JP2007204473A - Novel chondroitin sulfate proteoglycan, core protein thereof, dna encoding the same, and antibody against the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel proteoglycan in the brain and to provide an antibody for isolating the same. <P>SOLUTION: Provided are a chondroitin sulfate proteoglycan having an amino acid sequence identical with that part of a specified rat-derived amino acid sequence which covers from valine in position 1 to threonine in position 514 or having an amino acid sequence identical with that part of the specified amino acid sequence which covers from valine in position 31 to threonine in position 545, having bound chondroitin sulfate, having a molecular weight of about 150 kD, and existing in the brain; a DNA encoding the same; and an antibody that reacts with the proteoglycan. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel chondroitin sulfate proteoglycan, a core protein thereof, a DNA encoding the same, and an antibody thereto.

  Proteoglycan is a general term for compounds in which glycosaminoglycan is covalently bound to a protein (core protein). They are localized on the cell surface and in the extracellular space, and various on somatic cells by binding to chemotrophic factors, cell adhesion molecules, and extracellular matrix glycoproteins (Non-patent Documents 1 and 2). It is presumed to have a moderate adjustment effect.

  In addition, recent biochemical studies on mammalian brain proteins revealed that various proteoglycans are accurately regulated and expressed during brain development (Non-Patent Documents 3 and 4). Molecularly controlled expression of such proteoglycans is shown in glia fibers (Non-Patent Document 5), visual system (Non-Patent Document 6), and cerebral cortex barrel system (Non-Patent Documents 7-9). Is involved in the formation of some complex structures in the brain like. In vitro studies have also shown that nervous system proteoglycans are involved in neuronal morphogenesis, for example in axonal development. This proteoglycan is involved in both positive (Non-patent Documents 10 and 11) and negative (Non-patent Documents 12-15) effects on axonal growth in vitro.

  Regarding the function of nervous system proteoglycans, the primary structure of the core protein in other somatic cells has been elucidated, and various things have been elucidated accordingly. As a result of such research progress, it has been clarified that many proteoglycan core proteins have similar sequence motifs and can be grouped into several families. Neurocan (Non-patent document 16), brevican (Non-patent document 17), versican (Non-patent document 18) and BEHAB (Non-patent document 19) are members of the soluble chondroitin sulfate proteoglycan family. is there. These sequences contain a tandem repeat structure of the hyaluronic acid binding domain. N-syndecan (Non-Patent Document 20) is a member of the syndecan family, a heparan sulfate proteoglycan that contains many similar transmembrane and cytoplasmic domains. More recently, it has been clarified that phosphacan / 6B4 proteoglycan, a chondroitin sulfate proteoglycan, is an extracytoplasmic variant of the receptor-like protein tyrosine phosphatase, RPTPβ / ζ (Non-patent Documents 21 and 22). .

As described above, the primary structures of many core proteins have been recently reported. However, many proteoglycans remain unidentified in the brain. There are at least 16 core proteins in the membrane-bound fraction of rat brain (Non-Patent Document 4), but only the complete primary structure of only 3 types of membrane-bound proteoglycans has been reported. That is, NG2 (Non-patent Document 23), glypican (Non-patent Document 24), and cerebroglycan (Non-patent Document 25).
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  The discovery, purification, and identification of new proteoglycans will lead to progress in brain research, leading to the establishment of new diagnostic methods for brain diseases, and the development of new methods for treating and preventing brain diseases. In particular, the novel membrane-bound proteoglycan of the present invention is presumed to be involved in neuronal axon development and through these, to be involved in nervous system morphogenesis. The present inventors searched for a novel proteoglycan in the brain and found a novel proteoglycan. The object of the present invention is therefore to discover, purify and identify and provide new proteoglycans in the brain. Furthermore, the object of the present invention is to create an antibody against a membrane-bound proteoglycan specific to the brain as a tool for the discovery of such a novel proteoglycan, and provides such an antibody. There is. Furthermore, it is providing the DNA which codes the proteoglycan or protein of this invention.

  In order to solve the above problems, the present inventors first tried to isolate and identify a new proteoglycan. Next, an attempt was made to create an antibody against the isolated new proteoglycan. Using the obtained antibody, cDNA for the new proteoglycan was obtained, and after analyzing the entire gene structure, it was confirmed that the chondroitin sulfate proteoglycan was specific to the brain. As a result, the present invention has been completed.

  Since the novel chondroitin sulfate proteoglycan of the present invention is thought to contribute to the normal formation of neural networks, it can be used for the development of new diagnostic methods for neurological diseases and the prevention and treatment of neurological diseases.

Next, the present invention will be described in detail.
<1> Extraction and partial purification of chondroitin sulfate proteoglycan of the present invention A novel chondroitin sulfate proteoglycan can be extracted by the following method. A mammalian (eg, rat) brain is transformed into a proteolytic enzyme inhibitor (eg, 2 mM phenylmethylsulfonyl fluoride), a metal chelator (eg, 20 mM ethylenediaminetetraacetic acid (EDTA)) and a reducing agent (eg, 10 mM N-ethylmaleimide). Etc.) with a buffer solution (for example, phosphate buffered saline (PBS), etc.) using a homogenizer, etc., and the homogenate is subjected to high-speed centrifugation, etc. to obtain insoluble substances (repeat homogenization and high-speed centrifugation, etc. again). Also good). This insoluble substance is homogenized in a buffer solution containing a protease inhibitor and a surfactant (for example, Triton-based surfactant (Nonidet P-40 (trade name), etc.)), and then centrifuged to obtain a supernatant. As a result, a crude chondroitin sulfate proteoglycan fraction is obtained.

  From the obtained chondroitin sulfate proteoglycan crude fraction, partial purification of chondroitin sulfate proteoglycan can be performed. That is, the crude fraction is used as it is or after freeze-drying, etc., and then a surfactant (for example, Triton surfactant (Nonidet P-40 (trade name) etc.) etc.), metal chelating agent (eg EDTA etc.), reducing agent ( For example, N-ethylmaleimide etc.) and a buffer solution (urea buffer solution) containing a high concentration urea solution (eg 4M urea) containing a protease inhibitor (eg phenylmethylsulfonyl fluoride etc.) are dispersed. Then dialyze against the same urea buffer. After high-speed centrifugation (for example, 27,000 g, 30 minutes), the supernatant is mixed with an anion exchange resin (for example, DEAE Sephacel) equilibrated with urea buffer. The anion exchange resin is collected according to a conventional method, packed in a column, and chondroitin sulfate proteoglycan is eluted in a urea buffer solution by a salt concentration gradient (for example, a linear concentration gradient from 0.1 M to 0.7 M). The proteoglycan fraction (the fraction eluted at a concentration of 0.45 to 0.68 M NaCl) is collected and concentrated according to a conventional method (for example, ultrafiltration). Next, the gel sample is subjected to gel filtration chromatography using a gel filter agent such as Sepharose CL-4B (Pharmacia).

  The chondroitin sulfate proteoglycan fraction (Kav is 0.11 to 0.65) is collected and concentrated by a conventional method (for example, ultrafiltration or the like). Proteoglycans are precipitated from solution by the addition of aqueous potassium acetate (eg, about 1.3% (w / v) ethanol aqueous solution (eg, about 95% strength ethanol)) under ice cooling with potassium acetate (eg, 1% (W / V)), and then dried under reduced pressure.The dried product is dissolved in a buffer solution (guanidine buffer solution) containing an appropriate amount of guanidine hydrochloride (preferably 4M) and octyl. Hydrophobic chromatography is performed using sepharose, etc., and elution is performed with a concentration gradient of a surfactant (for example, Triton surfactant (Nonidet P-40 (trade name), etc.) etc.) Chondroitin sulfate proteoglycan fraction (for example, Nonidet P) In the case of -40, fractions of 0.3% to 0.6% nonidet P-40 concentration) were collected and The chondroitin sulfate proteoglycan thus obtained is centrifuged (for example, 150,000 × g) in a guanidine buffer by a cesium chloride (CsCl) density gradient centrifugation (for example, an initial density of 1.38 g / ml) according to a conventional method. The purified chondroitin sulfate proteoglycan can be obtained in this manner by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. A band of about 150 kilodaltons (kDa) is formed below.

  Further, since the amino acid sequence of the core protein of the chondroitin sulfate proteoglycan of the present invention has been clarified by the present invention, the chondroitin sulfate of the present invention can also be synthesized by synthesizing the core protein from the amino acid and covalently binding the sugar chain to the core protein. Proteoglycans can be obtained. In addition, since the base sequence of the DNA encoding the core protein of the chondroitin sulfate proteoglycan of the present invention has been clarified by the present invention, the core protein is expressed from this DNA, and the sugar chain is shared with the core protein obtained by the expression. The chondroitin sulfate proteoglycan of the present invention can also be obtained by binding.

<2> Purification of core protein Next, the core protein can be partially purified according to the method used for partial purification of tyrosine phosphatase, a proteoglycan type protein described by Maeda et al. (Non-patent Document 22). it can. That is, the nuclear removal fraction prepared from the whole mammalian brain is solubilized with 1% CHAPS or the like. The solubilized product is loaded onto a column of an anion exchange resin such as DEAE-resin (for example, DEAE Toyopearl) and eluted with a high-concentration sodium chloride (for example, about 0.6 M) solution by a conventional method. Subsequently, fractionation is performed by CsCl density gradient centrifugation. The obtained fraction can be digested with chondroitinase ABC and separated by SDS-PAGE (for example, 6% polyacrylamide etc.) to obtain the core protein. The core protein thus obtained forms a 120 kDa band by SDS-PAGE under reducing conditions. This core protein is recognized by a monoclonal antibody that recognizes a novel chondroitin sulfate proteoglycan described later using Western blot. Moreover, since the amino acid sequence of the core protein of the chondroitin sulfate proteoglycan of the present invention has been clarified by the present invention, the core protein can also be synthesized from the amino acid. Moreover, since the base sequence of DNA encoding the core protein of the chondroitin sulfate proteoglycan of the present invention has been clarified by the present invention, the core protein can be expressed from this DNA to obtain the core protein.

<3> Production of Antibody of the Present Invention The antibody of the present invention is an antibody that binds to either or both of the proteoglycan of the present invention and the protein of the present invention, and may be either polyclonal or monoclonal. The antibody of the present invention can be prepared according to a conventional method, for example, as described below, using the proteoglycan of the present invention produced as described above or a core protein thereof or a fusion protein of these and other proteins as an antigen. is there. The polyclonal antibody of the present invention can be obtained, for example, by immunizing an immunized animal such as mouse, rat, guinea pig, rabbit, goat or sheep with the above antigen, and collecting serum from these animals. When immunizing an animal to be immunized, it is desirable to use an adjuvant (adjuvant) in combination because it activates antibody-producing cells. The immunoglobulin fraction may be purified from the obtained antiserum by a conventional method.

  The monoclonal antibody of the present invention can be obtained, for example, as follows. That is, after the above antigen was administered intraperitoneally, subcutaneously, or footpad of an immunized animal such as mouse, rat, guinea pig, rabbit, goat, sheep, etc., the spleen or popliteal lymph node was removed and collected from these Cells and tumor cell lines, myeloma cells, are fused to establish hybridomas, the resulting hybridomas are continuously grown, and the resulting hybridomas continue to have specific antibodies to the proteoglycans of the present invention or the proteins of the present invention. Selective cell lines. By culturing the strain thus selected in a suitable medium, the antibody of the present invention which is monoclonal in the medium can be obtained. Alternatively, a large amount of the monoclonal antibody of the present invention can be produced by culturing the hybridoma in a living body such as the abdominal cavity of a mouse. As cells used for cell fusion, lymph node cells and lymphocytes in peripheral blood can be used in addition to spleen cells. The myeloma cell line is preferably derived from the same cell type as compared to that derived from a heterogeneous cell type, and a stable antibody-producing hybridoma can be obtained.

  Purification methods of the obtained polyclonal and monoclonal antibodies include salting out with sodium sulfate, ammonium sulfate, etc., low temperature alcohol precipitation, selective precipitation fractionation with polyethylene glycol or isoelectric point, electrophoresis, DEAE (diethylaminoethyl)- Derivatives, ion exchange chromatography using ion exchangers such as CM (carboxymethyl) -derivatives, affinity chromatography using protein A or protein G, hydroxyapatite chromatography, immunoadsorption chromatography with immobilized antigen, gel Examples thereof include a filtration method and an ultracentrifugation method. The antibody of the present invention may be a fragment containing Fab obtained by treatment with a protease that does not degrade the antigen binding site (Fab) (for example, plasmin, pepsin, papain, etc.). If the nucleotide sequence of the gene encoding the antibody of the present invention or the amino acid sequence of the antibody is determined, a fragment containing the Fab of the antibody of the present invention or a chimeric antibody (for example, including the Fab portion of the antibody of the present invention) is genetically engineered. Chimera antibodies, etc.) can be prepared, and such fragments and chimeric antibodies are also encompassed by the antibodies of the present invention.

  Although an example of the manufacturing method of the antibody of this invention is described below, it is not limited to this. The chondroitin sulfate proteoglycan of the present invention is administered to the peritoneal cavity of mice together with complete and incomplete Freund's adjuvant, for example, at 2-week intervals. After the fourth dose as a booster, the mice are sacrificed and the spleen cells are fused with myeloma cells using polyethylene glycol. After culturing the selected hybridoma in the medium, the hybridoma is screened by Western blotting using the culture supernatant in the presence or absence of an antibody that reacts with the chondroitin sulfate proteoglycan of the present invention. Positive hybridomas are cloned by limiting dilution. The established hybridoma cell line produced an antibody that reacts with chondroitin sulfate proteoglycan (molecular weight about 150 kDa) or core protein (about 120 kDa) by Western blot.

  Antibody production can, however, also be produced by other methods. That is, the antibody is a proteoglycan whose core protein is represented by an amino acid sequence having homology with the amino acid from the first valine (Val) to the 514th threonine (Thr) shown in SEQ ID NO: 2 in SEQ ID NO: 2; A non-human protein or peptide fragment thereof, which contains an amino acid sequence from the 252nd lysine (Lys) to the 398th cysteine (Cys) or an amino acid sequence having homology thereto, and may have a sugar chain An antibody that is immunized to a mammal and can be collected from a body fluid of the animal or produced by antibody-producing cells of the animal and reacts with the proteoglycan and / or the protein, such as Any antibody production method can be used as an antibody production method. The protein or fragment thereof recognized by the obtained antibody is localized in the brain. The antibody obtained by the above method may be further purified by a known method.

<4> Isolation of cDNA library and cDNA clone for chondroitin sulfate proteoglycan Prepare a cDNA library (Non-patent Document 22) to be used as a template and random primer using poly (A) ⁺ obtained from whole mammalian brain . Also, a poly (A) ⁺ obtained from the whole brain of a mammal is used to construct a cDNA library to be used as a template and oligo d (T) primer. These cDNA libraries can be screened immunologically using a monoclonal antibody that recognizes chondroitin sulfate proteoglycan obtained previously. Immunological screening can be performed as described in Sambrook et al. (Non-patent Document 29). By isolating positive clones by immunological screening and performing DNA sequencing or the like according to conventional methods, the DNA shown in SEQ ID NO: 1 can be obtained. Thus, a cDNA encoding a 514-residue amino acid encoding all of the 30-residue signal peptide and the core protein of the novel chondroitin sulfate proteoglycan could be obtained.

  The novel chondroitin sulfate proteoglycan found and identified in this way is localized in the brain and does not show homology with any other known proteoglycan, so the chondroitin sulfate proteoglycan of the present invention is a novel chondroitin sulfate proteoglycan ( Since these are considered to be involved in neuronal morphogenesis, they are expected to be used as diagnostic methods, therapeutic agents and preventive agents for nervous system diseases and the like. In addition, as an administration route in treatment or prevention, intravenous injection, arterial injection, intraventricular administration, genetic engineering administration means using a viral gene, or the like can be used.

<5> Proteoglycan of the Present Invention The proteoglycan of the present invention is a chondroitin sulfate proteoglycan comprising a core protein and a sugar chain having the following properties.
(A) Primary structure of the core protein: the amino acid sequence from the first valine (Val) to the 514th threonine (Thr) in SEQ ID NO: 2 in the sequence listing, or an amino acid sequence having homology thereto.
(B) Sugar chain: Contains chondroitin sulfate having a molecular weight of about 30 kilodaltons.
(C) When neuraminidase, O-glycosidase or N-glycosidase is allowed to act on the proteoglycan, sugar chains are released.

  Further, the chondroitin sulfate proteoglycan having a molecular weight of about 150 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions is also included in the present invention.

<6> Glycoprotein of the present invention Glycoprotein having the following properties is also included in the present invention.
(A) Molecular weight: (a) Forms a band of about 120 kilodaltons under reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) After enzymatic reaction of neuraminidase, O-glycosidase and N-glycosidase, dodecyl (B) Primary structure of protein that forms a band of about 100 kilodaltons under reducing conditions by sodium sulfate-polyacrylamide gel electrophoresis: first valine (Val) to 514 threonine shown in SEQ ID NO: 2 It has an amino acid sequence up to (Thr) or an amino acid sequence having homology thereto.

<7> Protein of the Present Invention The protein of the present invention comprises an amino acid sequence from the first valine (Val) to the 514th threonine (Thr) shown in SEQ ID NO. Proteins that may be included are also included in the present invention. Furthermore, since the amino acid sequence from the 252nd lysine (Lys) to the 398th cysteine (Cys) of SEQ ID NO: 2 is a characteristic of the proteoglycan and protein of the present invention, the 252nd of SEQ ID NO: 2 A protein that includes an amino acid sequence from lysine (Lys) to 398th cysteine (Cys) or an amino acid sequence having homology therewith and may have a sugar chain is also included in the present invention, and each of the above proteoglycans and proteins The DNA encoding is also included in the present invention.

  Such a protein includes, for example, an amino acid sequence from the -30th methionine (Met) to the 514th threonine (Thr) shown in SEQ ID NO: 2 in the sequence listing, or an amino acid sequence having homology thereto, and has a sugar chain. Proteins that may be included are included. In the present invention, homology in amino acid sequences means that a certain proportion of amino acid sequences are maintained in proteoglycans and proteins having the same function even if the biological species from which the proteoglycans and proteins of the present invention are derived are different. It means that it has. For example, in versican, which is a known chondroitin sulfate proteoglycan, the amino acid sequence of the active domain has 96% homology among the core protein of the proteoglycan derived from chicken and the proteoglycan derived from human. Moreover, it is known that the core protein of syndecan, which is a known membrane-bound proteoglycan, has a highly homologous portion in the amino acid sequence between mouse and human. Accordingly, the proteoglycans and proteins of the present invention are also expected to have about 60 to 90% or more amino acid sequence homology between species.

  Proteins deduced from the gene sequence of an example cDNA of the present invention include a signal peptide domain, a chondroitin sulfate binding domain (CS attachment), a cluster of basic amino acids, a cysteine-containing domain, a transmembrane domain, and a cytoplasmic domain. domain)) (FIG. 1). A novel chondroitin sulfate proteoglycan localized in the brain of the present invention was named neuroglycan C (hereinafter referred to as NGC). NGC mRNA is detected in Northern blot analysis as a single 3.1 kb mRNA in the brains of newborns and adult mammals, but not in any of kidney, liver, lung, or muscle. The core protein sequence did not show significant homology with any other known protein. This indicates that neuroglycan C (NGC) is a novel proteoglycan.

  Further, the present invention includes an amino acid sequence from the 31st valine (Val) to the 545th threonine (Thr) of SEQ ID NO: 4 in the sequence listing, or an amino acid sequence having homology thereto, and having a sugar chain. A good protein, an amino acid sequence from the 283rd lysine (Lys) to the 429th cysteine (Cys) in SEQ ID NO: 4 or an amino acid sequence having homology therewith, and a protein that may have a sugar chain Is included. Since these are human-derived proteins, they are particularly preferred as pharmaceuticals used for humans. Such a protein includes, for example, the amino acid sequence from the first methionine (Met) to the 545th threonine (Thr) shown in SEQ ID NO: 4 in the sequence listing, or an amino acid sequence having homology thereto, and has a sugar chain. Proteins that may be included are included.

  The protein of the present invention is not necessarily a single protein, and may be part of a fusion protein if necessary. A fusion protein can be produced by a known genetic engineering technique, and is usually expressed by expressing a DNA encoding the protein of the present invention from a recombinant DNA obtained by ligating the DNA encoding an arbitrary protein. Obtainable.

  In addition, the protein of the present invention has an amino acid sequence that includes amino acids that are common between the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: 4 in that order, and has a sugar chain. Also included are proteins that may be present. The amino acid that is not common between the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: 4 may be any amino acid. However, it is assumed that there is no amino acid in SEQ ID NO: 2 corresponding to the 282nd methionine (Met) of SEQ ID NO: 4.

<8> DNA of the present invention
The present invention includes the above proteoglycans, glycoproteins and DNAs encoding the proteins. For example, it encodes a protein that contains an amino acid sequence from the first valine (Val) to the 514th threonine (Thr) shown in SEQ ID NO: 2 or an amino acid sequence having homology thereto, and may have a sugar chain Specifically, the DNA having the nucleotide sequence to be exemplified is DNA containing the nucleotide sequence from the 103rd guanine (G) to the 1644th cytosine (C) in SEQ ID NO: 1 in the sequence listing. In addition, it encodes a protein that includes an amino acid sequence from the 252nd lysine (Lys) to the 398th cysteine (Cys) of Sequence Listing SEQ ID NO: 2 or an amino acid sequence having homology thereto, and may have a sugar chain Specific examples of the DNA to be performed include DNA containing the base sequence from the 856th adenine (A) to the 1296th cytosine (C) in SEQ ID NO: 1 in Sequence Listing. Such DNA includes DNA containing the base sequence from the 13th adenine (A) to the 1644th cytosine (C) shown in SEQ ID NO: 1 in the Sequence Listing.

  A base that encodes a protein that includes an amino acid sequence from the 31st valine (Val) to the 545th threonine (Thr) of the sequence listing SEQ ID NO: 4 or an amino acid sequence having homology thereto, and may have a sugar chain Specific examples of the DNA having a sequence include DNA containing the nucleotide sequence from 91st guanine (G) to 1635th cytosine (C) in SEQ ID NO: 3 in the Sequence Listing. A base that encodes a protein that includes an amino acid sequence from 283rd lysine (Lys) to 429th cysteine (Cys) in Sequence Listing SEQ ID NO: 4 or an amino acid sequence having homology thereto, and may have a sugar chain Specific examples of the DNA having a sequence include DNA containing a base sequence from the 847th adenine (A) to the 1287th cytosine (C) of SEQ ID NO: 3 in the sequence listing. Such DNA includes DNA containing the base sequence from the first adenine (A) to the 1635th cytosine (C) shown in SEQ ID NO: 3 in the sequence listing.

  It should be readily understood by those skilled in the art that DNAs of different base sequences due to the degeneracy of the genetic code are also included in the DNA of the present invention. Any of these DNAs are included in the DNA of the present invention. The present invention also includes DNA or RNA complementary to the DNA of the present invention. Further, the DNA of the present invention may be a single strand consisting only of the coding strand encoding the protein, or a double strand comprising this single strand and a DNA strand or RNA strand having a complementary sequence thereto. There may be. The DNA of the present invention also includes a DNA encoding an arbitrary protein and a recombinant DNA obtained by linking a functional DNA to the DNA of the present invention by a known genetic engineering technique. Note that the gene of the protein of the present invention derived from a chromosome is expected to contain an intron in the coding region, but even a DNA fragment fragmented by such an intron is not limited as long as it encodes the protein of the present invention. Included in the invention DNA. That is, the term “encode” in the present specification includes having a base sequence that can be processed at the time of transcription to finally produce a target protein.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

Extraction of membrane-bound chondroitin sulfate proteoglycan 10 day-old Sprague-Dawley rat brain (usually 100 rats per experiment; Slc: SD strain; manufactured by SLC Co.) as a protease inhibitor with 2 mM phenyl Homogenization was performed using a Teflon glass homogenizer in ice-cold phosphate-buffered saline (PBS) (2 ml / brain) containing methylsulfonyl fluoride, 20 mM EDTA and 10 mM N-ethylmaleimide. All homogenization and subsequent steps were performed at 4 ° C. unless otherwise indicated. The homogenate was centrifuged at 27,000 Xg for 40 minutes. The precipitate was rehomogenized with the same solution. After centrifugation, PBS-insoluble material was homogenized at 0 ° C. in PBS (2 ml / brain) containing the protease inhibitor and 1% Nonidet P-40. The homogenate was stirred with a magnetic stirrer for 60 minutes and then centrifuged at 27,000 Xg for 40 minutes. The supernatant was saved, and the precipitate was again treated with a surfactant-containing buffer and centrifuged. After centrifugation, these surfactant-containing supernatants were combined and lyophilized.

Partial purification of membrane-bound chondroitin sulfate proteoglycan The lyophilized residue obtained in Example 1 contains 0.2% nonidet P-40, 0.1 M NaCl, 2 mM EDTA, 1 mM N-ethylmaleimide and 0.2 mM phenylmethylsulfonyl fluoride. Dispersed in 4M urea, 50 mM Tris-HCl buffer, pH 7.5 (urea buffer) at a rate of 1 ml per brain, and this dispersion was dialyzed using the same buffer. After centrifugation at 27,000 Xg for 30 minutes, the supernatant was mixed with DEAE Sephacel resin (Pharmacia) equilibrated with urea buffer at a rate of 1 ml per brain, and the mixture was stirred with a magnetic stirrer for 2 hours. The DEAE Sephacel resin was recovered by centrifugation at 6,500 × g for 10 minutes and washed twice with urea buffer. The resin was packed in a glass column and the proteoglycan was eluted from the resin by a linear concentration gradient in urea buffer containing 0.1 M to 0.7 M NaCl.

  The proteoglycan fraction (eluted at a concentration of 0.45 to 0.68 M NaCl) was collected and concentrated to 5 ml using Diaflow YM-10 membrane (Amicon). The concentrated solution was applied to a Sepharose CL-4B (Pharmacia) column (diameter 1.6 cm × 100 cm) using 4M guanidine hydrochloride containing 0.2% nonidet P-40 and the protease inhibitor, and 50 mM Tris-HCl pH 7.5. And chromatographed. Proteoglycan fractions (Kav 0.11 to 0.65) were collected and concentrated to 3 ml using Diaflow YM-10 membrane. Proteoglycan was precipitated from the solution by adding 3 volumes of 95% ethanol containing 1.3% (W / V) potassium acetate. The precipitate was washed twice with 75% ethanol containing 1% (W / V) potassium acetate at 0 ° C. and dried over P 2 O 5 under reduced pressure. The obtained dried product was dissolved in 10 ml of 4M guanidine hydrochloride, 50 mM Tris hydrochloride, pH 7.5 (guanidine hydrochloride buffer) at room temperature. This solution was loaded onto a column (diameter 1.6 cm diameter × 5.0 cm) of octyl sepharose (Pharmacia). Elution was performed with a guanidine hydrochloride buffer containing nonidet P-40 from 0% to 0.8% (V / V) by a linear concentration gradient. Fractions eluted at 0.3% to 0.6% nonidet P-40 concentrations were collected, and the proteoglycans in the collected fractions were precipitated with ethanol and dried as described above.

  Further, the obtained proteoglycan was obtained at 150,000 Xg at 10 ° C using an RPS-65T rotor (manufactured by Hitachi) with a CsCl density gradient with an initial density of 1.38 g / ml (4M guanidine hydrochloride, 50 mM Tris hydrochloride, pH 7.5). Purified by ultracentrifugation for 30 hours. After centrifugation, the sample was collected in 7 fractions. Samples with fraction numbers 2 to 6 were collected and dialyzed against PBS at 4 ° C. The contents of hexuronate and protein in the proteoglycan solution were measured by Bitter and Muir method (Non-patent Document 26) and protein analysis kit (manufactured by Bio-Rad), respectively. By this purification method, proteoglycan having a hexuronic acid content of about 3 micromolar (protein content 2.2 mg) was obtained from 100 brains.

  Soluble proteoglycans were extracted from the brains of 10-day-old rats with PBS containing protease inhibitors by a known method (Non-patent Document 3) and purified. That is, proteoglycan was fractionated and purified by column chromatography using DEAE Sepharose and gel filtration using Sepharose CL-4B. Fractions with a Kav ranging from 0.45 to 0.60 were used for Western blot analysis.

Treatment of membrane-bound chondroitin sulfate proteoglycan with glycosidase To remove chondroitin sulfate side chain and heparan sulfate side chain from the core protein of proteoglycan, membrane-bound chondroitin sulfate proteoglycan (uronic acid content 50 nmol) is a chondro free of protease. Digested with itinase ABC (EC 4.2.2.4; manufactured by Seikagaku Corporation) and then heparitinase I (EC 4.2.2.8; manufactured by Seikagaku Corporation) Digested (see Non-Patent Document 8). The resulting protein (core glycoprotein) was subsequently added to neuraminidase 2mIU (EC) in 50 mM sodium acetate buffer, pH 5.0100 μl containing 2 mM EDTA, 1 mM N-ethylmaleimide, 0.2 mM phenylmethylsulfonyl fluoride and 0.07 mM pepstatin. 3.2.1.18; manufactured by Nacalai Tesque) at 37 ° C. for 1 hour. The enzymatic reaction was stopped by adding 300 μl of 95% ethanol containing 1.3% potassium acetate at 0 ° C. After 1 hour, core glycoprotein (hereinafter core glycoprotein) was precipitated by centrifugation. After denaturation of the core protein according to the instructions in the instructions, use 1 IU N-glycosidase F (EC 3.2.2.18; Boehringer Mannheim) or 1 mIU O-glycosidase (EC 3.2. 1.97; Boehringer Mannheim Co.) was used for digestion at 37 ° C. for 24 hours for deglycosylation.

Gel Electrophoresis and Western Blot Analysis Membrane-bound chondroitin sulfate proteoglycan was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 6% separation gel and 3% stacking gel under reducing conditions with dithiothrite. separated. Polyvinylidene difluoride (PVDF) membranes (Immobilon-P (trade name); Millipore) at 60 V overnight in 25 mM Tris, 192 mM glycine (pH 8.3) containing 20% (V / V) methanol at 4 ° C. Made by electrophoresis (transfer). In order to block non-specific binding of antibodies, the membrane was incubated in PBS containing 1% skim milk (DIFCO Laboratories). Next, this membrane was treated with the hybridoma culture supernatant (including mouse monoclonal antibody) of Example 5 described later at 4 ° C. overnight, and in PBS using a Vectastain ABC kit (manufactured by Vector Laboratory). Stained. In some experiments, the intensity of immunostaining was quantified using an image scanner (GT-6500; Epson) and a computer (LC520; Apple), application software, and NIH image program (public domain).

Production of monoclonal antibodies Various monoclonal antibodies were produced by known methods (see Non-Patent Document 27). That is, a preparation of membrane-bound chondroitin sulfate proteoglycan (50 μg protein / dose) was injected into the peritoneal cavity of BALB / c mice with complete and incomplete Freund's adjuvant at 2-week intervals. After administration as a booster for the fourth time, the mice were sacrificed, and the obtained spleen cells were fused with PAI myeloma cells using polyethylene glycol. Hybridomas selected in HAT medium were cultured in RPMI 1640 medium (Sigma) containing 10% fetal bovine serum. Cultured hybridomas were screened by Western blotting using the culture supernatant for the presence or absence of antibodies that react with proteoglycans. Positive hybridomas were cloned by limiting dilution. Monoclonal antibodies C1, C3, C5 and C15 obtained from the selected hybridomas reacted with the membrane-bound chondroitin sulfate proteoglycan of Example 2. All of these four antibodies were determined to be IgG1 using a mouse monoclonal antibody isotyping kit (Immunotype (trade name); manufactured by Sigma).

Purification of Core Glycoprotein Core glycoprotein was purified according to the method used for partial purification of tyrosine phosphatase, a proteoglycan type protein described by Maeda et al. (Non-patent Document 22). That is, the nucleus-removed fraction prepared from the whole brain of 8-day-old rats was solubilized with 1% CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid). The solubilized product was loaded onto a column of DEAE Toyopearl (manufactured by Tosoh Corporation), washed with 0.25 M NaCl, and then eluted with 0.6 M NaCl. In order to separate the proteoglycan from the eluate (negatively charged partially purified product obtained from the column), it was fractionated by CsCl density gradient ultracentrifugation. After centrifugation, the gradient was collected in 10 fractions. When these fractions were analyzed after digestion with chondroitinase ABC, proteins of 140 kDa and 120 kDa were detected as major components in the three lowest density fractions (see FIG. 2 in Non-Patent Document 22). Therefore, proteoglycans in these fractions were digested with chondroitinase ABC and the resulting core glycoprotein was separated by SDS-PAGE (6% polyacrylamide), which was transferred to a PVDF membrane. A 120 kDa core glycoprotein band was cut out with a scalpel, which was recognized by four monoclonal antibodies C1, C3, C5 and C15 in a Western blot.

  Amino acid sequence analysis of this purified 120 kDa core glycoprotein was performed. A 120 kDa core glycoprotein fragment was obtained by treatment with cyanogen bromide (CNBr) as described by Scott et al (28). The complete core glycoprotein and its fragments were separated by SDS-PAGE as described above. The protein and fragments thereof were transferred to a PVDF membrane at 10 ° C. for 3 hours at 4 ° C. at 0.3 A (ampere) in 10 mM CAPS (3- (cyclohexylamino) propanesulfonic acid) buffer (pH 11) containing 10% methanol. After staining with 0.5% Ponceau Red containing 1% acetic acid, protein bands were excised from the membrane and the amino acid sequence of the protein was determined with an automated protein sequencer (PPSQ-10; Shimadzu).

Isolation of cDNA library and cDNA clone λgt11 cDNA library constructed using poly (A) ⁺ RNA obtained from whole brain of 18-day-old (P18) Sprague-Dawley rats as a template and using random primers Was used. Another λgt11 cDNA library was constructed using oligo (dT) primers using poly (A) ⁺ RNA as a template from whole brains of 8-day-old (P8) sprag-Dawley rats. These cDNA libraries were immunologically screened for 1 × 10 ⁶ plaques using a mixture of monoclonal antibody C1, C3, C5 and C15 culture supernatants. Immunological screening was performed using the Vectastain ABC kit for immunodetection as described in Sambrook et al. (Non-Patent Document 29).

The first two clones (from random primer cDNA library) (from NGC1 and (from oligo-d (T) primer cDNA library) (from NGC3 were isolated by immunological screening) and subsequently cloned into ³² P Obtained by plaque hybridization using a labeled cDNA probe The cDNA probe for plaque hybridization and north blotting was labeled using the Megaprime DNA labeling system (Amersham International) (from gt11 clone). The cDNA insert was purified and subcloned into the pBluescript II plasmid vector (Stratogene Cloning System).

Sequencing of DNA Subcloning by restriction using restriction enzymes was performed for sequencing. DNA sequencing was performed using an automated DNA sequencer (AlfII / Alfred combined system; Pharmacia) using Taq polymerase and dye-labeled primers. Sequence editing and analysis was performed using GENETYX software (Software Development Corp.). The reading frame was supported by our data on the N-terminal amino acid sequence of the 120 kDa core glycoprotein and its fragments obtained by CNBr treatment. A comparison between the sequences in GeneBank (Release 87) and Swiss-Prot (Release 31) was performed using a partial sequence search tool (Non-patent Document 30).

Northern blot analysis Total RNA was extracted from the brains of 7-day-old and adult rats as described in Chomczynsky and Sacchi et al. Extracted from lung and muscle. Poly (A) ⁺ RNA was purified using Oligotex-dT30 (Takara Biochemical Co., Ltd.) according to the method procedure. Poly (A) ⁺ RNA (5 μg) was electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde and transferred (transferred) to a PVDF membrane (Immobilon-N, manufactured by Millipore). (The 682 base pair insert from NGC1 was labeled with ³² P-dCTP and used as a probe at a concentration of 3 × 10 ⁶ cpm / ml. This filter was finally washed with 0.2 × SSC (SSC: 0.15 NaCl and 0.015 M acetic acid. (Sodium) at 68 ° C. and exposed to X-ray film (Fuji Film) for autoradiography.

Characteristics of the monoclonal antibody The present inventors established four types of hybridoma cell lines (C1, C3, C5 and C15). Antibodies (monoclonal antibodies C1, C3, C5 and C15) were produced that each reacted with a membrane-bound chondroitin sulfate proteoglycan preparation in a Western blot. All four monoclonal antibodies (MAbs) recognized a very broad band corresponding to an average molecular weight of 150 kDa when the membrane-bound chondroitin sulfate proteoglycan preparation used as the antigen was tested (FIG. 3A lane 1, MAb C5). Immunoblots using When a membrane-bound chondroitin sulfate proteoglycan preparation was digested with chondroitinase ABC, a 120 kDa protein band was detected by immunoblot using MAb C5 (FIG. 3A lane 2 and FIG. 3B lane 1). This band did not migrate even when treated with heparitinase (Fig. 3A lane 3). When analyzed by Western blot using a large amount of PBS soluble proteoglycan preparation obtained from the brains of 10-day-old rats, 75 kDa core glycoprotein was weakly detected by MAb C5. MAb C5 did not react with any proteoglycan core glycoprotein prepared from other than the nervous system such as liver, lung, kidney, muscle and cartilage.

  To test the possibility that proteoglycans have oligosaccharide side chains in addition to chondroitin sulfate chains, the core glycoprotein was digested in turn with three glycosidases (neuramidase, O-glycosidase, N-glycosidase) as shown in FIG. 3B. The molecular weight of the deglycosylated core protein was measured by SDS-PAGE (FIG. 3B lanes 2, 3, and 4). Each treatment with glycosidase caused an increase in the mobility of the core protein band on SDS-PAGE. The ability of MAb C5 to recognize the core protein was not affected by digestion with any glycosidase. Therefore, the epitope recognized by MAb C5 appears to be in the polypeptide portion.

  Proteoglycan expression in rat cerebral cortex was examined by immunohistochemical staining with MAb C5. That is, the proteoglycan was associated with the developing neuron from the result of immunostaining with MAb C5 at 0 days after birth (FIG. 4A). A similar staining pattern was also seen in the cerebral cortex at 7 days of age (FIG. 4B). In the mature brain, the intensity of immunostaining in the cerebral cortex decreased. To quantify transient expression of proteoglycans in the cerebral cortex, PBS-insoluble extracts of cerebral cortex at various developmental stages (from 12 day embryo (E12) to maturation) were digested with chondroitinase ABC and immunoblotted And the intensity of the immunolabeled band was quantified. A small amount of proteoglycan was detected in 16-day embryos (E16) to 18-day embryos (E18). The amount of proteoglycan increased to reach the maximum level around 20 days after birth (P20) (FIG. 4C). After P20, the amount of proteoglycan decreased. And in the mature cerebral cortex, this proteoglycan was about half the maximum level of expression.

Isolation of cDNA clones The 120 kDa core glycoprotein was purified and digested into several peptides by treatment with CNBr, and the complete core glycoprotein and the partial N-terminal amino acid sequences of the two peptides were determined. The N-terminal sequence of the complete core glycoprotein was VPAREAGSAIEAEEL in single letter amino acid. Completely different N-terminal sequences were found in two products (15 kDa and 24 kDa) digested by CNBr. The 15 kDa and 24 kDa peptides were (M) VPGGSISLRRPPGDPGKDLA and (M) GRFPPGSP, respectively.

  One positive clone (λNGC1 from a gt11 cDNA library (random primer) derived from a rat brain of P18 by immunological screening with a mixture of four MAbs C1, C3, C5 and C15. ) Was isolated, and another positive clone (λNGC3) was first isolated from a rat brain of P8 (gt11 cDNA library (oligo-d (T) primer), λNGC1 and λNGC3 were 682 base pairs each. Each contains an insert cDNA of 1398 base pairs (FIG. 5) The fusion protein with β-galactosidase derived from λNGC1 reacted only with MAb C1, but not with MAbs C3, C5 and C15, in contrast to λNGC3. Fusion proteins derived from MAb C3, C5 and C15 ΛNGC1 and λNGC3 were subcloned into the pBluescript II plasmid vector (hereinafter referred to as pNGC1 and pNGC3, respectively) for further analysis, and the reliability of these clones was 120 kDa. The amino acid sequences of the core glycoprotein and the fragment obtained by CNBr treatment of 24 kDa match within the amino acid sequence deduced from the insert cDNA in pNGC1 (see the underlined part in FIG. 6), and obtained by CNBr treatment of 15 kDa. This was clearly demonstrated by the fact that the amino acid sequence of the obtained fragment matched within the amino acid sequence deduced from the insert cDNA in pNGC3 (see FIG. 6, underlined portion).

The insert cDNA in pNGC1 was labeled with [ ³² P] -dCTP and used as a probe to identify overlapping cDNA clones, derived from a rat brain of P18 (from a gt11 cDNA library (random primer)). Four types of cDNA clones (λNGC7, λNGC13, λNGC15, and λNGC19) obtained cDNA that contained the overlapping cDNA portion and encoded all of the core protein of proteoglycan, that is, proteoglycan, that is, neuroglycan C (NGC). The location of the different cDNA clones used for complete sequencing is indicated.

Predicted structure of NGC core protein FIG. 6 shows a cDNA sequence and a deduced amino acid sequence encoding all NGC core proteins. These clones cover more than 2,107 base pairs, including an open reading frame encoding 544 amino acids. The calculated molecular mass was 58,612 Da. The 3 ′ untranslated region consists of 463 nucleotides and contains a polyadenylation signal (AATAAA). In this coding region, two types of product sequences (underlined) by CNBr cleavage were identified. The N-terminal amino acid sequence of the 120 kDa core glycoprotein (sequence starting from amino acid residue 31 in FIG. 6 (underlined portion)) was determined. The nucleotide sequence near the ATG triplet starting from the 13th nucleotide corresponds to the sequence corresponding to the translation start site (Non-patent Document 32). The 12 base pairs of the 5 ′ region of the cDNA is a 5 ′ untranslated region. There is a hydrophobic amino acid sequence from the first methionine to the first amino acid residue (valine) of the mature core protein, which is probably the signal peptide sequence. From the analysis of whether the predicted protein amino acid sequence is hydrophobic or hydrophilic (Fig. 2) by the method of Kyte and Doolittle et al. (Non-patent Document 33), it consists of 24 amino acids following two basic residues ( A second hydrophobic sequence was revealed near the carboxyl terminus (amino acid residue numbers 426-450 in FIG. 6) (FIG. 6, dashed line). This sequence is consistent with the properties for the transmembrane domain proposed by Sabatini et al.

  NGC's core glycoprotein is rich in glycine (10.5%), leucine (9.9%), proline (9.0%), glutamic acid (8.1%), serine (7.9%) and threonine (7.7%) residues. The NGC pI obtained from the calculation is 4.9. A total of 10 cysteine residues were found in the core glycoprotein. All of these are located in the center of NGC and outside the cell from the transmembrane domain. The putative extracellular domain of NGC's core glycoprotein contains three N-glycosylation sites (Non-patent Document 35) and three serine-threonine clusters to which O-linked sugar chains can bind (FIG. 6). Amino acid residue numbers 143-144, 188-189, and 271-272) (Non-patent Document 36).

  The N-terminal region of amino acid number residues 31 to 281 in FIG. 6 contains eight serine-glycine (SG) or glycine-serine (GS) dipeptide sequences. These dipeptides are considered to be core-site sequences that match the binding site of chondroitin sulfate (Non-patent Documents 18 and 37). The amino acid sequences around these eight serine residues correspond to the glycosaminoglycan binding sites represented by SGXG (Non-patent Document 37) and (E / D) GSG (E / D) (Non-patent Document 18). It is different from the arrangement. However, these corresponding sequences are not the only sequences to which glycosaminoglycans can bind to the core protein. For example, in the neurocan, the sequence near the serine residue to which the chondroitin sulfate chain is bound is EEVASGQED (Non-patent Document 16). The amino acid sequence characteristic of the chondroitin sulfate binding site in proteoglycans such as aggrecan, versican, syndecan, decorin and type IX collagen has been reported to be preceded by SG / GS and acidic amino acid residues (FIG. 7). The importance of acidic amino acid residues in the binding of chondroitin sulfate is similarly described in other documents (Non-patent Documents 18, 37, 38). Sequences corresponding to these are arranged in the vicinity of the eight SG / GS-dipeptides of the NGC core protein. NGC contains two additional dipeptide sequences ((Serine residues 341 and 374 in FIG. 2) in the predicted extracellular domain, however, these dipeptides are sequences corresponding to the chondroitin sulfate binding site. Therefore, the N-terminal domain was the putative chondroitin sulfate binding domain, and the short sequence of basic amino acids KRRKRRRRRIR (amino acid residues 282 to 291 in FIG. 6) is in proximity to the temporary chondroitin sulfate binding domain. (A region consisting of 133 amino acids (amino acid residues 292 to 425 in FIG. 6) including 10 cysteine residues after a short basic amino acid sequence) Was found.

  The cytoplasmic domain of NGC contains serine and threonine residues that serve as sites that can be phosphorylated by protein kinases. The amino acid sequence near the threonine residue of amino acid residue number 465 in FIG. 6 (KLRRTNK) and the amino acid sequence near the serine residue of number 521 (SPK) are proposed by Graff et al (Non-patent Document 42). Is similar to the sequence corresponding to the phosphorylated site by protein kinase C (X (R / K) XX (T / S) X (R / K)). Although it has not been determined whether NGC is phosphorylated, NGC may be involved in signal conduction. The hypothetical cytoplasmic domain contains two tyrosine residues. However, the sequences connected to these tyrosine residues are different from the corresponding sequences reported for tyrosine phosphorylation (Non-patent Document 43). FIG. 8 is a diagrammatic representation of the proposed domain structure of the NGC core protein.

  By searching the database at the amino acid level, a small portion of NGC is similar to the core protein of aggrecan and chondroitin sulfate proteoglycan (Non-patent Document 44) of cartilage. All of these similar regions are distributed in the putative extracellular domain of NGC. In the putative chondroitin sulfate binding domain, 11 homologous clusters were identified: NGC (amino acid residues 45-94 in FIG. 2 and aggrecan residues 907-956 (32% identity / 48% chemical (Similarity); hereinafter, the residue numbers and their identities and similarities are shown as 26-70 and 967-1011 (29% / 56%); 53-103 and 955-1005 (22% / 47%); 88-113 and 1585-1610 (42% / 54%); 117-151 and 1649-1683 (31% / 40%); 43-92 and 1130-1179 (28% / 38%); 50-94 and 892 -936 (27% / 47%); 59-108 and 1233-1282 (22% / 44%); 122-163 and 1296-1337 (29% / 36%); 10 9-153 and 1705-1749 (31% / 40%); and 90-114 and 1108-1132 (32% / 56%) All of the homologous clusters in aggrecan are putative chondroitin sulfate binding site domains In the cysteine-containing domain of NGC, we found three homologous clusters: (amino acid residues 287-322, 1827 and 1862 in FIG. 2 (31% identity / 42% chemical similarity); 348-386 and 1898-1936 (23% / 38%); and 401-411 and 1998-2008 (55% / 55%), as well as the corresponding homology in aggrecan. All of the clusters in are also located in one of the cysteine-containing domains of aggrecan. An illustrative sequence of homologous sequences of aggrecan and NGC is shown in Figure 9. Aggrecan consists of several structurally distinct domains: an immunoglobulin-like domain and hyaluronic acid in the N-terminal site. It consists of two repeats of the binding region, a central chondroitin sulfate binding site domain, an EGF-like domain, a lectin-like domain, and a complement regulatory protein-like domain at the carboxy terminal site (Non-patent Document 44) (see FIG. 9). Some other chondroitin sulfate proteoglycans, such as versican (Non-patent document 18), neurocan (Non-patent document 16), brevican (Non-patent document 17) and BEHAB (Non-patent document 19) contain these domains as well. Therefore, they are all considered to be members of the aggrecan family. . NGC is not a member of this family because the NGC core protein does not have any of these domains except the chondroitin sulfate binding domain (FIG. 9).

Northern blot analysis The tissue distribution of NGC mRNA was examined by Northern blot analysis (Figure 10), and a single 3.1 kb transcript was detected by analysis of 7 day old and adult rat brains. Here, the difference in length between the NGC coding region and the NGC mRNA is attributed to the presence of a 5 ′ untranslated region and a 3 ′ uncoded region (FIG. 5, arrow) in the mRNA. This transcript was not detected by analysis of kidney, liver, lung and muscle.

Cloning of human-derived NGC
^{A 32} P-labeled cDNA probe was prepared based on a rat NGC DNA fragment (restriction enzyme XbaI-Hind III digested DNA fragment: base numbers 624 to 1735 in Sequence Listing 1). Using this as a probe, a clone having a highly homologous base sequence was isolated by plaque hybridization from a commercially available cDNA library (Stratagene Cloning System) produced from the temporal cortex of a 2-year-old healthy woman. Escherichia coli XL-1 blue MRF '(Stratagene Cloning System) was used as a plaque hybridization host. Hybridization was performed at 60 ° C. in 3 × SSC (SSC: 0.15 M NaCl, 0.015 M sodium acetate), and washing was performed at 60 ° C. and 0.1 × SSC. The isolated DNA clone was subcloned into pBluescriptII (Stratagene Cloning System).

  The DNA base sequence was determined in the same manner as in Example 8. The cDNA sequence encoding the entire NGC core protein and the deduced amino acid sequence are shown in SEQ ID NO: 2. These clones cover 1740 base pairs or more, including an open reading frame encoding 545 amino acids. The calculated molecular weight of the protein was 58538.89 (excluding three undefined amino acid residues). The homology with the rat NGC gene was 83.6%. In addition, the amino acid sequence deduced from the base sequence of DNA encoding human-derived NGC is also shown in SEQ ID NO: 2. The homology with rat NGC was 83.9%.

The schematic diagram of the chondroitin sulfate proteoglycan of the present invention is shown. It consists of CS-attachment domain, basic amino acid cluster domain, cysteine-containing domain, transmembrane domain and cytoplasmic amino acid sequence domain. The hydrophobicity and hydrophilicity analysis result of the amino acid sequence of the chondroitin sulfate proteoglycan of the present invention is shown. The abscissa indicates the amino acid residue number shown in FIG. 6, and the ordinate indicates the hydrophobicity and the positive indicates the hydrophilicity. The electrophoretic diagram on the polyacrylamide gel of the membrane bound type chondroitin sulfate proteoglycan partially purified from the 10-day-old rat is shown. The expression pattern of NGC in a rat cerebral cortex is shown. (A) Immunostained anterior jaw section of cerebral cortex of 0-day-old rat with MAb C5. (B) A 7-day-old rat cerebral cortex anterior jaw section immunostained with MAb C5. (C) Change in the relative generation amount of the core glycoprotein of the proteoglycan of the present invention. A clone encoding rat NGC of the present invention is shown. 2 shows the cDNA sequence and deduced amino acid sequence of rat NGC of the present invention. A single letter notation was used for amino acids. The amino acid sequences of the glycosaminoglycan binding or binding sites of rat NGC, versican, aggrecan, neurocan, syndecan, decorin and type IX collagen of the present invention are shown. The schematic diagram of the structure of NGC core protein is shown. The comparison between the amino acid sequence of rat NGC and the amino acid sequence of rat aggrecan (Non-patent Document 61) is shown. The central sites of similar clusters of NGC and rat aggrecan were connected with a line. CS, CS1, CS2 and CS3 are chondroitin sulfate binding domains, IG is an immunoglobulin-like domain, H1 and H2 are hyaluronic acid binding domains, KS is a keratan sulfate binding domain, and G (ELC) is an EGF-like domain. , A globular domain including a lectin-like domain and a complement regulatory protein-like domain, G a globular domain, T a transmembrane domain, and CP a cytoplasmic domain. The number of amino acid residues is shown on a scale. Northern blots of NGC mRNA from 7 day old rat brain (B7), adult rat brain (Ba), adult rat kidney (K), liver (Li), lung (Lu) and muscle (M) are shown.

Claims (5)

A chondroitin sulfate proteoglycan produced by the following method and having the following properties can be immunized to a mammal other than a human and collected from the body fluid of the animal, or produced by antibody-producing cells of the animal. An antibody having the following properties:
(Proteoglycan production method)
Step 1: Rat brain is homogenized in phosphate buffered saline containing 2 mM phenylmethylsulfonyl fluoride, 20 mM EDTA and 10 mM N-ethylmaleimide, and the homogenate is centrifuged at 27,000 × g for 40 minutes to collect the precipitate. (This step and the following steps are all performed at 4 ° C. unless otherwise specified.)
Step 2: The precipitate collected in Step 1 is homogenized again in the same manner as in Step 1 and centrifuged to collect insoluble substances.
Step 3: The insoluble material recovered in Step 2 is homogenized at 0 ° C. in phosphate buffered saline containing 2 mM phenylmethylsulfonyl fluoride and 1% nonidet P-40 to recover the homogenate.
Step 4: Stir the homogenate recovered in Step 3 for 60 minutes, then centrifuge it at 27,000 xg for 40 minutes to recover the supernatant and freeze-dry it.
Step 5: The lyophilized product obtained in Step 4 was mixed with 4M urea, 50 mM Tris-HCl buffer containing 0.2% nonidet P-40, 0.1 M NaCl, 2 mM EDTA, 1 mM N-ethylmaleimide and 0.2 mM phenylmethylsulfonyl fluoride. Disperse in solution (pH 7.5) and then dialyze against the same buffer.
Step 6: The solution after dialysis in Step 5 is centrifuged at 27,000 × g for 30 minutes, and the supernatant is collected.
Step 7: The supernatant collected in Step 6 is mixed with DEAE-resin equilibrated with urea buffer and the mixture is stirred for 2 hours.
Step 8: The DEAE-resin after stirring in Step 7 is recovered and washed with urea buffer.
Step 9: The washed DEAE-resin in Step 8 is packed into a column and then subjected to a linear NaCl gradient (0.1 M to 0.7 M) in urea buffer and eluted at a NaCl concentration of 0.45 to 0.68 M. Collect the minutes.
Step 10: The fraction collected in Step 9 is concentrated, and this concentrate is added to 4M guanidine hydrochloride, 50 mM Tris-HCl buffer (pH 7.5) containing 0.2% nonidet P-40 and 2 mM phenylmethylsulfonyl fluoride. Chromatography is performed on a Sepharose CL-4B column (diameter 1.6 cm × 100 cm) using the above, and a fraction having a Kav in the range of 0.11 to 0.65 is collected.
Step 11: Concentrate the fraction collected in Step 10.
Step 12: To the concentrate obtained in Step 11, 3 volumes of 95% ethanol containing 1.3% (W / V) potassium acetate is added to form a precipitate.
Step 13: The precipitate formed in Step 12 is washed with 75% ethanol containing 1% (W / V) potassium acetate at 0 ° C. and dried under reduced pressure.
Step 14: The dried product obtained in Step 13 is dissolved in guanidine hydrochloride buffer (4M guanidine hydrochloride, 50 mM Tris hydrochloride, pH 7.5) at room temperature.
Step 15: The solution obtained in Step 14 was loaded onto an octyl sepharose column (diameter 1.6 cm × 5.0 cm), and a linear concentration gradient (0% to 0.8% (V / V) of nonidet P-40 in guanidine hydrochloride buffer solution. V)) and collect the fraction eluted at a nonidet P-40 concentration of 0.3% to 0.6% (v / v).
Step 16: Ethanol is added to the fraction collected in Step 15 to form a precipitate, and this precipitate is dried under reduced pressure.
Step 17: The dried product obtained in Step 16 was subjected to cesium chloride density gradient centrifugation (initial density 1.38 g / ml; 10 ° C .; 150,000) in guanidine hydrochloride buffer (4 M guanidine hydrochloride, 50 mM Tris hydrochloride, pH 7.5). xg for 30 hours).
Step 18: The sample that has undergone the processing in Step 17 is fractionated into seven, and the second to sixth fractions are collected.
Step 19: The fraction collected in Step 18 is dialyzed against phosphate buffered saline to obtain a chondroitin sulfate proteoglycan fraction.
(Proteoglycan properties)
(A) Sugar chain: Contains chondroitin sulfate having a molecular weight of about 30 kilodaltons.
(B) When the neuraminidase, O-glycosidase or N-glycosidase is allowed to act on the proteoglycan, the sugar chain is released.
(C) The molecular weight is 150 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions.
(The nature of the antibody)
(1) It binds to the above proteoglycan.
(2) It binds to the product obtained by digesting the above proteoglycan with chondroitinase ABC.
(3) The product obtained by (2) above is further bound to the product obtained by digesting with heparitinase I.
(4) The product obtained in (3) above is further bound to the product obtained by digestion with neuraminidase.
(5) The product obtained by (4) above is further bound to a product obtained by digesting with any of N-glycosidase and O-glycosidase enzymes.
(6) An epitope is present in the polypeptide portion of the above proteoglycan. The antibody according to claim 1, which is a monoclonal antibody. The antibody according to claim 1 or 2, which is derived from a mouse and has an isotype of IgG1. A method for immunohistochemical staining of cerebral cortex, wherein the antibody according to any one of claims 1 to 3 is used. A method for detecting maturity of a cerebral cortex, comprising a step of detecting proteoglycans present in the cerebral cortex with the antibody according to any one of claims 1 to 3.

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