JP2006296429A - Novel polypeptide and gene encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel polypeptide having a homology with chondromodulin-I, and a gene encoding the polypeptide. <P>SOLUTION: The polypeptide having a specific amino acid sequence, the DNA encoding the polypeptide, and the antibody against the polypeptide are obtained by isolating genes from a human, mouse and rat cDNA libraries. The amino acid sequence has a homology with the chondromodulin-I that has an effect of controlling the growth and differentiation of chondrocytes and inhibiting angiogenesis. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention is a novel human, mouse, and rat that has homologous amino acid sequences with Chondromodulin-I (ChM-I), which is known to regulate chondrocyte proliferation / differentiation and to have an angiogenesis inhibitory effect. The present invention relates to a polypeptide, and human, mouse and rat genes encoding the same (hereinafter sometimes abbreviated as “ChM1L gene”).

  Most bones in mammals are made by a mechanism called “endochondral ossification”, in which calcification occurs through the proliferation and differentiation of chondrocytes, and finally they are replaced with bone. Various hormones and growth factors are involved in this series of processes, and insulin-like growth factors (IGF1, IGF2), fibroblast growth factor (FGF), cancer cell growth factor (TGF), growth hormone, etc. are known. It has been. Opened the ChM-I gene as a factor that promotes the proliferation and differentiation functions of chondrocytes in addition to the hormones and growth factors (Biochem. Biophys. Res. Commun., 175, 971-977, 1991, European Published Patent No. 473080). Human ChM-I is synthesized as a type II membrane protein consisting of 334 amino acid residues. After sugar chain modification, the C-terminal part consisting of 120 amino acid residues is secreted outside the cell after processing (Hirakiet). al, Eur. J. Biochem. 260, 869-878, 1999). ChM-I not only promotes the growth of cultured chondrocytes, but also strongly promotes proteoglycan synthesis and chondrocyte colonization in agarose (Inouet al, Biochem. Biophys. Res. Commun., 241, 395- 400, 1997). ChM-I also promotes not only cartilage but also osteoblast proliferation (Morietal, FEBS Letters, 406 310-314, 1997).

  On the other hand, it has long been pointed out that cartilage is not only an avascular tissue but also exhibits resistance to blood vessel invasion. Kai et al. Attempted to purify a growth inhibitory factor for vascular endothelial cells from cartilage tissue extract and succeeded in the complete purification. As a result, it was found that this is ChM-I (Hiraki et al, FEBS Letter, 415, 321-324, 1997, Hiraki et al, J. Biol. Chem., 272, 32419-32426, 1997). Cartilage tissue is usually characterized by being kept avascular. However, replacement with bone tissue is thought to require blood vessel invasion into cartilage tissue. Prior to the blood vessel invasion that prepares the formation of the primary ossification center, the chondrocyte hypertrophy and the cartilage matrix mineralization occur in the planned blood vessel invasion region. ChM-I is a region where hypertrophic cartilage and subsequent calcified cartilage appear, and its expression is dramatically lost. That is, ChM-I gene expression is cartilage-specific, but is limited to avascular cartilage that is resistant to vascular invasion. As already described, it is considered that ChM-I not only promotes cartilage proliferation and differentiation maturation, but also suppresses vascular invasion by inhibiting proliferation of vascular endothelial cells. Therefore, the expression in avascular cartilage and the disappearance of expression in the calcified layer prior to blood vessel invasion are in good agreement with the bifunctional action of ChM-I.

  In addition, bFGF, which is a potent angiogenesis-promoting factor, is accumulated in a large amount in the peripheral space in the cartilage tissue, but it has been clarified that ChM-I exists in the interspace to surround bFGF (Hiraki). et al, J. Biol. Chem., 272, 32419-32426, 1997). That is, in avascular cartilage, ChM-I exists in a form that masks angiogenesis-promoting factors, and the angiogenesis inhibitory action of ChM-I can explain the absence of blood vessels in cartilage. (Protein Nucleic Acid Enzyme Vol. 40 No. 5.1995). Further, it has been confirmed that ChM-I inhibits blood vessel invasion into human tumor cells in vivo and suppresses the growth of cancer cells (Hayami et al, FEBS Letters, 458, 436-440, 1999). . Analysis of the expression of ChM-I mRNA in each mouse tissue revealed that ChM-I is expressed in the eye and thymus in addition to cartilage. Regarding the function of ChM-I in these tissues, It is still unknown (Shukunami et al, Int. J. Dev. Biol. 43, 39-49, 1999).

  Proliferation of chondrocytes and expression of differentiation functions are important in the healing process of fractures and various cartilage diseases. Therefore, ChM-I, which is a factor that promotes the proliferation and differentiation of chondrocytes, is expected to be applied as a chondrocyte proliferation agent (Japanese Patent Laid-Open No. 7-138295). In the growth and metastasis of cancer cells, invasion of blood vessels into tissues is essential for energy acquisition. Therefore, ChM-I having an angiogenesis inhibitory action is also expected to be applied as an antitumor agent (Japanese Patent Laid-Open No. 7-138295). As described above, ChM-I is a molecule that regulates the proliferation and differentiation of chondrocytes and also has an angiogenesis inhibitory action, and is expected to be applied to pharmaceuticals because of its function.

  In recent years, biotechnology has continued to make rapid progress, and in connection with the progress of the human genome project, a large number of new genes have been cloned. It is said that there are about 100,000 human genes, and among them, a group of molecules having homology in amino acid sequence may form a family. It is known that there are various gene families such as TNF family, TNF receptor family, chemokine and G-protein coupled receptor as molecular groups in which homology is recognized in amino acid sequence. For example, the molecules belonging to the TNF family include Tumornecrosis factor α (TNFα, Pennica et al, Nature 312, 724, 1984), Fasligand (FasL, Suda et al, Cell 75, 1167, 1993), TNF-repaired adipandapino ligandapinodapino ligandapinodapino ligandapinodapino ligandapapido (in Japanese). It is known that there are about 20 types such as TRAIL, Steven et al, Immunity 3,673, 1995) and B lymphocyte simulator (BLYS, Moore et al, Science 285, 260-263, 1999).

  Molecules belonging to the TNF family are type II membrane proteins, and homology in amino acid sequence is recognized in the extracellular region. Although these molecules have homology in their amino acid sequences, it has been clarified that each molecule has a unique function, and attempts have been made as pharmaceuticals for various diseases. In addition, it has been clarified that each TNF family molecule has a unique receptor, and these receptors have been tried to be applied as pharmaceuticals, and some are actually approved as pharmaceuticals (examples). : Soluble TNF receptor, Immunex). In addition, antibodies to these molecules are being researched and developed as pharmaceuticals, and there are those that have actually been approved as pharmaceuticals (eg, anti-TNFα antibody, Centocore). The TNF family and the TNF receptor family are shown as examples above as examples in which molecules with homology in amino acid sequences are applied to drug development. The background that enabled these molecules to be applied to pharmaceuticals is that functional analysis of each molecule was performed and the similarities and differences were clarified.

  In addition, TNF family molecules have a type II membrane protein structure and are mainly expressed in blood and lymphoid cells. Many. Therefore, when a new gene belonging to the TNF family is discovered, the speed of its functional analysis seems to be faster than that of the molecule discovered at an early stage. Thus, the discovery of a new gene having homology to an amino acid sequence and its functional analysis not only help the functional analysis of the new gene to be discovered in the future, Since it can be compared with molecules, it is considered that more detailed knowledge can be obtained regarding the functions of known molecules.

  In general, when a novel gene encoding a protein having homology in amino acid sequence with a known molecule is cloned, the techniques and materials used for functional analysis can refer to examples of known molecules. However, even if the molecule has homology in the amino acid sequence, each molecule is considered to have a unique function like the above TNF family. It is necessary to clarify the expression and purification of recombinant proteins, the production of antibodies, the expression of mRNA and protein in various tissues, and the difference in structure and function from known molecules.

European Published Patent No. 473080 JP 7-138295 A Biochem. Biophys. Res. Commun. , 175, 971-977, 1991 Hirakiet al, Eur. J. et al. Biochem. 260, 869-878, 1999 Inouet et al, Biochem. Biophys. Res. Commun. 241, 395-400, 1997 Morietal, FEBS Letters, 406 310-314, 1997 Hirakiet al, FEBS Letter, 415, 321-324, 1997 Hirakiet al, J. et al. Biol. Chem. 272, 32419-32426, 1997 Hiraki et al, J. Org. Biol. Chem. 272, 32419-32426, 1997 Protein Nucleic Acid Enzyme Vol. 40 No. 5.1995 Hayamiet al, FEBS Letters, 458, 436-440, 1999 Shukunamiet al, Int. J. et al. Dev. Biol. 43, 39-49, 1999 TNFα, Pennicaet al, Nature 312, 724, 1984 FasL, Suda et al, Cell 75, 1167, 1993 TRAIL, Steven et al, Immunity 3,673, 1995 BLYS, Moore et al, Science 285, 260-263, 1999.

  Accordingly, an object of the present invention is to provide a new polypeptide similar to ChM-I and a gene encoding the same. Further, in the present invention, preparation of an antibody against the polypeptide, analysis of expression levels of the gene and polypeptide in various tissues, expression and structural analysis of recombinant protein, etc. are carried out, and similarity to ChM-I The purpose is to elucidate the function and to clarify the function, and to elucidate the pathophysiology, diagnose, and treat these related diseases.

  ChM-I is a type II membrane protein that regulates the proliferation and differentiation of chondrocytes and has an angiogenesis inhibitory effect, and is a molecule expected to be applied to pharmaceuticals. Therefore, if a gene encoding a new polypeptide similar to ChM-I can be provided, the expression level, structure, and function in various cells can be analyzed. Elucidation, diagnosis, treatment, etc. will be possible. However, at present, there is no report of a molecule showing homology with the amino acid sequence of ChM-I, and it is unclear whether ChM-I constitutes a gene family. Therefore, if the existence of a new polypeptide similar to ChM-I and the gene encoding it becomes clear, the similarity and difference with ChM-I can be examined by analyzing its structure and function. It will be possible to elucidate the physiological functions of each other's molecules, elucidate the pathological conditions involved in these molecules, and develop diagnostics and therapeutic agents.

  As a result of intensive studies from the above-mentioned object, the present inventors succeeded in isolating a gene (ChM1L gene) that meets the above-mentioned object newly from human, mouse and rat cDNA libraries. Analysis of the expression level of the polypeptide, production of an antibody against the polypeptide, expression of the polypeptide encoded by the gene in mammalian cells, detection and purification, etc. As a result, the present invention has been completed here.

  That is, the present invention is a gene encoding a polypeptide substantially comprising the amino acid sequence represented by SEQ ID NOs: 2, 4 and 6. As said gene, the base sequence represented by sequence number 1, 3, and 5 is mentioned, for example.

  Furthermore, the present invention is a polypeptide encoded by human, mouse and rat genes substantially comprising the amino acid sequences represented by SEQ ID NOs: 2, 4 and 6.

  Furthermore, the present invention is an oligonucleotide probe that hybridizes with at least a part of the gene.

  Furthermore, the present invention is a recombinant DNA containing the gene.

  Furthermore, the present invention is a transformant transformed with the recombinant DNA.

  Furthermore, the present invention is a method for producing the polypeptide, comprising culturing the transformant and collecting the polypeptide encoded by the gene of the present invention from the obtained culture.

  Furthermore, the present invention is a monoclonal antibody or a polyclonal antibody that specifically reacts with the polypeptide.

  Furthermore, the present invention is a hybridoma that produces the monoclonal antibody obtained by fusing an antibody-producing cell immunized with the polypeptide and a myeloma cell.

  Furthermore, the present invention is a gene detection reagent containing the oligonucleotide probe.

  Furthermore, the present invention is a diagnostic kit comprising the polypeptide and the monoclonal antibody or polyclonal antibody.

  Furthermore, the present invention is a pharmaceutical composition comprising a polypeptide encoded by a gene substantially comprising the amino acid sequence represented by SEQ ID NO: 2, 4 or 6.

  Furthermore, the present invention is a pharmaceutical composition comprising a monoclonal antibody or a polyclonal antibody that specifically reacts with the polypeptide.

  Furthermore, the present invention is a pharmaceutical composition comprising an antisense oligonucleotide that specifically hybridizes with a part of the gene.

  Furthermore, the present invention is a pharmaceutical composition comprising a nucleic acid that can be used for gene therapy, including at least a part of the gene.

  Furthermore, the present invention is a polypeptide characterized in that the polypeptide is a cell membrane-bound type.

  Furthermore, the present invention is a gene encoding the cell membrane-bound polypeptide.

  Furthermore, the present invention is a gene characterized in that the human gene is present on the X chromosome.

  Furthermore, the present invention is a polypeptide characterized in that the polypeptide has an angiogenesis inhibitory action.

  Furthermore, the present invention is a gene encoding the above polypeptide having an angiogenesis inhibitory action.

  In the present invention, “substantially contains” means that the gene or polypeptide of the present invention has the nucleotide sequence represented by SEQ ID NO: 1, 3 or 5 or SEQ ID NO: 2, 4 as long as it has the function. Or it means that mutation such as substitution, insertion or deletion may occur in the amino acid sequence represented by 6.

  The ChM1L gene sequence of the present invention is obtained by the RACE (RACE: Rapid amplification of cDNA ends; Frohman, MA et al, Proc. Natl. Acad. Sci. USA, 85, 8998-9002, 1988) method. However, the outline is as follows.

  In general, the RACE method is a method for efficiently obtaining a full-length cDNA based on a partial sequence of a cDNA. Primers are prepared so as to extend from the known sequence region in the 3′-end or 5′-end, respectively, and the cDNA is amplified by the PCR (Polymerase Chain Reaction, Science, 230, 1350-1354, 1985) method. When performing the PCR method, a primer that anneals specifically in a known region is used, and a primer that anneals to a sequence added by a ligation reaction or the like at the 3 'end and 5' end. Therefore, the region amplified by the PCR method includes a region whose sequence is unknown. Isolation and purification of the amplified DNA fragment can be carried out in accordance with a conventional method as described in Examples below, and may be performed, for example, by gel electrophoresis. Determination of the base sequence of the DNA fragment and the like thus obtained can also be carried out in accordance with a conventional method, for example, dideoxy method (Proc. Natl. Acad. Sci. USA, 74, 5463-5467, 1977), Maxam-Gilbert, etc. (Methods in Enzymology, 65, 499, 1980) and the like. Such a base sequence can be easily determined using a commercially available sequence kit or the like.

  More specifically, it will be described in detail in Example 2 described later in the present invention, and the outline is as follows. Using the amino acid sequence of human ChM-I, a TBLASTN search was performed on the EST database (dbEST, EST: Expressed sequence tag) in the Japan DNA Data Bank (DDBJ: DNAdata bank of Japan), and the EST file, Genbankaccession38 Detected. AI123839 is a base sequence fragment registered in dbEST, but it was revealed by the TBLASTN search that it was a novel gene fragment encoding an amino acid sequence similar to ChM-I. Therefore, a primer was synthesized from a partial sequence of cDNA obtained from dbEST, and the sequence of the human ChM1L gene was determined using the RACE method. Thereafter, the sequences of the mouse and rat ChM1L genes were determined in the same manner. The sequences of the human, mouse and rat ChM1L genes are shown in SEQ ID NOs: 1, 3 and 5, and the amino acid sequences of the polypeptides encoded by them are shown in SEQ ID NOs: 2, 4 and 6.

  The polypeptide encoded by the ChM1L gene of the present invention is composed of 317 amino acids (SEQ ID NOs: 2, 4, and 6). The amino acid sequence of ChM1L is homologous to ChM-I, but in particular has a very high homology with the C-terminal part that is secreted extracellularly after processing of ChM-I (FIG. 1 (a)). In addition, the amino acid sequence of ChM1L has very high homology among humans, mice and rats (FIG. 1 (b)). From the analysis of the hydrophobicity of the amino acid sequence, it is considered that ChM1L is a molecule having a type II membrane protein structure like ChM-I (FIG. 2). As shown in FIG. 2, both the polypeptide and ChM-I have a hydrophobic domain consisting of about 20 amino acids, characteristic of membrane-bound molecules, in the vicinity of several tens of amino acids from the N-terminus. . The fact that the polypeptide is a molecule having a type II membrane protein structure was also revealed from the results of Example 8 in which the polypeptide was expressed in COS7 cells (FIG. 4).

  It was revealed that the human ChM1L gene of the present invention exists in the human X chromosome as described in Example 12 described later (Genbank accession No. ALO35608).

  The ChM1L gene of the present invention includes cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof. The genomic DNA can also be isolated by hybridization to the ChM1L gene disclosed herein using standard techniques. RNA transcribed from the ChM1L gene is also encompassed by the present invention. The sequence of the gene of the present invention represented by SEQ ID NOs: 1, 3 and 5 is an example of one combination of codons indicating each amino acid residue encoded thereby, and the ChM1L gene of the present invention is not limited thereto, Of course, it is possible to have a DNA sequence selected by combining arbitrary codons for the residues. The selection of the codon can be performed according to a conventional method, for example, the codon usage of the host to be used can be considered (Nucleic Acids Research, 9, 43-74, 1981).

  Further, the ChM1L gene of the present invention also includes a DNA sequence encoding a mutant in which a part of the amino acid sequence represented by SEQ ID NOs: 2, 4 and 6 is substituted, deleted or added. The production, modification (mutation) and the like of these polypeptides may occur naturally, and may be carried out by post-translational modification or genetic engineering techniques, for example, Cytospecific Mutagenesis (Methodsin Enzymology, 154, 350, 367- 382, 1987; 100, 468, 1983; Nucleic Acids Research, 12, 9441, 1984; Secondary Biochemistry Experiment Course 1, “Gene Research Method II”, edited by the Japanese Biochemical Society, 105, 1986). it can.

  Production of the ChM1L gene of the present invention can be easily carried out by a general genetic engineering technique based on the sequence information of the ChM1L gene of the present invention (Molecular Cloning 2nd ED, Cold Spring Harbor Laboratory Press, 1989; Research Methods I, II, III ”, edited by Japanese Biochemical Society, 1986, etc.).

  This is carried out, for example, by selecting a desired clone from a cDNA library (prepared according to a conventional method from an appropriate source cell in which the ChM1L gene is expressed) using an appropriate probe or antibody specific to the gene of the present invention. (Proc. Natl. Acad. Sci. USA, 78, 6613, 1981; Science, 222, 778, 1983, etc.).

  In the above method, examples of the source cell include various cells, tissues, and cultured cells derived from them that express the ChM1L gene. Separation of total RNA, separation and purification of mRNA, conversion to cDNA ( Synthesis) and its cloning can be carried out according to conventional methods. Moreover, cDNA libraries are also commercially available, and in the present invention, these cDNA libraries, for example, various cDNA libraries commercially available from Clontech can be used.

  Screening of the ChM1L gene of the present invention from a cDNA library can be carried out according to the usual method. The screening method includes, for example, a method of selecting a corresponding cDNA clone by immunoscreening using a specific antibody against a polypeptide encoded by the ChM1L gene of the present invention against a polypeptide produced by cDNA, and a target base. Examples thereof include plaque hybridization, colony hybridization, and the like using probes that selectively bind to sequences. As the probe used here, the DNA sequence chemically synthesized based on the information on the DNA sequence of the ChM1L gene of the present invention, the ChM1L gene of the present invention already obtained, and fragments thereof can be used as such probes.

  Further, in obtaining the ChM1L gene of the present invention, a DNA / RNA amplification method by PCR can be suitably used. Primers used in adopting such a PCR method can be appropriately set based on the sequence information of the ChM1L gene of the present invention that has already been clarified by the present invention, and can be synthesized according to a conventional method.

  More specifically, although described in detail in Example 2, the outline is as follows. Primers are synthesized so as to include the coding sequence of the ChM1L gene, and the ChM1L gene is amplified by PCR using this primer. Thereafter, agarose electrophoresis is performed to cut out the target band, and then the DNA is purified. The purified DNA and plasmid vector are ligated and transformed with E. coli. Thereafter, the plasmid is purified from the E. coli culture solution, and it is confirmed that the target sequence has been incorporated by a DNA sequencer. The ChM1L gene thus cloned can be transferred to another plasmid vector or virus vector by using an appropriate restriction enzyme.

  By using the ChM1L gene (cDNA and genomic DNA) thus obtained, it is possible to prepare a genetically modified animal in which the expression of the ChM1L gene is increased, attenuated or eliminated in accordance with a conventional method.

  Based on the sequence information of the ChM1L gene of the present invention, the expression of the ChM1L gene of the present invention in various tissues can be detected by utilizing a part or all of the base sequence of the gene. This can be performed according to a conventional method, for example, RT-PCR (Reverse transcribed-polymerase chain reaction) (Kawasaki, ES, et al., Amplification of RNA. In PCR Protocol, A Guide to methods InP. , San Diego, 21-27, 1989), Northern blotting analysis (Molecular cloning, Cold Spring Harbor Laboratory, 1989) and the like. The RT-PCR primer and the northern blotting analysis probe are not limited as long as they are sequences that can specifically detect the ChM1L gene, and such sequences can be appropriately set according to the base sequence of the ChM1L gene of the present invention. It is. Therefore, the present invention provides primers and / or probes useful for the detection of ChM1L gene. The probe can also be used for detection of genomic DNA by Southern blotting analysis.

  As a means for detecting the expression of ChM1L mRNA, the RT-PCR method described in Example 6 can be exemplified. Details are described in Example 6, but the outline is as follows.

  After extracting each tissue and extracting RNA, cDNA is synthesized by reverse transcription reaction. PCR reaction was carried out using this cDNA as a template, and the resulting reaction solution was electrophoresed on an agarose gel, and the band was observed under ultraviolet irradiation to detect the expression level of the ChM1L gene in each tissue. As a result, expression of ChM1L mRNA in each tissue of adult mice was observed in the brain, eyeball, skeletal muscle, whole rib and thyroid gland (FIG. 3 (a)). On the other hand, the expression of ChM-I mRNA in mice has been confirmed in the eyeball, thymus, cartilage and whole ribs (Shukunami et al, Int. J. Dev. Biol 43, 39-49, 1999). Therefore, it became clear that ChM1L and ChM-I are expressed in different tissues in vivo, and their physiological functions were considered to be different. ChM1L was expressed in brain, skeletal muscle, and thyroid gland, which are tissues whose expression was not confirmed in ChM-I.

  In addition, since it is a tissue that is also expressed in ChM-I, and expressed in holerib including ocular and cartilage that is resistant to vascular invasion, ChM1L is involved in angiogenesis It is thought that there is. From these results, ChM1L is related to brain-related diseases such as Alzheimer's disease, skeletal muscle-related diseases such as muscular dystrophy, thyroid-related diseases such as Graves' disease, eye-related diseases such as diabetic retinopathy, deformation It is considered to be involved in diseases related to cartilage such as osteoarthritis and rheumatic diseases and diseases related to angiogenesis including cancer. Therefore, the ChM1L gene, ChM1L polypeptide, antagonists and agonists for ChM1L including antibodies that bind to ChM1L, substances that promote or attenuate the expression of the ChM1L gene, and the like can be applied as therapeutic agents for these diseases. Conceivable. The substances such as agonists and antagonists mentioned here may be peptides, proteins, low molecular compounds, and the like, and the properties of the substances are not limited as long as they have their functions.

  ChM1L mRNA expression in each fetal tissue was observed in the eyeball, kidney, stomach, holerib, and trachea (FIG. 3 (b)). In adult mice, expression of ChM1L mRNA in the kidney and stomach has not been observed, but the expression of ChM1L mRNA in these tissues in the fetus indicates that ChM1L is involved in the development and morphogenesis of these organs. This is considered to indicate that Therefore, ChM1L is considered to be involved in the repair and regeneration of these organs in adults. It was also revealed that ChM1L mRNA was expressed in the trachea. Therefore, the ChM1L gene, ChM1L polypeptide, antagonists and agonists for ChM1L including antibodies that bind to ChM1L, substances that promote or attenuate the expression of the ChM1L gene, and the like, diseases related to the kidney such as chronic renal failure, gastric cancer It can be applied as a therapeutic agent for diseases related to the stomach such as gastric ulcer and respiratory diseases related to the trachea such as chronic bronchitis and asthma.

  In the fetal development stage, the expression of ChM1L mRNA is very weak on the 10th day of pregnancy, and increases from the 11th day to the 13th day (FIG. 3 (c)). On the other hand, the expression of ChM-I increases with the development of the fetus like ChM1L, but clearly showed stronger expression than ChM1L on the 10th and 11th days of pregnancy. Therefore, it was revealed that the expression of ChM1L increases later than that of ChM-I in the fetal development stage, and it was revealed that both molecules have different functions in fetal development. In addition, an increase in ChM1L expression in the fetal development stage indicates that ChM1L is deeply involved in organ and skeleton formation. Accordingly, the ChM1L gene, ChM1L polypeptide, antagonists and agonists for ChM1L including antibodies that bind to ChM1L, substances that promote or attenuate the expression of the ChM1L gene, and the like are used for congenital diseases and acquired diseases caused by insufficient development of organs. It can be applied as a drug that regenerates and repairs an organ when the organ is damaged. In addition, ChM1L and ChM-I have differences in expression in adult and fetal tissues and in the fetal development stage, so these molecules and drugs targeting these molecules are applied as therapeutic agents for various diseases. In that case, the application may be different.

  If the sequence of the ChM1L gene of the present invention is used, a polypeptide encoded by the gene can be produced by genetic engineering techniques.

  The polypeptide is produced by preparing a recombinant DNA capable of expressing the ChM1L gene of the present invention in a host cell, introducing it into the host cell, transforming it, and culturing the transformant. . Here, as the host cell, either a eukaryotic host cell or a prokaryotic host cell can be used.

  Such eukaryotic host cells include vertebrates, yeast and insect cells. Examples of vertebrate cells include CHO cells and COS cells.

  As a vertebrate expression vector, a vector having a promoter, a polyadenylation site, a transcription termination sequence and the like which are usually located upstream of a gene to be expressed can be used. Examples of the expression vector include pSV2dhfr (Mol. Cell. Biol., 854, 1981), pcDNA3.1 (+) (Invitrogen) and pCAGGS (Gene, 108, 193-200, 1991), which possess the SV40 early promoter. ) Etc.

  As a means for expressing a polypeptide of interest in a eukaryotic cell, many systems are well known in the art.

  For example, a system for expression in yeast includes “expression of a polypeptide in yeast” described in JP-A-57-159589, and a system for expression in insect cells is disclosed in JP-A-60-37988. “A method for producing a recombinant baculovirus expression vector” described in Japanese Patent Application Publication No. JP-A-2-171198 is an example of a system for expression in mammalian cells. Of course, there are many other than these.

  The ChM1L gene of the present invention can also be expressed in prokaryotic host cells such as E. coli, Bacillus subtilis and Streptomyces. For example, Escherichia coli K12 strain or the like is often used as Escherichia coli as the host, and pBR322 and its improved vector are often used as vectors. However, it is not limited thereto, and various known strains and vectors can also be used. Examples of the promoter include, but are not limited to, promoters such as E. coli lactose (lac) and E. coli trp. In addition, all of the above promoters have already been characterized and are well known to those skilled in the art and can be synthesized synthetically or assembled from known plasmids.

  Many modifications and changes can be made to the exemplary DNA sequences, plasmids and viruses of the present invention. For example, due to the synonym of the genetic code, nucleotide substitutions can be made throughout the coding region of a polypeptide. Such a sequence can be deduced from the nucleotide sequence of the ChM1L gene of the present invention or the amino acid sequence of the polypeptide encoded by the gene, and can be assembled by the following conventional synthesis method. Such synthetic methods substantially follow the method of Itakura et al. (Itakura et al, Science 198, 1059, 1977) and the method of Claire et al. (Creaet al, Proc. Natl. Acad. Sci. USA 75, 5765, 1978). It can be carried out. Therefore, the present invention is not limited to the specifically exemplified base sequences, plasmids and viruses.

  As a method for introducing the desired recombinant DNA of the present invention thus obtained into a host cell and a transformation method therefor, various general methods can be employed. The obtained transformant can be cultured according to a conventional method, and the polypeptide encoded by the ChM1L gene of the present invention is produced by the culture. As the medium used for the culture, various media commonly used according to the employed host cells can be appropriately selected, and the culture can also be performed under conditions suitable for the growth of the host cells.

  As described above, the polypeptide is produced inside, outside the cell, or on the cell membrane of the transformant. If desired, the polypeptide may be subjected to various separation operations utilizing its physical properties, chemical properties, etc. ["Biochemical Data Book II", pages 1175-1259, first edition, first print, June 23, 1980. Published by Tokyo Chemical Co., Ltd .; Biochemistry, 25 (25), 8274-8277 (1986); Eur. J. et al. Biochem. , 163, 313-321 (1987) etc.]. Specific examples of the method include normal reconstitution treatment, treatment with a polypeptide precipitating agent (salting out method), centrifugation, osmotic shock method, ultrasonic disruption, ultrafiltration, gel filtration, and adsorption chromatography. , Ion exchange chromatography, affinity chromatography, various liquid chromatography such as high performance liquid chromatography (HPLC), dialysis method, combinations thereof, and the like. In addition, if a protein in which an affinity tag is fused to the polypeptide is expressed, affinity purification can be performed using this tag. Examples of the affinity tag described here include a polyhistidine tag (His tag, Sisket al, J. Virol. 68, 766, 1994) and a FLAG tag (Hopp et al, Biotechnology 6, 1204-1210, 1988). Expression and detection of ChM1L polypeptides fused to these affinity tags can be performed as described in Examples 8 and 9, and purification of ChM1L polypeptides using these tags can also be performed. .

  The method for producing the polypeptide encoded by the ChM1L gene of the present invention is more specifically described in detail in Example 8, but the outline is as follows.

  A human and mouse ChM1L gene of the present invention and a gene encoding a ChM1L protein fused with a His tag at the C-terminus were cloned into a pcDNA3.1 (+) vector (Example 4) and transfected into COS7 cells. After about 48 hours, the culture supernatant and cell components were collected and an attempt was made to detect the ChM1L recombinant protein by the Western blot method. However, ChM1L protein expression could not be detected in any of the culture supernatant and cell components.

  Then, when the conditions for detecting the expression of ChM1L recombinant protein were examined, it became possible to detect the expression of the polypeptide in COS7 cells by using pCAGGS as an expression vector. A human and mouse ChM1L gene of the present invention and a gene encoding a ChM1L protein having a His tag fused to the C terminus were cloned into a pCAGGS vector (Example 4), and this was transfected into COS7 cells. After about 48 hours, the culture supernatant and cell components were collected, and ChM1L recombinant protein was detected by the Western blot method. Expression of ChM1L protein was not confirmed in the culture supernatant, and two bands were detected around 40 kDa in the cell component.

  Therefore, it was revealed that ChM1L protein is a membrane-bound protein. On the other hand, it has been confirmed that when ChM-I is expressed in COS7 cells, it is secreted as a soluble protein in the culture supernatant (Hiraki et al, J. Biol. Chem., 272, 32419-32426, 1997). Therefore, analysis with COS7 cells revealed that ChM1L and ChM-I are proteins having different structures. That is, it was revealed that ChM1L is a cell membrane-bound protein, ChM-I is a secreted protein, and the processing mechanisms of both molecules are different. Of the two bands of the ChM1L protein, Example 10 described later revealed that the high molecular weight band was a foam modified with an N-linked sugar chain (FIG. 6).

  The ChM1L protein thus expressed can be affinity-purified using an antibody against a ChM1L-specific antibody or a tag (His tag) fused with 6 residues of histidine, a nickel column, and the like.

  The polypeptide encoded by the ChM1L gene of the present invention may be a membrane-bound polypeptide or a soluble polypeptide that does not have cell membrane-binding properties. For example, a case where it is expressed as a membrane-bound polypeptide on a cell membrane and then cleaved to become soluble is considered. ChM1L protein was detected as a membrane-bound protein in the expression in COS7 cells (Example 8), but may be processed into a soluble protein if the host cell or culture conditions are different. Alternatively, the soluble polypeptide lacking the transmembrane region can be expressed by fusing a heterologous signal peptide to the N-terminus.

  More specifically, although described in detail in Example 9, the outline of the method for expressing soluble ChM1L protein is as follows.

  A vector was constructed in which a pCAGGS vector was incorporated from the N-terminal side with a signal sequence of preprotrypsin, a FLAG tag, and a base sequence encoding a protein fused with the C-terminal side of the extracellular region of ChM1L (Example 5). The ChM1L protein expressed using this vector was secreted into the culture medium as a soluble protein after the signal sequence of preprotrypsin was cleaved (Example 9, FIG. 5).

  The soluble ChM1L polypeptide secreted into the culture medium in this manner can be purified using an anti-FLAG antibody (Sigma) since the anti-ChM1L antibody or the FLAG tag is fused. It is also possible to remove the FLAG tag by cleaving the FLAG fusion protein with enterokinase.

  More specifically, although described in detail in Example 13, the outline of the purification method of the soluble ChM1L protein is as follows.

  CSF7 cells were transfected with pSF-shChM1L according to the product instructions using Lipofectamine reagent (GIBCO BRL), and the culture supernatant was collected about 48 hours later. From this culture supernatant, the soluble human ChM1L protein was purified by affinity chromatography using anti-FLAGM2 affinity gel (Sigma) (FIG. 8).

  The ChM1L polypeptide of the present invention can be used as a polypeptide purification reagent. The polypeptide bound to a solid support material is useful for purification of a polypeptide that can bind to the polypeptide by affinity chromatography. Examples of the polypeptide that can bind to the ChM1L polypeptide include a soluble polypeptide, a membrane-bound polypeptide, and an antibody. Soluble ChM1L polypeptide can be easily applied to addition to cell culture medium in vitro, intravenous administration in vivo, and the like.

  For the purpose of searching for the activity of the ChM1L polypeptide of the present invention, the presence or absence of angiogenesis inhibitory effect was analyzed using human umbilical vein endothelial cells (HUVECs). Details will be described in Example 14 to be described later, but the outline is as follows. When HUVECs are cultured on a plate coated with Matrigel (BECTONDICKINSON), vascular endothelial cells form a lumen-like structure (FIG. 9). When ChM1L polypeptide purified by the above-mentioned affinity chromatography was added to this culture solution, the formation of lumen-like structures of HUVECs was inhibited (FIG. 9). Therefore, it has been clarified that ChM1L has an angiogenesis inhibitory action, and it has been clarified that a soluble ChM1L polypeptide can be applied as a therapeutic agent for diseases associated with angiogenesis such as diabetic retinopathy, cancer, and rheumatoid arthritis.

  The polypeptide encoded by the ChM1L gene of the present invention can be used to produce a specific antibody. Antigens used here include polypeptides produced in large quantities according to the above genetic engineering techniques or chemically synthesized polypeptides, and the antibodies obtained may be either polyclonal antibodies or monoclonal antibodies. Can be effectively used for purification, measurement, identification and the like of the polypeptide. Accordingly, polyclonal antibodies and monoclonal antibodies against the polypeptide can be used for the treatment of diseases mediated by the polypeptide (directly or indirectly) and the development of therapeutic methods. It is also possible to use it.

  An antibody that specifically binds to the polypeptide encoded by the ChM1L gene of the present invention can be prepared as shown in Example 7. It was confirmed by the result of Western blot of Example 8 that the prepared anti-ChM1L polypeptide antibody specifically bound to the polypeptide (FIG. 4).

  The anti-ChM1L polypeptide antibody can also be used for immunostaining of tissue sections as shown in Example 11. When the costal cartilage tissue was stained with the anti-ChM1L polypeptide antibody, cells having a flat fibroblast-like morphology existing surrounding the cartilage tissue were stained specifically (FIG. 7). On the other hand, ChM-I is specifically expressed in chondrocytes, and it has been revealed by immunostaining that it is accumulated in chondrocytes and the matrix outside the chondrocytes (Hiraki et al, J. Biol. Chem). , 272, 32419-32426, 1997). Therefore, it was revealed that ChM1L and ChM-I are expressed in different cells in tissues including cartilage. Therefore, it was revealed that ChM1L is a molecule having a function different from that of ChM-I.

  A tissue containing a group of cells showing a fibroblast-like morphology that has been revealed to express ChM1L protein by immunostaining has been conventionally called perichondrium (Sudaet al, bone formation) Bone resorption and their regulators 1, 2, 1995). Although there is no clear definition at present regarding the tissue of the perichondrium, in the present specification, it refers to a tissue containing cells having a fibroblast-like morphology surrounding a chondrocyte.

  Cells present in the perichondrium are considered to be a source of chondrocytes when cartilage tissue grows during the process of endochondral ossification. Therefore, the perichondrium is an important tissue that supplies chondrocytes during skeletal formation in the developmental process and bone and cartilage damage in adults. The cartilage tissue is characterized by the absence of blood vessels, nerves, and lymphatic vessels, but the perichondrium is located at the boundary between the cartilage tissue and other tissues. It is thought to control the invasion of nerves and lymphatic vessels. Thus, although the perichondrium is recognized as an important tissue, it is not a well-defined tissue as described above, and no detailed study has been conducted at present. The reason for this is that no molecule expressed specifically in the perichondrium has been clarified.

  Therefore, if the existence of a molecule specifically expressed in a tissue called perichondrium surrounding the periphery of the cartilage tissue is clarified, it can be used as a very important tool for research on the perichondrium and the cartilage tissue.

  ChM1L of the present invention is the only molecule that has been revealed to be expressed specifically in the perichondrium, and is thought to control the invasion of blood vessels, nerves, and lymphatic vessels into the cartilage tissue.

  Therefore, the results of the discovery of the ChM1L gene and the functional analysis of ChM1L included in the present invention will be used in the future to develop causes of diseases and other therapeutic methods involving other tissues in which ChM1L is expressed, including perichondrium and cartilage tissue. It is thought that it can give a new perspective.

  Therefore, the ChM1L gene, ChM1L polypeptide, antagonists and agonists for ChM1L including antibodies that bind to ChM1L, substances that promote or attenuate the expression of the ChM1L gene, etc. involve the cells expressing the above ChM1L. It is thought that it can be applied as a therapeutic agent for existing diseases.

  The ChM1L gene of the present invention and the polypeptide encoded by the gene have been analyzed from the results of the above mRNA expression analysis and immunostaining. It was revealed that it is expressed in cells that have a fibroblast-like flat shape that exists so as to surround. Therefore, the ChM1L gene of the present invention and the polypeptide encoded by the same are related to the above-mentioned tissues that are confirmed to be expressed, such as diabetic retinopathy, muscular dystrophy, Graves' disease, chronic renal failure, It is thought to be involved in gastric cancer, chronic bronchitis, osteoarthritis and rheumatic diseases.

  Therefore, the ChM1L gene, ChM1L polypeptide, antagonists and agonists for ChM1L including antibodies that bind to ChM1L, substances that promote or attenuate the expression of the ChM1L gene, and the like can be applied as therapeutic agents for these diseases. Conceivable.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples are not intended to limit the scope of the present invention.

Example 1. Analysis of ChM1L amino acid sequence The homology of the amino acid sequences of ChM-I and ChM1L was compared (FIG. 1 (a)). The amino acid sequence is indicated by a single letter of the alphabet. ChM1L has homology with ChM-I throughout the molecule, but has been found to have particularly high homology with the C-terminal side that is secreted extracellularly after processing of ChM-I.

  The homology of amino acid sequences of human, mouse and rat ChM1L was compared (FIG. 1 (b)). The ChM1L polypeptide is composed of 317 amino acids in both human, mouse and rat, but 300 amino acid residues were the same among the three species (about 95%).

  A diagram of the hydrophobicity of ChM-I and ChM1L is shown (FIG. 2). A large hydrophobic peak is observed on the N-terminal side of both ChM-I and ChM1L. This hydrophobic region is characteristically observed in cell membrane-bound proteins, and it was shown that ChM1L is a type II membrane-bound protein like ChM-I.

Example 2 Cloning of the ChM1L gene Using the amino acid sequence of human ChM-I (Genbank accession number M16441) from the Japan DNA Data Bank of Japan (DDBJ), an EXPRESSED SEQUENCE tag database (DBEST) search was performed on a BLEST. . As a result, an EST file, Genbank accession number AI123839 was detected as a novel gene fragment having homology with ChM-I.

The cDNA was amplified by the RACE method using Clonetech Human fetus Marathon-Ready ^™ cDNA according to the product instructions. Primers were synthesized from the base sequence obtained from the above dbEST, ExTaq polymerase (Takara Shuzo) was used according to the product instructions, GeneAmp® PCR System 9700 (PE Applied Biosystems) was used, and the reaction cycle was 96 ° C. 30 Second, 60 ° C. 30 seconds, 72 ° C. 1 minute was repeated 30 times, and finally incubated at 72 ° C. for 6 minutes to obtain a PCR reaction solution. 1/10 amount of this reaction solution was added as a template, and a second PCR was performed under the same conditions.

  The obtained PCR product was electrophoresed on a 1% agarose gel containing ethidium bromide, and the DNA band was examined by observing the gel under ultraviolet light. The amplified fragment was excised from the gel and purified using a QIAquick Gel Extraction Kit (QIAGEN) according to the product instructions.

The base sequence of the purified fragment was determined according to a product instruction using a DNA sequencer (ABI PRISM ^™ 310 Genetic Analyzer) and ABI PRISM ^™ BigDye Terminator Cycle Sequencing Reactor manufactured by PE Applied Blosystems.

  The nucleobase sequence of human ChM1L cDNA is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

  Since the amino acid sequence encoded by the human ChM1L gene represented by SEQ ID NO: 1 shows homology with human ChM-I, this gene was called a ChM1L gene (ChM-Ilike gene).

Coding sequence (CDS) of human ChM1L cDNA was amplified by PCR, purified after agarose electrophoresis, and cloned according to the product instructions using pCR-Script ^™ Amp cloning kit (Stratagene). The sequences of primers used for PCR are shown in SEQ ID NO: 7 (Forward primer) and SEQ ID NO: 8 (Reverse primer). The ChM1L gene sequence incorporated into the vector was determined using ABI PRISM ^™ 310 Genetic Analyzer (PE Applied Biosystems) and ABI PRISM ^™ BigDye Terminator Cycle Sequencing according to the product instructions.

Using the amino acid sequence of human ChM1L (SEQ ID NO: 2), a TBLASTN search was performed in the same manner as in the case of the human. As a result, an EST file and Genbank accession number AV009191 were detected as gene fragments encoding mouse ChM1L, and an EST file and Genbank accession number AI112003 were detected as gene fragments encoding rat ChM1L. The mouse and rat ChM1L gene sequences were determined by the RACE method in the same manner as the isolation of the human ChM1L gene using the Mouse 11-day Embryo Marathon-Ready ^™ cDNA and the Rat Skeletal muscle Marathon-Ready ^™ cDNA manufactured by Clontech.

  The nucleobase sequence of mouse ChM1L cDNA is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. The nucleobase sequence of rat ChM1L cDNA is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6.

Coding sequence (CDS) of mouse and rat ChM1L cDNA was amplified by PCR, purified after agarose electrophoresis, and cloned using pCR-Script ^™ Amp cloning kit (Stratagene) according to the product instructions. The primer sequences used for mouse gene PCR are SEQ ID NO: 9 (Forward primer) and SEQ ID NO: 10 (Reverse primer), and the primer sequences used for rat gene PCR are SEQ ID NO: 11 (Forward primer) and SEQ ID NO: 12 (Reverse primer). The ChM1L gene sequence incorporated into the vector was determined using ABI PRISM ^™ 310 Genetic Analyzer (PE Applied Biosystems) and ABI PRISM ^™ BigDye Terminator Cycle Sequencing according to the product instructions.

The following abbreviations are used for the vectors into which human, mouse and rat ChM1L genes have been prepared in this Example.
-Vector containing human ChM1L gene: pCR-hChM1L
-Vector containing mouse ChM1L gene: pCR-mChM1L
-Vector containing rat ChM1L gene: pCR-rChM1L

Example 3 Construction of a vector containing genes encoding human and mouse ChM1L proteins fused with 6 histidine residues at the C-terminus Coding sequence (CDS) of human and mouse ChM1L cDNA is amplified by PCR, purified after agarose electrophoresis, and purified. The product was cloned using a pCR-Script SK (+) vector (Stratagene) and a pCR-Script ^™ Amp cloning kit (Stratagene) modified so that 6 histidine residues (His tag) were fused to each other. The primer sequences used for PCR of the human gene are SEQ ID NO: 7 (Forward primer) and SEQ ID NO: 13 (Reverse primer), and the sequences of the primers used for PCR of the mouse gene are SEQ ID NO: 9 (Forward primer) and SEQ ID NO: 14 (Reverse primer). The incorporation of a base sequence encoding a protein in which a His tag is fused to the C-terminus of ChM1L is based on the description of ABI PRISM ^™ 310 Genetic Analyzer (PE Applied Biosystems) and ABI PRISM ^™ BigDye Recycle Cycle Recycler Used and confirmed according to the instructions. The amino acid sequences of human and mouse ChM1L fused with a His tag at the C-terminus are shown in SEQ ID NOs: 17 and 18, and the nucleobase sequences encoding them are shown in SEQ ID NOs: 15 and 16, respectively.

The following abbreviations are used for the vector in which the gene encoding the ChM1L protein fused with the His tag produced in this example is cloned.
A vector containing a gene encoding a protein in which human ChM1L and His tag are fused: pCR-hChM1LHis
A vector containing a gene encoding a protein in which mouse ChM1L and His tag are fused: pCR-mChM1LHis

Example 4 Construction of Expression Vector For the purpose of expressing ChM1L gene in mammalian cells, the above-described pCR-hChM1L was added to pcDNA3.1 (+) vector (Invitrogen) and pCAGGS vector (Gene, 108, 193-200, 1991). , PCR-mChM1L, pCR-hChM1LHis and pCR-mChM1LHis vectors were excised with restriction enzymes EcoRI and NotI, and after agarose electrophoresis, the band of interest was purified and ligation reaction was performed using Ligationhigh (Toyobo) according to the product instructions. Went. The solution after the ligation reaction was treated with E. coli. Transformation was performed using E. coli JM109 Competent cell (Takara Shuzo) according to the product instructions. After purification of the plasmid, the integration of the target gene was confirmed by restriction enzyme reaction and agarose electrophoresis.

The following abbreviations are used for the vectors prepared in this example.
PcDNA3.1 (+) vector containing hChM1L, mChM1L, hChM1LHis and mChM1L genes:
pcDNA-hChM1L, pcDNA-mChM1L, pcDNA-hChM1LHis and pcDNA-mChM1LHis
-PCAGGS vector containing hChM1L, mChM1L, hChM1LHis and mChM1L genes:
pCAGGS-hChM1L, pCAGGS-mChM1L, pCAGGS-hChM1LHis and pCAGGS-mChM1LHis

Example 5 FIG. Construction of vector expressing human soluble ChM1L protein fused with FLAG tag The FLAG tag (Sigma) described in this example is a hydrophilic marker peptide (Asp Tyr Lys Asp Asp Asp As Lys) consisting of 8 amino acids. 5 amino acids (Asp Asp Asp Asp Lys) are the recognition sequences for enterokinase. The vector produced by this example can express a protein in which the signal sequence of preprotrypsin, the FLAG tag, and the C-terminal side of the extracellular region of ChM1L are fused from the N-terminal side. The protein expressed using this vector is secreted into the culture solution as a soluble protein after the signal sequence of preprotrypsin is cleaved, as described in detail in Example 9 described later. In addition, since the protein expressed by this vector is fused with a FLAG tag, it can be purified using an anti-FLAG antibody (Sigma). By cleaving the fusion protein with enterokinase, It is also possible to remove the tag.

A pCAGGGS vector was constructed in which a nucleotide sequence (SEQ ID NO: 19, included in Sigma pFLAG-CMV-1 vector) encoding a preprotrypsin signal sequence and a FLAG tag (SEQ ID NO: 20) was constructed from the N-terminus. (Hereinafter referred to as pSF vector). A base sequence encoding the region containing amino acid numbers 212 to 317 of human ChM1L represented by SEQ ID NO: 2 and a translation stop codon (base sequence numbers 684 to 1020 of SEQ ID NO: 1) is amplified by the PCR method in the pSF vector, The amplified product was incorporated into the 3 ′ side of the base sequence encoding the FLAG tag of the pSF vector. The sequences of the primers used for PCR are shown in SEQ ID NO: 21 (Forward primer) and SEQ ID NO: 8 (Reverse primer). It was confirmed that the target base sequence was incorporated into the constructed vector using ABI PRISM ^™ 310 Genetic Analyzer (PE Applied Biosystems) and ABI PRISM ^™ BigDye Terminator Cycle Sequencing Ready Read product. In this example, the nucleobase sequence incorporated in the vector is shown in SEQ ID NO: 22, and the amino acid sequence encoded by it is shown in SEQ ID NO: 23. The vector produced in this example uses the abbreviation pSF-shChM1L.

Example 6 Expression analysis of ChM1L mRNA
Expression analysis of ChM1L mRNA in each tissue of adult (10 weeks old): FIG. 3 (a)
Ten week old C57BL / 6 mice were dissected and each tissue was removed and immediately frozen in liquid nitrogen. The frozen tissue was crushed and total RNA of each tissue was obtained using ISOGEN (Nippon Gene) according to the product instructions. Using 20 ug of the total RNA obtained from each tissue as a template, Superscript II preparation kit (GIBCO BRL) was used according to the product instructions to synthesize 20 uL of cDNA. In RT-PCR, the total volume of the reaction system is 50 uL, 0.5 μL of cDNA of each tissue, and 0.25 uL of ExTaq polymerase (Takara Shuzo) are used, and a forward primer (SEQ ID NO: 9) and reverse primer (SEQ ID NO: 10) Were added at 0.2 uM, respectively, and amplification was carried out for 30 cycles at 96 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute using GeneAmp registered trademark PCR System 9700 (PE Applied Biosystems). The obtained reaction solution was electrophoresed on a 1% agarose gel containing ethidium bromide, and the gel was photographed under ultraviolet irradiation to examine the expression of ChM1L mRNA in each tissue.

  As shown in FIG. 3 (a), the expression of ChM1L mRNA in each tissue of adult mice was observed in brain, eyeball, skeletal muscle, whole rib and thyroid gland. ChM-I expression in mice has been confirmed in the eyeball, thymus, cartilage and holerib. Therefore, it was revealed that ChM1L and ChM-I are expressed in different tissues in vivo, suggesting that their physiological functions are different.

Expression analysis of ChM1L mRNA in each tissue of fetus (17th day of pregnancy): FIG. 3 (b)
The fetus on the 17th day of pregnancy of C57BL / 6 mice was removed by caesarean section, and each tissue was removed and immediately frozen in liquid nitrogen. Extraction of total RNA from the frozen tissue, synthesis of cDNA, and RT-PCR were performed in the same manner as in the above-described <analysis of expression of ChM1L mRNA in each tissue of adult mouse>.

  As shown in FIG. 3B, the expression of ChM1L mRNA in each fetal tissue was observed in the eyeball, kidney, stomach, whole rib and trachea. In fetal mice, expression was observed in the kidney and stomach, where expression was not observed in adult mice. Therefore, ChM1L may be involved in the development and morphogenesis of these organs, and is thought to be involved in organ repair and regeneration. It was also revealed that ChM1L mRNA was expressed in the trachea.

Expression analysis of ChM1L mRNA in fetal development stage: FIG. 3 (c)
Each day-old fetus from the 10th day of pregnancy to the date of birth of C57BL / 6 mice was removed by caesarean section, and the whole fetus was frozen in liquid nitrogen. Extraction of total RNA from frozen fetuses, synthesis of cDNA, and RT-PCR were carried out in the same manner as in the above-described <analysis of expression of ChM1L mRNA in each tissue of adult mouse>.
The analysis of ChM-I mRNA was performed under the same conditions using Forward primer (SEQ ID NO: 23) and reverse primer (SEQ ID NO: 24).

  As shown in FIG. 3 (c), the expression of ChM1L mRNA in the fetal development stage is very weak on the 10th day of pregnancy, and the expression increases from the 11th day to the 13th day. On the other hand, the expression of ChM-I showed the same increase in expression as that of ChM1L, but clearly showed stronger expression than ChM1L on the 10th and 11th days of pregnancy. Therefore, in the fetal development stage, it became clear that ChM1L increased in expression later than ChM-I, and it was revealed that both molecules have different functions in fetal development.

Example 7 Preparation of anti-ChM1L peptide polyclonal antibody A peptide having a cysteine at the C-terminus of the sequence from 245 to 252 residues shown in SEQ ID NO: 2 of human ChM1L was chemically synthesized. MBS / KLH (m-maleimidebenzoyl-N-hydroxysuccinimide ester / keyhole limpet hemocyanin, Boehringer Mannheim) was coupled to this synthetic peptide. After this complex was dissolved in physiological saline, an equal amount of FCA (Freund's complete adjuvant) was added and sonicated to prepare an emulsion. This emulsion was administered subcutaneously to rabbits for initial immunization. Four weeks after the initial immunization, FIA (Freund's incomplete adjuvant) was used to boost the thigh muscle, and thereafter, the immunization was performed four times by subcutaneous administration at intervals of about 2 weeks or about 4 weeks. Partial blood was collected from the auricle during the booster immunization, and after the final immunization, whole blood was collected to separate the serum, and affinity purification using a peptide column was performed to obtain an anti-ChM1L peptide polyclonal antibody.

Example 8 FIG. Analysis of human and mouse ChM1L recombinant protein by Western blot method: FIG.
Using Lipofectamine reagent (GIBCO BRL) according to product instructions, pCAGGS, pCAGGS-hChM1L and pCAGGS-mChM1L (FIGS. 4 (a) and (c)) or pCAGGS, pCAGGS-hChM1LHis and pCAGGS1mCh Figures 4 (b) and (d)) were transfected into COS7 cells. About 48 hours after the transfection, the culture supernatant and cell components were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) with a 12.5% gel, and then transferred to a nitrocellulose membrane. A primary antibody reaction and a secondary antibody reaction were performed, and a color reaction was performed using ECLplus reagent (Amersham Pharmacia Biotech) according to the product instructions. The Western blot when pCAGGS, pCAGGS-hChM1L and pCAGGS-mChM1L were transfected were the primary antibody anti-ChM1L polyclonal antibody described in the above-mentioned Examples, and the secondary antibody labeled with the horseradish peroxidase (HRP). (Dako) is a Western blot when transfected with pCAGGS, pCAGGS-hChM1LHis and pCAGGS-mChM1LHis, the primary antibody is an anti-His tag antibody (Invitrogen), and the secondary antibody is an anti-mouse IgG antibody labeled with HRP (Amersham Pharmacia Biotech) was used.

SDS-PAGE was performed on the same sample as Western blot, and the results of staining with Coomassie Brilliant Blue (CBB) are shown in FIGS. 4 (a) and 4 (b).
As a result of Western blotting, no ChM1L band was confirmed in all culture supernatants. In the cell component, as shown in FIGS. 4B and 4D, the recombinant ChM1L protein is detected as two bands in the vicinity of 40 kDa regardless of whether the anti-ChM1L peptide antibody or the anti-His tag antibody is used. It was. As will be described in detail in Examples below, the analysis of the sugar chain structure confirmed that the high molecular weight band was a form in which N-linked sugar chains were bound.

Example 9 Analysis of soluble human ChM1L recombinant protein by Western blot: FIG.
COS7 cells were transfected with pCAGGS and pSF-shChM1L using Lipofectamine reagent (GIBCO BRL) according to the product instructions. The culture supernatant was subjected to SDS-PAGE with a 12.5% gel and then transferred to a nitrocellulose membrane. Product description using ECLplus reagent (Amersham Pharmacia Biotech) using anti-FLAGM2 antibody (Sigma) as primary antibody, anti-mouse IgG antibody (Amersham Pharmacia Biotech) labeled with HRP as secondary antibody The color reaction was carried out according to the instructions.

  As shown in FIG. 5, the soluble human ChM1L protein was detected as a single band around 17-18 kDa.

Example 10 Analysis of the sugar chain structure of ChM1L recombinant protein Using the lipofectamine reagent (GIBCO BRL), pCAGGS-mChM1LHis was transfected into COS7 cells according to the product instructions. PBS containing 2% SDS was added to the dish, and the cells were collected with a scraper. This suspension was heated at 95 ° C. for 60 minutes, and then the supernatant was used with SDS-OUT ^™ SDS Precipitation kit (Pierce). Processed to remove SDS. Using the protein solution thus obtained, the above-mentioned protein solution is treated with NANase II, O-Glycosidase DS and PNGase F according to the product instructions using Enzymatic Deglycylation Kit (BIO RAD), A digestion reaction was performed. This reaction solution was subjected to SDS-PAGE with a 12.5% gel and then transferred to a nitrocellulose membrane. Using an anti-His tag antibody (Invitrogen) as the primary antibody, an anti-mouse IgG antibody (Amersham Pharmacia Biotech) labeled with HRP as the secondary antibody, and using ECLplus reagent (Amersham Pharmacia Biotech) The color reaction was performed according to the instructions.

  As shown in FIG. 6, the high molecular weight band of ChM1L protein disappeared only when treated with PNGase F (lanes 2 and 5). Therefore, it was revealed that the ChM1L protein is modified with an N-linked sugar chain.

Example 11 Analysis of ChM1L protein in costal cartilage by immunostaining method About 10 weeks old C57BL / 6 mice were dissected and whole ribs were taken out in 10 mM phosphate buffer buffer containing 4% paraformaldehyde, pH 7.4 (PBS). After fixing and embedding with paraffin, sections were prepared. Each step of immunostaining was performed according to product instructions using a Histofine SAB-PO (R) kit (Nichirei), and the outline is as follows. After deparaffinization, endogenous peroxidase was digested with 3% aqueous hydrogen peroxide. After washing with PBS and blocking with 10% normal goat serum, the above-mentioned anti-ChM1L peptide antibody was added at 1/160 dilution and incubated at 4 ° C. overnight. Rabbit IgG was used as a negative control. After reacting biotin-labeled anti-rabbit IgG antibody and peroxidase-labeled streptavidin, 3,3-diaminobenzidine · 4HCl was added to carry out a color reaction. Nuclei were stained with hematoxylin and encapsulated before observation.

  As shown in FIG. 7, it was revealed that the ChM1L protein is expressed in cells having a flat fibroblast-like morphology existing around chondrocytes in the shark cartilage tissue. On the other hand, expression was not observed in chondrocytes reported to express ChM-I.

Example 12 Chromosome mapping of human ChM1L gene A BLASTN search was performed on all DDBJ data using the human ChM1L gene sequence (SEQ ID NO: 1) from the Japan DNA Data Bank (DDBJ). As a result, Genbank accession No. 5 was used as the genomic sequence of the ChM1L gene. ALO35608 was detected. ALO35608 is a sequence mapped to the human X chromosome. Therefore, it became clear that the human ChM1L gene exists on the X chromosome.

Example 13 Purified soluble human ChM1L recombinant protein COS7 cells were transfected with pSF-shChM1L using Lipofectamine reagent (GIBCO BRL) according to the product instructions, and the culture supernatant was collected after about 48 hours. An affinity column was prepared using an anti-FLAG M2 affinity gel (Sigma), and the culture supernatant was applied to the column. The column was washed 3 times with 25 mM Tris-HCl and 150 mM NaCl (pH 7.4), and then eluted with 0.1 M glycine-HCl (pH 3.5), and 1/20 volume of 1 M Tris-HCl (pH 9.5) was eluted. Was used to neutralize the eluate.

  FIG. 8 shows the results of SDS-PAGE using the culture supernatant and eluate, and Coomassie brilliant blue (CBB) staining. Although various proteins are present in the culture supernatant (FIG. 8, lane 1), the soluble human ChM1L protein is confirmed as a band of about 20 kDa in the eluate, and the soluble human ChM1L protein is concentrated and purified by the above operation. (FIG. 8, lane 2).

Example 14 Examination of angiogenesis inhibitory action using human umbilical vein endothelial cells Human umbilical vein endothelial cells (HUMECs, HUVECs, Clonetics) are endothelial cell-dedicated media (EGM®-2 Bullet Kit®, Clonetics). Cultured. Growth factor reduced Matrigel (BECTON DICKINSON) was added to a 12-well plate at 600 uL / well and incubated at 37 ° C. for 30 minutes. A cell suspension containing 5 × 10 ⁴ cells / mL of HUVECs using a medium obtained by diluting a medium dedicated to endothelial cells not containing heparin to 1/8 of an endothelial cell basic medium (EBM®-2, Clonetics). Was prepared.

Each test substance solution was prepared with a solution obtained by adding 1/20 volume of 1M Tris-HCl (pH 9.5) to 0.1 M glycine-HCl (pH 3.5), and treated with a volume of 200 uL / well. . The above buffer and BSA (bovin serum albumin) are 20 ug / well as a negative target, Pleletet factor 4 (PF-4, Chemicon) is 1 and 10 ug / well as a positive target substance, and soluble human ChM1L recombinant protein is an example. Thirteen elution fractions were treated with 10 and 20 ug / well. 2 mL (1 × 10 ⁵ cells) of cell suspension and 200 uL of each test substance solution were mixed and seeded on a 12-well plate coated with Growth factor reduced Matrigel. After 9 hours, the formation of a lumen-like structure was observed. I took a picture. The results are shown in FIG. HUVECs form a lumen-like structure in negative subjects (FIGS. 9 (a) and (b)), but when treated with 20 ug / well of ChM1L (FIG. 9 (d)), compared to negative subjects Thus, the formation of lumen-like structures was inhibited.

  Therefore, it has been clarified that ChM1L has an angiogenesis inhibitory action, and it has been clarified that a soluble ChM1L polypeptide can be applied as a therapeutic agent for diseases associated with angiogenesis such as diabetic retinopathy, cancer, and rheumatoid arthritis.

The result of having compared the homology of the amino acid sequence of human ChM1L and human ChM-I is shown. The result of having compared the homology of the amino acid sequence of human, mouse | mouth, and rat ChM1L is shown. The hydrophobic profiles of the amino acid sequences of human ChM-I, human ChM1L and mouse ChM1L are shown. The results of ChM1L mRNA expression analysis in adult mouse and fetal tissues, and ChM1L and ChM-I mRNA expression analysis results in mouse fetal developmental stages are shown. The result of expressing human and mouse ChM1L proteins in COS7 cells and detecting them by Western blot is shown. (A) shows the result of Mock (lane 1), human ChM1L (lane 2), and mouse ChM1L (lane 3) transfected, electrophoresed with cell components, and stained with Coomassie brilliant blue. The result of having detected the sample by Westernblot using an anti-ChM1L peptide antibody is shown. (B) Transfects Mock (lane 1), human ChM1L (with His tag) (lane 2) and mouse ChM1L (with His tag) (lane 3), electrophoresis of cell components, and Coomassie brilliant blue (D) shows the result of detecting the same sample by Western blot using an anti-His tag antibody. The result of having detected soluble ChM1L (lane 2) and Mock (lane 1) expressed in COS7 cells by Western blot method using anti-FLAGM2 antibody is shown. Mouse ChM1L (His-tagged) protein is expressed in COS7 cells, cell components are collected and subjected to sugar chain digestion, ChM1L protein is detected by Western blot using an anti-His tag antibody, and the structure of the sugar chain is analyzed The results are shown. Lane 1 is untreated, lane 2 is a nanase II + O-glycosidase DS + PNGase process, lane 3 is a nanase II process, lane 4 is an O-glycosidase DS process, and lane 5 is a result of Western blot of a PNGase process sample. The result of having detected the expression of ChM1L protein in the shark cartilage tissue of a mouse | mouth by the immuno-staining using an anti-ChM1L polypeptide antibody is shown. The results show that the soluble human ChM1L protein expressed in the culture solution of COS7 cells was purified by affinity chromatography using an anti-FLAG M2 affinity gel, stained with Coomassie brilliant blue after electrophoresis. Lane 1 shows the culture supernatant of COS7 cells, and Lane 2 shows the results of electrophoresis of purified ChM1L protein. Human umbilical vein endothelial cell luminal-like structure forming system includes (a) buffer only, (b) 20 ug of BSA (bovine serum albumin), (c) 10 ug of soluble human ChM1L, (d) 20 ug of soluble human ChM1L, (e) The results of processing 1 ug of PF-4 (platelet factor 4) and 10 ug of (f) PF-4 are shown.

Claims (27)

  A human gene encoding a polypeptide substantially comprising the amino acid sequence represented by SEQ ID NO: 2.   A mouse gene encoding a polypeptide substantially comprising the amino acid sequence represented by SEQ ID NO: 4.   A rat gene encoding a polypeptide substantially comprising the amino acid sequence represented by SEQ ID NO: 6.   The human gene according to claim 1, which has the base sequence represented by SEQ ID NO: 1.   The mouse gene according to claim 2, which has the base sequence represented by SEQ ID NO: 3.   The rat gene according to claim 3, which has the base sequence represented by SEQ ID NO: 5.   A polypeptide encoded by a human gene, substantially comprising the amino acid sequence represented by SEQ ID NO: 2.   A polypeptide encoded by a mouse gene substantially comprising the amino acid sequence represented by SEQ ID NO: 4.   A polypeptide encoded by a rat gene, substantially comprising the amino acid sequence represented by SEQ ID NO: 6.   An oligonucleotide probe that hybridizes with at least a portion of the gene according to any one of claims 1-6.   Recombinant DNA containing the gene according to any one of claims 1 to 6.   A transformant transformed with the recombinant DNA according to claim 11.   A method for producing the polypeptide, comprising culturing the transformant according to claim 12 and collecting a polypeptide encoded by a human, mouse, or rat gene from the resulting culture.   A monoclonal antibody that specifically reacts with the polypeptide according to any one of claims 7 to 9.   A polyclonal antibody that specifically reacts with the polypeptide according to any one of claims 7 to 9.   The hybridoma which produces the monoclonal antibody of Claim 14 obtained by fuse | melting the antibody producing cell immunized with the polypeptide as described in any one of Claims 7-9, and myeloma cell.   A gene detection reagent comprising the oligonucleotide probe according to claim 10.   A diagnostic kit comprising the polypeptide according to any one of claims 7 to 9, and the monoclonal antibody according to claim 14 and / or the polyclonal antibody according to claim 15.   A pharmaceutical composition comprising the polypeptide according to any one of claims 7 to 9.   A pharmaceutical composition comprising the monoclonal antibody according to claim 14 or the polyclonal antibody according to claim 15.   A pharmaceutical composition comprising an antisense oligonucleotide that specifically hybridizes with a part of the gene according to claim 1.   A pharmaceutical composition comprising a nucleic acid comprising at least a part of the gene according to claim 1 and usable for gene therapy.   A human gene which is present on the X chromosome according to claim 1 or 4.   A polypeptide according to claim 7, wherein the polypeptide is a cell membrane-bound type.   A gene encoding the cell membrane-bound polypeptide according to claim 24.   A polypeptide comprising the polypeptide according to any one of claims 7 to 9 having an angiogenesis inhibitory action.   A gene encoding a polypeptide having an anti-angiogenic activity according to claim 26.

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