JP2002272470A - Nucleic acid encoding lysyl oxidase-related protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide application technologies for forming collagen or elastin and for treating diseases in which the formation of collagen or elastin is involved. SOLUTION: A nucleic acid which has a specific base sequence derived from a human being or mouse or a derivative thereof and encodes a lysyl oxidase- related protein, an expression vector containing the nucleic acid, a transformant having the nucleic acid, a lysyl oxidase-related protein, and an antibody against the protein, are provided. The nucleic acid, the expression vector, the protein and the antibody are used as agents for promoting the reconstruction of bone/ cartilage, collagenous extracellular matrix production-regulating agents and as agents for treating diseases related with the formation of collagen or elastin. In addition, an oligonucleotide or antibody corresponding to the nucleic acid is used as an agent for testing the reconstruction state of bone/cartilage.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a lysyl oxidase-related protein, a nucleic acid encoding the same, an application to the production of collagen or elastin, and an application to the treatment of a disease involving the production of collagen or elastin.

[0002]

BACKGROUND OF THE INVENTION Endochondral bone formation is a programmed, multi-step phenomenon in skeletal development. For example, undifferentiated mesenchymal chondrocyte precursor cells differentiate into chondrocytes via cell aggregation.

[0003] The chondrocytes are composed of type II collagen and IX.
Surrounded by a multi-layered extracellular matrix containing type II collagen and type XI collagen, which is characteristic of cartilage [Castagnola, P. et al., J. Cell. B
iol., 106, 461-467 (1988); Mendler, M.
Et al., J. Cell. Biol., 108, 191-197 (1989)].

[0004] These cells undergo a continuous process of proliferation and maturation, transforming their genetic program into hypertrophic calcified chondrocytes and producing type X collagen. These phenomena are attributed to osteoinductive factors (BMP) [Tsumaki, N. et al., J. Cell. Biol., 144, 161-173.
(1999); Zou, H. et al., Genes Dev. 11, 2191-2203.
(1997)], fibroblast growth factor (FGF) [Corvin (C
olvin, JS) et al., Nat.Genet., 12, 390-397 (1996);
Dengue, C. et al., Cell, 84, 911-921 (1996)], parathyroid hormone-related protein (PTHrP) [Amizuka, N., et al., J. Cell. Biol., 126, 1611- 1623
(1994); Karaplis, AC, et al., Genes Dev.,
8, 277-289 (1994); Lanske, B. et al., Science,
273, 663-666 (1996)] and Indian hedgehog [Vortkamp, A. et al., Science, 273, 61.
3-622 (1996)].

The extracellular matrix components of cartilage play an important role in regulating / maintaining the chondrocyte phenotype. During the process of chondrocyte hypertrophy and calcification, mineralization of the extracellular matrix occurs before the chondrocytes are replaced by bone tissue.

[0006] It has been reported that the use of the cloned mouse cell line ATDC5 makes it possible to monitor multiple stages of cartilage differentiation in a single culture system [Akiyama
yama, H.) et al., J. Bone Miner. Res., 11, 22-28 (199
6); Akiyama et al., Biochem. Biophys. Res. Commun., 23.
5, 142-147, (1997); Akiyama et al., Cell Struct. Func
t., 25, 195-204, (2000); Ito, H. et al., Bioch.
im. Biophys. Acta., 1451, 263-270 (1999)]. When cultured in the presence of insulin, ATDC5 cells form cartilage-like nodules through cell aggregation. Upon completion of cartilage nodule formation, the cells migrate to hypertrophic chondrocytes that express type X collagen, followed by mineralization.

[0007] However, at present, little is known about the molecular mechanism related to these continuous events, and effective regulation means are desired.

[0008]

The present invention relates to a polypeptide having a lysyl oxidase activity, which is differentially expressed in cartilage precursor cells in a hypertrophic / calcified stage and cartilage precursor cells in an earlier differentiation stage, and A nucleic acid encoding it or its complementary strand; an expression vector that can be introduced into a cell, tissue or individual, and thereby regulate the endochondral ossification process; can be introduced into a cell, tissue or individual;
A cell-introducing carrier capable of regulating the endochondral ossification process; and a bone / cartilage repair promoter capable of promoting bone / cartilage repair; a cell having properties suitable for a substrate for cartilage regeneration, Collagenous extracellular matrix production regulator that can be used for the production of collagen suitable as a carrier during treatment; for diseases related to collagen or elastin production, such as fibrotic lesions, chondrosarcoma, osteosarcoma, etc. Therapeutic agents;
An object of the present invention is to provide a method for detecting a bone / cartilage repair state and a detection reagent used for the method.

[0009]

That is, the present invention provides:
[1] (i) a nucleotide sequence represented by SEQ ID NO: 1 or 3; (ii) a nucleotide sequence different from the nucleotide sequence of SEQ ID NO: 1 or 3 through degeneracy; (iii) a nucleotide sequence represented by SEQ ID NO: 1 or 3 In the base sequence, a base sequence having substitution, deletion, addition or insertion in at least one residue, (iv) a base sequence having a sequence identity with the base sequence of SEQ ID NO: 1 of more than 64.8% (V) a nucleotide sequence having a sequence identity of more than 63.2% with the nucleotide sequence of SEQ ID NO: 3, (vi) SEQ ID NO: 1
Or (iii) a nucleotide sequence that differs from the nucleotide sequence of (3) through a nucleic acid polymorphism under a stringent condition with a complementary strand of a sense strand nucleic acid having any one of the nucleotide sequences (i) to (vi). A base sequence of a nucleic acid capable of soybean, and (viii)
(I) to (vi) by alternative splicing
i) a nucleic acid having a base sequence selected from the group consisting of base sequences different from any of the base sequences, and the encoded polypeptide has lysyl oxidase activity; a lysyl oxidase-related protein; A sense strand nucleic acid to encode, [2] an antisense nucleic acid having a base sequence complementary to the nucleic acid according to [1],
[3] (I) the amino acid sequence of the polypeptide encoded by the sense strand nucleic acid according to [1], (II) the amino acid sequence shown in SEQ ID NO: 2 or 4, (III) the amino acid sequence of SEQ ID NO: 2 or 4, An amino acid sequence having a substitution, deletion, addition or insertion in at least one residue in the amino acid sequence; (IV) an amino acid sequence having a sequence identity with the amino acid sequence of SEQ ID NO: 2 of more than 55.5% , And (v)
The sequence identity with the amino acid sequence of SEQ ID NO: 4 is 5
A polypeptide having an amino acid sequence selected from the group consisting of more than 5.1% of amino acid sequences, and
A polypeptide having lysyl oxidase activity, [4]
An antibody that specifically binds to the polypeptide of [3], [5] the sense strand nucleic acid of [1],
An expression vector in which the polypeptide encoded by the nucleic acid is retained in a vector suitable for expression in a cell;
[6] The sense strand nucleic acid according to [1] or [5].
A carrier for cell introduction, which comprises a carrier containing the expression vector described above, wherein the carrier is a carrier selected from the group consisting of liposomes, calcium phosphate, and DEAE dextran; [7] the antisense nucleic acid according to [2]. And a carrier selected from the group consisting of liposomes, calcium phosphate and DEAE dextran, a carrier for cell introduction, [8] holding the sense strand nucleic acid according to [1]. Transforming or transducing cells,

[9] an oligonucleotide capable of specifically hybridizing to the sense strand nucleic acid according to [1] or the antisense nucleic acid according to [2] under stringent conditions, [10] the sense strand according to [1] A bone / bone containing one selected from the group consisting of a nucleic acid, the polypeptide according to [3], the expression vector according to [5], and the cell introduction carrier according to [7]; Cartilage repair promoter, [11] an active ingredient,
The bone / cartilage repair promoter according to the above [10], further comprising a substance that can be delivered to a site in need of cartilage repair,
[12] The sense strand nucleic acid according to [1], [2]
The antisense nucleic acid according to [3], the polypeptide according to [3], the antibody according to [4], the expression vector according to [5], the cell transfer carrier according to [6], and the [7]
The agent for controlling collagenous extracellular matrix production, comprising as an active ingredient one selected from the group consisting of the carrier for cell introduction according to the above, [13] The agent for regulating collagenous extracellular matrix production according to the above [12]. A reagent for producing a collagenous extracellular matrix, comprising, as an active ingredient, [14] the sense strand nucleic acid according to [1], the antisense nucleic acid according to [2], the polypeptide according to [3], 1 selected from the group consisting of the antibody according to [4], the expression vector according to [5], the carrier for cell introduction according to [6], and the carrier for cell introduction according to [7].
A therapeutic agent for a disease associated with collagen or elastin production, comprising a seed as an active ingredient, [15]
Diseases associated with the production of collagen or elastin,
The therapeutic agent according to [14], which is at least one selected from the group consisting of fibrotic lesions, chondrosarcoma, osteosarcoma, aortic sclerosis, hypertension and Ehlers-Danlos syndrome type V, [16] [1] The sense strand nucleic acid according to [2], the antisense nucleic acid according to [2], the antibody according to [4], and

[9] A reagent for detecting a bone / cartilage repair state, which comprises one selected from the group consisting of the oligonucleotides according to [9], [17] a reagent for detecting a bone / cartilage repair state according to [16]. Using, to detect the bone and cartilage repair state, a method for detecting the bone and cartilage repair state,
[18] (a) the sense strand nucleic acid according to [1], the antisense nucleic acid according to [2], and

Contacting the test sample with the reagent for detecting a bone / cartilage repair state according to the above [16], which contains one selected from the group consisting of the oligonucleotides according to the above [9]; and (b) 1) or (2): (1) The antisense nucleic acid according to the above [2] and the following (i)
To (viii): (i) the base sequence shown in SEQ ID NO: 1 or 3, (ii)
(Iii) a base sequence having substitution, deletion, addition or insertion in at least one residue in the base sequence of SEQ ID NO: 1 or 3; The sequence, (iv) a nucleotide sequence having a sequence identity with the nucleotide sequence of SEQ ID NO: 1 of more than 64.8%, (v)
The sequence identity with the nucleotide sequence of SEQ ID NO: 3 is 63.
A nucleotide sequence exceeding 2%, (vi) a nucleotide sequence different from the nucleotide sequence of SEQ ID NO: 1 or 3 through a nucleic acid polymorphism, and (vii) a nucleotide sequence of any of the above (i) to (vi) It consists of a nucleotide sequence of a nucleic acid capable of hybridizing under stringent conditions with a complementary strand of a sense strand nucleic acid, and (viii) a nucleotide sequence different from any one of the above (i) to (vii) by alternative splicing. A hybrid complex with a nucleic acid having a base sequence selected from the group or (2) the sense strand nucleic acid according to [1],
(viii) detecting the presence or absence of the formation of a hybrid complex with a complementary strand of the nucleic acid according to any of the above, wherein the formation of the hybrid complex expresses the gene having the nucleic acid according to the above [1]. And the indicator of bone / cartilage repair state, the detection method according to (17),
[19] The above

[9] using a primer pair consisting of the oligonucleotide according to the above, comprising a step of performing nucleic acid amplification using a nucleic acid derived from a test sample as a template, wherein the production of a product specifically amplified by the primer pair is [1] The detection method according to [17], wherein the gene having the nucleic acid according to [1] is expressed and is an indicator of a bone / cartilage repair state, and [20] (A) the antibody according to [4]; (B) contacting the antibody with the test sample; and (B) the amino acid sequence of the polypeptide encoded by the sense strand nucleic acid according to (1) above, ) An amino acid sequence represented by SEQ ID NO: 2 or 4, (III) an amino acid sequence having a substitution, deletion, addition or insertion in at least one residue in the amino acid sequence of SEQ ID NO: 2 or 4, (IV)
The sequence identity between the amino acid sequence of SEQ ID NO: 2 is 5
An amino acid sequence greater than 5.5%, and (V) SEQ ID NO:
Detecting the presence or absence of a complex with a polypeptide containing an amino acid sequence selected from the group consisting of amino acid sequences having an amino acid sequence of more than 55.1%, Here, the complex is formed when the gene having the nucleic acid according to the above [1] is expressed, and
The present invention relates to the detection method according to the above [17], which is an indicator of a cartilage repair state.

[0010]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to mRN from ATDC5 cells at the hypertrophic / calcified stage expressing type X collagen.
In A, the present inventors surprisingly found that a polypeptide having lysyl oxidase activity, that is, a gene encoding a lysyl oxidase-related protein, is expressed differently from mRNA derived from ATDC5 cells at an earlier differentiation stage. based on.

As used herein, the term “lysyl oxidase-related protein” refers to a cartilage progenitor cell having lysyl oxidase activity and in the hypertrophic / calcified stage, which is different from the chondrogenic precursor cell in the earlier differentiation stage. Mouse-derived protein and its human homolog. In this specification, such a protein is referred to as L
Also called OXC.

The sense strand nucleic acid of the present invention encodes a polypeptide having lysyl oxidase activity, and specifically includes (i) the nucleotide sequence shown in SEQ ID NO: 1 or 3, and (ii) SEQ ID NO: Nucleotide sequences that differ from the nucleotide sequence of 1 or 3 via degeneracy. The nucleic acid of the present invention has one major feature in the base sequence. Further, in the nucleic acid of the present invention, for example, in the case of a mouse, the cartilage precursor cells in the hypertrophic / calcified stage expressing X-type collagen by the nucleotide sequence shown in SEQ ID NO: 3 have a differentiating stage from the preceding stage. Is characterized in that it is differentially expressed from the chondrogenic progenitor cells. Therefore, the bone
Applications to cartilage repair promoters, promotion of bone formation, regulation of collagenous extracellular matrix production, therapeutic agents for diseases related to collagen or elastin production, detection of bone / cartilage repair status, and the like can be expected.

The nucleotide sequence shown in SEQ ID NO: 1 is the nucleotide sequence of a human-derived nucleic acid encoding a polypeptide having lysine oxidase activity, and the nucleotide sequence shown in SEQ ID NO: 3 is lysine oxidase. 1 is a nucleotide sequence of a mouse-derived nucleic acid encoding an active polypeptide.

As used herein, lysyl oxidase activity can be measured, for example, by the method described in Iguchi, H. et al., Toxicol. Appl.
Pharmacol. 62, 126-136, (1982). Hereinafter, an example of the measurement method will be described. A substrate solution obtained by dissolving the ^[3 H] -I collagen or type II collagen in 0.05M Tris-HCl buffer solution containing lysyl oxidase activity assay 0.15 M NaCl, mixing the measurement object The resulting mixture is incubated under conditions suitable for the reaction (eg, 3 hours at 37 ° C.) to react, the reaction is stopped (eg, frozen at −20 ° C.), and the method of Pinel et al. (Pinnell, SR), Proc. Natl. Ac
ad. Sci. USA, 61, 708-716 (1968)].
The radioactivity of ml is measured in a liquid scintillation counter.

As used herein, the term “nucleic acid” refers to
It is meant to include any of mRNA, cDNA and DNA. Further, the "sense strand" means a strand encoding a polypeptide having lysyl oxidase activity,
“Antisense (nucleic acid)” means a strand complementary to the “sense strand”.

The sense strand nucleic acid of the present invention is characterized in that, if the encoded polypeptide has lysyl oxidase activity, (iii) at least one residue in the nucleotide sequence of SEQ ID NO: 1 or 3 is substituted. It also includes a nucleic acid having a base sequence having a deletion, addition or insertion.

In the present invention, the number of mutations such as substitutions, deletions, additions and insertions in the nucleotide sequence of SEQ ID NO: 1 or 3 is at least one residue, specifically one or more, or more. It is. The number of such mutations, for the encoded polypeptide, for example, by measuring the activity according to the lysyl oxidase activity measurement method,
Any range may be used as long as lysyl oxidase activity is found, and preferably one or several ranges.

In the present invention, the location of the mutation may be any one as long as the encoded polypeptide has lysyl oxidase activity. When a mutation is artificially introduced by a mutagenesis method described below, the localization position of the mutation is expressed by a polypeptide encoded by a nucleic acid obtained by the mutagenesis, and the lysyl oxidase is used for the obtained polypeptide. The position may be any position where lysyl oxidase activity is found by measuring the activity according to the activity measurement method.

Examples of the mutagenesis method include a conventional site-specific mutagenesis method. Examples of the site-directed mutagenesis method include, for example, a method using an amber mutation (gapped duplex method, Nucleic Ac
ids Research, 12, 9441-9456 (1984)], dut (dU
Method using a host lacking TPase) and ung (uracil-DNA glycosylase) genes [Kunkel (K
unkel) method, Proc. Natl. Acad. Sci. USA, 82, 488-492 (1
985)], and a method by PCR using a primer for mutation introduction.

In the present specification, the nucleic acid having the base sequence of (iii) may be a nucleic acid having a naturally occurring mutation or a nucleic acid into which a mutation has been artificially introduced.

The sense strand nucleic acid of the present invention has (iv) sequence identity with the nucleotide sequence of SEQ ID NO: 1 if the encoded polypeptide has lysyl oxidase activity. -MAC 8.0 (Softw
are Development Corporation), it includes nucleic acids having a base sequence of more than 64.8%. More specifically, the sequence identity with the nucleotide sequence of SEQ ID NO: 1 is preferably 80% or more, more preferably 90% or more, and still more preferably
Desirably, it is 95% or more.

Furthermore, the sense strand nucleic acid of the present invention has the following (v) sequence identity with the nucleotide sequence of SEQ ID NO: 3 as long as the encoded polypeptide has lysyl oxidase activity. -When analyzed using MAC 8.0, it also includes a base sequence exceeding 63.2%. More specifically, the sequence identity with the nucleotide sequence of SEQ ID NO: 3 is preferably 80% or more, more preferably 90% or more, and still more preferably
Desirably, it is 95% or more.

The nucleic acid having the nucleotide sequence of (iv) or (v) is such that the encoded polypeptide is, for example,
By measuring the activity according to the lysyl oxidase activity measuring method, those in which the activity is found may be selected.

In the present invention, sequence identity refers to the sequence similarity of residues between two nucleic acids or between two polypeptides. Said "sequence identity" can be determined by comparing two optimally aligned sequences over the region of the sequence to be compared. Here, the nucleic acid or polypeptide to be compared has an addition or a deletion (for example, a gap or the like) as compared with a reference sequence (for example, a consensus sequence or the like) for optimal alignment of the two sequences. Is also good.

The numerical value (percentage) of sequence identity is
Determine the same nucleobases present in both sequences, determine the number of matching sites, then divide the number of matching sites by the total number of bases in the sequence region to be compared, and divide the resulting number by 1
It can be calculated by multiplying by 00. Algorithms for obtaining the optimal alignment and homology include, for example, the local homology algorithm of Smith et al. [Add. APL. Math., 2, 482 (1981)], and the homology alignment algorithm of Needleman et al. [J. Mol. Biol., 48, 443 (1970)], Parrson (P
earson) et al. [Proc. Natl. Acad. Sci. US
A, 85, 2444 (1988)], and more specifically, dynamic programming, gap penalty,
Smith-Waterman algorithm, Good-Kanehisa algorithm, BLAST algorithm, FASTA algorithm and the like.

The nucleic acid sequence identity can be determined, for example, by using sequence analysis software, specifically, GENETYX-MAC, BLAS.
It is measured using TN or the like. The BLASTN can be found at the homepage address http: // www. ncbi. n
lm. nih. gov / BLAST / is generally available.

The sense strand nucleic acid of the present invention further comprises (v
i) A nucleic acid having a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 1 or 3 via a nucleic acid polymorphism is also included.

As used herein, the term “nucleic acid polymorphism” refers to a difference or change that may be found between individuals of the same species, between identical individuals of an organism, or between organisms, and within a range exhibiting lysyl oxidase activity. , A difference or change in the structure, trait or morphology of a nucleic acid.

[0029] Such nucleic acid polymorphisms, single nucleotide polymorphisms (S
NP), and more specifically, cSNP that causes amino acid substitution, sSNP without amino acid substitution, gSNP that is a polymorphism in the genome, rSNP that is a polymorphism in the regulatory region, and the like. The SNP may be one kind or a plurality of kinds.

Further, the sense strand nucleic acid of the present invention has (vii) any one of the base sequences (i) to (vi) as long as the encoded polypeptide has lysyl oxidase activity. A nucleic acid having a nucleotide sequence of a nucleic acid capable of hybridizing with a complementary strand of the nucleic acid under stringent conditions is also included.

Here, “stringent conditions” refers to, for example, molecular cloning: A Laboratory Manual, 2nd edition [T. Maniatis (T.Man
iatis) et al., Molecular Cloning: A Laboratory Manual
2nd ed., Published by Cold Spring Harbor Laboratory, (1989)]
And the like. Specifically, for example, 6 × SSPE, 0.2% B
SA, 0.2% Ficoll 400, 0.2% polyvinylpyrrolidone, 0.1% SDS, 100 μg / m
l Conditions for incubation at 42 ° C. for 16 hours in denatured salmon sperm DNA.

The nucleic acid having the nucleotide sequence of (vii) can be obtained, for example, by subjecting an appropriate library to the hybridization method described in Molecular Cloning: A Laboratory Manual, 2nd ed. By selecting a clone exhibiting a signal associated with the formation of a hybrid, and measuring the activity of the obtained clone according to the lysyl oxidase activity measurement method, by selecting a clone in which lysyl oxidase activity is found. sell.

In general, in the process of protein expression in a series of transcription and translation, an isoform may be generated from the same genomic DNA by alternative splicing. In the present invention, if the obtained nucleic acid has lysyl oxidase activity, (v
iii) By alternative splicing, (i)
And base sequences different from any of the base sequences (vii) to (vii).

As described in Examples below, the sense strand nucleic acid of the present invention is derived from mRN from cartilage progenitor cells (ATDC5 cells) at the hypertrophic / calcified stage expressing type X collagen.
A and cartilage precursor cells (ATDC5
Cells), and Diachenko (Diachenko)
tchenko, L.) et al., Proc. Natl. Acad. Sci. USA, 93,602.
5-6030 (1996) and the like, and a nucleic acid that is differentially expressed in the cartilage precursor cells at the hypertrophic / calcified stage is isolated by performing a suppression subtractive hybridization method as described in In some cases,
By a conventional hybridization method, PCR method, etc.
It can be obtained by obtaining a fragment containing the full length, and evaluating the lysyl oxidase activity of the polypeptide expressed from the obtained fragment by the lysyl oxidase activity measurement method.

The sense strand nucleic acid and / or antisense nucleic acid of the present invention provides an oligonucleotide that specifically hybridizes to the nucleic acid under stringent conditions. Such oligonucleotides are also included in the present invention.

The oligonucleotide of the present invention has a continuous sequence in a sequence complementary to the sense strand or antisense nucleic acid of the present invention. Here, the length of the oligonucleotide can be appropriately selected according to the purpose, but is at least 10 consecutive nucleotides, and when used as a primer for PCR, preferably 10 to 30 nucleotides, more preferably 20 nucleotides. It is desirable to be before and after.

If the sequence of the oligonucleotide is one that specifically hybridizes to the nucleic acid of the present invention, its T
It can be appropriately selected depending on the m value and the specificity of the sequence.

Here, the stringent conditions refer to, for example, the conditions for oligonucleotides for probes or primers described in Molecular Cloning: Laboratory Manual, 2nd edition, for example, 6 × SSC, 0. 5% SDS, 5x Denhardt,
In a solution containing 0.01% denatured salmon sperm nucleic acid, [Tm-2
5 ° C.] overnight.

The Tm of the oligonucleotide probe is 1
If 8 is the nucleotide or more chain length, for example, the following formula (I): Tm = 81.5-16.6 ( log 10 [Na +] + 0.41 (% G + C) - (600 / N) (I) ( wherein, N is the length of the oligonucleotide probe, and% G + C is the content of guanine residues and cytosine residues in the oligonucleotide probe primer))
Required by Further, when the length of the oligonucleotide probe is shorter than 18 nucleotides, Tm is, for example, the product of the content of A + T (adenine + thymine) residue and 2 ° C. and the content of G + C residue and 4 ° C. The sum with the product [(A
+ T) × 2 + (G + C) × 4].

The oligonucleotide of the present invention is a probe for detecting the nucleic acid of the present invention (sense strand and / or antisense nucleic acid) or the nucleic acid of the present invention (sense strand and / or antisense nucleic acid).
Or an antisense nucleic acid) can be used as a primer for specifically amplifying the same. The primers and probes are useful in a method for detecting a bone / cartilage repair state described below.

As described above, the sense strand nucleic acid of the present invention encodes a polypeptide having lysyl oxidase activity. Such polypeptides are also included in the present invention.

The "polypeptide having lysyl oxidase activity" of the present invention comprises (I) the amino acid sequence of the polypeptide encoded by any of the nucleic acids (i) to (viii), and (II) SEQ ID NO: 2. Or, there is one major feature in the amino acid sequence shown in 4.

The amino acid sequence shown in SEQ ID NO: 2 is a polypeptide having lysine oxidase activity and is an amino acid sequence of a polypeptide derived from human, and the amino acid sequence shown in SEQ ID NO: 4 is A polypeptide having lysine oxidase activity,
2 is an amino acid sequence of a mouse-derived polypeptide.

The “polypeptide having lysyl oxidase activity” of the present invention is as follows: having a sequence presumed to be a signal peptide at its N-terminus, as shown in FIG. 3; And containing a four-fold repeating structure of a scavenger receptor cysteine-rich domain found in a membrane-bound protein; COS into which a sense nucleic acid of the present invention has been introduced by Western blot analysis using an antibody against the polypeptide of the present invention. It is recognized as a 82 kDa specific band in the culture supernatant of -7 cells, suggesting that it is an extracellular protein secreted as an 82 kDa active enzyme.

The "polypeptide having lysyl oxidase activity" of the present invention is, as shown in FIG.
Two sequence motifs important for tight binding to copper (II), a cofactor in lysyl oxidase: WXWHCHXHXH: involved four histidines involved in copper binding coordination and supplying nitrogen ligands; and GHK: another Lysine residue (amino acid number 639 in SEQ ID NO: 3) and tyrosine residue involved in the formation of lysine tyrosylquinone, a carbonyl cofactor in lysyl oxidase, Group (amino acid number: 67 in SEQ ID NO: 3)
5) is also included. Therefore, the "polypeptide having lysyl oxidase activity" of the present invention is expected to be involved in cross-linking of the extracellular matrix by lysyl oxidase activity.

The "polypeptide having lysyl oxidase activity" of the present invention has a property that collagen I and collagen II are used as substrates and the activity is inhibited by βAPN. In addition, the "polypeptide having lysyl oxidase activity" of the present invention is highly expressed in hypertrophic and calcified chondrocytes and has lysyl oxidase activity, thereby altering the formation of a collagenous extracellular matrix. This is expected to regulate endochondral bone formation.

Furthermore, the “polypeptide having lysyl oxidase activity” of the present invention is expressed in hypertrophic and calcified chondrocytes in vivo, in a cell line in vitro (cartilage precursor cell line ATDC5), and Because of its lysyl oxidase enzyme activity, LOXC is expected to play an important role in endochondral bone formation.

If the polypeptide of the present invention has a lysyl oxidase activity determined by measuring its activity in accordance with the lysyl oxidase activity measurement method described above, the amino acid sequence of (III) SEQ ID NO: 2 or 4 can be used. And a polypeptide having an amino acid sequence having a substitution, deletion, addition or insertion in at least one residue.

The introduction of a mutation such as substitution, deletion, addition, or insertion into the amino acid sequence of SEQ ID NO: 2 can be obtained by preparing a nucleic acid having a corresponding nucleotide sequence and expressing the nucleic acid. Such a nucleic acid can be prepared by the above-described mutagenesis method.

The number of the mutations is at least one residue, specifically, one or more, or more. The number of such mutations may be, for example, in a range where lysyl oxidase activity is found by measuring the activity according to the lysyl oxidase activity measurement method, and preferably one or several ranges.

The localization position of such a mutation may be any position at which lysyl oxidase activity is found by measuring the activity according to the lysyl oxidase activity measurement method.

The amino acid sequence having such a mutation may be an amino acid sequence having a naturally occurring mutation, or may be an amino acid sequence having an artificially introduced mutation.

The above-mentioned substitutions include hydrophobicity, charge, pK,
Substitution with an amino acid having similar properties such as characteristics on the three-dimensional structure; Substitution with an amino acid capable of changing only the three-dimensional structure and folding structure of the polypeptide only to maintain the physiological activity of the original polypeptide ( That is, β
-Substitution of residues easily taking a sheet, α-helix, etc.). Such substitutions include, for example,
Following: substitution within the group of glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.

Further, the polypeptide of the present invention, if its lysyl oxidase activity is found by measuring its activity in accordance with the lysyl oxidase activity measuring method described above, may have (IV) the amino acid sequence of SEQ ID NO: 2 When the sequence identity between them is analyzed using GENETYX-MAC 8.0, it also encompasses more than 55.5% of base sequences. More specifically, the sequence identity is preferably
It is preferably at least 70%, more preferably at least 80%, even more preferably at least 90%.

The polypeptide of the present invention, if its lysyl oxidase activity is found by measuring its activity in accordance with the lysyl oxidase activity measuring method described above, may have (V) the amino acid sequence of SEQ ID NO: 4 When the sequence identity between them is analyzed using GENETYX-MAC, it also includes a nucleotide sequence exceeding 55.1%. More specifically, the sequence identity is preferably at least 70%, more preferably at least 80%, even more preferably at least 90%.

The sequence identity between polypeptides can be determined by using sequence analysis software, specifically, GENETYX-MAC, BLA.
It is measured using STP, FASTA or the like. The BLA
The STP is at the homepage address http: // www
w. ncbi. nlm. nih. gov / BLAST /
In FASTA, homepage address http
p: // www. ddbj. nig. ac. jp is generally available.

Since the polypeptide of the present invention has the above-mentioned amino acid sequence, it has lysyl oxidase activity. For example, in the case of a polypeptide derived from a mouse having the amino acid sequence of SEQ ID NO: 4, a hypertrophy expressing type X collagen is obtained. In chondrogenic precursor cells at the calcification / calcification stage, they are differentially expressed with respect to chondrogenic precursor cells at an earlier differentiation stage. Therefore, application to bone / cartilage repair promoters, promotion of bone formation, regulation of collagenous extracellular matrix production, therapeutic agents for diseases related to collagen or elastin production, detection of bone / cartilage repair status, etc. are expected. sell.

The polypeptide of the present invention can be obtained by introducing the sense strand nucleic acid of the present invention into a suitable host cell and expressing it.

When the sense strand nucleic acid is introduced into a host cell, the polypeptide encoded by the nucleic acid is incorporated into a vector suitable for expression in the cell, an expression vector is obtained, and the expression vector is obtained by a conventional method. It can be introduced into a host cell, and the sense strand nucleic acid or the expression vector is retained on a carrier such as liposome, calcium phosphate, DEAE dextran, etc. to obtain a carrier for cell introduction, and the carrier for cell introduction can be obtained by a conventional method. Can be introduced into a host cell.

According to the present invention, 1) an expression vector in which the sense strand nucleic acid is carried by a vector suitable for expressing a polypeptide encoded by the nucleic acid in a cell, and 2) the sense strand nucleic acid or the Also included is a cell introduction carrier comprising a carrier containing an expression vector, wherein the carrier is a carrier selected from the group consisting of liposomes, calcium phosphate and DEAE dextran.

The expression vector of the above 1) can be prepared based on the above-mentioned molecular cloning: a laboratory manual. For example, a translation initiation codon is added upstream of a gene to be expressed and a translation stop codon is added downstream, and a control gene such as a promoter sequence (eg, trp, lac, T7) for controlling transcription is added, and an appropriate vector is added. By replicating in the host cell,
An operable expression vector can be obtained.

The vector used in the expression vector of the above 1) can be appropriately selected depending on the host cell to be used, the purpose, and the like.
Virus vectors and the like.

For example, when the host cell is an Escherichia coli cell, the vectors include pUC118, pUC119, pBR322, pCR3, and pCMV.
Plasmid vectors such as SPORT and phage vectors such as λZAPII and λgt11. When the host cell is a yeast cell, examples of the vector include pYES2 and pYEUra3. If the host cell is an insect cell, pAcSGHisNT
-A. When the host cell is an animal cell,
pKCR, pEFBOS, cDM8, pCEV4 and the like. Also,
A virus vector obtained from a retrovirus, a DNA virus, or the like can be used, and examples thereof include an adenovirus vector, a vaccinia virus vector,
Mouse leukemia virus vector and the like.

The vector may optionally have factors such as an inducible promoter, a selectable marker gene, and a terminator.

In order to facilitate isolation and purification, His
A tag and a sequence that can be expressed as a GST fusion protein may be added. In this case, GST (glutathion S-) having an appropriate promoter (lac, tac, trc, trp, CMV, SV40 early promoter, etc.) that functions in the host cell is used.
It can be used transferase) fusion protein vector (such as pGEX4T) and Tag (Myc, vectors having HisA etc.) sequence (pCDNA3.1 / Myc-His, etc.).

Examples of the carrier used for the carrier for cell introduction of 2) include liposomes, calcium phosphate, DEAE dextran and the like.

Then, the expression vector of the above 1) or 2)
Is introduced into a host cell to obtain a transformant. The present invention also includes such transformants.

The host cells used for expression of the polypeptide of the present invention include E. coli cells, yeast cells, animal cells,
Insect cells and the like. As Escherichia coli, Escheric
hiacoli K-12 strains HB101, C600, JM109, DH5
α strain, DH10B strain, XL-1BlueMRF ′ strain, TOP10F strain and the like. As yeast cells, Saccharomyces
Servisier and the like. As animal cells, L,
3T3, FM3A, CHO, COS, Vero, Hela and the like. Insect cells include sf9.

Examples of the method of introducing the expression vector of 1) or the carrier for cell introduction of 2) into host cells include the calcium phosphate method, the DEAE-dextran method, the electroporation method, lipofection, and infection. , Carrier and host cell.

The polypeptide of the present invention can be obtained by culturing the obtained transformant in a medium suitable for the host cell.

The obtained polypeptide can be further isolated and purified by general biochemical and purification means. Examples of the purification means include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, and gel filtration chromatography.

The polypeptide of the present invention can further provide an antibody capable of specifically binding to the polypeptide. According to such an antibody, it has a property of specifically binding to the polypeptide of the present invention, so that it is used for the treatment of diseases related to regulation of collagenous extracellular matrix production, collagenous extracellular matrix production, collagen or elastin production. It is expected to be applied to agents, bone / cartilage repair state detection, etc. Such antibodies are also included in the scope of the present invention.

The antibody of the present invention may be any antibody capable of specifically binding to the polypeptide, and may be a polyclonal antibody or a monoclonal antibody, or a fragment thereof. Furthermore, antibodies and antibody derivatives modified by known techniques, for example, humanized antibodies,
Fab fragments, single-chain antibodies and the like can also be used. The antibody of the present invention can be obtained, for example, in 1992 by John Wiely & Sons, Inc., edited by John E. Coligan, and edited by Current Protocols in Immunology (Current). Protocols
The method can be easily prepared by immunizing a rabbit, mouse, or the like using the entire polypeptide of the present invention or a region characteristic of the polypeptide of the present invention by the method described in In Immunology, for example. Antibodies can also be produced by genetic engineering. The present invention also includes an antibody or a fragment thereof capable of specifically binding to a partial fragment in the polypeptide.

The antibody fragment can be obtained by purifying the obtained antibody and treating it with peptidase or the like.

Further, when the antibody of the present invention is used for detecting a bone / cartilage repair state, etc., the antibody of the present invention is used to facilitate detection by enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay and the like. May be subjected to various modifications.

According to the nucleic acid (antisense nucleic acid) having a nucleotide sequence complementary to the sense strand nucleic acid of the present invention, lysyl oxidase activity can be regulated. It is expected to be involved in the regulation of extracellular matrix production and in the production of collagen or elastin, and it can also be expected to be applied to the detection of bone / cartilage repair status. The present invention also includes such antisense nucleic acids.

When the antisense nucleic acid is introduced into cells, the antisense nucleic acid is retained on a carrier selected from the group consisting of liposome, calcium phosphate and DEAE dextran, and the resulting carrier for cell introduction is used in a conventional manner. Can be introduced into cells by the method described above.

The present invention also includes a cell introduction carrier comprising a carrier containing an antisense nucleic acid, wherein the carrier is a carrier selected from the group consisting of liposomes, calcium phosphate and DEAE dextran.

According to any of the nucleic acids (i) to (viii) or a nucleic acid having a nucleotide sequence complementary thereto, the expression of lysyl oxidase activity can be regulated by introducing the nucleic acid into an appropriate cell. . Further, according to the polypeptide of the present invention, a polypeptide derived from a mouse having lysyl oxidase activity and having, for example, the amino acid sequence of SEQ ID NO: 4 is subjected to the hypertrophication / calcification stage expressing type X collagen. Utilizing the fact that certain cartilage progenitor cells are differentially expressed from cartilage progenitor cells at an earlier differentiation stage, to promote or assist in collagenous extracellular matrix production, collagen or elastin production, bone / cartilage It can be used to detect a repair state. Furthermore, according to the antibody of the present invention, the property of specifically binding to the polypeptide of the present invention is utilized to regulate the production of collagenous extracellular matrix, to participate in the production of collagen or elastin, and to repair bone / cartilage. Application to detection etc. is expected.

Therefore, according to the sense strand nucleic acid, antisense nucleic acid, polypeptide and antibody of the present invention, bone / cartilage repair promoter, collagenous extracellular matrix production regulator, disease associated with collagen or elastin production. Agent for detecting bone and cartilage repair state,
A method for detecting a cartilage repair state and the like can be provided, and these are also included in the scope of the present invention.

The bone / cartilage repair promoting agent of the present invention is selected from the group consisting of the sense strand nucleic acid, the polypeptide, the expression vector, and a carrier for cell introduction containing the sense strand nucleic acid or the expression vector. One feature is that one kind is contained as an active ingredient. According to the bone / cartilage repair promoting agent of the present invention, lysyl oxidase activity is expressed by introduction into cells, and the endochondral ossification process can be regulated. it can.

The content of the active ingredient in the bone / cartilage repair-promoting agent of the present invention is within a range capable of sufficiently exerting the bone / cartilage repair-promoting effect according to the purpose of administration, the age, weight, and condition of the individual. Can be set as appropriate. In addition, the number of administrations of the bone / cartilage repair promoter of the present invention can be appropriately set according to the purpose of administration, the patient's age, body weight, and the like, as long as the effect of promoting bone / cartilage repair can be sufficiently exerted. For example, in the case of the sense strand nucleic acid, 0.0001 to 100 mg, in the case of the polypeptide, 0.001 to 1000 mg, in the case of the expression vector or the cell introduction carrier, in the same degree as in the case of the sense strand nucleic acid. Desirably.

A dosage form can be produced according to a general method by mixing an active ingredient with usual pharmacologically acceptable carriers, excipients, binders, stabilizers and the like. When used in an injection dosage form, a buffer, a solubilizing agent, an isotonic agent and the like may be added.

In the present invention, when a bone / cartilage repair promoting agent containing a polypeptide as an active ingredient is introduced into cells, tissues or individuals, for example, oral administration, injection administration and the like are carried out according to conventional means. Can be. In order to sufficiently exhibit the effect of promoting bone and cartilage repair, it may further contain substances which may be delivered to the site in need of bone and cartilage repair. The polypeptide may be administered together with an adjuvant, or may be administered as a particulate dosage form. As the adjuvant, literature [Clin. M
icrobiol. Rev., 7, 277-289 (1994)]. In the present invention, it may be introduced as a liposome preparation, a particulate preparation bonded to beads having a diameter of several μm, or a preparation bonded to lipid. Such formulations, intradermal administration, subcutaneous administration, intravenous injection, intraarterial, intramuscular administration, local administration or the like, can be introduced. At the time of introduction, the amount of the bone / cartilage repair promoter used can be adjusted depending on the purpose.

Methods for introducing the sense strand nucleic acid or the expression vector into cells, tissues or individuals include: coprecipitation with calcium phosphate; direct injection of nucleic acid using a micro glass tube; gene transfer using encapsulated liposomes. Gene transfer method using electrostatic liposome; HVJ-liposome method,
Improved HVJ-liposome method (HVJ-AVE liposome method); receptor-mediated gene transfer method; method of transferring nucleic acid molecules into cells with a carrier (metal particles) using a particle gun; direct transfer of naked-DNA; An introduction method using a polymer and the like can be mentioned. Alternatively, the sense strand nucleic acid or the expression vector may be held in a carrier and introduced into cells as the cell introduction carrier.

Repair of bone and cartilage is described, for example, in The Journal.
of Pharmacology and ExperimentalTherapeutics, 290,
1054-1064 (1999).

The collagenous extracellular matrix production regulator of the present invention is selected from the group consisting of the sense strand nucleic acid, the antisense nucleic acid, the polypeptide, the antibody, the expression vector and the cell introduction carrier. Contains seeds as active ingredients.

According to the collagenous extracellular matrix production regulator of the present invention, the formation of crosslinks between collagens in the extracellular matrix (ie, lysine residue or hydroxyllysine in collagen fibrils) is achieved by regulating lysine oxidase activity. The amount of deamination of the residues to form aldehyde groups, thereby forming cross-links between the aldehyde groups and between the aldehyde groups and lysine or hydroxylysine residues) can be controlled. The tension and strength of collagen fibrils can be adjusted. Therefore, according to the collagenous extracellular matrix production regulator of the present invention, an extracellular matrix having properties suitable for a cartilage matrix can be obtained.

The content of the active ingredient in the collagenous extracellular matrix production regulator of the present invention depends on the amount of the crosslinks formed between the collagens in the collagen fibrils. What is necessary is just to set the property to a desired property.

Further, according to the present invention, there is also provided a reagent for producing a collagenous extracellular matrix, which comprises the above-mentioned agent for regulating the production of a collagenous extracellular matrix as an active ingredient.

[0091] The content of the active ingredient in collagenous extracellular matrix production reagent of the present invention, the amount of cross-linking between the collagen in the collagen fibrils, the collagen fibrils tensile strength adjusted to the extracellular matrix What is necessary is just to set the property to a desired property.

The therapeutic agent of the present invention contains, as an active ingredient, one selected from the group consisting of the sense strand nucleic acid, the antisense nucleic acid, the polypeptide, the antibody, the expression vector, and the cell introduction carrier. There is one feature in doing so.

The therapeutic agent of the present invention comprises the sense strand nucleic acid, the antisense nucleic acid, the polypeptide, the antibody, the expression vector,
Since the carrier for cell introduction and the like are included, lysyl oxidase activity can be regulated, whereby diseases associated with collagen or elastin production, that is, the process of collagen or elastin production and the matrix formed thereby can be used. Diseases in which abnormalities in the formation process can be the cause of disease, more specifically, improvement and treatment of diseases in which abnormal expression of lysyl oxidase activity is a cause of the disease are expected.

The diseases related to the production of collagen or elastin include diseases in which abnormal expression of lysyl oxidase activity causes the disease. Specific examples include fibrotic lesions, chondrosarcoma, osteosarcoma, and aortic sclerosis. , Hypertension, Ehlers-Danlos syndrome type V, and the like.

The amount of the active ingredient in the therapeutic agent of the present invention can be appropriately set according to the disease, the age and weight of the patient, and for example, in the case of the sense strand nucleic acid, in the case of the antisense strand nucleic acid, 0.0001 to 100 mg, 0.001 to 10 in the case of the polypeptide or the antibody.
In the case of 00 mg, the expression vector or the carrier for cell introduction, it is desirable that the amount is the same as that of the antisense strand nucleic acid.

When the active ingredient of the therapeutic agent is the sense strand nucleic acid, the antisense nucleic acid, the expression vector, or a carrier for cell introduction containing any of them, in vivo, the therapeutic agent is directly introduced into the body. The administration can be carried out by a method or an ex vivo method in which certain cells are removed from an animal, for example, a mammal such as a human, the antiretroviral agent is introduced into the cells outside the body, and the cells are returned to the body. For such techniques, for example,
Nikkei Science, April 1994, pp. 20-45; Monthly Pharmaceutical Affairs, 36
(1), 23-48, 1994; Experimental Medicine Extra Edition, 12 (15), 1994; Japanese Society of Gene Therapy, Gene Therapy Development Research Handbook, NTT, 1999, etc.

The dosage form can be appropriately selected according to the administration form. Specific examples include injections and liposome preparations. For example, in the case of an injection, the injection can be prepared by a conventional method. For example, after dissolving in an appropriate solvent (buffer, phosphate buffered saline, physiological saline, sterilized water, etc.), If desired, it can be prepared by sterilizing by filtration with a filter or the like, and then filling in a sterile container. Further, a conventional carrier or the like may be added.

The nucleic acid having the nucleotide sequence of (i) to (viii) and the polypeptide having the amino acid sequence of (I) to (V) are molecules having lysyl oxidase activity. The repair state of bone / cartilage can be detected using the expression of the polypeptide as an index. Therefore, the present invention can provide a reagent for detecting a bone / cartilage repair state, comprising one selected from the group consisting of the sense strand nucleic acid, the antisense nucleic acid, and the antibody, and a detection method using the same. . Detection reagents and detection methods are also included in the scope of the present invention.

The detection method is a method for detecting a bone / cartilage repair state using the detection reagent, and specifically includes three embodiments. That is, the method of aspect 1: (a) contacting the detection reagent (reagent containing the sense strand nucleic acid or antisense nucleic acid of the present invention) with a test sample; and (b) the following (1) or ( 2): (1) a hybrid complex of the antisense nucleic acid and a nucleic acid having a base sequence selected from the group consisting of (i) to (viii), or (2) the sense strand nucleic acid and the (i) A method comprising the step of detecting the presence or absence of the formation of a hybrid complex with the complementary strand of the nucleic acid according to any one of) to (viii); the method of Embodiment 2: a test sample using a primer pair comprising the oligonucleotide of the present invention; And a method comprising the step of performing nucleic acid amplification using a nucleic acid derived from as a template; and the method of Embodiment 3: (A) contacting the detection reagent (containing the antibody) with a test sample; and ) And antibody, comprising detecting the presence or absence of formation of a complex between the polypeptide containing the (I) ~ amino acid sequence selected from the group consisting of (V).

In the method of Embodiment 1, the test sample includes cells, tissues, products or extracts obtained therefrom, and more specifically, nucleic acids derived from bone / cartilage tissue, or those adjacent to bone / cartilage tissue. Nucleic acids derived from the connective tissue described above.

The contact between the detection reagent and the test sample is
For example, it can be performed by a conventional hybridization method, an antigen-antibody reaction method, or the like.

The detection of the hybrid complex is carried out, for example, by labeling a sense nucleic acid or an antisense nucleic acid used as a detection reagent with a conventional enzyme, fluorescent substance or radioactive label substance which can be used for labeling a nucleic acid. It is carried out by examining the presence or absence of a label derived from the complex formed by the hybridization method.

In the method of the first embodiment, detection of the hybrid complex is an indicator of the state of bone / cartilage repair.

In the method of Embodiment 2, the test sample is the same as in the method of Embodiment 1.

The nucleic acid amplification is carried out by a conventional PCR method, RT-
It can be performed by a PCR method or the like.

The primer pair is the oligonucleotide of the present invention, and is a pair of oligonucleotides capable of specifically amplifying the nucleic acid according to any one of the above (i) to (viii).

[0107] In the method of Embodiment 2, the production of a product specifically amplified by the primer pair is an indicator of a bone / cartilage repair state.

[0108] The test sample used in the methods of Embodiments 1 and 2 may be subjected to an operation of removing other components in the sample as long as the nucleic acid to be detected is not damaged.

Further, in the method of Embodiment 3, the test sample includes cells, tissues, products or extracts obtained therefrom, and more specifically, from bone / cartilage tissues and connective tissues adjacent thereto. The resulting product or extract is mentioned.

The detection of the complex between the antibody and the polypeptide
It can be performed by a conventional enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay or the like.

In the method of the third embodiment, the detection of the complex is an indicator of the state of bone / cartilage repair.

It is expected that the state of production of the collagenous extracellular matrix can be easily estimated by the method for detecting the state of bone / cartilage repair according to the present invention.

[0113]

EXAMPLE 1 Experimental method (1) Cells and culture conditions ATDC5 cells were seeded on a 6-well plate at 6 × 10 ⁴ cells per well and cultured according to a conventional method [Akiyama, H.] Et al., J. Bone Miner. Res., 11, 22-28
(1996); Akiyama et al., Biochem. Biophys. Res. Commu.
n., 235, 142-147, (1997); Akiyama et al., Cell Struct.
Funct., 25, 195-204, (2000); Ito, H. et al.,
Biochim. Biophys. Acta., 1451, 263-270 (1999); Shukunami, C. et al., J. Bone Miner. Res., 12,
1174-1188 (1997)].

Specifically, for 21 days after the start of the culture, 5%
Fetal bovine serum (FBS) [GIBCO
BRL) and 10 mM human transferrin (Roche Molecule Biochemicals).
ar Biochemiclas), 30 nM sodium selenate (Sigma) and 10 mM bovine insulin (Wako Pure Chemical Industries).
The cells were cultured under the conditions of 5% CO ₂ /95% air humidified with m's F12 medium. On day 21, the medium was supplemented with 5% FBS,
M containing 10 mM human transferrin, 30 nM sodium selenate, and 10 mM bovine insulin
EM medium [ICN Pharmaceuticals (I
CN Pharmaceuticals), and the cells were cultured with the CO ₂ concentration changed to 3% to promote cell hypertrophy and mineralization. Mouse embryonic fibroblast C3
H10T1 / 2 cells, mouse osteogenic cells MC3T3-E
One cell, mouse fibroblast NIH3T3 cells and mouse myoblast C2C12 cells (RIKEN cell bank) were seeded on a 6-well plate at a density of 6 × 10 ⁴ cells per well.
Cultured according to a conventional method [Akiyama et al., J. Biol. Chem., 27
4, 32192-32197 (1999)].

(2) Detection of Differentially Expressed Genes From the differentiated (day 21) ATDC5 cells and the calcified (day 42) ATDC5 cells, poly (A) ⁺
Single-step RNA [Ito et al., Biochem. B
iophys. Res. Commun., 260, 240-244 (1999)]. Next, using the poly (A) RNA, differentially expressed genes were concentrated by suppression subtractive hybridization. The suppression subtractive hybridization method includes a PCR-Select cDNA.
Using Subtraction Kit (Clonetech Laboratories),
Such an operation was performed according to the product instructions.

As a result, it was found that a cDNA fragment of about 600 bp was expressed at a high level in ATDC5 cells at the calcification stage.

(3) Identification and Isolation of Gene Next, the cDN of about 600 bp detected in the above (2) was used.
The A fragment was converted to a pCR2.1 vector [Invitogen
rogen).

In addition, poly-calcified ATDC5 cells
(A) Oligo (dT) primed c from ⁺ RNA using a ZAP expression vector [Stratagene].
A DNA library was constructed.

[0119] Such the cDNA library, as follows, wherein about using a cDNA fragment of 600bp as a probe, 1 × 10 ⁶ plaques were screened [Akiyamara, Biochem. Biophys. Res. Commu
n., 235, 142-147, (1997); Akiyama et al., J. Biol. Che.
m., 274, 32192-32197 (1999)]. In other words, the plaque a membrane (137mm nylon membrane, NEN Life Science Products (NEN Life Science Product
s). Then, the c of about 600 bp
The DNA fragment was labeled with ³² P, and the resulting labeled cDNA fragment was used as a probe to produce a DNA fragment, as described in Akiyama et al., Biochem.
Hybridization was performed as described in ys. Res. Commun., 235, 142-147, (1997). Then, after hybridization, 0.1 × SSPE (3M Na
Cl, 197 mM NaH ₂ PO ₄ , 25 mM EDT
A) and 0.1% SDS, washed under stringent conditions at 55 ° C. The nucleotide sequence is ALFred DNA
Analysis was performed with a sequencer (Amersham Pharmacia Biotech).

(4) Northern analysis ATDC5 cells, C3H10T1 / 2 cells, MC3T3
-E1 cells, NIH3T3 cells and C2C12 cells were each seeded on a 6-well plate, and cultured as described in (1) above.

The total RNA of the cultured cells cultured in vitro was compared with the polymorph of costal cartilage of 3-week-old ICR mice.
(A) ⁺ RNA was isolated. Obtained total RNA and p
For poly (A) ⁺ RNA, Akiyama et al. [Bi
ochem. Biophys. Res. Commun., 235, 142-147 (199
7)]; and Ito et al. [Biochem. Biophys. Res. Co.
mmun. 257, 814-820 (1999)]. That is, total RNA
(20 μg) and poly (A) ⁺ RNA (2 μg) were denatured, separated by 1% agarose gel electrophoresis, and transferred to a Nytran membrane (Schleicher & Schuell).
A 1.3 kb LOXC cDNA fragment, a 0.55 kb mouse type II collagen cDNA fragment and a 0.65 kb mouse X type collagen cDNA fragment were used as probes. Each of these cDNA fragments was labeled with ³² P.

In the analysis of tissue distribution in adult mice, labeled cDNA was analyzed using multiple tissue northern blot.
(Clonetech Laborat
ories). After hybridization, the resulting membrane is
htening plus intensifying screens (DuPont
X-Omat film (Eastman Kodak) was exposed at -80 ° C.

(5) In situ hybridization The tibia of a male newborn C57BL / 6J mouse was collected. The tibia was then fixed overnight at 4 ° C. in a 4% paraformaldehyde solution prepared with 10 mM phosphate buffered saline (pH 7.4). The obtained tibia,
Decalcified in 10% EDTA for 4 days. Furthermore, it was dehydrated with ethanol and embedded in paraffin. Using 6 μm-thick sections, literature [Akiyama et al., Biochem. Biophys. Res. C
ommun., 257, 814-820 (1999)].
Itu hybridization was performed.

That is, 1.3 kb mouse LOXC cDNA, 0.4 kb, subcloned into pBSII-KS (+) (Stratagene).
Mice Type II collagen cDNA and 0.55kb mouse type X collagen cDNA respective subclones body was linearized with the appropriate restriction enzymes and transferred to a sense or antisense ³⁵ S-labeled riboprobes.

Then, in a humidified chamber, 50% formamide, 10% dextran sulfate, 1 × Denhardt's solution, 600 mM NaCl, 10 mM Tris-H
Cl, 1 mM EDTA, 50 mM dithiothreitol, 0.25% SDS and 200 μg / ml tRN
In A, the section and ³⁵ S-labeled riboprobe were incubated at 55 ° C. for 18 hours.
Hybridization was performed by incubating for an hour. Thereafter, the obtained section was subjected to 5 × S
Wash with SC at 50 ° C., 50% formamide, 2 × SSC at 50 ° C. for 30 minutes, then 10 mM Tris-HCl, 500 mM NaC
1, 3 mM at 37 ° C. using 1 mM EDTA (TNE).
Washed for 0 minutes. Thereafter, the sections were prepared at 10 μg / ml R
The cells were treated with a TNE solution containing Nase A at 37 ° C. for 30 minutes. After washing with TNE, sections were incubated once with 2 × SSC at 50 ° C. for 20 minutes and 0.2 ×
Washing was performed twice at 50 ° C. for 20 minutes using SSC. The dried tissue sections were then immersed in Kodak NTB-22 emulsion and exposed at 4 ° C. Sections were counterstained with hematoxylin. In addition, hybridization using a sense probe was performed as a negative control. Also,
Sections treated with RNase prior to in situ hybridization with riboprobes showed no autographic signal, indicating that the hybridization signal was dependent on the presence of RNA.

(6) In vitro transcription / translation Rabbit reticulocyte lysate system [TNT Qui
ck CoupledTransscription / T
translation System; Promega (Prome
ga) Co., Ltd., and according to the product instructions, ³⁵ S-labeled methionine [Amersham Pharmacia Biotech (Amersham)
ham Pharmacis Biotech)] in the presence of a transcription / translation reaction. pcDNA3.1 vector [Invitrogen (I
5.4 kb mouse LOXC cDNA cloned into the company (Nvitrogen) was used as a template (pCMV / mLOXC). Translation products were electrophoresed on a 10-20% polyacrylamide gel and detected by autoradiography with X-Omat film (8 hours exposure at room temperature). Rainbow markers (Bio-Rad Laboratories) were loaded in adjacent lanes for molecular weight estimation.

(7) Preparation of Polyclonal Antibody C-terminal Sequence of Mouse LOXC (AELSLEQEQRLR)
Antisera were generated in rabbits using keyhole rimpet hemocyanin conjugated peptide corresponding to (NNL). Harlow, E.
Et al., Antibodies, Cold Spring Harbor Laboratory,
(1988).

(8) Introduction of DNA into COS-7 cells In a DMEM medium containing 10% FBS, COS-
Seven cells were cultured. For the measurement of lysyl oxidase activity, pDE using dextran (manufactured by Sigma) was used.
With CMV / mLOXC or pcDNA3.1, C
OS-7 cells were transiently transfected, and 7 days after transfection, a culture supernatant was prepared. For Western blot analysis, FuGene6 [Roche
Molecular Biochemicals Co., Ltd.] and according to the manufacturer's instructions, pCMV / mLOXC or pcD
COS-7 cells were transfected with NA3.1 and whole cell proteins were prepared two days after transfection.

(9) Western blot analysis Ito et al., Biochem. Biophys. Res. Commun., 260, 240
As described in -244 (1999), ATDC5 cells and pC
Whole cell proteins were prepared from COS-7 cells transfected with MV / mLOXC or pcDNA3.1. 40 μg of protein was separated on a 10-20% polyacrylamide gel and transferred to a nitrocellulose filter. Filter with 0.1% Tween2
Blocked in 3% gelatin solution prepared with Tris buffer containing 0 and incubated with anti-LOXC antibody. Rainbow markers (Bio-Rad Laboratories) were loaded in adjacent lanes for molecular weight estimation.

(10) Measurement of Lysyl Oxidase Activity Iguchi, H. et al., Toxicol. Appl. Pharmacol. 6
2, 126-136, (1982), collagen substrate preparation and lysyl oxidase activity measurement.

Specifically, 20 pairs of parietal or cartilage proximal end growth plates in 17-day-old chicken embryos were excised and suspended in 6 ml of lysine-free Eagle's minimal basal medium. To the obtained suspension, L- [4,5- ³ H] lysine [200 μCi, manufactured by N.N. Life Science Products] and β-aminopropionitrile (βAPN) -fumaric acid (50 μg / m as βAPN)
1, Sigma) and incubated at 37 ° C. for 24 hours.

Then, the obtained parietal bone or growth plate is
0.05 M Tris buffer containing 1 M NaCl (pH
The mixture was suspended in 7.4) and homogenized. The homogenate was stirred at 4 ° C. for 90 minutes and centrifuged at 20,000 × g for 20 minutes. The labeled collagen in the supernatant fraction was replaced with 20% NaC
and collected by centrifugation at 30,000 × g for 20 minutes. The resulting precipitate is suspended in a minimum amount of 0.05 M Tris buffer (pH) containing 0.15 M NaCl,
Dialysis was performed against the same buffer for 24 hours.

In an assay tube, 2.5 × 10 ⁵ cp
m dispensed considerable ^[3 H] -I collagen or a substrate solution containing a type II collagen minute of 0.9ml LOXC c
The culture supernatant of the DNA-introduced COS-7 cells was added.

The reaction was incubated at 37 ° C. for 3 hours, and the reaction was stopped by freezing at −20 ° C. Pinel et al. [Pinnel, SR, Proc. Natl. Aca
d. Sci. USA, 61, 708-716 (1968)], the generated tritium water is collected by distillation under reduced pressure,
The radioactivity of ml was measured with a liquid scintillation counter. As a control, preincubation was performed with [ ³ H] -labeled collagen substrate for 1 hour before the addition of the culture supernatant, and the assay was performed in the presence of βAPN (50 μg / ml), a specific inhibitor of lysyl oxidase. Done.

(11) Statistical analysis Statistical significance was determined by one-way analysis of variance and Student
Analysis was performed by t test of t.

Results (1) Cloning and Structure of Mouse LOXC cDNA In the presence of insulin, ATDC5 cells reached confluence 5 days after seeding and started to form cartilage. Akiyama et al., Biochem. Biophys. Res. Commun., 235, 142-147,
(1997); Ito, H. et al., Biochim. Biophys. Act
a., 1451, 263-270 (1999); and Shukunami,
C.) et al., J. Bone Miner. Res., 12, 1174-1188 (1997).
Reported that the cartilage formation process proceeded in an orderly manner in synchronization with the expression of a phenotypic marker gene characteristic of cartilage. On day 7, cell aggregation occurred, and from day 9 onwards 21
On day, cartilage nodule formation and growth was observed, followed by calcification on day 35.

In order to find a gene whose expression is promoted during the process of hypertrophy and calcification of chondrocytes, suppression subtraction was performed using poly (A) ⁺ RNA extracted from each of differentiated ATDC5 cells and calcified ATDC5 cells. Hybridization was performed. As a result, a differentially expressed 600 bp cDNA fragment was obtained.

Using the obtained fragment as a probe, calcification A
The mouse cDNA library prepared from TDC5 cells were screened under high stringency conditions. As a result, from 1 × 10 ⁶ plaques, 20 cDNs
A clone was obtained. Eight of these clones contained approximately 5.4 kb of cDNA. FIG. 1 shows the nucleotide sequence and amino acid sequence of the gene contained in the fragment. Such a gene was designated as LOXC (also referred to as lysyl oxidase-related protein) gene. The 600 bp cDNA fragment is a 3′-terminal of the LOXC gene.
It corresponds to the untranslated sequence.

FIG. 1 shows the nucleotide sequence of LOXC cDNA and the deduced amino acid sequence of LOXC protein. As shown in FIG. 1, it can be seen that the LOXC cDNA contains a 2271 bp open reading frame starting at the ATG codon. Therefore, it is estimated that the LOXC protein consists of 757 amino acid residues. LO
The XC protein is found to have a molecular weight of 84,705 daltons (Da) and an isoelectric point of 7.8. The 3 'end of the sequence contains a poly (A) stretch preceded by a putative polyadenylation signal (AATAAA).

FIG. 2 shows the result of analyzing the LOXC protein by the hydrophobicity plot. Kyte-Do
According to the hydrophobicity plot using the olittle algorithm, as shown in FIG. 2, the 25 amino acids starting from the first ATG start codon are characteristic of the signal peptide sequence. Based on these data, LOX
This suggests that the C cDNA may encode an extracellular protein. In addition, it can be seen that cleavage of the signal peptide yields a protein of 732 amino acid residues having a molecular weight of 81,864 Da.

For the amino acid sequence of the LOXC protein shown in FIG. 1, BLASTP (http: // www)
w. ncbi. nlm. nih. gov / BLAST
The homology search of the sequence was performed by /). The amino acid sequences of mouse lysyl oxidase (LO) and mouse lysyl oxidase-like protein 2 (Lor2)
Each Gene Bank Accession Number NM 01
This is a sequence deduced from the nucleotide sequences of 0728 and AF053368. As a result, as shown in FIG. 3, the amino acid sequence of the LOXC protein
or2 at the amino acid sequence level, respectively.
Shows 0% and 52% agreement. GENETYX-MA
When analyzed using C, the sequence identity of the amino acid sequence of the LOXC protein was 55.1% with respect to mouse lysyl oxidase-like protein 2 (Lor2), and human lysyl oxidase-like protein 2 (Lor2).
53.9% against 2).

(2) Expression of LOXC protein by in vitro transcription / translation To confirm that the 5.4 kb LOXC cDNA contains a functional open reading frame, rabbits were expressed in the presence of ³⁵ S-labeled methionine. In vitro transcription / translation was performed using the reticulocyte lysate system. As a result, as shown in FIG.
When cDNA 3.1) was used, no protein product was detected, but it was found that a single protein of the expected size could be detected in the clone retaining LOXC cDNA.

(3) Expression of Recombinant LOXC in COS-7 Cells and Western Blot Analysis Recombinant LOXC protein was expressed in COS-7 cells by transfection with pCMV / mLOXC. LOX
The protein was identified in the cell lysate by immunoblotting with the C antibody. The result is shown in FIG.

As shown in FIG. 5, no signal was observed in the mock-transfected cells, but a LOXC protein band was observed in the cell lysate, and a specific band of about 82 kDa was observed. It can be seen that it also corresponds to the calculated value of the molecular weight.

(4) Expression of LOXC in various cell lines ATDC5 cells, C3H10T1 / 2 cells, MC3T3
-Expression of LOXC mRNA in a cultured cell line was analyzed by Northern analysis using E1 cells, NIH3T3 cells and C2C12 cells.

As a result, as shown in FIG.
The major hybridization band of kb is C3H1
It can be seen that it is detected in 0T1 / 2 cells and MC3T3-E1 cells, but not in NIH3T3 cells and C2C12 cells. Also, as shown in FIG.
LOXC in ATDC5 cells was determined by Northern analysis.
It can be seen that mRNA levels increase in parallel with the induction of type X collagen mRNA. Further, as shown in FIG. 8, undifferentiated ATD
No LOXC protein was detected in C5 cells, indicating that LOXC protein levels in the culture system increased during the process of cartilage differentiation.

(5) Expression of LOXC mRNA in mature mouse tissues and growth plates of neonatal mice As shown in FIGS. 9 and 10, by Northern analysis,
Among various mature mouse tissues, LOXC was found in cartilage.
It turns out that mRNA is highly expressed. As shown in FIG. 11, by in situ hybridization, LOXCmRNA is seen to be localized in the hypertrophic and calcified cartilage cells growth plate of neonatal mice.

(6) Lysyl oxidase enzyme activity of LOXC Since the deduced amino acid sequence of LOXC showed homology to the amino acid sequence of lysyl oxidase, it was suggested that LOXC may have lysyl oxidase enzyme activity.

Using the type I collagen substrate and type II collagen substrate prepared from chicken embryos, the enzyme activity was measured by the tritium release method. As the LOXC protein, a culture supernatant obtained from COS-7 cells into which the LOXC cDNA was introduced was used to detect the LOXC protein by Western blot analysis.

As a result, as shown in FIG.
It can be seen that significant activity was detected in the culture supernatant of COS-7 cells into which XC cDNA had been introduced, but no activity was detected in the culture supernatant of cells into which the control vector had been introduced. In addition, as shown in FIG. 12, it can be seen that this enzyme activity is inhibited by βAPN which is an irreversible inhibitor of lysyl oxidase.

Lysyl oxidase is an extracellular copper-dependent enzyme that catalyzes the oxidative deamination of peptidyl lysine in a collagen protein to peptidyl aminoadipic semialdehyde, whereby intra- / inter-molecular units of collagen are produced. (Smith-Mungo, LI et al., Matrix Biol., 16,
387-398 (1998)]. Lysyl oxidase is 50 kD
It is secreted as a zymogen and undergoes proteolysis to become an active enzyme having a catalytic activity of 32 kDa.

The LOXC obtained in this example is as follows: As shown in FIG. 3, the LOXC protein has a sequence predicted to be a signal peptide at its N-terminus; LOXC is a four-fold repeat of the scavenger receptor cysteine-rich domain found in a variety of secreted and membrane-bound proteins [Resnick, D. et al., Trends. Bioc.
hem. Sci., 19, 5-8 (1994)]. In Western blot analysis using an anti-LOXC antibody, LOXC protein was found to be 82 kDa in the culture supernatant of COS-7 cells into which LOXC cDNA had been introduced. Is recognized as a specific band, indicating that it is an extracellular protein secreted as an 82 kDa active enzyme.

Lysyl oxidase is tightly bound to copper (II) as a cofactor [Smith-Mungo et al., Matr.
ix Biol., 16, 387-398 (1998)]. In LOXC, as shown in FIG. 3, two sequence motifs important for binding to copper: WXWHCHXHXH: four histidines involved in copper binding coordination and supplying nitrogen ligands GHK: another type A putative collagen-related copper affinity site has been identified. Such motifs are well conserved among various lysyl oxidase proteins.

Lysyl oxidase also contains lysine tyrosylquinone as a carbonyl cofactor [Wang,
SX) et al., Science, 273, 1078-1084 (1996)]. Tyrosine and lysine residues at the C-terminus of lysyl oxidase are both involved in the formation of this cofactor, and LOX
In C, the corresponding lysine residue (amino acid 639) and tyrosine residue (amino acid 675) are conserved (FIG. 3). These results suggest that LOXC has lysyl oxidase enzyme activity and may be involved in extracellular matrix crosslinking.

In fact, CO containing LOXC cDNA
Culture supernatants of S-7 cells showed significant lysyl oxidase enzyme activity in tests using tritium-labeled type I and type II collagen substrates. Furthermore, this enzyme activity was inhibited by βAPN.

Endochondral bone formation consists of a stream of events of chondrocyte proliferation, maturation, hypertrophy and calcification and replacement of cartilage by bone. During these processes, the collagenous component of the extracellular matrix transitions from type II collagen to type X collagen. Furthermore, at the cartilage-bone mineralization interface, type I collagen is expressed. Because LOXC is highly expressed in hypertrophic and calcified cartilage and has lysyl oxidase enzyme activity, LOXC regulates endochondral bone formation by altering the formation of collagenous extracellular matrix Is suggested.

In addition, LOXC is expressed in hypertrophic and calcified chondrocytes in vivo, in certain cell lines in vitro, and has lysyl oxidase enzyme activity. It suggests that it may play an important role in formation.

Example 2 Isolation of Nucleic Acid Encoding Human LOXC A human LOXC cDNA fragment was prepared by using the forward primer 1 (5′-ACAAG) with human chondroblastoma total RNA as a template.
AGACGGCGTACTTGG-3 '; SEQ ID NO: 7) and reverse primer 2 (5'-CTGGCAATCGATGTCATGC-3'; SEQ ID NO: 8)
And by using RT-PCR. After 5 minutes of denaturation at 94 ° C., amplification was carried out by 25 cycles of reaction in which 94 ° C. 30 seconds−55 ° C. 30 seconds−72 ° C. 30 seconds as one cycle.

Using the obtained fragment as a hybridization probe, a human testis 5′-stretch plus cDNA library (manufactured by Clontech) was screened, whereby the human LOXC c
DNA was obtained. In addition, SMART RACE cDN
A Amplification kit and reverse primer 3 (5'-GTCCAAGGAGCTCTCTGTGC-3 '; SEQ ID NO:
9) and reverse primer 4 (5'-CACGTTGTCCAGCCAGATG
G-3 ′; SEQ ID NO: 10) was used to perform RACE, whereby the full length sequence of the LOXC cDNA including the 5 ′ sequence was determined. In the RACE reaction, denaturation was performed at 94 ° C for 2 minutes, then at 94 ° C for 5 seconds-65 ° C for 10 seconds
Twenty-five cycles were performed with one minute at 72 ° C. Adapter-linked cD prepared from human chondroblastoma
The 5 'end of the human LOXC cDNA was amplified using a non-cloned library against NA. Obtained c
The DNA product was subcloned into a pCR2.1 vector (Invitrogen) and
LFred DNA sequencer [Amersham Pharmacia Biotech]
Was used to analyze the sequence.

As a result, a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1 was obtained. The amino acid sequence deduced from SEQ ID NO: 1 is shown in SEQ ID NO: 2.

As a result of analyzing the base sequence of SEQ ID NO: 1 using software GENETYX-MAC,
Mouse lysyl oxidase-like protein 2 (Lor2)
Shows a sequence identity of 57.5% to the nucleotide sequence of the nucleic acid that encodes human lysyl oxidase-like protein 2 (Lor2).
It showed 64.8% sequence identity.

The amino acid sequence of SEQ ID NO: 2 was analyzed using the software GENETYX-MAC, and showed 55.1% sequence identity with the amino acid sequence of mouse lysyl oxidase-like protein 2 (Lor2). Human lysyl oxidase-like protein 2 (Lor
It showed 55.5% sequence identity to the amino acid sequence of 2).

Sequence Listing Free Text SEQ ID NO: 7 is a sequence of a synthetic oligonucleotide having a sequence specific to LOXC.

SEQ ID NO: 8 is a sequence of a synthetic oligonucleotide having a sequence specific to LOXC.

SEQ ID NO: 9 is a sequence of a synthetic oligonucleotide having a sequence specific to LOXC.

SEQ ID NO: 10 is a sequence of a synthetic oligonucleotide having a sequence specific to LOXC.

[0167]

The sense nucleic acid and polypeptide of the present invention, for example, when derived from a mouse, are different from cartilage precursor cells at the stage of hypertrophy / calcification at the stage of hypertrophy / calcification and at the stage of differentiation before that. Thus, it has an excellent effect that it can be applied to the production of collagenous extracellular matrix, the promotion or assistance of collagen or elastin production, the detection of bone / cartilage repair state, and the like. Therefore, there is provided an application technique for production of a collagenous extracellular matrix, promotion or assistance of production of collagen or elastin, detection of a bone / cartilage repair state, and the like.

[0168]

[Sequence List] SEQUENCE LISTING <110> TAKASHI NAKAMURA <120> Nucleic acid encoding lysyl oxidase related protein <130> SP-13-003 <160> 10 <170> PatentIn Ver. 2.1 <210> 1 <211> 3607 <212 > DNA <213> Human <220> <221> CDS <222> (95) .. (2365) <400> Ser Pro Pro Ala 1 5 acc ctc ttt ctg ttc ctg ctg ctg cta ggc cag ccc cct ccc agc agg 163 Thr Leu Phe Leu Phe Leu Leu Leu Leu Gly Gln Pro Pro Pro Ser Arg 10 15 20 cca cag tca ctg ggc acc act aag ctc cgg ctg gtg ggc cca gag agc 211 Pro Gln Ser Leu Gly Thr Thr Lys Leu Arg Leu Val Gly Pro Glu Ser 25 30 35 aag cca gag gag ggc cgc ctg gag gtg ctg cc cac cag ggc cag tgg ggc 259 Lysly Glu Arg Leu Glu Val Leu His Gln Gly Gln Trp Gly 40 45 50 55 acc gtg tgt gat gac aac ttt gct atc cag gag gcc aca gtg gct tgc 307 Thr Val Cys Asp Asp Asn Phe Ala Ile Gln Glu Ala Thr Val Ala Cys 60 65 70 cgc cag ctg ggc ttc gaa gct gcc ttg acc tgg gcc cac agt gcc aag 355 Arg Gln Leu Gly Phe Glu Ala Ala Leu Thr Trp Ala His Ser Ala Lys 75 80 85 tac ggc caa ggg gag gga ccc atc tgg ctg gac aat gtg cgc tgt gtg Gly Gln Gly Glu Gly Gly Pro Ile Trp Leu Asp Asn Val Arg Cys Val 90 95 100 ggc aca gag agc tcc ttg gac cag tgc ggg tct aat ggc tgg gga gtc 451 Gly Thr Glu Ser Ser Leu Asp Gln Cys Gly Ser Asn Gly Trp Gly Val 105 110 115 agt gac tgc agt cac tca gaa gac gta ggg gtg ata tgc cac ccc cgg 499 Ser Asp Cys Ser His Ser Glu Asp Val Gly Val Ile Cys His Pro Arg 120 125 130 135 cgc cat cgt ggc tac ctt tct gaa act gtc tcc aat gcc ctt ggg ccc 547 Arg His Arg Gly Tyr Leu Ser Glu Thr Val Ser Asn Ala Leu Gly Pro 140 145 150 cag ggc cgg cgg ctg gag gag gtg cgg ctc aag ccc atc ctt gcc agt 595 Gln Gly Arg Arg Leu Glu Glu Val Arg Leu Lys Pro Ile Leu Ala Ser 155 160 165 gcc aag cag cat agc cca gtg acc gag gga gcc gtg gag gtg aag tat 643 Ala Lys Gln His Ser Pro Val Thr Glu Gly Ala Val Glu Val Lys Tyr 170 175 180 gag ggc cac tgg cgg cag gtg tgt gac cag ggc tgg acc atg aac aac 691 Glu Gly His Trp Arg Gln Val Cys Asp Gln Gly Trp Thr Met Asn Asn 185 190 195 agc agg gtg gtg tgc ggg atg ctg ggc ttc cccgc gc gc gc 739 Ser Arg Val Val Cys Gly Met Leu Gly Phe Pro Ser Glu Val Pro Val 200 205 210 215 gac agc cac tac tac agg aaa gtc tgg gat ctg aag atg agg gac cct 787 Asp Ser His Tyr Tyr Arg Lys Val Trp Asp Leu Lys Met Arg Asp Pro 220 225 230 aag tct agg ctg aag agc ctg acg aat aag aac tcc ttc tgg atc cac 835 Lys Ser Arg Leu Lys Ser Leu Thr Asn Lys Asn Ser Phe Trp Ile His 235 240 245 cag gtc acc tgc ctg gggca gag ccc cac atg gcc aac tgc cag gtg 883 Gln Val Thr Cys Leu Gly Thr Glu Pro His Met Ala Asn Cys Gln Val 250 255 260 cag gtg gct cca gcc cgg ggc aag ctg cgg cca gcc tgc cca ggt ggc 931 Gln Val Ala Pro Ala Arg Gly Lys Leu Arg Pro Ala Cys Pro Gly Gly 265 270 275 atg cat gct gtg gtc agc tgt gtg gca ggg cct cac ttc cgc cca ccg 979 Met His Ala Val Val Ser Cys Val Ala Gly Pro His Phe Arg Pro Pro 280 285 290 295 aag aca aag cca caa cgc aaa ggg tcc tgg gca gag gag ccg agg gtg 1027 Lys Thr Lys Pro Gln Arg Lys Gly Ser Trp Ala Glu Glu Glu Pro Arg Val 300 305 310 cgc ctg cgc tcc ggg gcc cag gtg ggc gag gtg gaa gtg ctc 1075 Arg Leu Arg Ser Gly Ala Gln Val Gly Glu Gly Arg Val Glu Val Leu 315 320 325 atg aac cgc cag tgg ggc acg gtc tgt gac cac agg tgg aac ctc atc 1123 Met Asn Arg Gln Trp Gly Thr Asp His Arg Trp Asn Leu Ile 330 335 340 tct gcc agt gtc gtg tgt cgt cag ctg ggc ttt ggc tct gct cgg gag 1171 Ser Ala Ser Val Val Cys Arg Gln Leu Gly Phe Gly Ser Ala Arg Glu 345 350 355 gcc ctc ttt gcc cgg ctg ggc caa ggg cta ggg ccc atc cac ctg 1219 Ala Leu Phe Gly Ala Arg Leu Gly Gln Gly Leu Gly Pro Ile His Leu 360 365 370 375 agt gag gtg cgc tgc agg gga tat gag cgg acc ctc agc gac t67 Ser Glu Val Arg Cys Arg Gly Tyr Glu Arg Thr Leu Ser Asp Cys Pro 380 385 390 gcc ctg gaa ggg tcc cag aat ggt tgc caa cat gag aat gat gct gct 1315 Ala Leu Glu Gly Ser Gln Asn Gly Cys Gln His Glu Asn Asp Ala Ala 395 400 405 gtc agg tgc aat gtc cct aac atg ggc ttt cag aat cag gtg cgc ttg 1363 Val Arg Cys Asn Val Pro Asn Met Gly Phe Gln Asn Gln Val Arg Leu 410 415 420 gct ggt ggg cgt atcct gag ggg cta ttg gag gtg cag gtg gag 1411 Ala Gly Gly Arg Ile Pro Glu Glu Gly Leu Leu Glu Val Gln Val Glu 425 430 435 gtg aac ggg gtc cca cgc tgg ggg agc gtg tgc agt gaa Valggg Ggg 14 Pro Arg Trp Gly Ser Val Cys Ser Glu Asn Trp Gly 440 445 450 455 ctc acc gaa gcc atg gtg gcc tgc cga cag ctc ggc ctg ggt ttt gcc 1507 Leu Thr Glu Ala Met Val Ala Cys Arg Gln Leu Gly Leu Gly Phe Ala 465 470 atc cat gcc tac aag gaa acc tgg ttc tgg tcg ggg acg cca agg gcc 1555 Ile His Ala Tyr Lys Glu Thr Trp Phe Trp Ser Gly Thr Pro Arg Ala 475 480 485 cag gag gtg gtg atg agt ggg gtg cgc tc aca gag ctg gcc 1603 Gln Glu Val Val Met Ser Gly Val Arg Cys Ser Gly Thr Glu Leu Ala 490 495 500 ctg cag cag tgc cag agg cac ggg ccg gtg cac tgc tcc cac ggt ggc 1651 Leu Gln Gln Cys Gln Arg His Gly Pro Val His Cys Ser His Gly Gly 505 510 515 ggg cgc ttc ctg gct gga gtc tcc tgc atg gac agt gca cca gac ctg 1699 Gly Arg Phe Leu Ala Gly Val Ser Cys Met Asp Ser Ala Pro Asp Leu 520 525 530 gt atg aac gcc cag cta gtg cag gag acg gcc tac ttg gag gac cgc 1747 Val Met Asn Ala Gln Leu Val Gln Glu Thr Ala Tyr Leu Glu Asp Arg 540 545 550 550 ccg ctc agc cag ctg tat tgt gcc tac gag gag tac aag 1795 Pro Leu Ser Gln Leu Tyr Cys Ala His Glu Glu Asn Cys Leu Ser Lys 555 560 565 tct gcg gat cac atg gac tgg ccc tac gga tac cgc cgc cta ttg cgc 1843 Ser Ala Asp His Met Asp Trp Pro Tyr Gly Tyr Ar Arg Leu Leu Arg 570 575 580 ttc tcc aca cag atc tac aat ctg ggc cgg act gac ttt cgt cca aag 1891 Phe Ser Thr Gln Ile Tyr Asn Leu Gly Arg Thr Asp Phe Arg Pro Lys 585 590 595 act gga cgc gat agc tgg tgg cac cag tgc cac agg cat tac cac 1939 Thr Gly Arg Asp Ser Trp Val Trp His Gln Cys His Arg His Tyr His 600 605 610 615 agc att gag gtc ttc acc cac tac gac ctc ctc act ctc aat ggc tcc 1987 Ser I le Glu Val Phe Thr His Tyr Asp Leu Leu Thr Leu Asn Gly Ser 620 625 630 aag gtg gct gag ggg cac aag gcc agc ttc tgt ctg gag gac aca aac 2035 Lys Val Ala Glu Gly His Lys Ala Ser Phe Cys Leu Glu Asp Thr Asn 635 640 645 tgc ccc aca gga ctg cag cgg cgc tac gca tgt gcc aac ttt gga gaa 2083 Cys Pro Thr Gly Leu Gln Arg Arg Tyr Ala Cys Ala Asn Phe Gly Glu 650 655 660 cag gga gtg act gta ggc acc tac cgg cat gac att gat 2131 Gln Gly Val Thr Val Val Gly Cys Trp Asp Thr Tyr Arg His Asp Ile Asp 665 670 675 tgc cag tgg gtg gat atc aca gat gtg ggc ccc ggg aat tat atc ttc 2179 Cys Gln Trp Val Asp Ile Thr Asp Val Gly Pro Gly Asn Tyr Ile Phe 680 685 690 695 cag gtg att gtg aac ccc cac tat gaa gtg gca gag tca gat ttc tcc 2227 Gln Val Ile Val Asn Pro His Tyr Glu Val Ala Glu Ser Asp Phe Ser 700 705 710 aac aat atg ctg cag tgc cgc tgc aag tat gat ggg cac cgg gtc tgg 2275 Asn Asn Met Leu Gln Cys Arg Cys Lys Tyr Asp Gly His Arg Val Trp 715 720 725 ctg cac aac tgc cac aca ggg aat tca tat cca gcc a gaa ctc 2323 Leu His Asn Cys His Thr Gly Asn Ser Tyr Pro Ala Asn Ala Glu Leu 730 735 740 tcc ctg gag cag gaa cag cgt ctc agg aac aac ctc atc tga 2365 Ser Leu Glu Gln Glu Gln Arg Leu Arg Asn Asn Ile 745 750 755 agctgtcact gcacactcct agctgctgcc gatacaccag atacctcagc ttattggagc 2425 catgcccttc acagagtccc aactcagagg aaaagggcca gtgccaaggg gcaccaagaa 2485 cctgctcagg aagccttttg atggcaagat caccaatcca gatggtattg ctccctcagg 2545 atggctctgg gcctgcccct aaaggcctgt ggcctatgga atatgtcctc caggctttgc 2605 tcagctgagc tcctcttctg taaggaaacc cagtcatccc tgaatcttgc cacagagatc 2665 cgggattcag gagctctcag tttcttaggg atggactatg gcccagtccc ccatctaagt 2725 ggtgctttgc aaatgtcttg gaggagtata ggacagagga ccaaaataca cagcaggtag 2785 tgttagctct ctgctaggag ctcaaagcaa cacaacttgt atcaaaatca caactggcag 2845 agaagctggt ggatccaatc ctttcttcat ctgttgttat ttagaactca cctctcacac 2905 tctgttcttt agtgtcctta cctttatctt accacacaca tgggtgtttc tattatcctt 2965 ggaagcacag acctcgggca tccccttatt gcctgatggg ccaacaccaa cagttacgga 3025 gtg cttgaga aggggcaagt ttcacagaaa tggccagata gggccttcct acagagcagc 3085 aagagtaggc caagcagaaa gactgctgag gtaacacgga ccccaccctg tcagggcctc 3145 tgccaaggaa ataatatgga ccatttacct ggcaggcagt ctgctctctc tcaggatcac 3205 cacgcatctc aggattggtc taaacttcaa gtctcaacca agtgtctgaa gtgaactttg 3265 cattgaataa atttttgcca tggaaagaac atcaaacaag ccactcatct ctacagagat 3325 aagaaaacaa gtttggcaga gcaagagaca gaagaccgtg gagaaatcag aagggggaac 3385 agtcagttta gttaaggatg gaacctggga aaggccacca ttcctgcttg atggggctct 3445 gatttgctct tgctcaagtg gaataaaacc ccatggtctt cttgacatga ttcttgatct 3505 tttctccact gagacacact taagtgatga tcttacagga ctgacaccct aatgccaata 3565 aaagttgctc attatggact gctacaaaaa aaaaaaaaaa aa 3607

<210> 2 <211> 756 <212> PRT <213> Human <400> 2 Met Ala Trp Ser Pro Pro Ala Thr Leu Phe Leu Phe Leu Leu Leu Leu 1 5 10 15 Gly Gln Pro Pro Pro Ser Arg Pro Gln Ser Leu Gly Thr Thr Lys Leu 20 25 30 Arg Leu Val Gly Pro Glu Ser Lys Pro Glu Glu Gly Arg Leu Glu Val 35 40 45 Leu His Gln Gly Gln Trp Gly Thr Val Cys Asp Asp Asn Phe Ala Ile 50 55 60 Gln Glu Ala Thr Val Ala Cys Arg Gln Leu Gly Phe Glu Ala Ala Leu 65 70 75 80 Thr Trp Ala His Ser Ala Lys Tyr Gly Gln Gly Glu Gly Pro Ile Trp 85 90 95 Leu Asp Asn Val Arg Cys Val Gly Thr Glu Ser Ser Leu Asp Gln Cys 100 105 110 Gly Ser Asn Gly Trp Gly Val Ser Asp Cys Ser His Ser Glu Asp Val 115 120 125 Gly Val Ile Cys His Pro Arg Arg His Arg Gly Tyr Leu Ser Glu Thr 130 135 140 Val Ser Asn Ala Leu Gly Pro Gln Gly Arg Arg Leu Glu Glu Val Arg 145 150 155 160 Leu Lys Pro Ile Leu Ala Ser Ala Lys Gln His Ser Pro Val Thr Glu 165 170 175 Gly Ala Val Glu Val Lys Tyr Glu Gly His Trp Arg Gln Val Cys Asp 180 185 190 Gln Gly Trp Thr Met Asn Asn Se r Arg Val Val Cys Gly Met Leu Gly 195 200 205 Phe Pro Ser Glu Val Pro Val Asp Ser His Tyr Tyr Arg Lys Val Trp 210 215 220 Asp Leu Lys Met Arg Asp Pro Lys Ser Arg Leu Lys Ser Leu Thr Asn 225 230 235 240 Lys Asn Ser Phe Trp Ile His Gln Val Thr Cys Leu Gly Thr Glu Pro 245 250 255 His Met Ala Asn Cys Gln Val Gln Val Ala Pro Ala Arg Gly Lys Leu 260 265 270 270 Arg Pro Ala Cys Pro Gly Gly Met His Ala Val Val Ser Cys Val Ala 275 280 285 Gly Pro His Phe Arg Pro Pro Lys Thr Lys Pro Gln Arg Lys Gly Ser 290 295 300 Trp Ala Glu Glu Pro Arg Val Arg Leu Arg Ser Gly Ala Gln Val Gly 305 310 310 315 320 Glu Gly Arg Val Glu Val Leu Met Asn Arg Gln Trp Gly Thr Val Cys 325 330 335 Asp His Arg Trp Asn Leu Ile Ser Ala Ser Val Val Cys Arg Gln Leu 340 345 350 Gly Phe Gly Ser Ala Arg Glu Ala Leu Phe Gly Ala Arg Leu Gly Gln 355 360 365 Gly Leu Gly Pro Ile His Leu Ser Glu Val Arg Cys Arg Gly Tyr Glu 370 375 380 Arg Thr Leu Ser Asp Cys Pro Ala Leu Glu Gly Ser Gln Asn Gly Cys 385 390 395 400 400 Gln His Glu Asn Asp Ala Ala Va l Arg Cys Asn Val Pro Asn Met Gly 405 410 415 Phe Gln Asn Gln Val Arg Leu Ala Gly Gly Arg Ile Pro Glu Glu Gly 420 425 430 Leu Leu Glu Val Gln Val Glu Val Asn Gly Val Pro Arg Trp Gly Ser 435 440 445 Val Cys Ser Glu Asn Trp Gly Leu Thr Glu Ala Met Val Ala Cys Arg 450 455 460 Gln Leu Gly Leu Gly Phe Ala Ile His Ala Tyr Lys Glu Thr Trp Phe 465 470 475 480 Trp Ser Gly Thr Pro Arg Ala Gln Glu Val Val Met Ser Gly Val Arg 485 490 495 Cys Ser Gly Thr Glu Leu Ala Leu Gln Gln Cys Gln Arg His Gly Pro 500 505 510 Val His Cys Ser His Gly Gly Gly Arg Phe Leu Ala Gly Val Ser Cys 515 520 525 Met Asp Ser Ala Pro Asp Leu Val Met Asn Ala Gln Leu Val Gln Glu 530 535 540 Thr Ala Tyr Leu Glu Asp Arg Pro Leu Ser Gln Leu Tyr Cys Ala His 545 550 555 560 Glu Glu Asn Cys Leu Ser Lys Ser Ala Asp His Met Asp Trp Pro Tyr 565 570 575 Gly Tyr Arg Arg Leu Leu Arg Phe Ser Thr Gln Ile Tyr Asn Leu Gly 580 585 590 Arg Thr Asp Phe Arg Pro Lys Thr Gly Arg Asp Ser Trp Val Trp His 595 600 605 Gln Cys His Arg His Tyr His Ser I le Glu Val Phe Thr His Tyr Asp 610 615 620 620 Leu Leu Thr Leu Asn Gly Ser Lys Val Ala Glu Gly His Lys Ala Ser 625 630 635 640 Phe Cys Leu Glu Asp Thr Asn Cys Pro Thr Gly Leu Gln Arg Arg Tyr 645 650 655 Ala Cys Ala Asn Phe Gly Glu Gln Gly Val Thr Val Gly Cys Trp Asp 660 665 670 Thr Tyr Arg His Asp Ile Asp Cys Gln Trp Val Asp Ile Thr Asp Val 675 680 685 Gly Pro Gly Asn Tyr Ile Phe Gln Val Ile Val Asn Pro His Tyr Glu 690 695 700 Val Ala Glu Ser Asp Phe Ser Asn Asn Met Leu Gln Cys Arg Cys Lys 705 710 715 720 Tyr Asp Gly His Arg Val Trp Leu His Asn Cys His Thr Gly Asn Ser 725 730 735 Tyr Pro Ala Asn Ala Glu Leu Ser Leu Glu Gln Glu Gln Arg Leu Arg 740 745 750 Asn Asn Leu Ile 755

<210> 3 <211> 2274 <212> DNA <213> Mouse <220> <221> CDS <222> (1) .. (2274) <400> 3 atg atg tgg ccc caa cca ccc acc ttc tcc ctg ttc ctg cta ctg ctg 48 Met Met Trp Pro Gln Pro Pro Thr Phe Ser Leu Phe Leu Leu Leu Leu 1 5 10 15 cta agc caa gcc cct tcc agt agg cca cag tca tca ggc acc aag aag 96 Leu Ser Gln Ala Pro Ser Ser Arg Pro Gln Ser Ser Gly Thr Lys Lys 20 25 30 ctc agg ctt gtg ggg cca gcg gac aga cca gag gag ggc cgc ttg gag 144 Leu Arg Leu Val Gly Pro Ala Asp Arg Pro Glu Glu Gly Arg Leu Glu 35 40 45 gtg ctg cac cag ggc cag tgg ggc acg gtg tgt gat gat gat ttc gct 192 Val Leu His Gln Gly Gln Trp Gly Thr Val Cys Asp Asp Asp Phe Ala 50 55 60 ctc cag gag gct act gtg gcc tgc cga cag ctg tca gcc 240 Leu Gln Glu Ala Thr Val Ala Cys Arg Gln Leu Gly Phe Glu Ser Ala 65 70 75 80 ttg acc tgg gca cac agt gcc aag tat ggt caa gga gag ggt ccc atc 288 Leu Thr Trp Ala His Ser Ala Lys Tyr Gly Gln Gly Glu Gly Pro Ile 85 90 95 tgg ctg gac aat gtt cgt tgt ttg ggc acc gag aag a cc cta gat cag 336 Trp Leu Asp Asn Val Arg Cys Leu Gly Thr Glu Lys Thr Leu Asp Gln 100 105 110 tgt ggc tct aac ggc tgg ggt atc agt gac tgc aga cac tca gaa gat 384 Cys Gly Ser Asn Gly Trp Gly Ile Ser Asp Cys Arg His Ser Glu Asp 115 120 125 gtt ggg gtg gta tgt cac cca cgg cgc cag cac gga tat cac tct gag 432 Val Gly Val Val Cys His Pro Arg Arg Gln His Gly Tyr His Ser Glu 130 135 140 aag gtc tcc aat gcc ctc ggg cct cag ggc cgg cgg cta gaa gag gta 480 Lys Val Ser Asn Ala Leu Gly Pro Gln Gly Arg Arg Leu Glu Glu Val 145 150 155 160 cgg ctg aaa ccc atc ctc gcc agt gcc aaa agg cac agc cca gtg act Arg Leu Lys Pro Ile Leu Ala Ser Ala Lys Arg His Ser Pro Val Thr 165 170 175 gaa ggg gct gtg gaa gta cgg tac gac ggc cac tgg agg cag gtg tgt 576 Glu Gly Ala Val Glu Val Arg Tyr Asp Gly His Trp Arg Gln Val Cys 180 185 190 gac cag ggc tgg acc atg aac aac agc agg gtt gta tgc ggg atg ctg 624 Asp Gln Gly Trp Thr Met Asn Asn Ser Arg Val Val Cys Gly Met Leu 195 200 205 ggc ttt ccc agt cag aca tct gtc aac a gc cac tac tac aga aaa gtc 672 Gly Phe Pro Ser Gln Thr Ser Val Asn Ser His Tyr Tyr Arg Lys Val 210 215 220 tgg aat ctg aag atg aag gat ccc aag tct agg ctc aac agc ctg aca 720 Trp Asn Leu Lys Met Lys Asp Pro Lys Ser Arg Leu Asn Ser Leu Thr 225 230 235 240 aaa aag aat tcc ttc tgg att cac cgg gtt gac tgt ttc ggg aca gag 768 Lys Lys Asn Ser Phe Trp Ile His Arg Val Asp Cys Phe Gly Thr Glu 245 250 255 ccc cac ttg gcc aag tgc cag gta cag gtg gct cca gga agg ggc aag 816 Pro His Leu Ala Lys Cys Gln Val Gln Val Ala Pro Gly Arg Gly Lys 260 265 270 ctt cgg cca gcc tgt cca ggc ggc atg cac gct gtg tgt gtg 864 Leu Arg Pro Ala Cys Pro Gly Gly Met His Ala Val Val Ser Cys Val 275 285 gca ggg ccc cac ttc cgc cga cag aag cca aag ccc acg cgc aag gag 912 Ala Gly Pro His Phe Arg Arg Gln Lys Pro Lys Pro Thr Arg Lys Glu 290 295 300 tcc cat gca gag gag ctg aaa gtg cgc ctg cgc tct ggg gct cag gtg 960 Ser His Ala Glu Glu Leu Lys Val Arg Leu Arg Ser Gly Ala Gln Val 305 310 315 320 ggt gag ggc cgt gtggaa gtg ctc atg aac cgc cag tgg ggc aca gtc 1008 Gly Glu Gly Arg Val Glu Val Leu Met Asn Arg Gln Trp Gly Thr Val 325 330 335 tgt gac cac agg tgg aac ctc atc tca gcc agc gtc gtg tgt cg cgcgccc His Arg Trp Asn Leu Ile Ser Ala Ser Val Val Cys Arg Gln 340 345 350 ctt ggc ttt ggc tct gcc cgg gag gcc ctt ttt ggg gcc cag ttg ggt 1104 Leu Gly Phe Gly Ser Ala Arg Glu Ala Leu Phe Gly Ala Gln Leu Gly 355 360 365 caa ggg cta gga ccc atc cac ctg agt gag gtg cgc tgc cgg ggg tat 1152 Gln Gly Leu Gly Pro Ile His Leu Ser Glu Val Arg Cys Arg Gly Tyr 370 375 380 gag cgg acc ctg ggt gac tgc cttgcc ct ggg tcc cag aat ggt 1200 Glu Arg Thr Leu Gly Asp Cys Leu Ala Leu Glu Gly Ser Gln Asn Gly 385 390 395 400 tgt caa cat gca aac gac gct gct gtc agg tgc aac atc cca gac atg 1248 Cys Gln His Ala Asn Asp Ala Ala Val Arg Cys Asn Ile Pro Asp Met 405 410 415 ggc ttc cag aac aag gtg cgc ttg gct ggt ggg cgc aac tcc gaa gag 1296 Gly Phe Gln Asn Lys Val Arg Leu Ala Gly Gly Arg Asn Ser Glu Glu 420 425 430 g ga gtg gtg gag gtg cag gtg gag gtg aat ggg ggt cca cga tgg ggg 1344 Gly Val Val Glu Val Gln Val Glu Val Asn Gly Gly Pro Arg Trp Gly 435 440 445 act gta tgc agt gac cac tgg ggg ctc acc gaa gat atg acc tgt 1392 Thr Val Cys Ser Asp His Trp Gly Leu Thr Glu Ala Met Val Thr Cys 450 455 460 cgg caa ctt ggt ctg gga ttt gcc aac ttt gct ctc aag gac acc tgg 1440 Arg Gln Leu Gly Leu Gly Phe Ala Asn Phe Ala Leu Lys Asp Thr Trp 465 470 475 480 tac tgg cag ggg aca cca gag gcc aaa gaa gtg gtg atg agt gga gtt 1488 Tyr Trp Gln Gly Thr Pro Glu Ala Lys Glu Val Val Met Ser Gly Val 485 490 495 cgc tgc tcc ccc gaa atg gcc ctg cag cag tgt cag agg cat ggg 1536 Arg Cys Ser Gly Thr Glu Met Ala Leu Gln Gln Cys Gln Arg His Gly 500 505 510 ccg gtg cac tgt tcc cac ggc cca ggg cgc ttc tcg gct ggc gtt gtt His Cys Ser His Gly Pro Gly Arg Phe Ser Ala Gly Val Ala 515 520 525 tgt atg aac agt gct cca gac ctc gtg atg aac gcc cag ctg gta caa 1632 Cys Met Asn Ser Ala Pro Asp Leu Val Met Asn Ala Gln Leu V al Gln 530 535 540 gag acg gcg tac ttg gag gat cgt cca ctc agc atg ctg tac tgt gct 1680 Glu Thr Ala Tyr Leu Glu Asp Arg Pro Leu Ser Met Leu Tyr Cys Ala 545 550 555 550 560 cac gag gaac tcc ag tac tcc gct gat cac atg gac tgg ccc 1728 His Glu Glu Asn Cys Leu Ser Lys Ser Ala Asp His Met Asp Trp Pro 565 570 575 tac ggg tac cgg cgc ttg ctg cgc ttc tcc tca cag atc tac aac ctt 1776 Tyr Gly Tyr Ar Leu Leu Arg Phe Ser Ser Gln Ile Tyr Asn Leu 580 585 590 ggc cgg gcc gac ttc cgt ccc aag gct gga cgc cac agc tgg att tgg 1824 Gly Arg Ala Asp Phe Arg Pro Lys Ala Gly Arg His Ser Trp Ile Trp 595 600 605 cac cag tgc cac agg cat aac cac agc atc gaa gtc ttc act cat tat 1872 His Gln Cys His Arg His Asn His Ser Ile Glu Val Phe Thr His Tyr 610 615 620 gac ctg ctc acg ctc aat ggc tcc aag gtg gcc gag gga aag gcc 1920 Asp Leu Leu Thr Leu Asn Gly Ser Lys Val Ala Glu Gly His Lys Ala 625 630 635 640 agc ttc tgt cta gaa gat aca aac tgc ccc tca gga gtg cag cgg cgc 1968 Ser Phe Cys Leu Glu Asp Thr Asn Cy s Pro Ser Gly Val Gln Arg Arg 645 650 655 tat gca tgt gcc aac ttt ggg gaa cag gga gtg gct gta ggc tgc tgg 2016 Tyr Ala Cys Ala Asn Phe Gly Glu Gln Gly Val Ala Val Gly Cys Trp 660 665 670 gac acc tac cgg cat gac atc gat tgc cag tgg gtg gat atc aca gat 2064 Asp Thr Tyr Arg His Asp Ile Asp Cys Gln Trp Val Asp Ile Thr Asp 675 680 685 gtg ggt cca ggg gac tat atc ttc cag gtg gtt gtg aac cccca Val Gly Pro Gly Asp Tyr Ile Phe Gln Val Val Val Asn Pro Thr Asn 690 695 700 gat gtt gca gag tcc gat ttc tcc aat aac atg ata cgg tgc cgc tgc 2160 Asp Val Ala Glu Ser Asp Phe Ser Asn Asn Met Ile Arg Cys Arg Cys 705 710 715 720 aag tat gat gga cag cga gtc tgg ttg cac aac tgc cac aca gga gat 2208 Lys Tyr Asp Gly Gln Arg Val Trp Leu His Asn Cys His Thr Gly Asp 725 730 735 tcc tac cga gcc ct gca gag ct tcc ctg gag cag gaa cag cga ctc 2256 Ser Tyr Arg Ala Asn Ala Glu Leu Ser Leu Glu Gln Glu Gln Arg Leu 740 745 750 agg aac aac ttg atc tga 2274 Arg Asn Asn Leu Ile 755

<210> 4 <211> 757 <212> PRT <213> Mouse <400> 4 Met Met Trp Pro Gln Pro Pro Thr Phe Ser Leu Phe Leu Leu Leu Leu 1 5 10 15 Leu Ser Gln Ala Pro Ser Ser Arg Pro Gln Ser Ser Gly Thr Lys Lys 20 25 30 Leu Arg Leu Val Gly Pro Ala Asp Arg Pro Glu Glu Gly Arg Leu Glu 35 40 45 Val Leu His Gln Gly Gln Trp Gly Thr Val Cys Asp Asp Asp Phe Ala 50 55 60 Leu Gln Glu Ala Thr Val Ala Cys Arg Gln Leu Gly Phe Glu Ser Ala 65 70 75 80 Leu Thr Trp Ala His Ser Ala Lys Tyr Gly Gln Gly Glu Gly Pro Ile 85 90 95 Trp Leu Asp Asn Val Arg Cys Leu Gly Thr Glu Lys Thr Leu Asp Gln 100 105 110 Cys Gly Ser Asn Gly Trp Gly Ile Ser Asp Cys Arg His Ser Glu Asp 115 120 125 Val Gly Val Val Cys His Pro Arg Arg Gln His Gly Tyr His Ser Glu 130 135 140 Lys Val Ser Asn Ala Leu Gly Pro Gln Gly Arg Arg Leu Glu Glu Val 145 150 155 160 Arg Leu Lys Pro Ile Leu Ala Ser Ala Lys Arg His Ser Pro Val Thr 165 170 175 Glu Gly Ala Val Glu Val Arg Tyr Asp Gly His Trp Arg Gln Val Cys 180 185 190 Asp Gln Gly Trp Thr Met Asn As n Ser Arg Val Val Cys Gly Met Leu 195 200 205 Gly Phe Pro Ser Gln Thr Ser Val Asn Ser His Tyr Tyr Arg Lys Val 210 215 220 Trp Asn Leu Lys Met Lys Asp Pro Lys Ser Arg Leu Asn Ser Leu Thr 225 230 235 240 Lys Lys Asn Ser Phe Trp Ile His Arg Val Asp Cys Phe Gly Thr Glu 245 250 255 Pro His Leu Ala Lys Cys Gln Val Gln Val Ala Pro Gly Arg Gly Lys 260 265 270 Leu Arg Pro Ala Cys Pro Gly Gly Met His Ala Val Val Ser Cys Val 275 280 285 Ala Gly Pro His Phe Arg Arg Gln Lys Pro Lys Pro Thr Arg Lys Glu 290 295 300 Ser His Ala Glu Glu Leu Lys Val Arg Leu Arg Ser Gly Ala Gln Val 305 310 315 320 Gly Glu Gly Arg Val Glu Val Leu Met Asn Arg Gln Trp Gly Thr Val 325 330 335 Cys Asp His Arg Trp Asn Leu Ile Ser Ala Ser Val Val Cys Arg Gln 340 345 350 Leu Gly Phe Gly Ser Ala Arg Glu Ala Leu Phe Gly Ala Gln Leu Gly 355 360 365 Gln Gly Leu Gly Pro Ile His Leu Ser Glu Val Arg Cys Arg Gly Tyr 370 375 380 Glu Arg Thr Leu Gly Asp Cys Leu Ala Leu Glu Gly Ser Gln Asn Gly 385 390 395 395 400 Cys Gln His Ala Asn Asp Ala A la Val Arg Cys Asn Ile Pro Asp Met 405 410 415 Gly Phe Gln Asn Lys Val Arg Leu Ala Gly Gly Arg Asn Ser Glu Glu 420 425 430 Gly Val Val Glu Val Gln Val Glu Val Asn Gly Gly Pro Arg Trp Gly 435 440 445 Thr Val Cys Ser Asp His Trp Gly Leu Thr Glu Ala Met Val Thr Cys 450 455 460 Arg Gln Leu Gly Leu Gly Phe Ala Asn Phe Ala Leu Lys Asp Thr Trp 465 470 475 480 Tyr Trp Gln Gly Thr Pro Glu Ala Lys Glu Val Val Met Ser Gly Val 485 490 495 Arg Cys Ser Gly Thr Glu Met Ala Leu Gln Gln Cys Gln Arg His Gly 500 505 510 Pro Val His Cys Ser His Gly Pro Gly Arg Phe Ser Ala Gly Val Ala 515 520 525 Cys Met Asn Ser Ala Pro Asp Leu Val Met Asn Ala Gln Leu Val Gln 530 535 540 Glu Thr Ala Tyr Leu Glu Asp Arg Pro Leu Ser Met Leu Tyr Cys Ala 545 550 555 560 His Glu Glu Asn Cys Leu Ser Lys Ser Ala Asp His Met Asp Trp Pro 565 570 575 Tyr Gly Tyr Arg Arg Leu Leu Arg Phe Ser Ser Gln Ile Tyr Asn Leu 580 585 590 Gly Arg Ala Asp Phe Arg Pro Lys Ala Gly Arg His Ser Trp Ile Trp 595 600 605 His Gln Cys His Arg His Asn His S er Ile Glu Val Phe Thr His Tyr 610 615 620 Asp Leu Leu Thr Leu Asn Gly Ser Lys Val Ala Glu Gly His Lys Ala 625 630 635 640 Ser Phe Cys Leu Glu Asp Thr Asn Cys Pro Ser Gly Val Gln Arg Arg 645 650 655 Tyr Ala Cys Ala Asn Phe Gly Glu Gln Gly Val Ala Val Gly Cys Trp 660 665 670 Asp Thr Tyr Arg His Asp Ile Asp Cys Gln Trp Val Asp Ile Thr Asp 675 680 685 Val Gly Pro Gly Asp Tyr Ile Phe Gln Val Val Val Asn Pro Thr Asn 690 695 700 Asp Val Ala Glu Ser Asp Phe Ser Asn Asn Met Ile Arg Cys Arg Cys 705 710 715 720 Lys Tyr Asp Gly Gln Arg Val Trp Leu His Asn Cys His Thr Gly Asp 725 730 735 Ser Tyr Arg Ala Asn Ala Glu Leu Ser Leu Glu Gln Glu Gln Arg Leu 740 745 750 Arg Asn Asn Leu Ile 755

<210> 5 <211> 754 <212> PRT <213> Mouse <400> 5 Met Arg Ala Val Ser Val Trp Tyr Cys Cys Pro Trp Gly Leu Leu Leu 1 5 10 15 Leu His Cys Leu Cys Ser Phe Ser Val Gly Ser Pro Ser Pro Ser Ile 20 25 30 Ser Pro Glu Lys Lys Val Gly Ser Gln Gly Leu Arg Phe Arg Leu Ala 35 40 45 Gly Phe Pro Arg Lys Pro Tyr Glu Gly Arg Val Glu Ile Gln Arg Ala 50 55 60 Gly Glu Trp Gly Thr Ile Cys Asp Asp Asp Phe Thr Leu Gln Ala Ala 65 70 75 80 His Val Leu Cys Arg Glu Leu Gly Phe Thr Glu Ala Thr Gly Trp Thr 85 90 95 His Ser Ala Lys Tyr Gly Pro Gly Thr Gly Arg Ile Trp Leu Asp Asn 100 105 110 Leu Ser Cys Arg Gly Thr Glu Gly Ser Val Thr Glu Cys Ala Ser Arg 115 120 125 Gly Trp Gly Asn Ser Asp Cys Thr His Asp Glu Asp Ala Gly Val Ile 130 135 140 Cys Lys Asp Gln Arg Leu Pro Gly Phe Ser Asp Ser Asn Val Ile Glu 145 150 155 160 Val Glu His Gln Leu Gln Val Glu Glu Val Arg Leu Arg Pro Ala Val 165 170 175 Glu Trp Gly Arg Arg Pro Leu Pro Val Thr Glu Gly Leu Val Glu Val 180 185 190 Arg Leu Pro Glu Gly Trp Ser Gln Val Cys Asp Lys Gly Trp Ser Ala 195 200 205 His Asn Ser His Val Val Cys Gly Met Leu Gly Phe Pro Gly Glu Lys 210 215 220 Arg Val Asn Met Ala Phe Tyr Arg Met Leu Ala Gln Lys Lys Gln His 225 230 235 240 Ser Phe Gly Leu His Ser Val Ala Cys Val Gly Thr Glu Ala His Leu 245 250 255 Ser Leu Cys Ser Leu Glu Phe Tyr Arg Ala Asn Asp Thr Thr Arg Cys 260 265 270 Ser Gly Gly Asn Pro Ala Val Val Ser Cys Val Leu Gly Pro Leu Tyr 275 280 285 Ala Thr Phe Thr Gly Gln Lys Lys Gln Gln His Ser Lys Pro Gln Gly 290 295 300 Glu Ala Arg Val Arg Leu Lys Gly Gly Gly Ala His Gln Gly Glu Gly Arg 305 310 315 320 Val Glu Val Leu Lys Ala Gly Thr Trp Gly Thr Val Cys Asp Arg Lys 325 330 335 Trp Asp Leu Gln Ala Ala Ser Val Val Cys Pro Glu Leu Gly Phe Gly 340 345 350 Thr Ala Arg Glu Ala Leu Ser Gly Ala Arg Met Gly Gln Gly Met Gly 355 360 365 Ala Ile His Leu Ser Glu Val Arg Cys Ser Gly Gln Glu Pro Ser Leu 370 375 380 Trp Arg Cys Pro Ser Lys Asn Ile Thr Ala Glu Asp Cys Ser His Ser 385 390 395 400 Gln Asp Ala Gly Val Arg Cys As n Leu Pro Tyr Thr Gly Val Glu Thr 405 410 415 Lys Ile Arg Leu Ser Gly Gly Arg Ser Arg Tyr Glu Gly Arg Val Glu 420 425 430 Val Gln Ile Gly Ile Pro Gly His Leu Arg Trp Gly Leu Ile Cys Gly 435 440 445 Asp Asp Trp Gly Thr Leu Glu Ala Met Val Ala Cys Arg Gln Leu Gly 450 455 460 Leu Gly Tyr Ala Asn His Gly Leu Gln Glu Thr Trp Tyr Trp Asp Ser 465 470 470 475 480 Gly Asn Val Thr Glu Val Val Met Ser Gly Val Arg Cys Thr Gly Ser 485 490 495 Glu Leu Ser Leu Asn Gln Cys Ala His His Ser Ser His Ile Thr Cys 500 505 510 Lys Lys Thr Gly Thr Arg Phe Thr Ala Gly Val Ile Cys Ser Glu Thr 515 520 525 Ala Ser Asp Leu Leu Leu His Ser Ala Leu Val Gln Glu Thr Ala Tyr 530 535 540 Ile Glu Asp Arg Pro Leu His Met Leu Tyr Cys Ala Ala Glu Glu Asn 545 550 555 560 Cys Leu Ala Ser Ser Ala Arg Ser Ala Asn Trp Pro Tyr Gly His Arg 565 570 575 Arg Leu Leu Arg Phe Ser Ser Gln Ile His Asn Leu Gly Arg Ala Asp 580 585 590 Phe Arg Pro Lys Ala Gly Arg His Ser Trp Val Trp His Glu Cys His 595 600 605 Gly His Tyr His Ser Met Asp Ile P he Thr His Tyr Asp Ile Leu Thr 610 615 620 Pro Asn Gly Thr Lys Val Ala Glu Gly His Lys Ala Ser Phe Cys Leu 625 630 635 640 Glu Asp Thr Glu Cys Gln Glu Asp Val Ser Lys Arg Tyr Glu Cys Ala 645 650 655 Asn Phe Gly Glu Gln Gly Ile Thr Val Gly Cys Trp Asp Leu Tyr Arg 660 665 670 His Asp Ile Asp Cys Gln Trp Ile Asp Ile Thr Asp Val Lys Pro Gly 675 680 685 Asn Tyr Ile Leu Gln Val Val Ile Asn Pro Asn Phe Glu Val Ala Glu 690 695 700 Ser Asp Phe Thr Asn Asn Ala Met Lys Cys Asn Cys Lys Tyr Asp Gly 705 710 715 720 His Arg Ile Trp Val His Asn Cys His Ile Gly Asp Ala Phe Ser Glu 725 730 735 Glu Ala Asn Arg Arg Phe Glu Arg Tyr Pro Gly Gln Thr Ser Asn Gln 740 745 750 Ile Val

<210> 6 <211> 411 <212> PRT <213> Mouse <400> 6 Met Arg Phe Ala Trp Ala Val Leu Leu Leu Gly Pro Leu Gln Leu Cys 1 5 10 15 Pro Leu Leu Arg Cys Ala Pro Gln Thr Pro Arg Glu Pro Pro Ala Ala 20 25 30 Pro Gly Ala Trp Arg Gln Thr Ile Gln Trp Glu Asn Asn Gly Gln Val 35 40 45 Phe Ser Leu Leu Ser Leu Gly Ala Gln Tyr Gln Pro Gln Arg Arg Arg 50 55 60 Asp Pro Ser Ala Thr Ala Arg Arg Pro Asp Gly Asp Ala Ala Ser Gln 65 70 75 80 Pro Arg Thr Pro Ile Leu Leu Leu Arg Asp Asn Arg Thr Ala Ser Thr 85 90 95 Arg Ala Arg Thr Pro Ser Pro Ser Gly Val Ala Ala Gly Arg Pro Arg 100 105 110 Pro Ala Ala Arg His Trp Phe Gln Ala Gly Phe Ser Pro Ser Gly Ala 115 120 125 Arg Asp Gly Ala Ser Arg Arg Ala Ala Asn Arg Thr Ala Ser Pro Gln 130 135 140 Pro Pro Gln Leu Ser Asn Leu Arg Pro Pro Ser His Ile Asp Arg Met 145 150 155 160 Val Gly Asp Asp Pro Tyr Asn Pro Tyr Lys Tyr Ser Asp Asp Asn Pro 165 170 175 Tyr Tyr Asn Tyr Tyr Asp Thr Tyr Glu Arg Pro Arg Pro Gly Ser Arg 180 185 190 Asn Arg Pro Gly Tyr Gly Thr Gly Tyr Phe Gln Tyr Gly Leu Pro Asp 195 200 205 Leu Val Pro Asp Pro Tyr Tyr Ile Gln Ala Ser Thr Tyr Val Gln Lys 210 215 220 Met Ser Met Tyr Asn Leu Arg Cys Ala Ala Glu Glu Asn Cys Leu Ala 225 230 235 240 Ser Ser Ala Tyr Arg Ala Asp Val Arg Asp Tyr Asp His Arg Val Leu 245 250 255 Leu Arg Phe Pro Gln Arg Val Lys Asn Gln Gly Thr Ser Asp Phe Leu 260 265 270 Pro Ser Arg Pro Arg Tyr Ser Trp Glu Trp His Ser Cys His Gln His 275 280 285 Tyr His Ser Met Asp Glu Phe Ser His Tyr Asp Leu Leu Asp Ala Asn 290 295 300 Thr Gln Arg Arg Val Ala Glu Gly His Lys Ala Ser Phe Cys Leu Glu 305 310 315 320 Asp Thr Ser Cys Asp Tyr Gly Tyr His Arg Arg Phe Gly Cys Thr Ala 325 330 335 His Thr Gln Gly Leu Ser Pro Gly Cys Tyr Asp Thr Tyr Ala Ala Asp 340 345 350 Ile Asp Cys Gln Trp Ile Asp Ile Thr Asp Val Gln Pro Gly Asn Tyr 355 360 365 Ile Leu Lys Val Ser Val Asn Pro Ser Tyr Leu Val Pro Glu Ser Asp 370 375 380 Tyr Thr Asn Asn Val Val Arg Cys Asp Ile Arg Tyr Thr Gly His His 385 390 395 400 Ala Tyr Ala Ser Gly Cys Thr Il e Ser Pro Tyr 405 410

<210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized oligonucleotide ha ving a specific sequence to LOXC. <400> 7 acaagagacg gcgtacttgg 20

<210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized oligonucleotide ha ving a specific sequence to LOXC. <400> 8 ctggcaatcg atgtcatgc 19

<210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized oligonucleotide ha ving a specific sequence to LOXC. <400> 9 gtccaaggag ctctctgtgc 20

<210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized oligonucleotide ha ving a specific sequence to LOXC. <400> 10 cacgttgtcc agccagatgg 20

[Brief description of the drawings]

FIG. 1 is a diagram showing the nucleotide sequence and deduced amino acid sequence of mouse LOXC.

FIG. 2 shows a hydrophobicity plot of the putative LOXC protein sequence. Positive values indicate hydrophobicity and negative values indicate hydrophilicity.

FIG. 3 shows the amino acid sequence of mouse LOXC (sequence
No. 4) and lysyl oxidase-related protein 2 (L
or2) amino acid sequence (SEQ ID NO: 5) and lysyl oxy
Amino acid sequence of Sidase (LO) (SEQ ID NO: 6)
FIG. 4 is a diagram showing a comparison of amino acid sequences in the present invention. Between each array
Are shown in gray. Between each array
Conserved cysteine residues are shown in black. Putative cleavage site,
Indicated by arrows. Four scavenger receptors cysteine
Switch domains with a superscript line in parentheses.
To display the number. A putative copper binding site and a GHK site,
Display in a box. Cross-linking to Rizil Chiro
Lysine and tyrosine producing silquinone
(Tyr) residues are indicated by arrowheads. Mouse LO and
The amino acid sequence of mouse Lor2 is
Link accession number NM 010728 and AF053
This is a sequence deduced from the base sequence of 368.

FIG. 4 is a diagram showing LOXC gene translation in vitro. Using the mouse LOXC cDNA cloned into the pcDNA3.1 vector as a template, a ligation transcription / translation reaction [TnT Reticulocy
tes; Promega). Translation products were separated by electrophoresis on gradient polyacrylamide gels (10-20%) and detected by autoradiography. Three independent experiments were performed with the same results.

FIG. 5 is a diagram showing expression of LOXC protein. COS-7 cells were transiently transfected with the pCMV / mLOXC vector or the pcDNA3.1 vector as a control. Using an anti-LOXC antibody, LO
XC protein was detected. Original magnification is 100
Times. Three independent experiments were performed with the same results.

FIG. 6 shows the results of examining LOXC expression in various cell lines. C3H10T1 / 2, MC3
T3-E1 (undifferentiated and calcified), NIH3T3 and C2
20 μg of total RNA extracted from each of C12 was used per lane. 1.3 kb LOXC cDN
A was analyzed by Northern blot hybridization using A.

FIG. 7 shows the results of examining LOXC expression in various cell lines. ATDC5 cells were cultured according to the examples. 20 μg of total RNA and conditioned media were prepared at the times indicated in the figure, followed by 1.3 kb LOX.
C cDNA, 0.55 kb type II collagen cDN
A, using 0.65 kb X type collagen cDNA,
Analyzed by Northern blot. Three independent experiments were performed with the same results.

FIG. 8 shows the results of examining LOXC expression in various cell lines. ATDC5 cells were cultured according to the examples. 20 μg of total RNA and conditioned media were prepared at the times indicated in the figure and then analyzed by Western blot using an anti-LOXC antibody. Three independent experiments were performed with the same results.

FIG. 9 shows LOX in various mature mouse tissues.
FIG. 3 is a view showing expression of C mRNA. LOXC cDN
A (upper row) and 2 μg of poly (A) ⁺ RNA of various mouse tissues using β-actin probe (lower row)
Mutiple tissue blot containing [Clonte
ch)). MRN of each lane
A is 1: heart, 2: brain, 3: spleen, 4:
Isolated from lung, 5: liver, 6: skeletal muscle, 7: kidney, 8: testis. As a control, a DNA fragment of β-actin was used to hybridize to the same blot.

FIG. 10 shows L in various mature mouse tissues.
FIG. 9 is a view showing expression of OXC mRNA. LOXC c
Using DNA (upper) and β-actin (lower), hybridization was performed with 2 μg of poly (A) ⁺ RNA derived from costal cartilage. Three independent experiments were performed with the same results.

FIG. 11 shows L in the growth plate of a newborn mouse.
It is a figure which shows the localization of OXC gene, type II collagen gene, and type X collagen gene expression. Neonatal cartilage growth plates were fixed, dehydrated and embedded in paraffin. The sections (6 μm thick) were in situ by the method described in the examples.
u was subjected to hybridization. Silver particles were accumulated in hypertrophic and calcified chondrocytes of the cartilage growth plate. Three
Independent experiments were performed with the same results.

FIG. 12 is a graph showing the lysyl oxidase activity of LOXC. A plasmid containing LOXC cDNA or a control plasmid was introduced into COS-7 cells, and the lysyl oxidase activity in the culture supernatant was measured by the method described in the method section. Data are presented as the mean and standard deviation of six measurements. The figure shows a representative example of three independent tests showing the same results. In the figure, * indicates a statistically significant difference of p <0.001 from the corresponding control and βAPN group.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 47/02 A61K 47/42 4B065 47/36 48/00 4C076 47/42 A61P 3/00 4C084 48/00 9/10 101 4C085 A61P 3/00 9/12 4C086 9/10 101 19/08 4H045 9/12 35/00 19/08 43/00 105 35/00 C07K 16/40 43/00 105 C12N 1/15 C07K 16/40 1/19 C12N 1/15 1/21 1/19 9/06 B 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z 9/06 33/50 T C12Q 1/68 Z G01N 33 / 15 33/53 D 33/50 M 33/566 33/53 C12P 21/08 (C12N 9/06 B 33/566 C12R 1:91) // C12P 21/08 C12N 15/00 ZNAA (C12N 9/06 A61K 37/02 C12R 1:91) C12N 5/00 A F (Ref.) DA13 4B065 AA90X AA91Y AA93Y AB01 BA01 CA28 CA44 CA46 4C076 AA95 BB11 CC26 CC41 DD26 EE30 EE60 FF68 4C084 AA01 AA02 AA13 AA16 BA01 BA08 BA22 BA23 BA35 BA42 MA65 NA13 NA14 ZA421 ZA422 ZA01 ZA422 ZA1 452 KA04 KB82 LL20 4C086 AA01 AA02 MA01 MA04 MA07 NA14 ZA96 4H045 AA11 AA30 DA89 EA50 FA71

Claims (20)

[Claims] (1) a base sequence represented by SEQ ID NO: 1 or 3; (ii) a base sequence different from the base sequence of SEQ ID NO: 1 or 3 through degeneracy; (iii) SEQ ID NO: 1 or In the nucleotide sequence of No. 3, a nucleotide sequence having a substitution, deletion, addition or insertion in at least one residue, (iv) the sequence identity with the nucleotide sequence of SEQ ID NO: 1 exceeds 64.8% A base sequence, (v) a base sequence having a sequence identity of more than 63.2% with the base sequence of SEQ ID NO: 3, (vi) a SEQ ID NO:
(Vii) a base sequence different from the base sequence of 1 or 3 through a nucleic acid polymorphism, under a stringent condition with a complementary strand of a sense strand nucleic acid having any one of the base sequences (i) to (vi). A nucleotide sequence of a nucleic acid capable of hybridizing, and (viii)
(I) to (vi) by alternative splicing
i) a nucleic acid having a base sequence selected from the group consisting of base sequences different from any of the base sequences, and the encoded polypeptide has lysyl oxidase activity; a lysyl oxidase-related protein; The sense strand nucleic acid to encode. 2. An antisense nucleic acid having a base sequence complementary to the nucleic acid according to claim 1. 3. The amino acid sequence of the polypeptide encoded by the sense strand nucleic acid according to claim 1, (II) the amino acid sequence represented by SEQ ID NO: 2 or 4, and (III) the amino acid sequence represented by SEQ ID NO: 2 or 4. In the amino acid sequence of
An amino acid sequence having substitutions, deletions, additions or insertions at residues, (IV) an amino acid sequence having a sequence identity of more than 55.5% with the amino acid sequence of SEQ ID NO: 2, and (v)
The sequence identity with the amino acid sequence of SEQ ID NO: 4 is 5
A polypeptide having an amino acid sequence selected from the group consisting of more than 5.1% of amino acid sequences, and
A polypeptide having lysyl oxidase activity. 4. An antibody that specifically binds to the polypeptide according to claim 3. 5. An expression vector, wherein the sense strand nucleic acid according to claim 1 is retained in a vector suitable for expressing a polypeptide encoded by the nucleic acid in a cell. 6. A carrier comprising the sense strand nucleic acid according to claim 1 or the expression vector according to claim 5, wherein the carrier is a carrier selected from the group consisting of liposomes, calcium phosphate and DEAE dextran. Carrier for cell introduction. 7. A cell introduction carrier comprising a carrier containing the antisense nucleic acid according to claim 2, wherein the carrier is a carrier selected from the group consisting of liposomes, calcium phosphate, and DEAE dextran. 8. A transformed or transduced cell, comprising the sense strand nucleic acid according to claim 1. 9. An oligonucleotide capable of specifically hybridizing to the sense strand nucleic acid according to claim 1 or the antisense nucleic acid according to claim 2 under stringent conditions. (10) An effective one selected from the group consisting of the sense strand nucleic acid according to (1), the polypeptide according to (3), the expression vector according to (5), and the carrier for cell introduction according to (7). An agent for promoting bone and cartilage repair, which is contained as an ingredient. 11. The bone / cartilage repair promoting agent according to claim 10, further comprising a substance capable of delivering an active ingredient to a site requiring bone / cartilage repair. 12. The sense strand nucleic acid according to claim 1, the antisense nucleic acid according to claim 2, the polypeptide according to claim 3, the antibody according to claim 4, the expression vector according to claim 5, and the expression vector according to claim 6. A collagenous extracellular matrix production regulator comprising, as an active ingredient, one selected from the group consisting of a cell introduction carrier and the cell introduction carrier according to claim 7. 13. A reagent for producing a collagenous extracellular matrix, comprising the collagenous extracellular matrix production regulator according to claim 12 as an active ingredient. 14. The sense strand nucleic acid according to claim 1, the antisense nucleic acid according to claim 2, the polypeptide according to claim 3, the antibody according to claim 4, the expression vector according to claim 5, and the expression vector according to claim 6. A therapeutic agent for a disease associated with the production of collagen or elastin, which comprises, as an active ingredient, one selected from the group consisting of the carrier for cell introduction and the carrier for cell introduction according to claim 7. 15. The disease associated with collagen or elastin production is at least one selected from the group consisting of fibrotic lesions, chondrosarcoma, osteosarcoma, aortic sclerosis, hypertension, and Ehlers-Danlos syndrome type V. , Claim 1
4. The therapeutic agent according to 4. 16. A nucleic acid comprising a sense strand nucleic acid according to claim 1, an antisense nucleic acid according to claim 2, an antibody according to claim 4, and an oligonucleotide selected from the group consisting of oligonucleotides according to claim 9. , A reagent for detecting the state of bone and cartilage repair. 17. A method for detecting a bone / cartilage repair state, comprising detecting the bone / cartilage repair state using the bone / cartilage repair state detection reagent according to claim 16. (18) the sense strand nucleic acid according to (1),
The reagent for detecting a bone / cartilage repair state according to claim 16, comprising one selected from the group consisting of the antisense nucleic acid according to claim 2 and the oligonucleotide according to claim 9.
(B) following (1) or (2): (1) the antisense nucleic acid according to claim 2 and the following (i) to
(viii): (i) the base sequence shown in SEQ ID NO: 1 or 3, (ii)
(Iii) a base sequence having substitution, deletion, addition or insertion in at least one residue in the base sequence of SEQ ID NO: 1 or 3; The sequence, (iv) a nucleotide sequence having a sequence identity with the nucleotide sequence of SEQ ID NO: 1 of more than 64.8%, (v)
The sequence identity with the nucleotide sequence of SEQ ID NO: 3 is 63.
A nucleotide sequence exceeding 2%, (vi) a nucleotide sequence different from the nucleotide sequence of SEQ ID NO: 1 or 3 through a nucleic acid polymorphism, and (vii) a nucleotide sequence of any of the above (i) to (vi) It consists of a base sequence of a nucleic acid capable of hybridizing with a complementary strand of a sense strand nucleic acid under stringent conditions, and (viii) a base sequence different from any of the base sequences (i) to (vii) by alternative splicing. A hybrid complex with a nucleic acid having a base sequence selected from the group; or (2) the sense strand nucleic acid according to claim 1, and (i) to (v).
iii) detecting the presence or absence of the formation of a hybrid complex with the complementary strand of the nucleic acid according to any of the above, wherein
18. The detection method according to claim 17, wherein the formation of the hybrid complex serves as an indicator that the gene having the nucleic acid according to claim 1 is expressed and the bone / cartilage is repaired. 19. A step of performing nucleic acid amplification using a primer pair consisting of the oligonucleotide according to claim 9 and a nucleic acid derived from a test sample as a template, wherein a product specifically amplified by the primer pair is obtained. Generates
A gene having the nucleic acid according to claim 1 is expressed, and
The detection method according to claim 17, which serves as an indicator of a cartilage repair state. 20. (A) a step of contacting the antibody according to claim 4 with a test sample; and (B) the antibody according to the following (I) to (V): (I) the sense according to claim 1 Amino acid sequence of the polypeptide encoded by the strand nucleic acid, (II) the amino acid sequence shown in SEQ ID NO: 2 or 4, (III) SEQ ID NO: 2 or 4
In the amino acid sequence of at least one residue,
An amino acid sequence having a deletion, addition or insertion, (IV) a sequence identity to the amino acid sequence of SEQ ID NO: 2
A polypeptide comprising an amino acid sequence selected from the group consisting of an amino acid sequence having an amino acid sequence of more than 55.1% and (V) an amino acid sequence of more than 55.1%; Detecting the presence or absence of the formation of the complex of claim 1, wherein the formation of the complex is
The protein encoded by the described nucleic acid is expressed and serves as an indicator of a bone / cartilage repair state.
7. The detection method according to 7.