DNA, and then 3 of 1st strand DNA ' After attaching a homopolymer tail (for example, G residue) to the end or attaching an adapter to the DNA, the cDNA is converted to P-DNA using an oligo (dT) primer and an oligo (dC) primer or an adapter primer.
CR amplification can also be performed. Commercially available kits suitable for this include the SuperScript ^TM pre-amplification system (G
ibco-BRL); cDNA Cycle ^™ kit (Invitrogen) and the like.

This will be described in more detail below. Probe DN
Using A, a novel human cathepsin Y gene is screened and identified from a gene library, and the nucleotide sequence of a DNA insert encoding the novel human cathepsin Y gene obtained by cloning is determined. Probe DNA
As the ovarian cancer-derived cDNA sequence (H39591; Hill
Using primers designed based on the WashU-Merck EST Project registered by ier et al., commercial human prostate cDNA
PCR amplification is performed using λgt11-phage DNA of a library (Clontech, Palo Alto, CA) as a template, and the thus amplified DNA can be cloned as necessary, labeled, and used as a probe. For labeling, a DNA labeling kit can be used. For example, random-pri
Use the ming kit (Pharmacia LKB, Uppsala, Sweden) etc. to convert the probe DNA to [α- ³² P] dCTP (Amersham, U
By labeling with K) or the like, a probe having radioactivity can be obtained. A gene library prepared from the labeled DNA fragment and human tissues / cells, for example, human P1 artificial
Chromosome genomic library (Human Genome Ma
Hybridization is performed using a human brain or prostate cDNA library (for example, available from Clontech, PaloAlto, CA, etc.). Human brain
The cDNA library can be constructed, for example, in a phage such as λgt10, and can be obtained by infecting host E. coli such as E. coli C600hfl strain to form plaques.

In the hybridization, the plaque formed as described above was transferred to a membrane such as a nylon filter and, if necessary, subjected to denaturation treatment, immobilization treatment, washing treatment and the like, and then transferred to the membrane. The reaction is carried out by reacting with a denatured labeled probe DNA fragment in a hybridization buffer if necessary. The hybridization process is usually performed at about 35 ° C. to about 80 ° C., more preferably about 50 ° C. to about 65 ° C., for about 15 minutes to about 36 hours, more preferably about 1 hour to about 24 hours. It can be carried out by selecting optimal conditions as appropriate. For example, the hybridization treatment is performed at about 55 ° C. for about 18 hours. The hybridization buffer can be selected from those commonly used in the art, and for example, a Rapid hybridization buffer (Amersham) or the like can be used. The denaturation of the transferred membrane
A method using an alkali denaturing solution is mentioned, and after the treatment, it is preferable to treat with a neutralizing solution or a buffer solution. The immobilization treatment of the membrane is usually performed at about 40 ° C. to about 100 ° C., more preferably about 70 ° C. to about 90 ° C., for about 15 minutes to about 24 hours, more preferably about
Baking is carried out for 1 hour to about 4 hours, but it can be carried out by appropriately selecting preferable conditions. For example, the immobilization is performed by baking the filter at about 80 ° C. for about 2 hours. The washing treatment of the transferred membrane includes a washing liquid commonly used in the art, for example, 1M N
aCl, 1mM EDTA and 0.1% SodiumDodecyl sulfate
It can be performed by washing with (SDS) -containing 50 mM Tris-HC1 buffer, pH 8.0, etc. The membrane such as a nylon filter can be selected from those commonly used in the art, and examples thereof include a nylon filter [Hybond-N, Amersham].

The above-mentioned alkali denaturing solution, neutralizing solution and buffer solution can be selected from those commonly used in the art, and examples of the alkali denaturing solution include:
A solution containing 0.5M NaOH and 1.5M NaCl can be mentioned. As the neutralizing solution, for example, a solution containing 1.5M NaCl
0.5M Tris-HCl buffer, pH 8.0 and the like. As the buffer, for example, 2 × SSPE (0.36M NaCl,
20 mM NaH ₂ PO ₄ and 2 mM EDTA). Prior to the hybridization treatment, the transferred membrane is preferably subjected to a pre-hybridization treatment, if necessary, in order to prevent a non-specific hybridization reaction. This pre-hybridization treatment is performed, for example, using a pre-hybridization solution [50%
formamide, 5 × Denhardt's solution (0.2% bovine serum albumin, 0.2% polyvinylpyroldone), 5 × SSPE, 0.
1% SDS, 100 μg / ml heat-denatured salmon sperm DNA] and the like, and allowed to react at about 35 ° C. to 50 ° C., preferably about 42 ° C., for about 4 to 24 hours, preferably for about 6 to 8 hours. However, those skilled in the art can determine more preferable conditions by repeating experiments as appropriate. Labeled probe DNA used for hybridization
The denaturation of the fragments is carried out, for example, at about 70 ° C to 100 ° C, preferably at about
It can be carried out by heating at 100 ° C. for about 1 minute to about 60 minutes, preferably for about 5 minutes. The hybridization can be carried out by a method known per se or a method analogous thereto.
50 mM, preferably about 19-40 mM, more preferably about 19-20
In mM, the temperature is about 35-85 ° C, preferably about 50-70 ° C.
C, more preferably about 60-65C.

After the completion of the hybridization, the filter is sufficiently washed to remove the labeled probe other than the labeled probe DNA fragment that has undergone the specific hybridization reaction. The filter can be washed and selected from those commonly used in the art. For example, 0.5 × SSC containing 0.1% SDS (O.15M NaCl,
It can be carried out by washing with a 15 mM citric acid) solution or the like. The hybridized plaque can be typically detected by autoradiography, but can also be appropriately selected from methods used in the art and used for plaque detection. The plaque corresponding to the detected signal is added to an appropriate buffer such as SM solution (100 mM N 2
aCl and 10 mM MgSO ₄ containing 50 mM Tris-HCl buffer, pH 7.
5), and then appropriately dilute the phage suspension to infect Escherichia coli, cultivate the obtained Escherichia coli, and obtain a desired recombinant phage from the cultured Escherichia coli. The process of screening for a target recombinant phage from a gene library or a cDNA library by hybridization using the above probe DNA as necessary can be repeatedly performed. The target recombinant phage can be obtained by subjecting the cultured Escherichia coli to extraction treatment, centrifugation treatment and the like.

The obtained phage particles can be purified and separated by a method commonly used in the art.
Glycerol gradient ultracentrifugation (Molecular cl
oning, a laboratory manual, ed.T. Maniatis, Cold
Spring Harbor Laboratory, 2nd ed. 78, 1989). From the phage particles, DNA can be purified and separated by a method commonly used in the art.
O ₄ containing 50 mM Tris-HCl buffer, pH 7.8) were suspended in such,
After treatment with DNase I and RNase A, 20mM EDTA, 50
Add a mixture of μg / ml Proteinase K and 0.5% SDS, incubate at about 65 ° C for about 1 hour, extract with phenol and diethyl ether, precipitate DNA with ethanol.
Then, the obtained DNA is washed with 70% ethanol, dried, and dissolved in a TE solution (10 mM Tris-HC1 buffer containing 10 mM EDTA, pH 8.0). The target DNA can be obtained in large quantities by subcloning or the like. For example, subcloning can be performed using Escherichia coli as a host and a plasmid vector. The DNA obtained by such subcloning can also be purified and separated by methods such as centrifugation, phenol extraction, and ethanol precipitation in the same manner as described above.

The nucleotide sequence of the DNA thus obtained, for example, the nucleotide sequence of single-stranded DNA, can be sequenced in the same manner as described above. For example, the fluorescent DNA sequencer Mo
del373A (Applied Biosystems), Taq Dye Primer Cycle Sequencing Kit (Applied Biosystems)
s) can be used to determine the sequence. By analyzing the determined sequence, it is possible to identify and analyze a gene that has a structural characteristic typical of the known human cathepsin sequence but is presumed to be a novel human cathepsin. Based on the nucleotide sequence of the analyzed gene, primers are designed to obtain the cDNA of the novel putative human cathepsin. The sense primer is
Preferably, it can be synthesized by selecting from the exon site at the 5 ′ end of the putative novel human cathepsin analyzed, and the antisense primer is preferably putatively analyzed as the novel human cathepsin. Gene
It can be synthesized by selecting from the 3 'end exon site. The cDNA of a gene may aim to obtain the full length at once, but using the analyzed exon site (multiple exon sites), design and synthesize multiple primers, and perform multiple PCRs. The DNA fragment whose nucleotide sequence was determined in this way was analyzed,
The entire nucleotide sequence of the cDNA is determined, and the cDNA of the gene can be obtained from the cloned DNA fragment based on the determined nucleotide sequence. The primer is preferably an oligonucleotide consisting of 5 or more bases, more preferably an oligonucleotide consisting of 10 to 50 bases, and even more preferably an oligonucleotide consisting of 18 to 25 bases. As described above, primer synthesis is performed by an automatic DNA synthesizer,
For example, model 381A DNA synthesizer, Applied Biosys
Synthesized using tems.

Using mRNA isolated from human tissues, particularly human brain, human prostate, etc., the first transcriptase
Make strand cDNA. Brain or prostate tissue
Use a commercially available mRNA extraction / separation kit such as izolReagent, Life Technologies, etc.
Prepare NA. For the 1st strand cDNA, use Oligo dT _12-18 primer, Super Script kit, Life Technolog
Using a commercially available cDNA synthesis kit such as ies, the mRNA can be used as a template for synthesis. 1st strand cDNA synthesis, for example,
Poly (A) ⁺ RNA was added to 5 × RT buffer (250 mM Tris-HCl (pH 8.
_{3), 50mM MgCl 2, 375mM} KCl, 50mM DTT -containing), dNTPs
(Deoxynucleoside triphosphate dATP, dGTP, dCTP, dT
TP mixture), oligo dT ₁₆ primer, RNase inhibitor
(Boehringer), MMLV RT (Gibco-BRL) or Supersc
This can be achieved by mixing with ript RT plus (Life Technologies) and deionized distilled water and incubating at about 37 ° C. for about 1 hour.

The obtained 1st strand DNA and a primer designed based on the exon of the analyzed gene were combined with 10 × reaction buffer (attached to Taq DNA polymerase), dNTPs (deoxynucleoside triphosphate dAT
P, dGTP, dCTP, dTTP), Taq DNA polymerase and deionized distilled water. The mixture, for example,
The cycle is repeated 25 to 60 times under general PCR cycle conditions using an automatic thermal cycler such as the GeneAmp 2400 PCR system, Perkin-Elmer / Cetus, and the number of cycles for amplification is appropriately determined according to the purpose. It can be the number of times. PCR cycle conditions include, for example, denaturation
90 ~ 95 ° C 5 ~ 100 seconds, annealing 40 ~ 60 ° C 5 ~ 150
Cycle, elongation 65-75 ° C 30-300 seconds, preferably denaturation 94 ° C 15 seconds, annealing 58 ° C 15 seconds, elongation 72
℃ 45 seconds cycle, but the reaction temperature and time of annealing can be appropriately selected by appropriate experiment, and the time of denaturation reaction and extension reaction may also be expected PC
An appropriate value can be selected according to the chain length of the R product. The annealing reaction temperature is usually preferably changed according to the Tm value of the hybrid of the primer and the template DNA. The time for the extension reaction is generally about 1 minute per 1000 bp chain length, but a shorter time can be selected in some cases.

The obtained PCR product is usually subjected to 1 to 2% agarose gel electrophoresis, cut out from the gel as a specific band, and the DNA is extracted using a commercially available extraction kit such as a gene clean kit or Stratagene. The extracted DNA is cleaved with an appropriate restriction enzyme, purified if necessary, and further, if necessary, phosphorylated at the 5 ′ end with T4 polynucleotide kinase or the like.
Ligation is performed with a suitable plasmid vector such as a C-type vector, and a suitable competent cell is transformed. The nucleotide sequence of the cloned PCR product is analyzed. If it is necessary to obtain a sequence at the 5 'end and / or 3' end as a result of the nucleotide sequence analysis, a rapid amplification of cDNA end (RACE)
s) can be achieved.

Further, the primers of the cDNA of the gene are utilized by using primers designed based on the nucleotide sequence of the exon site at the 5 'end analyzed among the exon sites of the gene.
Obtain the cDNA at the 5 'end, while using primers designed based on the nucleotide sequence of the exon site at the 3' end analyzed among the exon sites of the gene, the 3 'end of the cDNA of the gene Side cDNA, and then, if necessary, these primers and the cDNA of the 5 'end of the cDNA of the gene obtained.
Using a primer designed by using information on the base sequence of the NA and the 3′-end cDNA, 1st strand cDNA prepared by reverse transcriptase from mRNA isolated from human tissues, particularly human brain tissues, etc. (Or human cDNA library) as a template and amplify by PCR,
NA can be obtained.

The primer at the 5 ′ end contains at least a start codon or is selected so as to be capable of amplifying including the start codon, and the primer at the 3 ′ end contains at least a stop codon. Alternatively, it is preferable to select so that amplification can be performed including the stop codon. In obtaining the full-length cDNA of the gene, PCR can be performed as described above. Preferred PCR cycle conditions include, for example,
~ 95 ° C for 10-20 seconds, annealing 55-60 ° C for 10-30 seconds,
Elongation 65-75 ° C 150-300 seconds cycle, more preferably denaturation 94 ° C 15 seconds, annealing 58 ° C 15 seconds, elongation 68
Cycle for 4 minutes. The obtained PCR product is cloned in the same manner as described above, and its nucleotide sequence is analyzed and determined. In addition, primers are designed based on the determined base sequence of DNA, and screening is performed by using these primers and cDNA libraries derived from various animal cells (for example, cDNA libraries derived from various human cells). Similarly, a desired clone can be obtained. In addition, PCR amplification can be performed using these primers to obtain a target gene, a novel gene, a fragment thereof, and the like. Thus, it is possible to search for a PCR product which has homology to human cathepsin Y but has a novel sequence.

Thus, according to the present invention, the desired DNA
(For example, as a recombinant phage). For example, we screened a human brain cDNA library and a human prostate cDNA library, cloned a novel cysteine protease, and clarified the entire nucleotide sequence of the cDNA encoding it. The determined total length of the nucleotide sequence of the DNA of the isolated gene is 1517 bp
It can be seen that the sequence was obtained as shown in SEQ ID NO: 2 in the sequence listing. GENBANK / EMBL DNA D
Using ata Base, the nucleotide sequence described in SEQ ID NO: 2 in the sequence listing was searched, and no identical sequence was found.
In this isolated and identified 1517 bp DNA sequence,
The presence of an open reading frame encoding a putative 484 amino acids is confirmed, and the deduced amino acid sequence is as shown in SEQ ID NO: 1 in the sequence listing. This putative protein has less than 40% homology to other known human cathepsins, ranging from about 37% homology to human cathepsin L2 to about 26% homology to human cathepsin B ( In addition, Schistosoma ma
nsoni's cysteine protease is 48% homologous), which shows the structural features typical of such cathepsins, a novel human cathepsin that is referred to as "human cathepsin Y". I named it.

It is clear that the human cathepsin Y gene encodes a protein belonging to the papain family of a novel cysteine protease. Transformants and transfectants obtained by transformation or transfection with the plasmid are also novel. The nucleic acid having all or a part of the base sequence represented by SEQ ID NO: 2 in the sequence listing can be obtained by chemical synthesis. In that case, the fragments may be chemically synthesized and ligated with an enzyme. In addition, as described above, a chemically synthesized fragment can be used as a primer or a probe to obtain a target sequence. The primer used in the PCR method is not particularly limited as long as it can amplify a DNA fragment containing the above site. Typically, the primer is (a) an oligonucleotide having a nucleotide sequence corresponding to an arbitrary region of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing; and Oligonucleotides having a complementary base sequence to any of the indicated base sequences can be used, and more preferably (1) 5 ′ of the base sequence shown in SEQ ID NO: 2 in the sequence listing. An oligonucleotide having a base sequence corresponding to an arbitrary region on the terminal side and an oligonucleotide having a complementary base sequence to an arbitrary region on the 3 ′ end side of the base sequence shown in (2) SEQ ID NO: 2 of (2) Sequence Listing Nucleotides can be used, for example, 3 to 100, preferably 10 to
Those containing 50, more preferably 15 to 35 nucleotides are included. In addition, the PCR conditions are not particularly limited, and may be well-known conditions that are usually performed. In PCR, one cycle consisting of heat denaturation of DNA strands, annealing of primers and synthesis of complementary strands by polymerase is repeated, for example, 10 to 50 times, preferably 20 to 35 times, more preferably 25 to 30 times. Done.

Human cathepsin Y consists of 484 amino acid residues, has all the structural features of existing cathepsins, but has a long propeptide not found in other cysteine proteases. In Escherichia coli, cathepsin Y produced as a fusion protein with glutathione-S-transferase is a synthetic substrate benzyloxycarbo, which is common to the cysteine protease papain family.
nyl-Phe-Arg-7-amido-4-methylcoumarin (Z-Phe-Arg-AM
C) can be decomposed. This synthetic substrate degrading activity is
Blocked by E-64, a cysteine protease inhibitor. Therefore, from this, the newly obtained cD
It was confirmed that NA encodes a functional protease. In addition, screening of human-rodent somatic cell hybrids based on fluorescence in situ hybridization and PCR confirmed that the gene encoding human cathepsin Y was located on chromosome 11q13, and that human cathepsin W was encoded. Have been found to be in close proximity to the region. Analysis by Northern blot shows that human cathepsin Y expression sites are widely distributed in human tissues, suggesting that they are involved in normal intracellular protein metabolism. On the other hand, high expression is seen in skeletal muscle and testis, suggesting that they play some special role in these tissues. In addition, characteristically high expression was observed in some cancer cells, suggesting that this novel enzyme may be involved in cancer extension through the destruction process of deviated tissues.

The DNA fragment obtained according to the present invention is converted into a suitable vector, for example, a plasmid, as described in detail below.
It can be integrated into a vector such as pEX, pMAMneo, or pKG5 and expressed in a suitable host cell as described in detail below, for example, E. coli, yeast, CHO cells, COS cells, and the like. The DNA fragment may be used as it is or as a DNA fragment to which an appropriate control sequence is added, or may be incorporated into an appropriate vector and introduced into an animal to produce a transgenic animal expressing the cathepsin Y gene, for example, human cathepsin Y. Can be created. As an animal,
Examples include warm-blooded animals, such as mice, rats, rabbits, guinea pigs, and cows. Preferably,
A transgenic animal can be prepared by introducing the DNA fragment into a fertilized egg of an animal such as a mouse.

Confirmation of the human cathepsin Y gene product was carried out using the COS-transfected human cathepsin Y gene.
This can be performed using animal cells suitable for it, such as one cell. This foreign gene can be introduced into animal cells such as mammals by a method known in the art or a method substantially similar thereto. For example, the calcium phosphate method (for example, FL Graham et al., V.
irology, 52: 456, 1973), DEAE-dextran method (for example, D. Warden et al., J. Gen. Virol., 3:37).
1, 1968, etc.), electroporation method (for example,
E. Neumann et al., EMBO J, 1: 841, 1982), microinjection method, ribosome method, virus infection method, phage particle method and the like. Gene products produced by animal cells transfected with the human cathepsin Y gene can be analyzed by immunoprecipitation experiments using an anti-human cathepsin Y monoclonal antibody or by Western blotting.

As plasmids incorporating the human cathepsin Y gene, host cells commonly used in genetic engineering (eg, prokaryotic host cells such as Escherichia coli and Bacillus subtilis, eukaryotic host cells such as yeast, CHO cells and COS cells, Sf21, etc.) Any plasmid can be used as long as it can express the DNA in an insect cell host. In such a sequence, for example, a codon suitable for expression in a selected host cell can be introduced, a restriction enzyme site can be provided, and expression of a target gene can be easily performed. Linkers and adapters that are useful for binding the target gene, such as regulatory sequences and promoter sequences, and sequences that are useful for controlling antibiotic resistance, metabolism, selecting, etc. Etc. can be included. Preferably, a suitable promoter, for example, a plasmid using Escherichia coli as a host, tryptophan promoter (trp), lactose promoter (lac), tryptophan lactose promoter (tac), lipoprotein promoter (lpp), λ phage PL promoter, etc.
SV40 rate promoter, MMTV LTR promoter, RSV LTR promoter, CMV promoter, SRα promoter and the like can be used for a plasmid using animal cells as a host, and GAL1, GAL10 promoter and the like can be used for a plasmid using yeast as a host.

As a plasmid using Escherichia coli as a host,
For example, pBR322, pUC18, pUC19, pUC118, pUC119, pSP6
4, pSP65, pTZ-18R / -18U, pTZ-19R / -19U, pGEM-3, pG
EM-4, pGEM-3Z, pGEM-4Z, pGEM-5Zf (-), pBluescrip
t KS ^TM (Stratagene), pGEX-3X (Pharmacia LKB) and the like. Plasmid vectors suitable for expression in E. coli include pAS, pKK223 (Pharmacia), pMC1403,
pMC931, pKC30, pRSET-B (Invitrogen) and the like are also included. Plasmids using animal cells as hosts include SV40 vectors, polyoma virus vectors, vaccinia
Viral vectors, retroviral vectors and the like, for example, pcD, pcD-SRα, CDM8, pCEV4, pME18S,
pBC12BI, pSG5 (Stratagene) and the like. As plasmids using yeast as a host, YIp-type vectors, YE
Examples include a p-type vector, a YRp-type vector, and a YCp-type vector, such as pGPD-2. When the host cell is E. coli, examples of the host cell include those derived from E. coli K12 strain, such as NM533 XL1-Blue, C6
00, DH1, DH5, DH11S, DH12S, DH5α, DH10B, HB
101, MC1061, JM109, STBL2, BL21 (DE3) pLysS and the like. When the host cell is an animal cell, for example, African green monkey fibroblast-derived COS-7 cells, COS-1 cells, CV-1 cells, mouse fibroblast-derived COP cells, MOP
Cells, WOP cells, Chinese hamster cells
CHO cells, CHO DHFR ^- cells, human HeLa cells, mouse cell-derived C127 cells, mouse cell-derived NIH 3T3 cells, and the like. Insect cells include silkworm nuclear polyhedrosis virus
(Bombyx mori nuclear polyhedrosis virus) or a vector derived therefrom, using silkworm larvae or silkworm culture cells, such as BM-N cells.

In the genetic engineering method of the present invention, a restriction enzyme, reverse transcriptase, or a DNA fragment known or widely used in the art is modified or converted into a structure suitable for cloning. DNA modifying / degrading enzymes, DNA polymerases, terminal nucleotidyl transferases, DNA ligases, etc. can be used. Restriction enzymes include, for example, RJ Roberts, Nuc
leic Acids Res., 13: r165, 1985; S. Linn et al. ed.
Nucleases, p. 109, Cold Spring Harbor Lab., Cold
Spring Harbor, New York, 1982; RJ Roberts, D. M
acelis, Nucleic Acids Res., 19: Suppl. 2077, 1991
And the like. As a reverse transcriptase,
For example, mouse Moloney leukemia virus (mouse Moloney
leukemiavirus (MMLV) derived reverse transcriptase (reverse tra
nscriptase), chicken myeloblastosis virus (avian mye
loblastosis virus (AMV) -derived reverse transcriptase. As the reverse transcriptase, an RNase H-deficient mutant or the like can be preferably used, and particularly, a modified MM lacking RNase H activity.
LV RT can be preferably used, and more preferably those having high thermal stability. Suitable reverse transcriptases include MMLV RT (G
ibco-BRL), Superscript RT plus (Life Technologie
s).

As DNA polymerase, for example, E. coli
DNA polymerase, its derivative Klenow fragment, Escherichia coli phage T4 DNA polymerase, Escherichia coli phage T7 DNA polymerase, thermostable DNA polymerase and the like. Examples of the terminal nucleotidyltransferase include R. Wu et al. Ed., "Methods in Enz
ymology ", Vol. 100, p. 96, Academic Press, New Yor
k (1983) 3'-OH terminal deoxynucleotide
TdTase to which (dNMP) is added. DNA modification
Examples of the degrading enzyme include exonuclease, endonuclease, and the like. , Micrococcus (Micr
ococcus) nuclease and the like. Examples of the DNA ligase include Escherichia coli DNA ligase and T4 DNA ligase. Cloning DNA gene and DN
A Suitable vectors for constructing the library include plasmids, lambda phage, cosmids, P1 phage,
Factor F, YAC and the like, preferably a vector derived from λ phage, such as Charon4A and Charon 21
A, λgt10, λgt11, λDASHII, λFIXII, λEMBL3,
λZAPII ^™ (Stratagene) and the like.

The transformant transformed with the expression vector containing the nucleic acid encoding the protein of the present invention can stably maintain high expression ability by repeatedly cloning using an appropriate selection marker if necessary. Cell lines can be obtained. For example, in a transformant using an animal cell as a host cell, when the dhfr gene is used as a selection marker, the protein of the present invention is encoded by gradually increasing the MTX concentration and culturing to select a resistant strain. By amplifying DNA, a cell line that can obtain higher expression can be obtained. The transformant of the present invention can be cultured under conditions under which the nucleic acid encoding the protein of the present invention can be expressed to produce and accumulate the desired product. The transformant can be cultured in a medium commonly used in the art. For example, a transformant using a prokaryotic host such as Escherichia coli or Bacillus subtilis or a yeast as a host can suitably use a liquid medium. The medium contains a carbon source, a nitrogen source, inorganic substances and the like necessary for the growth of the transformant. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn steep liquor, peptone, casein,
Inorganic or organic substances such as meat extract, malt extract, soybean meal, and potato extract, and examples of the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, calcium carbonate, and the like. Further, yeast, vitamins, casamino acids, growth promoting factors and the like may be added. If necessary, an agent such as 3β-indolylacrylic acid can be added to make the promoter work efficiently. The pH of the medium is preferably about 5 to 8.

Cultivation is usually carried out at about 15 to 45 ° C. for E. coli, for example.
For about 3 to 75 hours, and if necessary, ventilation or stirring may be added. When culturing a transformant whose host is an animal cell, for example, a MEM medium containing about 5 to 20% fetal bovine serum, a PRMI1640 medium, a DMEM medium, or the like is used as a medium. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 72 hours, and if necessary, aeration or stirring is added. When extracting from the cultured cells,
After culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and disrupted by ultrasonication, lysozyme, and / or freeze-thawing, etc., followed by centrifugation or filtration. A method of obtaining an extract can be used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (trade name) or Tween-80 (trade name). When the target product is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected. The target product contained in the culture supernatant or the extract thus obtained can be purified by appropriately combining known separation and purification methods, for example, by a salt such as ammonium sulfate precipitation method. Precipitation, gel filtration by Sephadex, etc., for example, ion-exchange chromatography using a carrier having a diethylaminoethyl group or a carboxymethyl group, etc., for example, a carrier having a hydrophobic group such as a butyl group, an octyl group, or a phenyl group. The hydrophobic chromatography method used, the dye gel chromatography method,
It can be obtained by purification by electrophoresis, dialysis, ultrafiltration, affinity chromatography, high performance liquid chromatography, and the like. Preferably, it can be purified and separated by treatment with polyacrylamide electrophoresis, affinity chromatography in which an antibody specifically reacting with an antigen such as a monoclonal antibody is immobilized, or the like. For example, gelatin-agarose affinity
Chromatography, heparin-agarose chromatography and the like.

Further, the human cathepsin Y according to the present invention
By using a method commonly used in genetic engineering based on the nucleotide sequence of the gene, substitution, deletion, insertion, transfer, or addition of one or more amino acids was appropriately performed in the amino acid sequence of human cathepsin Y. Corresponding proteins having such mutations introduced can be produced. Such mutation, conversion, and modification methods are described in, for example, The Society of Biochemistry, Japan, “Seismological Chemistry Laboratory Course 1, Gene Research Method II”, p105 (Susumu Hirose), Tokyo Kagaku Dojin (1986);
"New Chemistry Experiment Course 2, Nucleic Acid III (Recombinant DNA Technology)", p233 (Susumu Hirose), Tokyo Chemical Dojin (199)
2); R. Wu, L. Grossman, ed., "Methods in Enzymol
ogy ", Vol. 154, p. 350 & p. 367, Academic Press, N
ew York (1987); R. Wu, L. Grossman, ed., "Methods i
n Enzymology ", Vol. 100, p. 457 & p. 468, Academic
Press, New York (1983); JA Wells et al., Gen
e, 34: 315, 1985; T. Grundstroem et al., Nucleic Ac
ids Res., 13: 3305, 1985; J. Taylor et al., Nucle
ic Acids Res., 13: 8765, 1985; R. Wu ed., "Methods
in Enzymology ", Vol. 155, p. 568, Academic Press,
New York (1987); AR Oliphant et al., Gene, 4
4: 177, 1986 and the like. For example, position-directed mutagenesis using synthetic oligonucleotides (site-directed mutagenesis), Kunkel method, dNTP [αS]
Method (Eckstein), a region-directed mutagenesis method using sulfurous acid, nitrous acid, or the like.

The obtained protein of the present invention can be modified by modifying the amino acid residues contained in the protein by a chemical method. Can be modified or partially decomposed into a derivative thereof using the above enzyme. Proteins of the present invention, C-terminal, usually a carboxyl group (-COOH) or carboxylate (-COO ^-) is a, C-terminal, may be an amide (-CONH ₂₎ or ester (-COOR). Here, R in the ester is, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl and cyclohexyl, for example, phenyl A C _6-12 aryl group such as α-naphthyl, for example, a phenyl-C _1-2 alkyl group such as benzyl and phenethyl or α-naphthylmethyl such as α-naphthylmethyl
In addition to a C _7-14 aralkyl group such as -naphthyl-C _1-2 alkyl group, a pivaloyloxymethyl group widely used as an oral ester and the like are used. The protein of the present invention
When the compound has a carboxyl group (or carboxylate) at a position other than the C-terminal, the protein of the present invention includes a carboxyl group amidated or esterified. Examples of the ester in this case include those described above.
C-terminal esters and the like are used.

Further, in the protein of the present invention, the amino group of the N-terminal methionine residue in the above-mentioned protein may have a protecting group (for example, C-formyl group, acetyl or the like).
C _1-6 acyl group such as a carbonyl group) - _1-5 alkyl
, A glutamyl group formed by cleavage of the N-terminal side in vivo and pyroglutamylation, a substituent on the side chain of an amino acid in the molecule (for example, -OH, -COOH,
Amino group, imidazole group, indole group, guanidino group, etc.) are protected with a suitable protecting group (eg, formyl group, C _1-6 acyl group such as acetyl group, etc.),
Alternatively, a complex protein such as a so-called glycoprotein having a sugar chain bonded thereto is also included. Alternatively, it may be expressed as a fusion protein when produced by a genetic recombination method, and converted and processed into a substance having a biological activity substantially equivalent to that of natural human cathepsin Y in vivo or in vitro. . A fusion production method commonly used in genetic engineering can be used, and such a fusion protein can be purified by affinity chromatography or the like using the fusion portion. For example, modification and alteration of the structure of a protein are described in, for example, “The New Chemistry Experiment Course 1, Protein VII, Protein Engineering” edited by The Biochemical Society of Japan, Tokyo Chemical Dojin (19)
93), the method described therein or the method described in the literature cited therein, or a method substantially similar thereto can be used. As described below, the biological activity may include having an immunological activity, for example, having antigenicity.

Thus, the human-derived protein of the present invention is one in which one or more amino acid residues differ from the natural protein in terms of identity, and one in which the position of one or more amino acid residues differs from the natural protein. There may be. The human-derived protein of the present invention has one or more amino acid residues unique to human cathepsin Y (for example, 1 to 100, preferably 1 to 60).
, More preferably 1 to 40, more preferably 1 to
A deletion analog lacking 20, particularly 1 to 10, etc., one or more (eg, 1 to 100, preferably 1 to 60, more preferably 1 to 90 amino acid residues specific to the human cathepsin Y) Is 1 to 40, more preferably 1 to 20, especially 1 to
Substituted analogs in which 10 residues are substituted with other residues, 1
Or more (e.g., 1 to 100, preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20,
In particular, addition analogs to which 1 to 10 amino acid residues have been added are also included. As long as the domain structure or enzymatic activity center structure characteristic of natural human cathepsin Y is maintained, all of the above mutants are included in the present invention. It is considered that the human cathepsin Y of the present invention may also include those having a primary structural conformation substantially equivalent to that of natural human cathepsin Y or a part thereof. It is considered that those having substantially the same biological activity (eg, cysteine protease activity or antigenicity) may be included.

Further, it may be one of naturally occurring mutants. The human-derived protein of the present invention has, for example, the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, those having the 271st to 484th amino acid sequences, and the 20th to 484th amino acid sequences. An amino acid sequence selected from the group consisting of those having an amino acid sequence and those having the first to 484th amino acid sequences;
% Or more, preferably 80% or more homology, more preferably 90% or more.
% Or more.
A part of the human-derived protein of the present invention is a partial peptide of the human-derived protein (that is, a partial peptide of the protein), which is substantially equivalent to the human cathepsin Y of the present invention. Any substance may be used as long as it has activity. For example, the partial peptide of the protein of the present invention has at least five or more, preferably at least five, of the constituent amino acid sequences of human cathepsin Y of the present invention.
20 or more, more preferably 50 or more, more preferably
70 or more, more preferably 100 or more, in some cases
Peptides having an amino acid sequence of 200 or more, for example, those having the same homology as described above with respect to homology to the corresponding region in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.

As used herein, “substantially equivalent” means that the activities of the proteins, for example, catalytic activity, physiological activity, and biological activity are substantially the same. Furthermore, the meaning of the term may include the case where the substance has substantially the same activity. Examples of the substantially same activity include, for example, protease activity, degradation to a synthetic substrate for cysteine protease. Activity and the like. The substantially equivalent activity indicates that those activities are the same in nature, for example, physiologically,
Indicates that it is pharmacologically or biologically equivalent. For example, activities such as protease activity are equivalent (for example, about 0.001 to 1000 times, preferably about 0.01 to 100 times,
More preferably about 0.1 to 20 times, even more preferably about 0.5 times.
However, the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different. Second, amino acid substitutions, deletions, or insertions often do not result in significant changes in the physiological or chemical properties of the polypeptide, in which case the substitutions, deletions, or insertions are made. The polypeptide is
Would be substantially identical to those without such substitutions, deletions, or insertions. Substantially identical substitutions of amino acids in the amino acid sequence may be selected from other amino acids of the class to which the amino acid belongs. For example, non-polar (hydrophobic)
Amino acids include alanine, phenylalanine, leucine, isoleucine, valine, proline, tryptophan, methionine, and the like, and polar (neutral) include glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, and the like. Amino acids having a positive charge (basic amino acids) include arginine, lysine, histidine and the like, and amino acids having a negative charge (acidic amino acids) include aspartic acid and glutamic acid.

The protein of the present invention and a partial peptide thereof can be synthesized by a method known in the field of peptide synthesis,
For example, a chemical synthesis method such as a liquid phase synthesis method and a solid phase synthesis method can be used. In such a method, for example, a resin for protein or peptide synthesis is used, and appropriately protected amino acids are sequentially bonded to a desired amino acid sequence on the resin by various condensation methods known per se. For the condensation reaction, various known activating reagents are preferably used. As such a reagent, for example, carbodiimides such as dicyclohexylcarbodiimide can be preferably used. When the product has a protecting group, the desired product can be obtained by appropriately removing the protecting group.
When the protein of the present invention and some of its peptides are obtained in a free form, they can be converted into salts by a method known per se or a method analogous thereto, and they can be converted into salts. When obtained, it can be converted to a free form or another salt by a method known per se or a method analogous thereto. The salts of the protein of the present invention and some of its peptides are preferably physiologically acceptable or pharmaceutically acceptable, but are not limited thereto. Such salts include:
For example, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., for example, acetic acid, formic acid, maleic acid, fumaric acid, succinic acid, citric acid, tartaric acid, malic acid, benzoic acid, methanesulfonic acid And salts with organic acids such as p-toluenesulfonic acid and benzenesulfonic acid. Examples of the salt further include ammonium salts, for example, salts with organic bases such as ethylamine, dimethylamine, trimethylamine, and hydroxyethylamine.

The human cathepsin Y of the present invention and its mutants, modifications, derivatives and the like can be subjected to the separation / purification treatment as described above. The thus-obtained natural human cathepsin Y of the present invention or a protein or a salt thereof having substantially the same activity as the protein or a salt thereof, or a partial peptide thereof can be used to develop or search for an enzyme inhibitor. Research, drug development research, and biological phenomena and reactions that may involve human cathepsin Y, and can be used to generate antibodies against it, as well as specific assays. Alternatively, it can be used for researching an object to be measured. Human cathepsin Y is a type of cysteine protease that is thought to be involved in many normal cellular processes, including intracellular protein metabolism, activation of hormone precursors, and bone modification. And is useful for studying reactions. Human cathepsin Y is also useful for elucidating its function in many diseases, such as Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, neurodegenerative diseases and cancer invasion and metastasis, especially in relation to cancer It is expected to be extremely useful for elucidating the pathogenesis of these diseases, treating and developing therapeutic drugs. On the one hand, the invention thus also encompasses the DNA sequences coding for the above-mentioned polypeptides and the human cathepsin Y polypeptides having all or part of the natural properties, as well as the DNA sequences coding for their analogs or derivatives.

Since the DNA sequence of the present invention provides information on the amino acid sequence of a previously unknown mammalian protein, the use of such information is also encompassed by the present invention. Such uses include, for example, the design of probes for the isolation and detection of genomic DNA and cDNA from mammals, especially humans, encoding human cathepsin Y and related proteins.
The DNA sequences of the present invention are useful, for example, as probes for the isolation and detection of genomic DNA and cDNA from mammals, particularly preferably mice and humans, encoding human cathepsin Y and related proteins. The probe can be provided with the label described in relation to the antibody, if necessary. For the isolation of the gene, a PCR method and a PCR method using reverse transcriptase (RT) (RT-PCR) can be used. Human cathepsin Y cDNA and its related DNA
Is cloned and sequenced human cathepsin
Y Select a characteristic sequence region based on the amino acid sequence deduced from the cDNA sequence, design a DNA primer, synthesize chemically, and use the obtained DNA primer to perform PCR, RT
-It can be used for the isolation and detection of human cathepsin Y-related gene using PCR and other methods. For example,
Expression of human cathepsin Y mRNA in human tissues can be examined by Northern blot analysis on poly (A) ⁺ RNA derived from various tissues. When the cDNA of the present invention is used as a probe, the expression and expression of human cathepsin Y mRNA in human tissues and the human cathepsin Y gene itself can be detected and measured by, for example, Northern blotting, Southern blotting, in situ hybridization, and the like. The role of cysteine proteases involved in many normal cellular processes, including intracellular protein metabolism in human tissues, activation of hormone precursors, and bone modification, Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis And contribute to the development of research on many diseases such as neurodegenerative diseases and cancer invasion / metastasis. It can also be used for genetic diagnosis of diseases related to human cathepsin Y. Such a diagnosis can be for diagnosing an abnormality in the nucleic acid encoding the human cathepsin Y and related proteins, such as damage, mutation, decreased expression, overexpression, and the like.

When transferring the DNA obtained in the present invention (for example, DNA encoding human cathepsin Y) to a subject animal, it is ligated as a DNA fragment or downstream of a promoter capable of expressing the DNA in animal cells. It is generally advantageous to use them. For example, when human cathepsin Y DNA is introduced into a mouse, a gene construct in which human cathepsin Y DNA derived from an animal having a high homology to the gene is bound downstream of various promoters capable of expressing the same in animal cells is used as a fertilized egg of the target animal. For example, microinjection into mouse fertilized eggs can produce transgenic mice that produce high human cathepsin Y. Although the mouse is not particularly limited to a pure mouse, for example, C57BL / 6, Balb / C, C3H,
(C57BL / 6 × DBA / 2) F ₁ (BDF ₁ ). Examples of the promoter include a virus-derived promoter,
Ubiquitous expression promoters such as metallothionein and the like can be preferably used. The human cathepsin Y DNA
When introducing a recombinant retrovirus,
It can also be performed using it. Preferably, a mouse fertilized egg into which the target DNA has been introduced can be grown using a foster parent mouse such as ICR. Transfer of the DNA obtained in the present invention (for example, DNA encoding human cathepsin Y) at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target animal. The presence of the DNA encoding human cathepsin Y in the germ cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the DNA encoding the human cathepsin Y in all of the germ cells and somatic cells. means. The offspring of this type of animal that inherited the gene have the human cathepsin Y in all of its germinal and somatic cells.
Has the potential to be expressed.

The animal into which the human cathepsin Y DNA was introduced was confirmed to stably maintain the gene by mating.
The breeding subculture can be performed in a normal breeding environment as a holding animal. Furthermore, by crossing male and female animals having the target DNA, homozygous animals having the transgene on both homologous chromosomes are obtained, and by crossing the male and female animals, all offspring have the DNA. Breeding can be subcultured. Since the animal into which the human cathepsin Y DNA is introduced has high expression of the human cathepsin Y protein, it is useful as an animal for screening for an inhibitor (inhibitor) for the human cathepsin Y protein. It is also useful as an antisense oligonucleotide capable of inhibiting the replication of the human cathepsin Y gene, for example, an animal for screening antisense DNA and the like. This transgenic animal can also be used as a cell source for tissue culture. For example, a protein related to the papain family of cysteine protease can be analyzed by directly analyzing DNA or RNA in the tissue of a transgenic mouse or by analyzing a protein tissue expressed by the gene. The cells of the tissue having the cathepsin Y are cultured by standard tissue culture techniques and used to determine the function of cells from generally difficult-to-cultivate tissues from, for example, the thymus, testis, brain, intestine, kidney and other tissues. Can be studied. Further, by using the cells, it is possible to contribute to the development of a drug that enhances the functions of various tissues, for example. In addition, if there is a high expression cell line, cathepsin Y can be isolated and purified therefrom. Techniques related to transgenic mice and the like include, for example, Brinster, RL, et al.,;
Proc. Natl. Acad. Sci. USA, 82: 4438, 1985; Costan
tini, F. & Jaenisch, R. (eds): Genetic manipulatio
n of the early mammalian embryo, Cold Spring Harbo
The method can be carried out by a method described in a document such as r Laboratory, 1985, a method described in a document cited therein, or a modified method thereof.

The gene obtained in the present invention (for example, coding for mouse cathepsin Y corresponding to human cathepsin Y)
Mutant mice (knockout mice) that have mutations in DNA) and do not express mouse cathepsin Y at all can be produced. For example, before and after the translation initiation codon of the gene
Neo resistance gene-polyA is located in the exon near the center of the genomic DNA of about 8 kb including 4 kb and near the translation initiation codon.
A targeting vector having a mutant gene into which a gene cassette consisting of an additional signal has been inserted can be constructed. The gene cassette to be inserted includes a DT-A cassette, a tk cassette, a lacZ cassette and the like in addition to the neo resistance gene cassette. The targeting vector is opened linearly, and established mouse embryonic stem cells
s: ES cells) by electroporation and further culturing to select ES cells that have acquired neo resistance. ES cells can be prepared by selecting from mouse strains such as 129, C57BL / 6 and F1 (C57BL / 6 × CBA) mice. It is assumed that the ES cells that have acquired neo resistance have undergone homologous recombination with the targeting vector into which the gene cassette has been inserted in the mouse cathepsin Y gene region. Cannot be expressed normally. For the selection, an appropriate method is selected depending on the inserted gene cassette, and the introduction of the mutation can be confirmed by a method such as PCR, Southern hybridization or Northern hybridization.

The ES cells into which the mutation was introduced were C57BL / 6, BALB
/ c, injected into 8-cell stage embryos taken from ICR mice, etc.
Individuals can be grown by culturing for one day and then developing the blastocyst and transplanting it to a foster parent such as ICR. Born offspring mice are chimeric mice derived from ES cells having mutations and normal host embryos, and the amount of cells derived from ES cells is determined by the coat color of the individual. Therefore, it is desirable that the ES cell and the host embryo are a combination of lineages having different coat colors. Mutations in the obtained chimeric mice are heterozygous, and homozygous mutant mice can be obtained by crossing them appropriately.
The homozygous mutant mouse thus obtained has only the mouse cathepsin Y gene disrupted in all germ cells and somatic cells and does not express mouse cathepsin Y at all. Have. This knockout mouse is useful for analyzing the role of cathepsin Y in the life cycle of an individual such as development, growth, reproduction, aging and death, and the function of cathepsin Y in various organs and tissues, as compared with normal mice. In addition, it can be applied to drug development related to cysteine protease. Knockout mice can be used not only as model animals but also as a cell source for tissue culture, and can be used for functional analysis of cathepsin Y at the cell level. Techniques relating to knockout mice and the like include, for example, Mansour, SL, et al.,; Nature, 3
36: 348-352, 1988; Joyner, AL, ed .; Gene target
ing, IRL Press, 1993; Shinichi Aizawa, Generation of Mutant Mice Using Gene-Targeting ES Cells, Methods Described in Literatures such as Yodosha, 1995, or Methods Described in Literatures Cited There, It can be performed by a modified method.

According to the present invention, an antisense oligonucleotide (nucleic acid) capable of inhibiting the replication or expression of the cathepsin Y gene is used as the base sequence information of the cloned or determined DNA encoding cathepsin Y. Can be designed and synthesized based on Such an oligonucleotide (nucleic acid) is the cathepsin Y gene RNA (or
DNA) and can inhibit the synthesis or function of the RNA, or regulate and control the expression of the cathepsin Y gene through interaction with a cathepsin Y-related RNA. . Cathepsin
Oligonucleotides that are complementary to the selected sequence of the Y-related gene and that can specifically hybridize to the cathepsin Y-related gene regulate and control cathepsin Y gene expression in vivo and in vitro It is also useful for treating or diagnosing diseases related thereto. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. "Corresponding" between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) as directed by the nucleotide (nucleic acid) sequence or its complement. . 5 'untranslated region of the gene, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon, and
The 3 'end untranslated region can be selected as a preferred target region, but any region within the gene can be selected as a target.

The relationship between the target nucleic acid and the oligonucleotide complementary to at least a part of the target region means the relationship between the target nucleic acid and the oligonucleotide capable of hybridizing with the target, which is “antisense”. It can be said. Antisense oligonucleotides are 2-
Deoxy-D-ribose-containing polydeoxynucleotides, D-ribose-containing polydeoxynucleotides, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or others having a non-nucleotide backbone Polymers (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds, provided that the polymers are capable of base pairing and base pairing as found in DNA and RNA. Containing nucleotides having a configuration permitting attachment). They are double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and
DNA: RNA hybrids, and further unmodified polynucleotides or unmodified oligonucleotides, or even those with known modifications, such as those with a label known in the art, capped, Methylated, one or more natural nucleotides replaced by analogs, modified with intramolecular nucleotides, such as uncharged bonds (eg, methylphosphonate,
Those having a phosphotriester, phosphoramidate, carbamate, etc., those having a charged or sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as proteins (nucleases, nuclease inhibitors, toxins, Antibodies, signal peptides, poly-L-lysine, etc.) and sugars (for example,
Monosaccharides, etc.), those having side chain groups, such as those having an interactive compound (eg, acridine, psoralen, etc.), chelating compounds (eg,
Metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, and those having modified bonds (eg, α-anomeric nucleic acids, etc.) Good. Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing known purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications include methylated purines and pyrimidines,
It may contain acylated purines and pyrimidines, or other heterocycles. The modified nucleosides and modified nucleotides may also be modified at the sugar moiety, e.g., one or more hydroxyl groups are replaced with halogens, aliphatic groups, etc., or converted to functional groups such as ethers, amines, etc. May have been.

The antisense nucleic acid of the present invention can be used for RNA, DNA
Or a modified nucleic acid. Specific examples of modified nucleic acids include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is,
Making antisense nucleic acids more stable in cells,
Increase the cell permeability of the antisense nucleic acid, increase the affinity for the target sense strand, and, if toxic, reduce the toxicity of the antisense nucleic acid. Many such modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Ja
pan, 8: 247, 1992; 8: 395, 1992; ST Crooke et a
l. ed., Antisense Research and Applications, CRC Pr
ess, 1993. The antisense nucleic acid of the present invention may contain altered or modified sugars, bases or bonds, may be provided in a special form such as liposomes or microspheres, may be applied by gene therapy, It can be provided in an added form. Such additional forms include polycations such as polylysine, which acts to neutralize the charge of the phosphate backbone, and lipids, which enhance the interaction with cell membranes and increase the uptake of nucleic acids ( For example, hydrophobic substances such as phospholipid and cholesterol can be mentioned. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Examples of other groups include cap groups specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

The inhibitory activity of the antisense nucleic acid can be measured by using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention,
Alternatively, it can be examined using an in vivo or in vitro translation system of cathepsin Y. The nucleic acid can be applied to cells by various methods known per se. As described above, the research results of the present inventors provide a method for transferring a human cathepsin Y gene and a recombinant DNA molecule into a host, expressing human cathepsin Y, and obtaining a desired human cathepsin Y. Thus, according to the present invention, a recombinant or transfectant that substantially expresses the human cathepsin Y gene, a method for producing the same, and uses thereof are also provided. In another aspect, the present invention provides a protein or a salt thereof, wherein the protein or the salt thereof has substantially the same activity as natural cathepsin Y.
More preferably, a polypeptide having at least a part or all of human cathepsin Y or a salt thereof and having substantially the same activity or having substantially the same primary structure conformation as E. coli or the like is used. The present invention relates to nucleic acids such as DNA and RNA that can be expressed in prokaryotes or eukaryotes such as mammalian cells. Also such nucleic acids, especially DNs
A is (a) a sequence capable of encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing or a sequence complementary thereto;
(B) a sequence capable of hybridizing with the DNA sequence of (a) or a fragment thereof, and (c) a sequence having a degenerate code capable of hybridizing with the sequence of (a) or (b). Can be Here, the conditions for hybridization can be stringent conditions. Prokaryotes such as Escherichia coli or eukaryotes such as mammalian cells which can be transformed with such a nucleic acid and express the polypeptide of the present invention also feature the present invention.

The antibodies (for example, antisera and monoclonal antibodies) according to the present invention can be used to immunize animals by a known method using human cathepsin Y or a fragment thereof obtained according to the present invention as an immunogen, and are known in the art. Alternatively, it can be produced by a commonly used method, for example, the method of Keller and Milstein (Nature, 256: 495-97, 1975). In this method, natural human cathepsin Y, recombinant human cathepsin Y is used as the immunogen.
And a fragment obtained from a synthetic peptide or protein having an amino acid sequence selected from a region having low homology to a cysteine protease belonging to another papain family in human cathepsin Y, for example, consisting of at least three consecutive amino acids. A synthetic peptide having a partial amino acid sequence of human cathepsin Y, for example, 4-250
, Preferably a synthetic peptide having an amino acid sequence having 6 to 45, more preferably 10 to 35, amino acid residues, preferably a peptide having a sequence portion characteristic of human cathepsin Y. Can be. For example, the propeptide region of human cathepsin Y (e.g., the region from position 20 to position 270 of SEQ ID NO: 1 in the sequence listing, or a part thereof, or its vicinity),
A region containing a propeptide cleavage site (for example, the region at positions 260 to 280 in SEQ ID NO: 1 of the sequence listing, or a part or vicinity thereof), a region of active human cathepsin Y (for example, SEQ ID NO: 271 of the column list to position 271
Region 292, 295th to 326th, 333th to 365th
366th to 378th, 379th to 440th, 441th
Region or the region of the 484th position or a part thereof or its vicinity)
And so on.

Human cathepsin Y can be obtained from production cells inside and outside the body, for example, cultured cells, excised tissues, cultured tissues and the like. Tissues and cells such as brain, placenta, lung, liver, skeletal muscle, kidney and pancreas; leukemia such as leukemia K-562; lung cancer such as A549; melanoma cell lines such as G361; cultured tumor cells such as HeLa cell S3 Etc. can be obtained. Furthermore, human cathepsin Y can be obtained as recombinant human cathepsin Y, and can be obtained from human cathepsin Y-producing cells and tissues as described above by using gene recombination techniques. Human cathepsin Y prepared according to the present invention or one derived therefrom can be suitably used as an immunizing antigen. These human cathepsins
Y 2 can be purified and separated from various raw materials, for example, antigen-producing materials such as transformed cells such as cultured cells and cultured tissues by a conventionally known method as described above. The purified recombinant human cathepsin Y can be suitably used as an immunizing antigen for producing a monoclonal antibody.

Further, a peptide is synthesized based on the information of the human cathepsin Y gene obtained in the present invention, and the synthesized peptide can be suitably used as an immunizing antigen for producing an antibody (for example, a monoclonal antibody). Further, the antibodies (including monoclonal antibodies) can be appropriately labeled by a commonly used method. As the label, luminescent substances such as enzymes, radioisotopes (radioactive substances), and chemiluminescent compounds, fluorescent substances, metal colloids, prosthetic molecules, coloring substances, biotin, and the like can be used. Hereinafter, production of the antibody will be described in detail.

The monoclonal antibody of the present invention may, of course, be a monoclonal antibody obtained by utilizing a cell fusion technique using myeloma cells. The monoclonal antibody of the present invention can be produced, for example, by the following steps. 1. 1. Preparation of immunogenic antigen 2. Immunization of animals with immunogenic antigens. 3. Preparation of myeloma cells (myeloma cells) 4. Cell fusion between antibody-producing cells and myeloma cells 5. Selection and monocloning of hybridomas (fused cells) Production of monoclonal antibodies

1. Preparation of immunogenic antigenAs the antigen, recombinant human procathepsin Y prepared according to the method described above can be used.
Based on the determined information on cathepsin Y, an appropriate oligopeptide can be chemically synthesized and used as an antigen. Human cathepsin Y is also a precursor human cathepsin.
Y and activated human cathepsin Y can be used. Furthermore, it may be an immunogenic conjugate or the like, but it can be used as it is for immunizing an animal by mixing it with an appropriate adjuvant. For example, an antigen used as an immunogen is obtained by fragmenting human cathepsin Y or selecting a characteristic sequence region based on an amino acid sequence deduced from a cloned and sequenced cDNA sequence, and designing a polypeptide. It may be a synthetic polypeptide fragment obtained by chemical synthesis. In addition, the fragment is bound to various carrier proteins via an appropriate condensing agent to form an immunogenic conjugate such as a hapten-protein, which can be used to react with only a specific sequence (or only a specific sequence). Can be used to design monoclonal antibodies. A cysteine residue or the like can be added to the designed polypeptide in advance so that the immunogenic conjugate can be easily prepared. Upon binding to the carrier proteins, the carrier proteins can first be activated. For such activation, introduction of an activation bonding group may be mentioned. Examples of the activated bonding group include (1) an activated ester or an activated carboxyl group such as a nitrophenyl ester group, a pentafluorophenyl ester group, a 1-benzotriazole ester group, an N-succinimide ester group, and the like. A dithio group, for example, a 2-pyridyldithio group and the like can be mentioned. Carrier proteins include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, globulin, polypeptides such as polylysine, and bacterial cell components such as BCG.

2. Immunization of animals with immunogenic antigens To immunize animals, for example, Shigeru Muramatsu, et al., Experimental Biology Course 14, Immunobiology, Maruzen Co., Ltd., 1985, Ed. 5. Immunobiochemical research method,
This can be performed according to the method described in Tokyo Kagaku Dojin, 1986, edited by The Biochemical Society of Japan, New Chemistry Laboratory Course 12, Molecular Immunology III, antigen / antibody / complement, Tokyo Kagaku Dojin, 1992, and the like. Adjuvants used with the antigen include, for example, Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG, lipid A, liposomes, aluminum hydroxide, silica and the like. Immunization is performed using animals such as mice such as BALB / c. The dose of the antigen is, for example, about 1 to 400 μg / animal for a mouse, and is generally injected intraperitoneally or subcutaneously into a host animal, and thereafter, intraperitoneally every 1 to 4 weeks, preferably every 1 to 2 weeks. Booster immunization is repeated about 2 to 10 times internally, subcutaneously, intravenously or intramuscularly. As the mouse for immunization, besides BALB / c mice, F1 mice of BALB / c mice and other mice can also be used. If necessary, an antibody titer can be prepared, and the antibody titer can be measured to confirm the degree of animal immunity. The antibodies of the present invention may be those obtained from the immunized animals thus obtained, and include, for example, antisera, polyclonal antibodies and the like.

3. Preparation of myeloma cells (myeloma cells) Infinitely proliferative strains (tumor cell lines) used for cell fusion can be selected from cell lines that do not produce immunoglobulin. For example, P3-NS-1-Ag4-1 ( NS-1, Eur.J. Immuno
l., 6: 511-519, 1976), SP2 / 0-Ag14 (SP2, Nature, 27
6: 269-270,1978), P3-X63-Ag8-U1 derived from mouse myeloma MOPC-21 cell line (P3U1, Curr.
biol. Immunol., 81: 1-7, 1978), P3-X63-Ag8 (X63,
Nature, 256: 495-497, 1975), P3-X63-Ag8-653 (653,
J. Immunol., 123: 1548-1550, 1979). 8-Azaguanine-resistant mouse myeloma cell line is prepared by adding antibiotics such as penicillin and amikacin, fetal calf serum (FCS), etc. to cell culture medium such as Dulbecco's MEM medium (DMEM medium) and RPMI-1640 medium, and The cells are passaged in a medium supplemented with azaguanine (for example, 5 to 45 μg / ml), and the cells can be passaged in a normal medium 2 to 5 days before cell fusion to prepare a required number of cell lines. The cell line used was RPMI-
After washing with a normal medium such as 1640 medium three times or more, the cells may be cultured in a normal medium to prepare a required number of cell lines.

4. Cell fusion between antibody-producing cells and myeloma cells. An animal, for example, a mouse immunized according to the above step is spleen removed 2 to 5 days after the final immunization to obtain a spleen cell suspension. In addition to spleen cells, lymph node cells from various parts of the body can be obtained and used for cell fusion. The thus obtained spleen cell suspension and 3. The myeloma cell line obtained according to the steps of
(MEM medium), DMEM medium, RPMI-1640 medium, etc., and add a cell fusion agent such as polyethylene glycol. Other various cell fusion agents known in the art can be used, such as inactivated Sendai virus (HVJ: Hemagglutina
ting Virus of Japan). Preferably,
For example, 0.5 to 2 ml of 30 to 60% polyethylene glycol
Polyethylene glycol having a molecular weight of 1,000 to 8,000 can be used.
000-4,000 polyethylene glycol can be more preferably used. It is preferable that the concentration of polyethylene glycol in the fusion medium be, for example, 30 to 60%. If necessary, fusion can be promoted by adding a small amount of, for example, dimethyl sulfoxide. The ratio of spleen cells (lymphocytes) to myeloma cell line used for the fusion may be, for example, 1: 1 to 20: 1, and more preferably 4: 1 to 7: 1. The fusion reaction is performed for 1-10 minutes, and then a cell culture medium such as RPMI-1640 medium is added.
The fusion reaction treatment can be performed plural times. After the fusion reaction treatment, the cells are separated by centrifugation or the like, and then transferred to a selection medium.

5. Selection and Monocloning of Hybridomas (Fused Cells) As a selection medium, for example, FCS-containing MEM medium containing hypoxanthine, aminopterin and thymidine, RPMI-1
A medium such as 640 medium, a so-called HAT medium can be used. The method of replacing the selective medium can be generally performed by adding an equal volume to the volume dispensed to the culture plate on the next day, and thereafter replacing half the amount with the HAT medium every 1 to 3 days. Changes can be made to this. On the 8th to 16th day after the fusion, the so-called aminopterin was removed.
The medium can be replaced with the HT medium every 1 to 4 days.
As a feeder, for example, mouse thymocytes can also be used, which may be preferred. Culture supernatants from culture wells of hybridoma growth can be analyzed, for example, by radioimmunoassay (RIA), enzyme immunoassay (ELISA), and fluorescence immunoassay.
(FIA) or a fluorescence-induced cell separation device (F
Screening is performed by using human cathepsin Y or a fragment peptide thereof as an antigen, or by measuring a target antibody using a labeled anti-mouse antibody, for example, by ACS). The hybridoma producing the desired antibody is cloned. Cloning can be done by picking up colonies in an agar medium or by limiting dilution. It can be more preferably performed by a limiting dilution method. Cloning is preferably performed multiple times.

6. Production of monoclonal antibody The obtained hybridoma strain was used in MEM medium containing FCS, RPMI
The cells are cultured in a suitable growth medium such as -1640 medium, and the desired monoclonal antibody can be obtained from the culture supernatant. In order to obtain a large amount of antibodies, ascites of the hybridoma can be mentioned. In this case, each hybridoma is transplanted into the peritoneal cavity of a histocompatible animal of the same strain as the myeloma cell-derived animal and grown, or for example, nude or
Each hybridoma can be transplanted and expanded into a mouse or the like, and the monoclonal antibody produced in the ascites of the animal can be collected and obtained. Animals can be intraperitoneally administered mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) prior to hybridoma transplantation, after which the hybridomas are allowed to grow and ascites are collected You can also. Ascites fluid as it is or conventionally known methods such as salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, etc., ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography And purified by high performance liquid chromatography or the like and used as a monoclonal antibody. Preferably, the ascites containing the monoclonal antibody can be purified and separated by fractionating with ammonium sulfate, followed by treatment with an anion exchange gel such as DEAE-Sepharose and an affinity column such as a protein A column. Particularly preferred are affinity chromatography in which an antigen or antigen fragment (eg, a synthetic peptide, a recombinant antigen protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized, and affinity chromatography in which protein A is immobilized. No.

It is also possible to determine the sequence of the antibody obtained in such a large amount, and to prepare an antibody by a gene recombination technique using a nucleic acid sequence encoding an antibody obtained from a hybridoma strain. Furthermore, these antibodies are treated with enzymes such as trypsin, papain, and pepsin, and optionally reduced to obtain Fab, Fab ', F (ab') _2.
May be used. As the antibody to be labeled, an IgG fraction, and a specific binding portion Fab ′ obtained by digestion with pepsin and then reduction can be used. Examples of labels in these cases include enzymes (such as peroxidase, alkaline phosphatase or β-D-galactosidase), chemical substances, fluorescent substances, radioisotopes, and the like, as described below. Detection and measurement in the present invention can be performed by immunostaining, for example, tissue or cell staining, immunoassay, for example, a competitive immunoassay or a non-competitive immunoassay, and a radioimmunoassay, ELISA, or the like can be used.
The measurement may be performed with or without BF separation. Preferred are a radioimmunoassay and an enzyme immunoassay, and further include a sandwich type assay. For example, in a sandwich-type assay, one of the antibodies to human cathepsin Y is detectably labeled. Another antibody that can recognize the same antigen is immobilized on a solid phase.

Incubation is performed to react the sample with the labeled antibody and the immobilized antibody sequentially as necessary. After separating the unbound antibody, the labeled substance is measured. The amount of label measured is proportional to the amount of antigen, ie, human cathepsin Y. This assay is referred to as a simultaneous sandwich-type assay, a forward sandwich-type assay, or a reverse sandwich-type assay, depending on the order of addition of the insolubilized antibody or the labeled antibody. For example, washing,
Stirring, shaking, filtration, or pre-extraction of the antigen, etc., are appropriately employed in these measurement steps under specific circumstances. Other measurement conditions such as the concentration of specific reagents and buffers, temperature and incubation time can be changed according to factors such as the concentration of antigen in the sample and the properties of the sample. A person skilled in the art can perform the measurement by appropriately selecting the optimal conditions effective for each measurement while using a usual experimental method.

Many carriers on which an antigen or an antibody can be immobilized are known. In the present invention, they can be appropriately selected and used. As the carrier, various carriers used for antigen-antibody reactions and the like are known, and in the present invention, of course, any of these known carriers can be used. Particularly preferably used are, for example, glass, such as activated glass, porous glass, silica gel, silica-
Inorganic materials such as alumina, alumina, magnetized iron, magnetized alloys, polyethylene, polypropylene, polyvinyl chloride,
Crosslinking such as polyvinylidene fluoride, polyvinyl acetate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, cross-linked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer Natural or denatured cellulose such as albumin, collagen, gelatin, dextran, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethyl cellulose, cellulose acetate, polyamide such as cross-linked dextran, nylon, and organic polymers such as polyurethane and polyepoxy resin Substances and those obtained by emulsion polymerization of these substances, cells, erythrocytes, etc. Which are introduced are mentioned. Further, filter paper, beads, inner walls of test containers, for example, test tubes, titer plates, titer wells, cells made of synthetic materials such as glass cells, synthetic resin cells, glass rods, rods made of synthetic materials, and thicker ends. Alternatively, the surface of a solid material (object) such as a thinned rod, a rod having a round or flat projection at its end, or a thin plate-shaped rod may be used.

An antibody can be bound to these carriers, and preferably, a monoclonal antibody that specifically reacts with human cathepsin Y obtained in the present invention can be bound thereto. The binding between the carrier and those involved in the antigen-antibody reaction is performed by a physical method such as adsorption, a condensing agent, or a chemical method using an activated material, or a mutual method. It can be performed by a method utilizing a chemical binding reaction or the like. Labels include enzymes, enzyme substrates, enzyme inhibitors, prosthetic molecules,
Coenzyme, zymogen, apoenzyme, fluorescent substance, dye substance,
Examples include a chemiluminescent compound, a luminescent substance, a coloring substance, a magnetic substance, a metal particle such as a gold colloid, and a radioactive substance. As enzymes, dehydrogenase,
Oxidoreductases such as reductases and oxidases, for example, transferases that catalyze the transfer of amino groups, carboxyl groups, methyl groups, acyl groups, phosphate groups, etc., for example, ester bonds, glycoside bonds, ether bonds, peptide bonds Hydrolases, lyases, isomerases, ligases, and the like, which hydrolyze and the like. Enzymes can be used for detection by using a plurality of enzymes in combination. For example, enzymatic cycling can be used.

Representative radioisotope labeling isotopes include [ ³² P], [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C], [ ³⁵ S] and the like. . Typical enzyme labels include peroxidase such as horseradish peroxidase, E. coli β-D
-Galactosidase such as galactosidase; maleate dehydrogenase; glucose-6-phosphate dehydrogenase; glucose oxidase; glucoamylase; acetylcholinesterase; catalase; When alkaline phosphatase is used, 4-
Umbelliferone derivatives such as methyl umbelliferyl phosphate, phosphorylated phenol derivatives such as nitrophenyl phosphate, enzymatic cycling systems using NADP, luciferin derivatives, and fluorescence generated by using substrates such as dioxetane derivatives, It can be measured by light emission or the like. Luciferin and luciferase systems can also be used. When catalase is used, it reacts with hydrogen peroxide to generate oxygen, and the oxygen can be detected by an electrode or the like. The electrode may be a glass electrode, an ion electrode using a poorly soluble salt film, a liquid film electrode, a polymer film electrode, or the like. The enzyme label can be replaced with a biotin label and an enzyme-labeled avidin (streptavidin). The sign may use a plurality of different kinds of signs. In such a case, it may be possible to make multiple measurements continuously or discontinuously, and simultaneously or separately.

In the present invention, 4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine and the like and horseradish peroxidase,
Umbelliferyl galactoside, nitrophenyl galactoside, etc. and β-D-galactosidase, glucose-6-
Combinations of enzyme reagents such as phosphate and dehydrogenase can also be used. Can be used. Examples of the fluorescent substance or the chemiluminescent compound include fluorescein isothiocyanate, for example, rhodamine derivatives such as rhodamine B isothiocyanate and tetramethylrhodamine isothiocyanate, dansyl chloride, dansyl fluoride, fluorescamine, phycobiliprotein, acridinium salt, lumiferin, and luciferase. And luminols such as equorin, imidazole, oxalate, rare earth chelate compounds, coumarin derivatives and the like. Labeling can be carried out using a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, and the like. A possible method,
Further, the method can be applied by appropriately selecting from methods in which they are modified. In addition, a condensing agent that can be used for preparing the immunogenic complex, a condensing agent that can be used for binding to a carrier, and the like can be used.

Examples of the condensing agent include formaldehyde, glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, N, N'-polymethylenebisiodoacetamide, N, N'-ethylenebismaleimide, ethyleneglycolbissuccinimide Zirlsuccinate, bisdiazobenzidine, 1-ethyl-3- (3-
Dimethylaminopropyl) carbodiimide, succinimidyl 3- (2-pyridyldithio) propionate (SPD
P), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl ( Four-
Iodoacetyl) aminobenzoate, N-succinimidyl 4- (1-maleimidophenyl) butyrate, N- (ε
-Maleimidocaproyloxy) succinimide (EMC
S), iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3- (4'-dithiopyridyl) propionimidate, methyl-4-mercaptobutyrylimidate, methyl-3-mercaptopropionimidate, N- And succinimidyl-S-acetylmercaptoacetate.

According to the measurement method of the present invention, a labeled antibody reagent such as a monoclonal antibody in which a substance to be measured is labeled with an enzyme or the like and an antibody bound to a carrier can be reacted sequentially or simultaneously. You can also. The order in which the reagents are added depends on the type of carrier system chosen. In the case of using sensitized beads such as plastic, a labeled antibody reagent such as a monoclonal antibody labeled with an enzyme or the like is first put together with a sample containing the substance to be measured in an appropriate test tube, and then the sample is put into an appropriate test tube. The measurement can be performed by adding beads such as sensitized plastic. In the quantification method of the present invention, an immunological measurement method is used.In this case, as a solid support, polystyrene, polycarbonate, polypropylene or polyvinyl balls that well adsorb proteins such as antibodies,
Various materials and forms such as a microplate, a stick, a microparticle, or a test tube can be arbitrarily selected and used. For measurement, optimal pH, for example, pH about 4
It can be carried out in a suitable buffer system so as to keep at ~ 9. Particularly suitable buffers include, for example, acetate buffers, citrate buffers, phosphate buffers, Tris buffers, triethanolamine buffers, borate buffers, glycine buffers, carbonate buffers, Tris-HCl. Buffers and the like can be mentioned. Buffering agents can be used by mixing them at an arbitrary ratio. The antibody-antigen reaction is performed at about 0 ° C to 60 ° C.
It is preferred to work at a temperature between 0 ° C.

An antibody reagent such as a monoclonal antibody labeled with an enzyme or the like and an antibody reagent bound to a carrier;
In addition, the incubation of the substance to be measured
The reaction can be performed until equilibrium is reached, but at a much earlier point in time when the equilibrium of the antibody-antigen reaction is achieved, the solid phase and the liquid phase can be separated and the reaction can be stopped after a limited incubation treatment. The extent of the presence of a label, such as an enzyme, in either the phase or the solid phase can be measured. The measurement operation can be performed using an automated measurement device, and the measurement is performed by detecting a display signal generated by the substrate being converted by the action of an enzyme using a luminescence detector, a photo detector, or the like. You can also. In the antibody-antigen reaction, reagents to be used, substances to be measured, and further stabilize labels such as enzymes,
Appropriate measures can be taken to stabilize the antibody-antigen reaction itself. In addition, proteins, stabilizers, surfactants, chelating agents, etc. should be added to the incubation solution to remove non-specific reactions and reduce inhibitory effects, or to activate measurement reactions. Can be added. As a chelating agent, ethylenediaminetetraacetic acid salt (EDTA) is more preferable. It may be subjected to a blocking treatment to prevent non-specific binding reactions commonly used in the art or known to those skilled in the art, for example, normal serum proteins such as mammals, albumin, skim milk, milk fermented substances , Collagen, gelatin and the like. These methods can be used without any particular limitation as long as the purpose is to prevent a non-specific binding reaction.

As the sample to be measured by the measuring method of the present invention, any form of solution, colloid solution, non-fluid sample, etc. can be used, but a sample derived from an organism, such as thymus, testis, intestine, kidney, Brain, breast cancer, ovarian cancer, colorectal cancer, blood, serum, plasma, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, urine, other body fluid, cell culture, tissue culture, tissue homogenate, biopsy sample, Examples include a tissue and a cell. In applying these individual immunological assay methods to the assay method of the present invention, no special conditions, operations, and the like need to be set. The normal human cathepsin Y, which is a factor having cysteine protease activity of the present invention, or a human having substantially the same activity as the factor having cysteine protease activity of the present invention, by adding ordinary technical considerations of those skilled in the art to the ordinary conditions and operation methods in each method. What is necessary is just to construct the measurement system related to the protein of origin. For more information on these common technical measures,
Reviews and books can be referred to [for example, edited by Hiroshi Irie, "Radio Immunoassay", Kodansha, published in 1974; Hiroshi Irie, edited by "Radio Immunoassay", published in Kodansha, 1979; Eiji Ishikawa et al. Ed., "Enzyme Immunoassay", Medical Shoin, published in 1978; Eiji Ishikawa et al., Ed., Enzyme Immunoassay (Second Edition), Medical Shoin, published in 1982; Eiji Ishikawa, et al., "Enzyme Immunity". "Measurement Methods" (Third Edition), published by Medical Shoin, 1987; "Methods in Enzymology" Vol. 7
0 (Immunochemical Techniques (Part A)); Ibid. Vol.
73 (Immunochemical Techniques (Part B))
l. 74 (Immunochemical Techniques (Part C)); ibid
Vol. 84 (Immunochemical Techniques (Part D: Select
ed Immunoassays)); Ibid. Vol. 92 (Immunochemical Te
chniques (Part E: Monoclonal Antibodiesand General
Immunoassay Methods)); Ibid. Vol. 121 (Immunochemi
cal Techniques (Part I: Hybridoma Technology and M
onoclonal Antibodies)) (Academic Press (US
A) issuance). Epitope mapping can also be performed using the anti-cathepsin Y antibody of the present invention, particularly a monoclonal antibody, and detection and measurement of proenzymes, active enzymes, and the like can be performed using antibodies that recognize each epitope.

Thus, typically, the object of the present invention is to use a monoclonal antibody against human cathepsin Y and a monoclonal antibody against human cathepsin Y for use as a solid support, or furthermore, if necessary, a human cathepsin Y.
An object of the present invention is to provide an excellent method for fractionating and quantifying a free precursor or active human cathepsin Y in a test sample by using an inhibitor of the present invention, and a reagent kit therefor. It is understood that the present invention includes all the reagents of the reagent kit capable of differentially quantifying such free precursor and active human cathepsin Y in its embodiments. Further, an object of the present invention is to separate and quantify free precursors and active human cathepsin Y using the above-described quantification method, thereby enabling many methods such as intracellular protein metabolism, activation of hormone precursors, and bone modification. Methods and reagents that can monitor the role of cysteine proteases involved in normal cellular processes, Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, neurodegenerative diseases and many diseases such as cancer invasion / metastasis, etc. It is to provide a diagnostic agent. Therefore, various uses of the above reagents in the medical and physiological fields, and the use of the above reagents for the purpose of research, analysis and measurement of cells and tissues of animals such as humans such as tumors and cancers are all practicing the present invention. It is understood to be included in the embodiments.

The human cathepsin Y or a salt thereof of the present invention has protease activity and is considered to be an indispensable factor in, for example, protein metabolism, antigen presentation, bone modification, activation of hormone precursor, etc. in vivo. . The protein is considered to be useful for the treatment of human cathepsin Y-related dysfunction, which has pathological conditions such as human cathepsin Y aplasia, cathepsin Y expression deficiency, and cathepsin Y gene deficiency. That is, human cathepsin Y,
Due to insufficient activity by cathepsin Y, if a drug containing a mutant, a modified product, or a derivative is used,
It is possible to make a disease patient healthy. The protein of the present invention is one of proteases, and is useful as a medicament such as an agent for treating and / or preventing various diseases caused when the expression level of the protein or the protease activity is reduced. Further, the protein of the present invention is useful as a medicament such as an agent for treating and / or preventing various diseases caused when the degrading activity for the protein is reduced. For example, if the biological activity in cells is not sufficiently obtained due to a decrease or lack of human cathepsin Y in the living body, or if the patient has abnormal symptoms, (A) the present invention (B) by administering a nucleic acid such as a DNA of the present invention to the patient and expressing the protein or the like of the present invention in vivo, (C) By transplanting cells into which the nucleic acid such as the DNA of the present invention can be expressed so that the protein of the present invention can be replenished in a living body, the symptoms in the patient can be improved.

The compound (agonist or promoter) for promoting a function such as biological activity of human cathepsin Y (eg, protease activity) or a salt thereof according to the present invention can be used for human cathepsin Y dysfunction, emphysema, muscular dystrophy, etc. It can be used as a medicament useful as an agent for treating and / or preventing various diseases such as osteoporosis, Alzheimer's disease, rheumatoid arthritis and cancer invasion / metastasis. On the other hand, a compound (antagonist or inhibitor) or a salt thereof that inhibits a function such as a biological activity of human cathepsin Y (eg, protease activity) of the present invention or a salt thereof can be used for hyperhuman cathepsin Y dysfunction, emphysema, muscular dystrophy,
It can be used as a medicament such as an agent for treating and / or preventing various diseases such as osteoporosis, Alzheimer's disease, rheumatoid arthritis and cancer invasion / metastasis. For example, human cathepsin
Y has a degrading activity on cysteine protease substrates, which activity is inhibited by cysteine protease specific inhibitors. Therefore, the inhibitor is useful as a medicament for treating a disease caused by excessive degradation by cathepsin Y.

Thus, the protein such as human cathepsin Y according to the present invention is a compound (agonist) that promotes a function such as biological activity (eg, protease activity) such as the protein such as human cathepsin Y according to the present invention. It is useful as a reagent for screening a compound (antagonist) or a salt thereof that inhibits or inhibits. Thus, using the protein such as human cathepsin Y of the present invention, a partial peptide thereof or a salt thereof, the protein such as the cathepsin Y of the present invention, a partial peptide thereof or a biological activity such as a salt thereof. Compound promoting function (eg, protease activity) (agonist)
And a method of screening for a compound that inhibits or inhibits (antagonist) or a salt thereof. In the screening, for example, (i) a case where a substrate is brought into contact with the protein of the present invention, a partial peptide thereof, or a salt thereof (which may include a transformant expressing the protein; the same applies hereinafter); (ii) A comparison is made with the case where a substrate and a test sample are brought into contact with the protein of the present invention, some of its peptides, or salts thereof. Specifically, in the above-mentioned screening, the biological activity (for example, protease activity or the like) is measured and compared. As the substrate, any substrate can be used as long as it can serve as a substrate for the protein of the present invention. For example, casein, collagen,
It can be used by selecting from those used for measuring protease activity such as synthetic oligopeptides, but preferably a fluorescently labeled synthetic peptide substrate for cysteine protease can be used, and as an example of such a peptide substrate Is, for example, benzyloxycarbonyl-L-phen
ylalanyl-L-arginine-7-amido-4-methylcoumarin (Z-Ph
e-Arg-AMC). The substrate can be used as it is, but preferably a substrate labeled with a fluorescent, enzymatic or radioactive substance such as fluorescein can be used.

The test sample includes, for example, proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, plant extracts, tissue extracts of animals and the like, and cell extracts. Examples of test compounds used in test samples may include cysteine protease inhibitors, compounds having inhibitory activity against the cysteine protease family, preferably synthetic compounds. These compounds may be novel compounds,
Known compounds may be used. The screening can be performed according to a usual method for measuring protease activity, for example, Biochemistry, 32, pp. 4330-4337 (1993)
The method can be carried out with reference to the method described in, for example. In addition, various labels, buffer systems and other appropriate reagents described in the above-mentioned measurement method using the antibody of the present invention can be used, or the measurement can be performed according to the operation described there. In the screening, the protein or the like of the present invention may be treated with an activator such as mercury aminophenylacetate, or its precursor or latent form may be converted into the active form in advance. The measurement can be usually performed in a buffer such as Tris-HCl buffer or phosphate buffer which does not adversely affect the reaction, for example, at pH 4 to 10 (preferably pH about 6 to 8). . In each of these screenings, the human cathepsin Y of the present invention or a protein or peptide having substantially equivalent activity to the human cathepsin Y of the present invention is added to the ordinary conditions and operation methods of each method with ordinary technical considerations of those skilled in the art. What is necessary is just to build a related measurement system. For details of these general technical means, reference can be made to reviews, books, and the like (for example, Methods in Enzymology, Vol.
1, 2, 5 & 6 (Preparation and Assay of Enzymes); Ibid, Vol. 3 (Preparation and Assay of Substrates);
Ibid, Vol. 4 (Special Techniques for the Enzymolog
ist); Ibid, Vol. 19 (Proteolytic Enzymes); Ibid, Vo
l. 45 (Proteolytic Enzymes, Part B); ibid, Vol. 8
0 (Proteolytic Enzymes, Part C)
ss (USA)).

Compounds or salts thereof obtained by using the screening method or screening kit of the present invention include:
Test compounds described above, for example, peptides, proteins,
It is a compound selected from non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like, and compounds that promote or inhibit the functions of the protein and the like of the present invention. Examples of the salt of the compound include pharmaceutically acceptable salts. For example,
Examples thereof include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Preferred examples of the salt with an inorganic base include, for example,
Alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt,
And aluminum salts and ammonium salts. Preferred examples of salts with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine,
Salts with dicyclohexylamine, N, N'-dibenzylethylenediamine and the like are mentioned. Preferable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. Preferred examples of salts with organic acids include, for example, formic acid, acetic acid, propionic acid, fumaric acid,
Oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid,
Salts with malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid and the like can be mentioned. Preferred examples of the salt with a basic amino acid include, for example, salts with arginine, lysine, ornithine, and preferred examples of the salt with an acidic amino acid include, for example, salts with aspartic acid, glutamic acid, and the like. Is mentioned.

Compounds which promote or inhibit the function of the protein or the like of the present invention or salts thereof are safe and low toxic to various diseases as well as the above-mentioned proteins of the present invention, partial peptides or salts thereof. Or it is useful as a prophylactic agent. When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic and / or prophylactic agent, the compound of the present invention, a partial peptide thereof or a salt thereof may be used in a conventional manner. It can be implemented according to the means. The active ingredient of the present invention (for example, (a) the protein of the present invention, a partial peptide thereof or a salt thereof, (b) a nucleic acid such as the DNA of the present invention, (c) the protein of the present invention, a part thereof (C) an antisense oligonucleotide to a nucleic acid such as DNA of the present invention, (d) a protein of the present invention, a partial peptide thereof or a salt thereof. A compound or a salt thereof that promotes or inhibits biological activity) as a medicine, for example, a protein such as human cathepsin Y of the present invention, a partial peptide thereof or a salt thereof is usually used alone or pharmacologically. Can be administered as a pharmaceutical composition or a pharmaceutical preparation by mixing with various formulation auxiliaries that are acceptable. It is preferably administered in the form of a pharmaceutical preparation suitable for use such as oral administration, topical administration, or parenteral administration, and may be administered in any form (including inhalation or rectal administration) depending on the purpose. Good.

The parenteral administration form may include topical, transdermal, intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal administration, but direct administration to the affected area is also possible.
It is also suitable in some cases. Preferably orally to mammals including humans, or parenterally (eg, intracellular,
Tissue, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathoracic, intraspinal, infusion, enema, rectal, ophthalmic or nasal,
Tooth, skin or mucous membranes). Specific forms of pharmaceutical preparations include solution preparations, dispersion preparations, semi-solid preparations, granular preparations, molded preparations, leaching preparations, and the like, for example, tablets, coated tablets, sugar-coated preparations, and pills. Agents, troches, hard capsules, soft capsules, microcapsules, implants, powders, powders, granules, fine granules, injections, liquids, elixirs, emulsions, irrigants, syrups, Solutions, emulsions, suspensions, liniments, lotions, aerosols, sprays,
Inhalants, sprays, ointments, plasters, patches, pastes, cataplasms, creams, oils, suppositories (eg, rectal suppositories), tinctures, skin solutions, eye drops, nasal drops, Powders for ear drops, liniments, infusions, injections, etc.
Lyophilized preparations, gel preparations and the like can be mentioned.

The pharmaceutical composition can be formulated according to a usual method. For example, if necessary, a physiologically acceptable carrier, a pharmaceutically acceptable carrier, an adjuvant, an excipient, a excipient, a diluent, a flavor, a flavor, a sweetener, an excipient, a vehicle, Preservatives, stabilizers, binders, pH regulators, buffers, surfactants, bases, solvents,
Fillers, extenders, solubilizers, solubilizers, isotonic agents, emulsifiers, suspending agents, dispersants, thickeners, gelling agents, curing agents,
Using an absorbent, an adhesive, an elastic agent, a plasticizer, a disintegrant, a propellant, a preservative, an antioxidant, a light-blocking agent, a humectant, an emollient, an antistatic agent, a soothing agent or the like alone or in combination, In addition, by mixing the protein of the present invention and the like, it can be produced in a unit dosage form generally required for the practice of preparations. Formulations suitable for parenteral use include sterile solutions or suspensions of the active ingredients with water or other pharmaceutically acceptable vehicles, such as injections. In general, water, saline, aqueous dextrose, other related sugar solutions, and glycols such as ethanol, propylene glycol and polyethylene glycol are preferred liquid carriers for injections. When preparing an injection, distilled water, Ringer's solution, a carrier such as physiological saline, a suitable dispersant or wetting agent and a suspending agent, etc., using a method known in the art, , Suspensions and emulsions.

Examples of the aqueous liquid for injection include physiological saline, isotonic liquid containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). A suitable acceptable solubilizer such as an alcohol (eg, ethanol),
It may be used in combination with a polyalcohol (eg, propylene glycol, polyethylene glycol, etc.), a nonionic surfactant (eg, Polysorbate 80 (TM), HCO-50, etc.). The oily liquid includes sesame oil, soybean oil and the like, and may be used in combination with a solubilizing agent such as benzyl benzoate and benzyl alcohol. Also, buffers (eg, phosphate buffer, sodium acetate buffer, etc.) or reagents for adjusting osmotic pressure, soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants such as ascorbic acid, absorption promoters and the like may be blended. The prepared injection is usually filled in a suitable ampoule.

For parenteral administration, in a sterile pharmaceutically acceptable liquid such as water, ethanol or oil, with or without the addition of surfactants and other pharmaceutically acceptable auxiliaries. In the form of a solution or suspension. Examples of the oily vehicle or solvent used in the preparation include natural or synthetic or semi-synthetic mono- or di- or triglycerides, natural, semi-synthetic or synthetic oils or fatty acids, such as peanut oil, corn oil, Vegetable oils such as bean oil and sesame oil are included. For example, this injection can be usually prepared so as to contain about 0.1 to 10% by weight of the compound of the present invention. Formulations suitable for topical, e.g., oral, or rectal use include, for example, mouthwashes, dentifrices, mouth sprays,
Examples include inhalants, ointments, dental fillers, dental coatings, dental pastes, suppositories and the like. Mouthwashes and other dental agents are prepared by a conventional method using a pharmacologically acceptable carrier. The oral spray or inhalant can be dissolved in a solution for aerosol or nebulizer together with the compound of the present invention itself or a pharmacologically acceptable inert carrier, or can be administered as fine powder for inhalation to teeth. The ointment is prepared by a conventional method by adding a commonly used base, for example, an ointment base (white petrolatum, paraffin, olive oil, macrogol 400, macrogol ointment, etc.).

Drugs for topical application to teeth and skin can be formulated as solutions or suspensions in suitably sterilized aqueous or non-aqueous vehicles. Additives include, for example, buffers such as sodium bisulfite or disodium edetate; preservatives including bactericidal and antifungal agents such as phenylmercuric acetate or benzalkonium chloride or chlorohexidine; And thickeners. Suppositories are carriers well known in the art, preferably non-irritating suitable excipients, such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc., which are preferably solid at room temperature but at intestinal temperature. It is prepared by a conventional method using a liquid which melts in the rectum and releases the drug, and is usually prepared so as to contain about 0.1 to 95% by weight of the compound of the present invention. The drug, depending on the excipient and concentration used, can either be suspended or dissolved in the excipient. Adjuvants such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle. Formulations suitable for oral use include, for example, solid compositions such as tablets, pills, capsules, powders, granules and troches, and liquid compositions such as solutions, syrups and suspensions. No. When preparing a formulation,
A formulation auxiliary known in the art is used. Tablets and pills can also be prepared with enteric coating. When the preparation unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material.

Further, when the nucleic acid such as the DNA of the present invention is used as a therapeutic and / or prophylactic agent as described above, the nucleic acid can be used alone or can be obtained by the above-mentioned genetic recombination technique. It can be used by binding to an appropriate vector to be used, for example, a virus-derived vector such as a retrovirus-derived vector, or the like. The nucleic acid such as the DNA of the present invention can be administered by a commonly known method, and until then, or together with a suitable auxiliary agent or a physiologically acceptable carrier, for example, so that uptake into cells is promoted. And can be used as a pharmaceutical composition or a pharmaceutical preparation as described above. Also, a method known as gene therapy can be applied. The active ingredient of the present invention can be administered by selecting its dosage over a wide range.
Age, weight, general health, diet, administration time, administration method, excretion rate, combination of drugs, the degree of medical condition of the patient being treated at that time, and these or other factors are determined. . In the manufacture of pharmaceuticals, their additives and preparation methods are described, for example, in the Japanese Pharmacopoeia Commentary Editing Committee, 13th Revised Japanese Pharmacopoeia Commentary, published on July 10, 1996, Hirokawa Shoten Co., Ltd .; Takashi Ichigase Other editions Development of Pharmaceuticals, Volume 12, Pharmaceutical Ingredient [I], Published October 15, 1990, Hirokawa Shoten Co., Ltd .; Development of Pharmaceuticals, Volume 12, Pharmaceutical Materials [II] It can be appropriately selected and applied as necessary from the description of Hirokawa Shoten Co., Ltd. issued on October 28, 2010 as needed.

By utilizing the above-mentioned various aspects of the present invention, the present invention relates to the diagnosis of many diseases such as Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, neurodegeneration and cancer invasion / metastasis. Various technical means can be provided as a test / measurement means useful for research or applied to other medical and physiological uses. In the description and drawings, the terms are based on IUPAC-IUB Commission on Biochemical Nomenclatu
re, or based on the meaning of a term commonly used in the art. The main abbreviations used in this specification are shown below. bp: base pair (s); E-64: trans-epoxysuccinyl-L-leucylamido- (4-guanidi
no) butane; EST: expressed sequence tag; GST: glutathione S-transferase; IPTG: isopropyl-1-thio- β-D-galactopyranoside; PAGE: polyacrylamide gel electrophoresis; PCR: polymerase chain reaction; SDS: sodium dodecyl sulfate; Z -Phe-Arg-AMC: benzyloxycarbonyl-L-phenylalanyl-L-
arginine-7-amido-4-methylcoumarin

[0116]

The present invention will be described in detail with reference to the following examples. However, it is understood that the present invention is not limited to these examples, and that various embodiments based on the concept of the present specification are possible. It should be. In the following examples, unless otherwise specified, specific operations and processing conditions are not limited to those described in T. Maniatis et al., "Molec
ular Cloning ", 2nd ed., Cold Spring Harbor Laborat
ory, Cold Spring Harbor, NT (1989) and DM G
lover et al. ed., "DNA Cloning", 2nd ed., Vol. 1 t
o 4, (The Practical Approach Series), IRL Press, Ox
ford University Press (1995)
Erlich ed., PCR Technology, Stockton Press, 1989;
DM Glover et al. Ed., "DNA Cloning", 2nd ed.,
Vol. 1, (The Practical Approach Series), IRL Pres
s, Oxford University Press (1995) and MA Innis
et al. ed., "PCR Protocols", Academic Press, New Y
This method is performed according to the method described in ork (1990), and when commercially available reagents or kits are used, attached instructions (protocols) or attached chemicals are used.

Example 1 Preparation of Probe, Screening of cDNA Library, and Analysis of Human Cathepsin Y Sequences having homology to the papain family of human cysteine proteases were expressed in human expressed sequence tags (ES
Ovarian cancer cDNA showing significant similarity to existing cathepsin amino acid sequences, retrieved from GenBank ^TM database
Clone (H39591; registered by Hillier et al., WashU-Merc
k EST Project). Searching for ESTs similar to H39591, we found 6 overlapping ESTs of 680 bp in length useful for the construction of a novel human cysteine protease cloning probe. To prepare this probe, a prostate cDNA library (Clontech, Palo Alt
o, CA), two specific primers designed from the aforementioned ESTs, 5′-GGACTGTGGACAAGATGGAC (primer Y1; SEQ ID NO: 3 and 5′-AGGCTGTTCTTGATGGCCCA (primer
PCR was performed using Y2; SEQ ID NO: 4) to amplify DNA.
Primers were synthesized by the phosphoramidite method using a DNA synthesizer from Applied Biosystems (model 392A). The PCR reaction was performed using the Perkin-Elmer / Cetus Gene
Amp 2400 PCR system allows for denaturation (94 ° C, 15 seconds), annealing (56 ° C, 15 seconds) and extension (72 ° C, 15 seconds).
Performed in 35 cycles. The obtained PCR product was cloned and its nucleotide sequence was confirmed, and then used for screening a prostate cDNA library.

For labeling of double-stranded DNA, Pharmacia LKB (Upp
sala, Sweden) random-priming kit and Amersham (Am
Ersham, it was used UK) of ^{[α- 32 P] dCTP (3000} Ci / mmol). Hybridization with a radioactively labeled probe was performed using 6 x SSC (1 x = 150 mM NaCl, 15 mM sodium citr
ate, pH 7.0), 5 × Denhardt's (1 × = 0.02% bovine
serum albumin, 0.02% polyvinylpirrolidone, 0.02%
Ficoll), 0.1% SDS, 100 μg / mL denatured herring sperm DNA at 65 ° C. for 18 hours. 65 at 0.1 × SSC, 0.1% SDS
The autoclave was washed twice at 1 ° C. for 1 hour. About 1 × 10 ⁶ plaques were screened to isolate 10 independent clones that hybridized with the probe.

From these plaques, phage was purified.
Insert the insert into EcoRI (Boehringer Mannheim (Mannheim, Ge
rmany) digestion and subcloned into the EcoRI site of pUC18. The cloned DNA insert was excised and inserted into the polylinker region of the phage vector M13mp19, and the M13 universal primer or cDNA-specific primer and Sequenase Versio
The nucleotide sequence was determined by the dideoxy chain termination method using the n 2.0 kit (US Biochemicals, Cleveland, OH). DITP was used instead of dGTP to eliminate ambiguous sequence analysis. All sequence analysis was performed on both chains and confirmed. Computer analysis of DNA base sequence and protein amino acid sequence is available from Wisconsin University Genet
Performed by the GCG software package of the ics Computer group. As a result of sequence analysis, these clones
It was confirmed to encode a protein belonging to the papain family of a novel cysteine protease. Of these, the open reading frame encoding 484 amino acid residues (estimated molecular weight 53,365) was found from the clone with the longest insertion (Figures 1-3 and SEQ ID NO: 1).

In order to confirm that the obtained cDNA derived from the prostate encodes cysteine protease, the database was searched for a similar amino acid sequence. As a result, the one with the highest homology was Schistosoma manson
i cysteine protease (48%). The homology with the known human cysteine protease was characteristically high, ranging from 37% of cathepsin L2 to 26% of cathepsin B. FIGS. 4 to 6 show diagrams in which the amino acid sequences of the novel protein and the known cysteine protease are aligned based on homology. The novel protein had all the features of domain organization and structural motifs typical of other papain-like cysteine proteases. That is, a sequence rich in hydrophobic amino acids starting with Met corresponds to a signal peptide that is also found in all other members. At the end of the 19 residue signal sequence is an Ala-Val-Ala sequence that perfectly matches the Ala-X-Ala cleavage site motif for eukaryotic preproproteins. The algorithm of Nielsen et al. (Nielsen, H, et al., Protein Eng., 10; 1-
6, 1997) confirmed that this site was most likely to be cleaved and was likely to be a signal peptide cleavage site. The active form of the novel protein can be identified from the pro-region of the known cysteine protease and the cleavage site of the active form enzyme. As indicated by the arrows in FIGS. 4-6, the N-terminus of the active cysteine protease has the second amino acid Pro in all cases. According to this, the active form of the novel protein is 271
It was presumed that the starting position was Ala residue.

Furthermore, the novel protein has a Cys residue present in the active site of the cysteine protease, a His residue and an Asn residue which are important for the catalytic mechanism, each having a No. 295 residue.
In the 431st and 451st places. These three amino acid residues and their neighboring sequences are very well conserved compared to other cathepsins (Kamphuis, IG,
et al., J. Mol. Biol., 182: 317-329, 1985, Musil,
D., et al., EMBO J., 10: 2321-2330, 1991, Coulomb
e, R., et al., EMBO J., 15: 5492-5503, 1996). In addition, an oxyanion hole is formed in the N-terminal region.
Along with the Gln residue (position 289), a Trp residue and a hydrophobic segment are located immediately adjacent to the Cys active site. further,
The C-terminal region contains a series of conserved aromatic residues and a Gly residue near the His and Asn residues in the active site. The novel protein has five potential N-linked glycosylation sequences (Asn-Glu-Thr at position 160, Asn-Ar at position 195)
g-Thr, Asn-Phe-Ser at position 367, Asn-Asp- at position 378
Ser and Asn-Arg-Ser at position 440). All previously reported mammalian cysteine proteases are glycosylated and have at least one glycosylation required for lysosomal induction by the mannose-6-phosphate receptor system. It is estimated that

Summarizing all these structural features, it can be concluded that the novel protein is a novel human cysteine protease. This novel human cysteine protease is called "cathepsin Y" (cathepsin
Y). NEIG of PHYLIP software package
According to the phylogenetic tree created by the HBOR program (FIG. 7), cathepsin Y was evolutionally shown to be closely related to cathepsin W among human cysteine proteases. The most characteristic structure of cathepsin Y is a long propeptide not found in other papain-like cysteine proteases. Structural features can divide the propeptides of these enzymes into two groups (Karrer, KM,
et al., Proc. Natl. Acad. Sci. USA, 90: 3063-3
067, 1993, Vernet, T., et al., J. Biol. Chem., 270:
10838-10846, 1995). The first group consists of a cathepsin L-like enzyme, which has a pro-region of about 90 amino acid residues containing two motifs called ERFNIN and GNFD.

The second is a group consisting of cathepsin B, which has a slightly shorter proregion of about 60 amino acid residues lacking the ERFNIN motif. In addition there are two cysteine proteases that do not belong to either. The pro-region of cathepsin C has 206 amino acid residues and the pro-region of cathepsin Z has only 41 amino acid residues, lacking both motifs described above. The pro-region of cathepsin Y consists of 251 amino acid residues and clearly deviates from these groups. At this time, the function of this long pro domain is unknown. It is well known that propeptides function as endogenous inhibitors of proteolytic activity in papain-like enzymes. Propeptides may be required for correct refolding of these cysteine proteases, may contribute to stability in the pH environment, or may be a structural marker required for binding to microsomal membranes and translocation to lysosomes. (Fox, T., et al., Biochemistry, 31:
12571-12576, 1992, Tao, K., et al., Arch.Biochem.
Biophys., 311: 19-27, 1994, McIntyre, GF, eta
l., J. Biol. Chem., 269: 567-572, 1994, Cuozzo, J.
W., et al., J. Biol. Chem., 270: 15611-15619, 1995,
Carmona, E., et al., Biochemistry, 35: 8149-8157,
1996). In addition, this nucleic acid sequence is EMBL accession numbe
r: Registered as AJ007331.

Example 2 Construction of Expression Vector and Expression in E. coli In order to express recombinant cathepsin Y in E. coli, a 647 bp DNA fragment containing a sequence encoding active human cathepsin Y was prepared. Primer 5'-ATGGCCCCACCTGA
ATGGGACT (primer Y3; SEQ ID NO: 5) and 5'-TCAG
PCR amplification was performed using TCCACCACCGCCGAG (primer Y4; SEQ ID NO: 6) as full-length cDNA. The PCR reaction is
Expand ^TM Long Template PCR System to reduce errors
m (Boehringer Mannheim), denaturation (95 ° C, 30 seconds)
, Annealing (60 ° C, 30 seconds) and elongation (68 ° C, 1
Min) for 20 cycles. The PCR product is phosphorylated with T4 polynucleotide kinase (Boehringer Mannheim), repaired with Klenow fragment (Boehringer Mannheim), and previously SmaI and alkaline phosphatase (Boehrer).
inger Mannheim) -treated expression vector pGEX-3X (Pha
rmacia LKB). The resulting plasmid (pGEX-3X
(CTSY) was used to transform E. coli BL21 (DE3) strain. 37 ° C. Transformants with 100 [mu] g / mL ampicillin-containing LB medium, and cultured for about 16 hours, then, with the same medium diluted 1/100, A ₆₀₀ is 1.
They were further grown until they reached zero. Isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to a final concentration of 1 mM and the culture was continued for another 3 hours. The cells were collected by centrifugation, washed and resuspended in 0.05 volume of PBS, lysed with a French press, and centrifuged at 20,000 g (4 ° C, 20 minutes). The soluble extract of the supernatant was applied to a glutathione Sepharose 4B gel column, and the glutathione elution buffer (1
50 mM Tris-HCl buffer containing 0 mM reduced glutathione, pH
8.0). E. coli extracts and purified recombinant proteins were analyzed by SDS-PAGE. As can be seen in FIG.
E. coli transformed with the recombinant plasmid pGEX-3X CTSY contained an approximately 52 kDa fusion protein not found in E. coli transformed with the control plasmid pGEX-3X. A 29 kDa band derived from glutathione-S-transferase (GST) was observed in the control E. coli extract. The purified fusion recombinant human cathepsin Y was purified by SDS-PAGE.
Showed a single band of the expected size (about 52 kDa).

Example 3 Analysis of Enzymatic Activity of Recombinant Human Cathepsin Y Recombinant human cathepsin produced and purified in Escherichia coli
The enzyme activity of Y was 20 μM Z-Phe-Arg-AMC, Z
-Arg-Arg-AMC or Z-Arg-AMC (Bubendorf, Switzerl
and) and reported by Barrett and Kirschke (Barrett, A.
J., et al., Methods Enzymol., 80: 535-561, 1981) according to a slightly modified method. Analysis of 8 at 30 ° C
mM dithiothreitol, 2 mM EDTA, 0.05% Brij35
The test was performed with a 0.1 M sodium acetate buffer (pH 5.5). Substrate hydrolysis was performed using the Cytofluor 2350 fluorometer (Millipore,
Bedford, MA) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Inhibition analysis was performed with recombinant human cathepsin Y at 20 μM.
E-64 (Sigma, St. Louis, MO) was previously incubated at 30 ° C. for 15 minutes, and the residual activity of recombinant human cathepsin Y was measured using the fluorescent substrate Z-Phe-Arg-AMC as described above. . As a positive control for enzyme activity measurement, recombinant human cathepsin L2 produced in Escherichia coli was used.

Recombinant human cathepsin Y is a synthetic peptide Z-Phe-Ar which is an optimal substrate for other cysteine proteases.
It showed a significant substrate degradation activity of 3.75 μmol / min / mol enzyme against g-AMC. The substrate degradation activity was slightly higher than the activity of the GST-fused recombinant human cathepsin L2 measured under the same conditions (FIG. 9). On the other hand, the fluorescent substrates of recombinant human cathepsin Y Z-Arg-Arg-AMC, Z-Arg-AMC
Substrate-degrading activity was substantially undetectable. Furthermore, it was examined whether the degradation activity of recombinant human cathepsin Y against Z-Phe-Arg-AMC is inhibited by an inhibitor specific to cysteine protease. E-64 is an inhibitor that specifically inhibits cysteine protease. Z-
The substrate-degrading activity of recombinant human cathepsin Y against Phe-Arg-AMC was inhibited by E-64 (FIG. 9), but the inhibitors of metalloprotease, serine protease and aspartic protease were EDTA and phenylmethyl, respectively. It was not inhibited by sulfonyl fluoride and pepstatin A. These enzymological analyzes concluded that human cathepsin Y is a cysteine protease with substrate specificity and sensitivity to inhibitors specific to the cysteine protease family.

Example 4 Chromosomal Mapping of Human Cathepsin Y In order to clarify the structural or evolutionary relationship between human cathepsin Y and other members of the cysteine protease papain family, a chromosomal locus of the cathepsin Y gene was searched. Was. 24 human single chromosome panel cells (fused cells of human somatic cells containing human single chromosomes and mouse or hamster cell lines; Human Genome Mapping R
esource Center, courtesy of Cambridgeshire, UK) and a cathepsin Y-specific primer 5'-
GTGCTGATCAGAAGTGCTGCTGC (Primer Y5; SEQ ID NO:
7) and 5'-AGTTTCCTGGACATGGATAGGGAC (primer Y
6; SEQ ID NO: 8). PCR amplification includes denaturation (94 ° C, 15 seconds), annealing (68 ° C, 15 seconds)
And extension (72 ° C., 1 minute) for 35 cycles. As a result, positive amplification was observed from the fused cells containing human chromosome 11 and chromosome 15 (FIG. 10). But this 15
Fusion cells containing chromosome 11 also contained a fragment of chromosome 11, suggesting that the chromosomal locus of human cathepsin Y is human chromosome 11.

To confirm this result, the fluorescence ins
Itu hybridization (FISH) mapping was performed. Human P1 artificial chromosome (PAC) library (Human Genom
Hybridization was performed using the full length cDNA of human cathepsin Y as a probe, and two independent chromosomal clones containing human cathepsin Y were isolated. After confirming that they contained cathepsin Y by PCR and Southern blot, DNA was prepared from one of the obtained PAC clones (designated as 123H18) by the alkaline lysis method and used for FISH mapping. Two
μg of PAC DNA was nick-translated in the presence of biotin-16-dUTP and hybridized to metaphase chromosomes prepared from normal male fetohemagglutinin-stimulated lymphocytes. Biotinylated probes were detected with the two avidin-dyes. The chromosomes are banded with diamidine-2-phenylindole dihydrochloride (DAPI), and the CCD camera (Photome
trics) and observed under a fluorescence microscope (Zeiss).

Biotinylation on chromosome banded with DAPI
A yellow fluorescent signal derived from the PCA clone was clearly observed at q13 of chromosome 11 (FIG. 11). Cathepsin W is present in this region (Wex, T., et al., Bi
ochem. Biophys. Res. Commum., 248: 255-261, 1998)
It was strongly suggested that cathepsins Y and W form clusters on human chromosomes and are related to each other. In addition,
Human cathepsin C is adjacent to cathepsin Y and W regions
11q14.1-14.3 (Rao, NV, et al., J. Biol.
Chem., 272: 10260-10265, 1997), but the remaining family members reside on separate chromosomes. Similar to the analysis by the phylogenetic tree showing evolutionary relationships (Fig. 7), human cathepsin Y and human cathepsin W
Has been shown to be the most closely related papain family of cysteine proteases. The mapping of human cathepsin Y on the chromosome is extremely useful for searching for genetic diseases caused by abnormalities in this gene.
Interestingly, other studies have reported that this 11q13 region is frequently altered in various human cancers (Chuaqui, RF, et al., Am. J. Pathol.,
150: 297-303,1997, Newsham, IF, Am. J. Patho
l., 153: 5-9, 1998). Therefore, the human cathepsin Y gene can be an index for examining the state of a gene abnormality that occurs in human cancer.

Example 5: Analysis of human cathepsin Y expression in tissues and cancer cell lines 2 μg poly (A) ⁺ RN from different human tissues and cancer cell lines
A (Clontech, Palo Alto, CA) was subjected to Northern blot analysis. Prehybridization is 50%
formamide, 5 × SSPE (1 × = 150 mM NaCl, 10 mM NaH
_{2 PO 4, 1 mM EDTA,} pH 7.4), 10 × Denhardt's, 2% SDS and 100 μg / mL 42 ℃ in denatured herring sperm DNA, and was carried out for 3 hours. After prehybridization, the filter hybridized with the full-length cDNA of human cathepsin Y. After hybridization, the filters were washed with 0.1 × SSC, 0.1% SDS at 50 ° C. for 2 hours and subjected to autoradiography. The quality and quantity of the prepared RNA was evaluated by hybridization using actin as a probe.

Poly (A) ⁺ RNA from various human tissues
The results of Northern blot analysis for, were varying in degree, but most of the tissues examined (colon, small intestine, ovary, testes, prostate, spleen, pancreas, kidney, skeletal muscle, liver,
A transcript of about 2.1 kb was observed in the lung, placenta, brain and heart) (FIG. 12). The major sites of expression of cathepsin Y were skeletal muscle and testis, virtually undetectable in leukocytes and thymus. Broad expression of cathepsin Y in human tissues is consistent with cathepsin Y being a lysosomal enzyme involved in the digestion of intracellular proteins that occurs in all cells. Cathepsin Y has a housekeeping role like lysosomal enzymes, but is expected to have some special function in specific tissues such as skeletal muscle and testis, which are highly expressed.

In the expression mode, human cathepsin Y is
It is classified into a group that is widely expressed in human tissues, such as human cathepsins B, L, H, O, and Z, and one that is excessively or exclusively expressed in specific tissues, such as human cathepsins K, S, W, and L2. Are distinguished. Human cathepsin Y expression is very high in skeletal muscle and testis, reaching 20 times that in kidney and colon. High expression in skeletal muscle is due to the function of cysteine proteases involved in muscle proteolysis observed in diseases including normal and muscular dystrophy (Takeda, A., et al., Biochem. J., 28
8: 643-648, 1992, Richard, I., et al., Cell, 81: 2
7-40, 1995, Tsujinaka, T., J. Clin. Invest., 97: 24
4-249, 1996). High expression in the testicles, together with the recently discovered cathepsin L2, is expected to be involved in the fertilization process. In addition, cancer cell lines (leukemia HL-60, HeLa cells, leukemia K-562, leukemia MOLT-4,
Burkitt's lymphoma Raji, poly (A) ⁺ RNA from colorectal cancer SW480, lung cancer A549 and melanoma G361)
Was subjected to Northern blot analysis. A transcript of approximately 2.1 kb, which was strong in HeLa cells and weakly expressed in melanoma, K-562 and lung cancer, was observed (FIG. 13). Cathepsin Y, which is highly expressed in multiple cancer cell lines, especially HeLa cells, may play a role in its progression in some human cancers.

Example 6 Preparation of Monoclonal Antibody The purified fusion recombinant cathepsin Y obtained in Example 2 can be used as an antigen for immunization. Alternatively, a recombinant protein obtained by treating the fusion recombinant cathepsin Y with Factor Xa and removing the glutathione-S-transferase (GST) portion can be used. Further, as an antigen used for immunization, it is possible to use recombinant cathepsin Y obtained by ligating the cDNA obtained in Example 1 to an animal cell expression vector and expressing this in CHO cells or COS cells. . These antigenic proteins can be purified by ion exchange, gel filtration or other various types of chromatography. The purified antigen for immunization is immunized by a general method, antibody-producing cells are induced, and the antibody-producing cells can be obtained as hybridomas by cell fusion. Furthermore, based on the reactivity with the purified antigen for immunization, it can be cloned and established as a monoclonal antibody-producing hybridoma. Since the hybridoma obtained using the fused recombinant cathepsin Y as an immunogen contains an antibody-producing strain against GST, the antibody-producing hybridoma that reacts with GST must be removed.

As an immunogen for obtaining a cathepsin Y-specific monoclonal antibody, a haptenylated synthetic peptide having an amino acid sequence characteristic of cathepsin Y can be used. For example, the propeptide region at positions 20-270 (SEQ ID NO: 9) is a specific sequence not found in other cysteine protease papain families, and is suitable for preparing a pro-type human cathepsin Y-specific antibody. In addition, positions 300-325 (SEQ ID NO: 10) located in the N-terminal region of activated human cathepsin Y, positions 367-397 (SEQ ID NO: 11) or 404-423 (located in the central region) (SEQ ID NO: 12) and C-
Positions 473-484 (SEQ ID NO: 13) located in the terminal region have low homology to other cysteine protease papain families. Synthetic peptides containing these polypeptides, the sequences near the polypeptides and some of them, are cathepsins.
It is suitable as an immunogen for obtaining Y-specific monoclonal antibodies.

(A) Preparation of Antigen Polypeptide The above-mentioned polypeptide was subjected to Fmoc synthesis using a peptide synthesizer (Peptide Synthesizer 9600, MilliGen / Biosearch).
Synthesized by -bop method. Cysteine is introduced at the N-terminal or C-terminal of the polypeptide. The synthesized peptide is μBo
Purification by high performance liquid chromatography using ndasphere, C18 column (Waters). (B) Preparation of a complex of polypeptide and BSA The antigen was conjugated with bovine serum albumin (BSA) via a cysteine residue. BSA dissolved in 0.1 M phosphate buffer (pH 7.5) and N- (6-maleimidcaproyl
(oxy) succinimide mixed with dimethylformamide dissolved therein, reacted at 30 ° C. for 30 minutes, and then the above mixture was equilibrated with 0.1 M phosphate buffer (pH 7.0) PD-10.
(Pharmacia). Maleimide-bonded BS
A is collected, concentrated to 1.5 mL or less, and maleimide or BS
Each of the polypeptides synthesized in the above (a) in a 50-fold molar amount with respect to A was mixed with each dissolved in a 0.1 M phosphate buffer (pH 7.0), and incubated at 4 ° C. for 20 hours. A polypeptide complex is prepared. (C) Preparation of antibody-producing cells A complex of the polypeptide prepared in (b) and BSA,
200 μg with complete Freund's adjuvant for 8 weeks
A lb / c female mouse is intraperitoneally administered and immunized for the first time. On the 19th day
Complex dissolved in 0.1 M phosphate buffer (pH 7.5) on day 34
200 μg of mice are intraperitoneally administered to the immunized mice and boosted. Further, on the 69th day, 200 μg of the conjugate was intravenously administered to make the final immunization. Three days later, the spleen is removed and a spleen cell suspension is prepared.

(D) Cell fusion (1) The following materials and methods are used. RPMI-1640 medium: RPMI-1640 (Flow Lab.) And sodium bicarbonate (24m
M), sodium pyruvate (1 mM), potassium penicillin G (50 U / ml), amikacin sulfate (100 μg / mL), adjust the pH to 7.2 with dry ice, and remove bacteria with a 0.2 μm Toyo membrane filter. NS-1 medium: RPMI-1 above
Fetal bovine serum (FCS, MA Biop.
roducts) to a concentration of 15% (v / v). PEG-
4000 solution: RPMI-1640 Polyethylene glycol-4 in medium
000 (PEG-4000, Merk & Co.) is prepared at 50% (w / w) to prepare a serum-free medium. Fusion with 8-azaguanine-resistant myeloma cell SP2 (SP2 / 0-Ag14)
hod in culture immunology pp351-372 (ed.BB M
ishell and SN Shiigi), WH Freeman and Compan
y (1980) described in Oi & Herzenberg et al. (2) Nucleated splenocytes prepared in (c) above (viable cell rate 100
%) And myeloma cells (100% viable cell rate) were fused at a ratio of 5: 1 by the following procedure. Each polypeptide-immunized splenocyte suspension and myeloma cells were converted to RPMI1640
Wash with medium. The cells are then suspended in the same medium and the nucleated splenocytes and myeloma cells are mixed for fusion. The cells are then sedimented by centrifugation and the supernatant is completely aspirated off. A PEG-4000 solution heated to 37 ° C. is added dropwise to the precipitated cells over 1 minute, and the mixture is stirred for 1 minute to resuspend and disperse the cells. Next, after dropping the RPMI1640 medium heated to 37 ° C over 2 minutes, the medium was dropped while constantly stirring for 2 to 3 minutes.
Disperse the cells. This is centrifuged and the supernatant is completely aspirated off. Next, NS was heated to 37 ° C.
Add 1 medium quickly and carefully disperse large cell clumps by pipetting. Further, 64 mL of the same medium was added to dilute the solution, and the solution was added to a polystyrene 96-well microwell at 6.
Add 0 × 10 ⁵ cells / 0.1 ml cells. The microwells containing the cells were placed in 7% carbon dioxide / 93% air at a temperature of 37 ° C.
Incubate at 100% humidity.

(E) Selective growth of hybridoma on selective medium (1) The medium to be used is as follows. HAT medium: NS-1 medium described in (d) (1) above was further supplemented with hypoxanthine (100 μM), aminopterin (0.4 μM) and thymidine.
(16 μM). HT medium: The same composition as the above HAT medium except that aminopterin was removed. (2) The next day (day 1) after the start of the culture described in the above (d), 2 drops (about 0.1 ml) of HAT medium are added to the cells using a Pasteur pipette. On days 2, 3, 5, and 8, half of the medium (about 0.1 ml) is replaced with fresh HAT medium, and on day 11, half of the medium is replaced with fresh HT medium. On the 14th day, positive wells are examined by solid phase-antibody binding test (ELISA) in all the wells where hybridoma growth was visually observed. That is, a polystyrene 96-well plate was coated with each polypeptide using the antigen, and then washed with PBS for washing (0.05% T
Washing using ween20) to remove unadsorbed peptides. In addition, the uncoated portion of each well is blocked with 1% BSA. 0.1 mL of the supernatant of the well in which the growth of the hybridoma has been confirmed is added to each well, and the mixture is allowed to stand at room temperature for about 1 hour. Horseradish peroxidase (HR) as secondary antibody
P) A labeled goat anti-mouse immunoglobulin (Cappel Lab.) Is added, and the mixture is allowed to stand at room temperature for about 1 hour. Next, hydrogen peroxide and o-phenylenediamine as substrates are added, and the degree of color development is measured at 492 nm using a microplate absorbance meter (MRP-A4, Tosoh).

(F) Cloning of hybridomas The hybridomas in the wells positive for the antigenic peptide obtained in the above (e) are monocloned using the limiting dilution method. That is, the cloning medium containing 10 ⁷ mouse thymocytes were prepared as NS-1 medium 1ml per feeder, five wells per hybridomas in 96 microwells, one, diluted to 0.5 or, respectively
Add to 36, 36, and 24 holes. On days 5 and 12, add about 0.1 mL of NS-1 medium to all wells. Approximately two weeks after the start of cloning, macroscopically sufficient growth of hybridomas was observed, and the ELISA described in (e) was performed for the group in which colony formation negative wells were 50% or more. If all the wells examined are not positive, 4 to 6 wells with one colony in antibody-positive wells are selected and recloned. Finally, a hybridoma producing a monoclonal antibody against each polypeptide is obtained.

(G) Culture of hybridoma and purification of monoclonal antibody The obtained hybridoma cells are cultured in NS-1 medium, and a monoclonal antibody having a concentration of 10 to 100 µg / mL can be obtained from the supernatant. Moreover, mice with pristane before advance one week hybridoma ¹⁰ cells obtained was administered intraperitoneally (Balb / c system, female, 6 weeks old) was also administered intraperitoneally, 1
Two weeks later, ascites containing 4-7 mg / ml of the monoclonal antibody can be obtained from the ascites. 40% of ascites obtained
After salting out with saturated ammonium sulfate, an IgG class antibody is adsorbed on Protein A Affigel (Bio-Rad) and purified by elution with 0.1 M citrate buffer (pH 5.0). (H) Determination of Class and Subclass of Monoclonal Antibody According to the above-mentioned ELISA, the supernatant of the hybridoma obtained in (f) is added to a 96-well polystyrene plate coated with each polypeptide. Next, after washing with PBS, isotype-specific rabbit anti-mouse IgG antibody (Zyme
d Lab.). After washing with PBS, horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) was added, and the class and subclass were determined using hydrogen peroxide and 2,2'-azino-di (3-ethylbenzothiazolic acid) as a substrate. decide.

Example 7: Sandwich EIA A sandwich EIA system for specifically detecting and measuring cathepsin Y from the anti-cathepsin Y monoclonal antibody prepared in Example 6 can be constructed by appropriately combining two kinds. The EIA system can be either a one-step method or a two-step method, and the labeled antibody is not limited to Fab'-HRP. The composition and reaction conditions of each reaction buffer can be adjusted such as shortening or extending according to the purpose of measurement. (A) Preparation of labeled antibody Anti-human cathepsin Y monoclonal antibody was added to 0.1 M acetate buffer (pH 4.2) containing 0.1 M NaCl, and 2% (W / W) of pepsin was added to the antibody, and digested at 37 ° C for 24 hours. I do. 3M Tris- for digest
The reaction is stopped by adding HCl buffer (pH 7.5). 0.1M
Ultrogel AcA54 equilibrated with phosphate buffer (pH 7.0)
The F (ab ') 2 fraction is collected by gel filtration using a column. this
Cysteinamine hydrochloride was added to the F (ab ') 2 fraction to a final concentration of 0.01M, reduced at 37 ° C for 1.5 hours, and equilibrated with 0.1M phosphate buffer (pH 6.0) containing 5mM EDTA. The Fab ′ fraction is collected by gel filtration using an Ultrogel AcA54 column.
Separately from the above operation, add HRP to 0.1 M phosphate buffer (pH 7.0)
And add 25 times the molar amount of HRP to EMCS as a DMF solution and react at 30 ° C for 30 minutes. This is gel-filtered through a NICK-5 column (Pharmacia) equilibrated with a 0.1 M phosphate buffer (pH 6.0), and a maleimide-labeled HRP fraction is collected.

The Fab ′ fraction and the maleimide-labeled HRP were mixed in equal amounts to give equimolar fractions and reacted at 4 ° C. for 20 hours.
Block unreacted thiol groups with N-ethylmaleimide 10 times the molar amount of Fab '. Add this to a 0.1 M phosphate buffer (pH
The solution is subjected to gel filtration using an Ultrogel AcA54 column equilibrated in 6.5) to collect Fab′-HRP-labeled antibodies. Add 0.1% BSA and 0.001% chlorhexidine to this and store at 4 ° C. (B) Preparation of Monoclonal Antibody-Binding Carrier The anti-human cathepsin Y monoclonal antibody is dissolved in a 0.1 M phosphate buffer (pH 7.5) and adjusted to a concentration of 50 μg / mL.
This monoclonal antibody solution is added to a 96-well microplate in an amount of 100 μL per well, and left at 4 ° C. for 18 hours. Remove the monoclonal antibody solution and add 1
Times, 0.05% Tween20, 0.1M NaCl, 5mM CaCl 2 containing Tris
-HCl buffer (pH8.0) 3 times, then 1% BSA, 0.1M NaCl
Then, Tris-HCl buffer (pH 8.0) containing 5 mM CaCl ₂ is added to perform blocking.

(C) One-step sandwich EIA method Cathepsin Y was purified using the purified cathepsin Y fraction as a standard antigen.
Y Create a standard curve for quantification. 1% BSA, 0.05% Brij35,
Tris-HCl containing 0.05% Tween 20, 0.1 M NaCl, 5 mM CaCl ₂
Standard human cathepsin Y serially diluted with buffer (pH 8.0)
Dispense 60 μL each, 1% BSA, 0.05% Brij35, 0.
Labeled antibody Fab'-H adjusted to 100 ng / 50 μL with Tris-HCl buffer (pH 8.0) containing 05% Tween20, 0.1 M NaCl, 5 mM CaCl ₂
Add 60 μL of RP and mix well. Prepare the prepared antibody binding microplate with 0.05% Tween20, 0.1M NaCl, 5mM.
After washing three times with a CaCl ₂ -containing Tris-HCl buffer (pH 8.0), 100 μL / well of a standard antigen / labeled antibody mixture is added.
After reacting for 1 hour at room temperature, 0.05% Tween20, 0.1M NaCl,
Wash several times with Tris-HCl buffer (pH 8.0) containing 5 mM CaCl ₂ . Next, 100 μL per well of 1.2 mg / mL o-phenylenediamine dissolved in 0.1 M citrate-phosphate buffer (pH 4.9) containing 0.02% hydrogen peroxide was added, and reacted at room temperature for 20 minutes. Stop the reaction by adding 100 μL of sulfuric acid. The A ₄₉₂ of the reaction mixture was measured using a microplate reader to determine the standard curve. The measurement sample is a body fluid component derived from human serum, plasma, synovial fluid, urine and saliva,
It is prepared from extracts of various human tissues, cell extracts of various cultured cells such as human-derived or recombinant cells, and culture supernatants. Each measurement sample is subjected to the above-mentioned one-step sandwich EIA in place of the standard human cathepsin Y, and the reaction proceeds simultaneously with the standard cathepsin Y. The value of _A492 obtained from the measurement sample is fitted to the obtained standard curve, and the amount of human cathepsin Y contained in the measurement sample is calculated.

[0143]

As described above, the full-length gene encoding human cathepsin Y belonging to the papain family of cysteine proteases was cloned from a human cDNA library, and its nucleotide sequence was analyzed, whereby intracellular protein metabolism, antigen presentation, and hormone precursors were obtained. Of many normal cellular processes, such as activation of bone and bone modification, as well as many diseases such as Alzheimer's disease, emphysema, rheumatoid arthritis, muscular dystrophy, osteoporosis, neurodegenerative diseases and cancer invasion and metastasis Its use has become possible in research. Furthermore, expression of this gene in Escherichia coli and other hosts can be used to clarify the enzymatic properties of the recombinant protein, and it is also possible to study substances that inhibit its activity. Also, human cathepsin Y
The chromosomal locus of the gene can be determined and its expression in human tissues examined. An antibody specifically reacting with human cathepsin Y, in particular, a monoclonal antibody can be prepared to provide a means for detecting human cathepsin Y such as immunological tissue staining and a reagent therefor. Also provided is a method for detecting and measuring human cathepsin Y, for example, an EIA system. The reagent has a possibility for use in detection and diagnosis of cancer, elucidation of the pathogenesis of the above-mentioned diseases, development of therapeutics and therapeutic drugs, and the like.

[0144]

[Sequence List] SEQUENCE LISTING <110> Fuji Yakuhin Kogyo Kabushiki Kaisha <120> Novel Human Cathepsin Y Protein, Gene Encoding Said Protein And Use Thereof <130> P-98NF272 <140> <141> <160> 13 <170> PatentIn Ver. 2.0 <210> 1 <211> 484 <212> PRT <213> Human <400> 1 Met Ala Pro Trp Leu Gln Leu Leu Ser Leu Leu Gly Leu Leu Pro Gly 1 5 10 15 Ala Val Ala Ala Pro Ala Gln Pro Arg Ala Ala Ser Phe Gln Ala Trp 20 25 30 Gly Pro Pro Ser Pro Glu Leu Leu Ala Pro Thr Arg Phe Ala Leu Glu 35 40 45 Met Phe Asn Arg Gly Arg Ala Ala Gly Thr Arg Ala Val Leu Gly Leu 50 55 60 Val Arg Gly Arg Val Arg Arg Ala Gly Gln Gly Ser Leu Tyr Ser Leu 65 70 75 80 Glu Ala Thr Leu Glu Glu Pro Pro Cys Asn Asp Pro Met Val Cys Arg 85 90 95 Leu Pro Val Ser Lys Lys Thr Leu Leu Cys Ser Phe Gln Val Leu Asp 100 105 110 Glu Leu Gly Arg His Val Leu Leu Arg Lys Asp Cys Gly Pro Val Asp 115 120 125 Thr Lys Val Pro Gly Ala Gly Glu Pro Lys Ser Ala Phe Thr Gln Gly 130 135 140 Ser Ala Met Ile Ser Ser Leu Ser Gln Asn His Pro Asp Asn Arg Asn 145 150 155 160 Glu Thr Phe Ser Ser Val Ile Ser Leu Leu Asn Glu Asp Pro Leu Ser 165 170 175 Gln Asp Leu Pro Val Lys Met Ala Ser Ile Phe Lys Asn Phe Val Ile 180 185 190 Thr Tyr Asn Arg Thr Tyr Glu Ser Lys Glu Glu Ala Arg Trp Arg Leu 195 200 205 Ser Val Phe Val Asn Asn Met Val Arg Ala Gln Lys Ile Gln Ala Leu 210 215 220 Asp Arg Gly Thr Ala Gln Tyr Gly Val Thr Lys Phe Ser Asp Leu Thr 225 230 235 240 Glu Glu Glu Phe Arg Thr Ile Tyr Leu Asn Thr Leu Leu Arg Lys Glu 245 250 255 Pro Gly Asn Lys Met Lys Gln Ala Lys Ser Val Gly Asp Leu Ala Pro 260 265 270 Pro Glu Trp Asp Trp Arg Ser Lys Gly Ala Val Thr Lys Val Lys Asp 275 280 285 Gln Gly Met Cys Gly Ser Cys Trp Ala Phe Ser Val Thr Gly Asn Val 290 295 300 Glu Gly Gln Trp Phe Leu Asn Gln Gly Thr Leu Leu Ser Leu Ser Glu 305 310 315 320 Gln Glu Leu Leu Asp Cys Asp Lys Met Asp Lys Ala Cys Met Gly Gly 325 330 335 Leu Pro Ser Asn Ala Tyr Ser Ala Ile Lys Asn Leu Gly Gly Leu Glu 340 345 350 Thr Glu Asp Asp Tyr Ser Tyr Gln Gly His Met Gln Ser Cys Asn Phe 355 360 365 Ser Ala Glu Lys Ala Lys Val Tyr Ile Asn Asp Ser Val Glu Leu Ser 370 375 380 Gln Asn Glu Gln Lys Leu Ala Ala Trp Leu Ala Lys Arg Gly Pro Ile 385 390 395 400 400 Ser Val Ala Ile Asn Ala Phe Gly Met Gln Phe Tyr Arg His Gly Ile 405 410 415 Ser Arg Pro Leu Arg Pro Leu Cys Ser Pro Trp Leu Ile Asp His Ala 420 425 430 Val Leu Leu Val Gly Tyr Gly Asn Arg Ser Asp Val Pro Phe Trp Ala 435 440 445 445 Ile Lys Asn Ser Trp Gly Thr Asp Trp Gly Glu Lys Gly Tyr Tyr Tyr 450 455 460 Leu His Arg Gly Ser Gly Ala Cys Gly Val Asn Thr Met Ala Ser Ser 465 470 475 475 480 Ala Val Val Asp <210> 2 <211> 1517 <212> DNA <213> Human <400> 2 gaattccggg tctgttgggt ccgggccgcc ggcttcgccc tcgccatggc gccctggctg 60 cagctcctgt cgctgctggg gctgctcccg ggcgcagtgg ccgcccccgc ccagccccga 120 gccgccagct ttcaggcctg ggggccgccg tccccggagc tgctggcgcc cacccgcttc 180 gcgctggaga tgttcaaccg cggccgggct gcggggacgc gggccgtgct gggccttgtg 240 cgcggccgcg tccgccgggc gggtcagggg tcgctgtact ccctggaggc caccctggag 300 gagccaccct gcaacgaccc catggtgtgc cggctccccg tgtccaagaa aaccctgctc 360 tgcagcttcc aagtcctgga tgagctcgga agacacgtgc tgctgcggaa ggactgtggc 420 ccagtggaca ccaaggttcc aggtgctggg gagcccaagt cagccttcac tcagggctca 480 gccatgattt cttctctgtc ccaaaaccat ccagacaaca gaaacgagac tttcagctca 540 gtcatttccc tgttgaatga ggatcccctg tcccaggact tgcctgtgaa gatggcttca 600 atcttcaaga actttgtcat tacctataac cggacatatg agtcaaagga agaagcccgg 660 tggcgcctgt ccgtctttgt caataacatg gtgcgagcac agaagatcca ggccctggac 720 cgtgggacag ctcagtatgg agtcaccaag ttcagtgatc tcacagagga ggagttccgc 780 actatctacc tgaatactct cctgaggaaa gagcctggca acaagatgaa gcaagccaag 840 tctgtgggtg acctcgcccc acctgaatgg gactggagga gtaagggggc tgtcacaaaa 900 gtcaaagacc agggcatgtg tggctcctgc tgggccttct cagtcacagg caatgtggag 960 ggccagtggt ttctcaacca ggggaccctg ctctccctct ctgaacagga gctcttggac 1020 tgtgacaaga tggacaaggc ctgcatgggc ggcttgccct ccaatgccta ctcggccata 1080 aagaatttgg gagggctgga gacagaggat gactacagct accagggtca catgcagtcc 1140 tgcaacttct cagcagagaa ggccaaggtc tacatcaatg actcc gtgga gctgagccag 1200 aacgagcaga agctggcagc ctggctggcc aagagaggcc caatctccgt ggccatcaat 1260 gcctttggca tgcagtttta ccgccacggg atctcccgcc ctctccggcc cctctgcagc 1320 ccttggctca ttgaccatgc ggtgttgctt gtgggctacg gcaaccgctc tgacgttccc 1380 ttttgggcca tcaagaacag ctggggcact gactggggtg agaagggtta ctactacttg 1440 catcgtgggt ccggggcctg tggcgtgaac accatggcca gctcggcggt ggtggactga 1500 agaggggccc cgaattc 1517 <210> 3 <211> 20 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 3 ggactgtgga caagatggac 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 4 agctgttctt gatggccca 19 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 5 atggccccac ctgaatggga ct 22 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 6 tcagtccacc accgccgag 19 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 7 gtgctgatca gaagtgctgc tgc 23 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 8 agtttcctgg acatggatag ggac 24 <210> 9 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Protein to act as an immunogen <400> 9 Ala Pro Ala Gln Pro Arg Ala Ala Ser Phe Gln Ala Trp Gly Pro Pro 1 5 10 15 Ser Pro Glu Leu Leu Ala Pro Thr Arg Phe Ala Leu Glu Met Phe Asn 20 25 30 Arg Gly Arg Ala Ala Gly Thr Arg Ala Val Leu Gly Leu Val Arg Gly 35 40 45 Arg Val Arg Arg Ala Gly Gln Gly Ser Leu Tyr Ser Leu Glu Ala Thr 50 55 60 Leu Glu Glu Pro Pro Cys Asn Asp Pro Met Val Cys Arg Leu Pro Val 65 70 75 80 Ser Lys Lys Thr Leu Leu Cys Ser Phe Gln Val Leu Asp Glu Leu Gly 85 90 95 Arg His Val Leu Leu Arg Lys Asp Cys Gly Pro Val Asp Thr Lys Val 100 105 110 Pro Gly Ala Gly Glu Pro Lys Ser Ala Phe Thr Gln Gly Ser Ala Met 115 120 125 Ile Ser Ser Leu Ser Gln Asn His Pro Asp Asn Arg Asn Glu Thr Phe 130 135 140 Ser Ser Val Ile Ser Leu Leu Asn Glu Asp Pro Leu Ser Gln Asp Leu 145 150 155 160 Pro Val Lys Met Ala Ser Ile Phe Lys Asn Phe Val Ile Thr Tyr Asn 165 170 175 Arg Thr Tyr Glu Ser Lys Glu Glu Ala Arg Trp Arg Leu Ser Val Phe 180 185 190 Val Asn Asn Met Val Arg Ala Gln Lys Ile Gln Ala Leu Asp Arg Gly 195 200 205 Thr Ala Gln Tyr Gly Val Thr Lys Phe Ser Asp Leu Thr Glu Glu Glu 210 215 220 Phe Arg Thr Ile Tyr Leu Asn Thr Leu Leu Arg Lys Glu Pro Gly Asn 225 230 235 240 Lys Met Lys Gln Ala Lys Ser Val Gly Asp Leu 245 250 <210> 10 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 10 Val Thr Gly Asn Val Glu Gly Gln Trp Phe Leu Asn Gln Gly Thr Leu 1 5 10 15 Leu Ser Leu Ser Glu Gln Glu Leu Leu Asp 20 25 <210> 11 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 11 Asn Phe Ser Ala Glu Lys Ala Lys Val Tyr Ile Asn Asp Ser Val Glu 1 5 10 15 Leu Ser Gln Asn Glu Gln Lys Leu Ala Ala Trp Leu Ala Lys Arg 20 25 30 <210> 12 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Desinged peptide to act as an immunogen <400> 12 Ile Asn Ala Phe Gly Met Gln Phe Tyr Arg His Gly Ile Ser Arg Pro 1 5 10 15 Leu Arg Pro Leu 20 <210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed peptide to act as an immunogen <400> 13 Gly Val Asn Thr Met Ala Ser Ser Ala Val Val Asp 1 5 10

[Brief description of the drawings]

FIG. 1 shows the nucleic acid sequence and the predicted amino acid sequence of human cathepsin Y cloned from a human prostate cDNA library (continuation of FIGS. 2 and 3). Amino acid residues typical of the active site of a cysteine protease are boxed. Arrows indicate signal peptide and propeptide cleavage sites, and propeptide and active enzyme cleavage sites, respectively. EcoRI cloning sites are underlined. Amino acid residues are indicated in conventional single letter notation.

FIG. 2 shows the nucleic acid sequence and the predicted amino acid sequence of human cathepsin Y cloned from a human prostate cDNA library (FIG. 3 is continued).

FIG. 3 shows the nucleic acid sequence and the predicted amino acid sequence of human cathepsin Y cloned from a human prostate cDNA library.

FIG. 4 shows the amino acid sequence alignment of human cathepsin Y and human cysteine protease (FIGS. 5 and 6).
Continuation is shown). The amino acid sequence of a known human cysteine protease was retrieved from the SwissProt database and analyzed with the PILEUP program in the GCG package.
The numbers correspond to human cathepsin Y. Amino acids common to human cathepsin Y and the cathepsin family are shown in bold. Gaps are indicated by hyphens. The arrow is
The cleavage sites for the propeptide and the active enzyme, which are common to human cathepsin Y and human cysteine protease, are shown.

FIG. 5 shows the amino acid sequence alignment of human cathepsin Y and human cysteine protease continued from FIG. 4 (continued in FIG. 6).

FIG. 6 shows the amino acid sequence alignment of human cathepsin Y and human cysteine protease continued from FIGS. 4 and 5.

FIG. 7 illustrates the evolutionary implications of human cysteine protease. The phylogenetic tree is based on the PHYLIP software package.
Created by NEIGHBOR program. The distance between systems is Ki
mura method (Kimura, M., Proc. Natl. Acad. Sci. USA,
78: 454-458, 1981).

FIG. 8 shows the results of purification of recombinant human cathepsin Y expressed in E. coli. 5 μL of an extract of E. coli transformed with each of pGEX-3X and pGEX-3X CTSY was added per lane, and analyzed by SDS-PAGE. CTSY contains 1 μL of purified fusion protein per lane.
The arrow indicates GST (29 kDa) and the fusion protein (about 52 kDa). The molecular weight indicated by the molecular weight marker (MWM) is shown on the right side of the figure.

FIG. 9 shows the results of enzyme activity analysis of recombinant human cathepsin Y expressed in Escherichia coli. Recombinant human cathepsin Y (C
TSY) and human cathepsin L2 (CTSL2) at 20 μM Z-Phe-Arg-
Figure 3 shows the hourly substrate hydrolysis (30 ° C) in the presence and absence of 20 μM E-64 mixed with AMC.

FIG. 10 shows the results of chromosome mapping of human cathepsin Y. According to the method described in Example 4,
PCR was performed using 100 ng of DNA extracted from 24 chromosome panel cell lines as a template. HaeIII digest of pBR322 (Boehr
inger Mannheim) was used as a marker.

FIG. 11 shows the results of chromosome mapping of human cathepsin Y. Fluorescence in situ hybridization was performed using a biotinylated human cathepsin Y specific probe. Metaphase chromosomes were stained with diamine-2-phenylindole dihydrochloride.

FIG. 12 shows the results of Northern blot analysis of human cathepsin Y mRNA in human tissues and cultured cell lines. 2 μg poly (A) ⁺ prepared from the indicated tissue was replaced with human cathepsin
This is the result of hybridization and analysis using cDNA containing the full length of Y as a probe. RNA markers are shown beside the figure. After washing the probe, the filter
Hybridization was repeated with a human actin probe.

FIG. 13 shows the results of Northern blot analysis of human cathepsin Y mRNA in human tissues and cultured cell lines. 2 μg poly (A) ⁺ prepared from the indicated cancer cell lines and 20 μg of total RNA prepared from various cancer tissues were hybridized with the aforementioned human cathepsin Y probe. Next to the figure
RNA markers showed. After washing the probe, the filter hybridized with the human actin probe.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 31/00 621 A61K 31/00 621 4C084 625 625 4C085 626 626N 4H045 629 629A 635 635B 38/55 39/395 D39 / 395 N 45/00 45/00 48/00 48/00 C07K 16/40 C07K 16/40 C12N 1/19 C12N 1/19 1/21 1/21 C12P 21/08 5/10 C12Q 1/68 A 15/09 ZNA G01N 33/53 D C12P 21/08 33/573 A C12Q 1/68 A61K 37/64 G01N 33/53 C12N 5/00 B 33/573 15/00 ZNAA // (C12N 1/21 C12R 1:19) (C12N 5/10 C12R 1:91) (72) Inventor Gloria Velasco 33400 Astorias Salinas Pablo Rocks Izda 10−60 (72) Inventor Albert M. Pendas Spain 33004 Oviedo Cervantes 25 7-D (72) Inventor Takanori Aoki 530 Chokeiji, Takaoka City, Toyama Prefecture Inside Fuji Pharmaceutical Industry Co., Ltd. (72) Invention Person Kazushi Iwata 530 Chokeiji Temple, Takaoka City, Toyama Prefecture F-term in Fuji Pharmaceutical Industry Co., Ltd. 4B064 AG01 CA02 CA06 CA10 CA19 CA20 CC24 DA03 DA13 4B065 AA26X AA72X AA90X AA91X AA93Y AB01 CA33 CA44 CA46 4C084 AA01 AA07 AA13 AA17 BA01 BA22 BA44 DA39 DC07 MA13 MA17 MA22 MA23 MA28 MA37 MA63 MA57 MA63 MA63 MA37 MA63 MA63 MA63 MA63 MA63 MA37 MA63 MA37 MA57 MA67 ZA162 ZA592 ZA942 ZA962 ZA972 ZB152 ZB262 ZC192 ZC202 4C085 AA13 AA14 BB22 CC02 DD63 4H045 AA10 AA30 BA10 CA40 DA89 EA22 EA28 FA74

Claims (16)

[Claims] 1. A novel protein having at least 49% homology with a human-derived cathepsin Y protein or an amino acid sequence of the cathepsin Y protein and having cysteine protease activity or equivalent antigenicity. Human-derived cathepsin protein or a salt thereof. 2. Among the amino acid sequences represented by SEQ ID NO: 1 in the sequence listing, those having the 271st to 484th amino acid sequences, those having the 20th to 484th amino acid sequences, 1st to 484th amino acid sequences, amino acid sequences having substantially the same amino acid sequence as any one of them, and amino acids having substantially the same biological activity as any one of them A protein or a salt thereof, which is a protein selected from the group consisting of those having a sequence. 3. A partial peptide of the protein according to claim 1, or a salt thereof. 4. A nucleic acid having a base sequence encoding the protein according to claim 1 or 2. 5. The nucleic acid according to claim 4, wherein the nucleic acid has an open reading frame portion or a nucleotide sequence substantially equivalent thereto in the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing. 6. A vector comprising the nucleic acid according to claim 4 or 5. 7. A transformant comprising the vector according to claim 6. 8. The transformant according to claim 7, wherein the host cell is selected from the group consisting of Escherichia coli, yeast, CHO cells and COS cells. 9. The protein according to claim 1, which is obtained by being expressed by the transformant according to claim 7. 10. Antibodies against: (i) the protein of claim 1 or a salt thereof, (ii) the protein of claim 2 or a salt thereof, or (iii) the partial peptide of claim 3 or Its salt. 11. An immunological method for a protein or a salt thereof according to claim 1 or 2, wherein a monoclonal antibody that specifically immunoreacts with the protein or a salt thereof according to claim 1 or 2 is used as a measurement reagent. Measuring method. 12. A reagent for immunologically measuring a protein or a salt thereof according to claim 1 or 2, which comprises a monoclonal antibody that specifically immunoreacts with the protein or a salt thereof according to claim 1 or 2. 13. A hybridization probe for a human-derived cathepsin Y gene, which comprises the whole or a part of the base sequence represented by SEQ ID NO: 2 in the sequence listing. 14. The protein according to any one of claims 1 to 3, a partial peptide thereof or a salt thereof; or the nucleic acid according to claim 4 or 5, or the antibody according to claim 10. A medicament characterized in that: 15. A compound which promotes or inhibits the biological activity of the protein according to any one of claims 1 to 3, a partial peptide thereof or a salt thereof, or a salt thereof. Medicine. 16. A screening method and a screening kit for a compound that promotes or inhibits the biological activity of the protein according to claim 1, a partial peptide thereof, or a salt thereof.