JP4514708B2 - Identification and utilization of chondroitin polymerization factor - Google Patents

Description

  The present invention relates to identification of a polymerization factor (polymerization promoting factor) that promotes a polymerization reaction of a chondroitin chain by a chondroitin synthase, a method for synthesizing a chondroitin sugar chain using the same factor, and the like.

Chondroitin sulfate is a type of sulfated glycosaminoglycan that is widely distributed on the cell surface and extracellular matrix. It is a cell-cell interaction, cell recognition mechanism in immunity, specific homing phenomenon of lymphocytes, cancer cell adhesion and It is considered to be involved in various biological phenomena such as metastasis, and is widely used in pharmaceuticals, cosmetics, foods, etc. due to its various physiological functions. Also, chondroitin sulfate, like heparan sulfate, has been revealed from recent studies to play an important role in the formation of neural networks in the brain in vertebrates and the like.
Chondroitin sulfate biosynthesis begins with the transfer of xylose (Xyl) to a specific serine (Ser) residue of the core protein, followed by two galactose (Gal) and glucuronic acid (GlcA) in monosaccharide units. The tetrasaccharide binding region (GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser) is synthesized. Thereafter, N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) are alternately and repeatedly transferred to the binding region, whereby a polymerization reaction of chondroitin chains occurs, and a disaccharide repeating region of chondroitin sulfate is formed. Then, the constituent sugars are sulfated by sulfate group transferase to become chondroitin sulfate.
As a conventional method for obtaining chondroitin sulfate, a method of preparing from a natural product such as whale cartilage has been known. However, the amount of chondroitin sulfate that can be prepared from whale cartilage or the like is limited. In recent years, whales are restricted from whaling from the viewpoint of animal welfare, and it is difficult to procure whales themselves.
Accordingly, development of a technique for synthesizing chondroitin sugar chains and development of a method for producing chondroitin sulfate by sulfating the obtained chondroitin sugar chains are desired. There are following reports on the synthesis of chondroitin sugar chains. That is, α-thrombomodulin, which is a part-time proteoglycan in which the growth of a sugar chain is stopped in a tetrasaccharide-binding region (GlcAβ1-3Galβ1-3Galβ1-4Xyl) using the culture supernatant of human malignant melanoma G361 cells as an enzyme source. When α-TM) is used as a sugar acceptor substrate, GalNAc and GlcA disaccharides are alternately and repeatedly polymerized, and the polymerized sugar chain has been reported to extend to the same length as natural chondroitin. (Nadanaka, S., Kitagawa, H., Goto, F., Tamura, J., Neumann, K.W., Ogawa, T., and Sugawara, K. (1999) Biochem. J. 340, 353-. 357).
On the other hand, cDNA cloning of enzymes involved in the polymerization of chondroitin was delayed, but recently, N-acetylgalactosamine transferase-II (GalNAcT-II) and glucuronic acid transfer involved in the synthesis of the disaccharide repeat region of chondroitin chain The cDNA of chondroitin synthase (ChSy) having both activities of enzyme-II (GlcAT-II) has been cloned (Kitagawa, H., Uyama, T., and Sugahara, K. (2001) J. Biol. Chem. 276, 38721-38726). However, ChSy expressed as a recombinant protein in vitro showed activity to transfer GalNAc and GlcA, respectively, but could not polymerize chondroitin chains on α-TM, and culture of the above G361 cells. The polymerization reaction of chondroitin chains detected in the supernatant could not be reproduced.

Based on the above findings, the present inventor predicted that there may be an unidentified protein factor that is essential for polymerizing chondroitin chains in cooperation with ChSy, and searches for the same molecule. It was.
An object of the present invention is to identify a novel molecule involved in polymerization of a chondroitin chain, and to provide a method for synthesizing a chondroitin sugar chain using the same molecule, a method for producing chondroitin sulfate, and the like.
Most recently, five glycosyltransferases, including ChSy, have been identified as glycosyltransferases involved in the biosynthesis of chondroitin sulfate. Since these all show high homology with ChSy, it was considered that factors involved in the polymerization of the chondroitin chain also show homology with ChSy, and the database was searched based on the amino acid sequence of ChSy. As a result of further analysis based on the obtained data, the existence of a novel human gene consisting of 775 amino acids and showing 23% homology with human ChSy at the amino acid level was identified. Furthermore, as a result of functional analysis, it was found that this gene product has an action of promoting the polymerization of chondroitin chains in vitro in cooperation with ChSy, and the present invention has been completed.
That is, the present invention includes the following inventions A) to P) as industrially useful methods and products.
A) A polynucleotide encoding a protein comprising the amino acid sequence of any one of (a) to (d) below.
(A) the amino acid sequence shown in SEQ ID NO: 2,
(B) the amino acid sequence shown in SEQ ID NO: 4,
(C) an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 or 4,
(D) An amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in any one of the amino acid sequences of (a) to (c) above.
B) The polynucleotide according to A), which has the base sequence represented by SEQ ID NO: 1 or 3.
C) A recombinant expression vector comprising the polynucleotide described in A) or B) above. For the production of the recombinant expression vector, a plasmid, phage, cosmid or the like can be used, but it is not particularly limited.
D) A transformant in which the polynucleotide described in A) or B) is introduced. Here, “the polynucleotide has been introduced” means that the polynucleotide (gene) is introduced into the target cell (host cell) so as to be expressed by a known genetic engineering technique (gene manipulation technique). To do. The above-mentioned “transformant” means not only cells / tissues / organs but also animals (transformed animals such as nematodes, Drosophila melanogaster, mice, rats, rabbits, pigs, monkeys).
E) A protein comprising the amino acid sequence of any one of (a) to (d) below.
(A) the amino acid sequence shown in SEQ ID NO: 2,
(B) the amino acid sequence shown in SEQ ID NO: 4,
(C) an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 or 4,
(D) An amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in any one of the amino acid sequences of (a) to (c) above.
F) A chondroitin polymerization accelerator comprising the protein described in E) above.
G) A complex obtained by co-expressing the protein described in E) above and chondroitin synthase.
H) An antibody against the protein described in E) above. The antibody can be obtained as a polyclonal antibody or a monoclonal antibody by a known method using the full-length protein or a partial peptide thereof as an antigen.
I) A method for promoting polymerization of chondroitin chains using the protein described in E) above.
J) A chondroitin chain polymerization method as described in I) above, wherein a complex obtained by co-expressing the protein as described in E) and chondroitin synthase is used.
K) The chondroitin chain polymerization method as described in I) or J) above, wherein a chondroitin chain is synthesized on a core protein such as α-thrombomodulin to which a tetrasaccharide is bound.
L) A chondroitin chain produced using the method according to any one of I) to K) above.
M) Chondroitin sulfate produced using the method according to any one of I) to K) above.
N) Dermatan sulfate produced using the method according to any one of I) to K) above.
O) A method for determining the presence or absence of a genetic disease by detecting a mutation on a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2. As described later, this protein is a human-derived chondroitin polymerization factor (ChPF) that plays an important role in the polymerization of chondroitin chains in cooperation with ChSy, and its mutation causes an abnormality in the biosynthesis of chondroitin sulfate, It may be the cause of the disease. About such a disease, the presence or absence of the genetic disease can be determined by detecting a mutation on the ChPF gene.
P) A knockout non-human animal obtained by modifying all or part of the chondroitin polymerization factor (ChPF) gene sequence in the genome, wherein one or both of the ChPF genes on the homologous chromosome are disrupted .
Other objects, features, and advantages of the present invention will be fully understood from the following description. The benefits of the present invention will become apparent from the following description with reference to the accompanying drawings.

FIG. 1 is a diagram showing the sequence and structure of human-derived chondroitin polymerization factor (ChPF) according to the present invention in comparison with human-derived chondroitin synthase (ChSy) and human-derived chondroitin glucuronyltransferase (Chandroitin GlcAT). It is. In the figure, the box indicates the transmembrane domain, and the putative N-glycosylation site of ChPF is indicated by *.
FIG. 2 is a diagram showing the genome structure of the human-derived chondroitin polymerization factor (ChPF) in comparison with chondroitin glucuronyltransferase (Chondroitin GlcAT). In the figure, boxes represent exons, bars represent introns, white boxes represent untranslated regions, and black boxes represent translated regions. ATG represents a start codon and TGA • TAG represents a stop codon.
3A and 3B show experimental results of conducting a polymerization reaction using a binding domain oligosaccharide, an artificial substrate and α-thrombomodulin (α-TM) as a receptor substrate, and comparing the sizes of the obtained chondroitin chains. It is a graph.
4A to 4C are graphs showing experimental results of conducting a polymerization reaction using (GlcAβ1-3GalNAc) ₃ or a chondroitin polymer as a receptor substrate and comparing the sizes of the obtained chondroitin chains.
5A and 5B are graphs showing the analysis results of the hexasaccharide in the sugar-protein binding region derived from the chondroitin sugar chain synthesized on α-TM.
FIG. 6 is a diagram showing the results of an interaction analysis between ChPF and ChSy using the pull-down method.
FIG. 7 shows the results of Northern blot analysis of ChPF and ChSy.

〔実施例４：様々な糖受容体基質を用いた重合化反応〕
前述のように、組換え型タンパク質として発現させたＣｈＳｙはＧａｌＮＡｃとＧｌｃＡのそれぞれを単糖で転移させるＧａｌＮＡｃＴ−ＩＩおよびＧｌｃＡＴ−ＩＩ両活性を保持しているにもかかわらず、α−ＴＭ上にＣｈｎ鎖を重合させる活性は保持していなかった。
そこで、ＣｈＰＦとＣｈＳｙを共発現させたものに糖転移活性の上昇が見られたことより、ＣｈＰＦとＣｈＳｙを共発現させたものがＣｈｎ鎖を重合させる活性を保持しているのではないかと考えた。この点について解析するため、ＣｈＳｙとＣｈＰＦを共発現させた組換え型タンパク質を酵素源として、α−ＴＭ、ＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−Ｓｅｒ、四糖結合領域にα−ＴＭ由来のペプチドが結合したＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−（Ｇｌｙ）Ｓｅｒ−Ｇｌｙ−Ｇｌｕ、四糖結合領域にβ−ｇｌｙｃａｎとよばれるプロテオグリカン（ＰＧ）由来のペプチドが結合したＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−Ｓｅｒ−Ｇｌｙ−Ｔｒｐ−Ｐｒｏ−Ａｓｐ−Ｇｌｙおよび人工基質であるＧｌｃＡβ１−３Ｇａｌβ１−Ｏ−Ｃ_２Ｈ_４ＮＨ−ｂｅｎｚｙｌｏｘｙｃａｒｂｏｎｙｌを糖受容体基質として用い、重合活性を測定した。その結果が図３のグラフＡ、Ｂに示される。
図３の説明：Ａ．Ｃｈｎ鎖の重合反応の基質としてＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−Ｓｅｒ（●）、ＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−（Ｇｌｙ）Ｓｅｒ−Ｇｌｙ−Ｇｌｕ（×）、ＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−Ｓｅｒ−Ｇｌｙ−Ｔｒｐ−Ｐｒｏ−Ａｓｐ−Ｇｌｙ（△）、ＧｌｃＡβ１−３Ｇａｌβ１−Ｏ−Ｃ_２Ｈ_４ＮＨ−ｂｅｎｚｙｌｏｘｙｃａｒｂｏｎｙｌ（◇）、α−ＴＭ（○）を用いた。［^３Ｈ］標識重合反応生成物をＳｕｐｅｒｄｅｘ ｐｅｐｔｉｄｅカラムを用いてゲルろ過クロマトグラフィーを行うことにより分析した。α−ＴＭを受容体基質に用いた場合には、［^３Ｈ］標識重合反応生成物をアルカリ還元処理した後、分析に用いた。矢印はＣｈｎ由来八糖〜十糖の溶出位置を示す。Ｖｔはフラクション（Ｆｒ．）６０付近である。Ｂ．重合反応によりα−ＴＭ上で合成された糖鎖（○）をＳｕｐｅｒｄｅｘ ７５カラム（１．６×６０ｃｍ）を用いて分析した。ゲルろ過クロマトグラフィー後の溶出液の放射活性を測定した。分子量スタンダードとしてＣｈｎポリマーについても分析し、溶出液の２１０ｎｍにおける紫外吸収を測定した（●）。
図３に示すように、用いた様々な基質上にＣｈｎ鎖の重合が起ったが、α−ＴＭ上には最も効率よく、より長くＣｈｎ鎖が伸長した。
また、既に糖鎖が伸長したＣｈｎ鎖にもＣｈＳｙとＣｈＰＦを共発現させた融合タンパク質により、Ｃｈｎ鎖が伸長されるかを確かめるため、Ｃｈｎ由来の六糖（ＧｌｃＡβ１−３ＧａｌＮＡｃ）_３およびＣｈｎポリマーを受容体基質として、Ｃｈｎ鎖の重合反応を行った。その結果が図４のグラフＡ〜Ｃに示される。
図４の説明：Ａ．Ｃｈｎ鎖の重合反応の受容体基質としてＣｈｎ由来のオリゴ糖（ＧｌｃＡβ１−３ＧａｌＮＡｃ）_３を用いた。［^３Ｈ］標識重合反応生成物をＳｕｐｅｒｄｅｘ ｐｅｐｔｉｄｅカラムを用いてゲルろ過クロマトグラフィーにより分離し、溶出液の放射活性を測定した（●）。矢印はＣｈｎ由来八糖〜十糖の溶出位置を示す。ＶｔはＦｒ．６０付近である。Ｂ．Ｃｈｎポリマーを受容体基質としてＣｈＰＦとＣｈＳｙを共発現させたもの（○）およびＣｈＳｙ単独のもの（●）を酵素源に用い重合反応を行い、［^３Ｈ］重合反応生成産物をゲルろ過クロマトグラフィーにより単離した。単離した［^３Ｈ］標識重合反応生成物をＳｕｐｅｒｄｅｘ ２００カラム（１．６×６０ｃｍ）を用いたゲルろ過クロマトグラフィーを行い、溶出液の放射活性を測定した。矢印は分子量マーカーとして用いたデキストランにより求めたそれぞれの分子量における溶出位置を示している。Ｃ．上記Ｂで単離した反応生成物をＣＳａｓｅＡＣＩＩ消化し、消化後Ｓｕｐｅｒｄｅｘ Ｐｅｐｔｉｄｅカラムを用いてゲルろ過クロマトグラフィーを行うことによって分離し、液体シンチレーションカウンターで測定した。ＣｈＰＦとＣｈＳｙを共発現させたものを酵素源に用いたときの反応生成物のＣＳａｓｅ消化後（○）およびＣｈＳｙ単独で発現させたものを酵素源に用いたときの反応生成物のＣＳａｓｅ消化後（●）のクロマトグラムを示す。矢印はＣｈｎ由来の二糖およびＧａｌＮＡｃの溶出位置を示す。
図４に示すように、Ｃｈｎ由来の六糖（ＧｌｃＡβ１−３ＧａｌＮＡｃ）_３を受容体基質として使用した場合は八〜十糖の位置にピークが検出されたことより、二糖〜四糖程度の糖鎖の伸長が起ったと予想された（同図Ａ）。Ｃｈｎポリマーを受容体基質とした場合、伸長した糖鎖の長さは、ｃｏｎｔｒｏｌとしてＣｈＳｙのみを酵素源として得られた生成物と比べて、ゲルろ過クロマトグラフィーによる分析で大きな差異は見られなかった（同図Ｂ）。しかし、ＣＳａｓｅＡＣＩＩ消化の結果、ＣｈＳｙを用いて調製した反応生成物からは単糖の位置のみに放射活性のあるピークが検出されたのに対して、ＣｈＰＦとＣｈＳｙを共発現させたものを酵素源に用いた場合の反応生成物からは、二糖の位置にも顕著なピークが検出された（同図Ｃ）。このことより、ＣｈＳｙ単独時よりも、Ｃｈｎポリマー基質上に少なくとも数糖程度の糖鎖の伸長反応が起ったことが明らかになった。
〔実施例５：α−ＴＭの四糖結合領域に重合したＣｈｎ鎖の分析〕
次に、α−ＴＭの四糖結合領域をプライマーとしてＣｈｎ鎖の伸長反応が起ったかを確認した。その結果が図５のグラフＡ、Ｂに示される。
図５の説明：Ａ．α−ＴＭを受容体基質として重合反応を行い、［^３Ｈ］標識重合反応生成物をゲルろ過により単離した。単離した［^３Ｈ］標識重合反応生成物をアルカリ処理した後、２−ａｍｉｎｏｂｅｎｚａｍｉｄｅ（ＡＢ）により蛍光標識した。蛍光標識した糖鎖をＣＳａｓｅＡＢＣで完全に消化し、不飽和二糖標準品としてΔＤｉ−０Ｓを添加し、Ｓｕｐｅｒｄｅｘ ｐｅｐｔｉｄｅカラムを用いたゲルろ過クロマトグラフィーにより分画し、溶出液の放射活性（●）を測定した。矢印は不飽和二糖（ΔＨｅｘ α １−３ＧａｌＮＡｃ）の溶出位置を示す。Ｂ．結合領域を含む放射標識された画分（グラフＡのＦｒ．１）を回収し、陰イオン交換カラムを用いたＨＰＬＣで分析した。Ｆｒ．１のｃｈｏｎｄｒｏ−ｇｌｙｃｕｒｏｎｉｄａｓｅによる消化前（○）および消化後（●）のクロマトグラムを示す。溶出液の放射活性を測定した。矢印は２−ＡＢで標識された結合領域五糖と六糖の溶出位置を示す。波線はＮａＨ_２ＰＯ_４の濃度勾配を示す。
図５Ａに示すように、放射標識された糖鎖がＣＳａｓｅＡＢＣにより消化され、結合領域を含むＦｒ．１および二糖を含むＦｒ．２を得た。Ｆｒ．１とＦｒ．２の放射活性の比は１：４７であるため、合成されたＣｈｎ鎖は平均約百糖から成っていると予想された。
その後、Ｆｒ．１を陰イオン交換ＨＰＬＣで分析したところ、放射活性をもった１本のピーク（図５Ｂの矢印２）が検出され、その溶出位置は、結合領域六糖標準品ΔＨｅｘＡ α １−３ＧａｌＮＡｃβ１−４ＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌ−２−ＡＢのものと一致した。また、このピークはｃｈｏｎｄｒｏ−ｇｌｙｃｕｒｏｎｉｄａｓｅによる消化によって非還元末端のΔＨｅｘＡが消化され、ＧａｌＮＡｃβ１−４ＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌ−２−ＡＢの溶出位置（図５Ｂの矢印１）へ移動した。これらの結果より、糖鎖の伸長はα−ＴＭの結合領域四糖上で起ったことが示された。
興味深いことに、ＣｈＳｙとＣｈＰＦの複合体がＧａｌＮＡｃＴ−ＩＩ、ＧｌｃＡＴ−ＩＩ活性だけでなく、結合領域に最初にＧａｌＮＡｃを転移するＧａｌＮＡｃＴ−Ｉ活性をも保持していることが明らかとなった。この結果は、以前報告された、ヒトの悪性黒色腫Ｇ３６１細胞の培養上清中に見出した重合活性と同様の結果であった。
〔実施例６：ＣｈＰＦとＣｈＳｙの相互作用解析〕
以上の結果より、ＣｈＳｙとＣｈＰＦを同じ細胞内で共発現させると、糖転移活性の上昇およびＣｈｎ鎖の重合が起こることが明らかとなった。さらに、それぞれを単独に発現させた融合タンパク質を混合したものでは、糖転移活性の上昇は見られなかった。このことより、単独で発現させたときには複合体は形成されず、同じ細胞内でＣｈＳｙとＣｈＰＦを共発現させることにより、複合体が形成されることが示唆された。そこで、ＣｈＳｙとＣｈＰＦが実際に複合体を形成しているかを確認するため、ＣｈＰＦおよびＣｈＳｙを６×Ｈｉｓあるいはｐｒｏｔｅｉｎ Ａとの分泌型融合タンパク質として同一細胞内で共発現させ、ｐｕｌｌ−ｄｏｗｎ法によりＣｈＰＦおよびＣｈＳｙ間の相互作用を調べた。その結果が図６に示される。
図６の説明：まず、ＣｈＰＦおよびＣｈＳｙを６×Ｈｉｓあるいはｐｒｏｔｅｉｎ Ａとの分泌型融合タンパク質として単独および同一細胞内で発現させ、Ｎｉ−ＮＴＡ ａｇａｒｏｓｅを用いたｐｕｌｌ−ｄｏｗｎ ａｓｓａｙを行った。その後、ＳＤＳ−ＰＡＧＥにより分離し、ｐｒｏｔｅｉｎ Ａとの融合タンパク質をＩｇＧ抗体を用いたｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇにより検出した。Ｌａｎｅ１ではｐｒｏｔｅｉｎ Ａ−ＣｈＰＦの大きさと予想される約１１０ｋＤａのバンドが検出された。Ｌａｎｅ４ではｐｒｏｔｅｉｎ Ａ−ＣｈＳｙの大きさと予想される約１２０ｋＤａのバンドが検出された。各レーンのサンプルは以下のとおりである。Ｌａｎｅ１：ｐｒｏｔｅｉｎ Ａ−ＣｈＰＦ／Ｈｉｓ−ＣｈＳｙ、ｌａｎｅ２：Ｈｉｓ−ＣｈＳｙ、ｌａｎｅ３：ｐｒｏｔｅｉｎ Ａ−ＣｈＳｙ＋Ｈｉｓ−ＣｈＰＦ（単独に発現させたタンパク質を混合したもの）、ｌａｎｅ４：ｐｒｏｔｅｉｎ Ａ−ＣｈＳｙ／Ｈｉｓ−ＣｈＰＦ、ｌａｎｅ５：Ｈｉｓ−ＣｈＰＦ。
このように、ｐｒｏｔｅｉｎ Ａ−ＣｈＰＦ／Ｈｉｓ−ＣｈＳｙおよびｐｒｏｔｅｉｎ Ａ−ＣｈＳｙ／Ｈｉｓ−ＣｈＰＦの組合せでＣｈＰＦとＣｈＳｙを共発現させたものでは、それぞれｐｒｏｔｅｉｎ Ａ−ＣｈＰＦ（約１１０ｋＤａ）およびｐｒｏｔｅｉｎ Ａ−ＣｈＳｙ（約１２０ｋＤａ）と予想されるバンドが検出された。しかし、単独で発現させたものや、単独で発現させそれぞれのタンパク質を混合したものでは、バンドは検出されなかった。このことより、ＣｈＰＦおよびＣｈＳｙは同一細胞内で相互作用していることが示された。
〔実施例７：Ｎｏｒｔｈｅｒｎ ｂｌｏｔによるＣｈＰＦの発現パターン解析〕
ＣｈＰＦの発現パターンを調べるため、ｎｏｒｔｈｅｒｎ ｂｌｏｔ分析を行った。その結果が図７に示される。
図７の説明：ヒトの様々な組織から抽出したｍＲＮＡを用いて、ＣｈＰＦ（上段）およびＣｈＳｙ（下段）の発現を調べた。各レーンのサンプルは以下のとおりである。
Ｌａｎｅ１，ｂｒａｉｎ；ｌａｎｅ２，ｈｅａｒｔ；ｌａｎｅ３，ｓｋｅｌｅｔａｌ ｍｕｓｃｌｅ；ｌａｎｅ４，ｃｏｌｏｎ；ｌａｎｅ５，ｔｈｙｍｕｓ；ｌａｎｅ６，ｓｐｌｅｅｎ；ｌａｎｅ７，ｋｉｄｎｅｙ；ｌａｎｅ８，ｌｉｖｅｒ；ｌａｎｅ９，ｓｍａｌｌ ｉｎｔｅｓｔｉｎｅ；ｌａｎｅ１０，ｐｌａｃｅｎｔａ；ｌａｎｅ１１，ｌｕｎｇ；ｌａｎｅ１２，ｐｅｒｉｐｈｅｒａｌ ｂｌｏｏｄ ｌｅｕｋｏｃｙｔｅｓ。
図７に示すように、ｍＲＮＡのサイズは約３．４ｋｂであり、末梢白血球を除き調べた組織全てに発現が認められた。このことは、コンドロイチン硫酸がほとんどすべての組織に発現しているという結果と一致した。しかし、組識により発現レベルは異なり、胎盤および心臓では発現がもっとも強く、続いて脳、骨格筋、腎臓および肝臓で強い発現が見られた。さらに、ＣｈＰＦはＣｈＳｙのｍＲＮＡの発現パターンと非常に類似していた。このことより、両者が共同してコンドロイチン硫酸の生合成に関与していることが示された。
〔実験方法〕
（１）ＣｈＰＦの機能解析のためのｓｏｌｕｂｌｅ ｆｏｒｍの発現プラスミドの作製
内在性の糖転移酵素の混入をさけるために、培養上清中に可溶性の組換え型タンパク質としてＣｈＰＦを発現させるための発現プラスミドを作製した。ＣｈＰＦのＮ末端側から膜貫通ドメインと予想される最初の６２アミノ酸を除いた配列をＰＣＲ法によって増幅させた。５’−Ｐｒｉｍｅｒは５’末端にＢａｍＨＩ ｓｉｔｅ（下線）を含む５’−ＣＧＧＧＡＴＣＣＡＡＣＴＣＧＧＴＧＣＡＧＣ−３’を、３’−ｐｒｉｍｅｒは５’末端にＢａｍＨＩ ｓｉｔｅ（下線）を含む５’−ＣＧＧＧＡＴＣＣＧＣＴＣＴＧＧＴＴＴＴＧＧＧＧＧＡＧＡＡＧ−３’を用いた。ＰＣＲはＫＯＤ ＤＮＡ ｐｏｌｙｍｅｒａｓｅを使い、鋳型としてヒト悪性黒色腫細胞株Ｇ３６１（ＡＴＣＣ ＣＲＬ−１４２４）由来ｃＤＮＡ ｌｉｂｒａｒｙ ５０ｎｇを使用した。反応は、５％ＤＭＳＯ存在下で９４℃，３０ｓｅｃ；５５℃，３０ｓｅｃ；６８℃，１８０ｓｅｃを３２ｃｙｃｌｅ行った。増幅したｆｒａｇｍｅｎｔをＢａｍＨＩで消化し、ｐＥＦ−ＢＯＳ／ＩＰベクターに組込み発現プラスミドとした（ｐＥＦ−ＢＯＳ／ＩＰ−ＣｈＰＦ）。
（２）培養細胞への遺伝子の導入と組換えタンパク質の精製
上記発現プラスミドｐＥＦ−ＢＯＳ／ＩＰ−ＣｈＰＦ ６．０μｇをＦｕＧＥＮＥ^ＴＭ６ｔｒａｎｓｆｅｃｔｉｏｎ ｒｅａｇｅｎｔを用いてアフリカミドリザル腎臓由来ＣＯＳ−１細胞に遺伝子導入した。共発現の場合には、ｐＥＦ−ＢＯＳ／ＩＰ−ＣｈＰＦとｐＥＦ−ＢＯＳ／ＩＰ−ＣｈＳｙを３．０μｇずつ同様にＦｕＧＥＮＥ^ＴＭ６ｔｒａｎｓｆｅｃｔｉｏｎ ｒｅａｇｅｎｔを用いて、ＣＯＳ−１細胞に両方のプラスミドを導入した。Ｔｒａｎｓｆｅｃｔｉｏｎ後、ＤＭＥＭ／１０％ ＦＢＳ培地で２日間、３７℃／５％ ＣＯ_２の条件でｉｎｃｕｂａｔｉｏｎした後、培養上清をＩｇＧ−Ｓｅｐｈａｒｏｓｅと４℃で１時間混合し、ｐｒｏｔｅｉｎ Ａとの融合タンパク質を結合させた。その後、１，０００ｒｐｍで２ｍｉｎ遠心し上清を除き、沈殿した樹脂を０．１５Ｍ ＮａＣｌ／０．０２％ Ｔｗｅｅｎ−２０／５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）で洗浄した。これを３回繰り返し、精製された融合タンパク質／ＩｇＧ−Ｓｅｐｈａｒｏｓｅを酵素源とした。
（３）糖転移酵素活性の測定条件
（３−１）ＧａｌＮＡｃＴ−ＩＩ活性の測定
最終濃度として、５０ｍＭ ＭＥＳ−ＮａＯＨ（ｐＨ６．５）、１０ｍＭ ＭｎＣｌ_２、８．５７μＭ ＵＤＰ−［^３Ｈ］ＧａｌＮＡｃ（３．６０ｘ１０^５ｄｐｍ）を加えた条件で１７１μｇのＣｈｎを糖受容体基質として加えた。酵素源は上記方法（２）で精製したものを使用した。この反応溶液を３７℃で２時間ｉｎｃｕｂａｔｉｏｎした。シリンジカラムを用いて反応産物から遊離のアイソトープを除去し、溶出液の放射活性を液体シンチレーションカウンターで測定した。
（３−２）ＧｌｃＡＴ−ＩＩ活性の測定
最終濃度として、５０ｍＭ ｓｏｄｉｕｍ ａｃｅｔａｔｅ ｂｕｆｆｅｒ（ｐＨ５．６）、１０ｍＭ ＭｎＣｌ_２、１４．３μＭ ＵＤＰ−［^１４Ｃ］ＧｌｃＡ（１．４６ｘ１０^５ｄｐｍ）を加えた条件で１７１μｇのＣｈｎを糖受容体基質として加えた。酵素源は上記方法（２）で精製したものを使用した。この反応溶液を３７℃で２時間ｉｎｃｕｂａｔｉｏｎした。シリンジカラムを用いて反応産物から遊離のアイソトープを除去し、溶出液の放射活性を液体シンチレーションカウンターで測定した。
（３−３）Ｐｏｌｙｍｅｒｉｚａｔｉｏｎ活性の測定
最終濃度として１００ｍＭ ＭＥＳ−ＮａＯＨ（ｐＨ６．５）、１０ｍＭ ＭｎＣｌ_２、２５０μＭ ＵＤＰ−［^３Ｈ］ＧａｌＮＡｃ（５０．０ｘ１０^５ｄｐｍ）、２５０μＭ ＵＤＰ−ＧｌｃＡを加えた条件で１ｎｍｏｌのα−ＴＭ、ＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−Ｓｅｒ、ＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−（Ｇｌｙ）Ｓｅｒ−Ｇｌｙ−Ｇｌｕ、ＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−Ｓｅｒ−Ｇｌｙ−Ｔｒｐ−Ｐｒｏ−Ａｓｐ−Ｇｌｙ、１００ｎｍｏｌ ＧｌｃＡβ１−３Ｇａｌβ１−Ｏ−Ｃ_２Ｈ_４ＮＨ−ｂｅｎｚｙｌｏｘｙｃａｒｂｏｎｙｌ、１０ｎｍｏｌ（ＧｌｃＡβ１−３ＧａｌＮＡｃ）_３または１７１μｇ Ｃｈｎをそれぞれ糖受容体基質として加えた。酵素源は上記方法（２）で精製したものを使用した。この反応溶液を３７℃で１２時間ｉｎｃｕｂａｔｉｏｎした。
（４）Ｐｏｌｙｍｅｒｉｚａｔｉｏｎ ｒｅａｃｔｉｏｎ ｐｒｏｄｕｃｔの同定
（４−１）α−ＴＭを糖受容体基質に用いた際のｐｒｏｄｕｃｔの同定
α−ＴＭの重合反応後の精製物を、シリンジカラムを用いて、遊離のアイソトープを除去することにより単離した。α−ＴＭ上の［^３Ｈ］ＧａｌＮＡｃで標識された糖鎖を０．５Ｍ ＬｉＯＨを用いたアルカリ処理によりコアタンパク質より切り出し、４分の１をＳｕｐｅｒｄｅｘ７５カラムを用いたゲルろ過クロマトグラフィーにかけ、分画した画分の放射活性を液体シンチレーションカウンターで測定した。アルカリ処理した残りの４分の３を２−ａｍｉｎｏｂｅｎｚａｍｉｄｅ（ＡＢ）により蛍光標識した。得られた蛍光標識された糖鎖を５０ｍＩＵのＣＳａｓｅＡＢＣを用いて１時間、完全に消化した。このＣＳａｓｅＡＢＣによる消化物をＳｕｐｅｒｄｅｘ Ｐｅｐｔｉｄｅカラムを用いたゲルろ過クロマトグラフィーにかけ、２−ＡＢで標識された結合領域六糖と考えられる画分を分取し、ポリアミン結合シリカカラム（ＰＡ−０３）を用いたＨＰＬＣで分析した。また、ｃｈｏｎｄｒｏ−ｇｌｙｃｕｒｏｎｉｄａｓｅによる消化を行い、ＨＰＬＣで分析した。反応生成物の同定は２−ＡＢで標識された結合領域六糖標準品および結合領域五糖標準品とのｃｏ−クロマトグラフィーにより行った。
（４−２）糖鎖または糖ペプチドを受容体基質に用いた際のｐｒｏｄｕｃｔの同定
重合反応後の生成物をＳｕｐｅｒｄｅｘ Ｐｅｐｔｉｄｅカラムを用いたゲルろ過クロマトグラフィーによって分離し、分画した溶出液の放射活性を液体シンチレーションカウンターで測定した。
（４−３）Ｃｈｎポリマーを受容体基質に用いた際のｐｒｏｄｕｃｔの同定
重合反応後の生成物を、シリンジカラムを用いて遊離のアイソトープを除去することにより単離した。その後、精製物をそのままＳｕｐｅｒｄｅｘ ２００カラムを用いたゲルろ過クロマトグラフィーで分析した。また、単離した精製物をＣＳａｓｅＡＣＩＩ消化し、消化後Ｓｕｐｅｒｄｅｘ Ｐｅｐｔｉｄｅカラムを用いたゲルろ過クロマトグラフィーを行うことによって分離し、分画した画分の放射活性を液体シンチレーションカウンターで測定した。
（５）ＣｈＰＦとＣｈＳｙの相互作用解析
（５−１）相互作用解析用のＨｉｓ−ｔａｇ融合タンパク質発現プラスミドの作製
ＣｈＰＦとＣｈＳｙとの相互作用を確認する際、内在性の糖転移酵素の影響を除去するため、培養上清中にＨｉｓ−ｔａｇされたＣｈＰＦおよびＣｈＳｙを分泌型組換えタンパク質として発現させるための発現プラスミドを作製した。ＣｈＰＦおよびＣｈＳｙの膜貫通ドメイン以降の配列をＰＣＲ法によって以下のように増幅した。
ａ）ＣｈＰＦ：５’−Ｐｒｉｍｅｒは５’末端にＥｃｏＲＩ ｓｉｔｅ（下線）を含む５’−ＣＧＧＡＡＴＴＣＡＡＣＴＣＧＧＴＧＣＡＧＣＣＣＧＧＡＧＣ−３’を、３’−ｐｒｉｍｅｒは５’末端にＢａｍＨＩ ｓｉｔｅ（下線）を含む５’−ＣＧＧＧＡＴＣＣＧＣＴＣＴＧＧＴＴＴＴＧＧＧＧＧＡＧＡＡＧ−３’を用い、上記方法（１）と同条件でＰＣＲを行った。増幅したｆｒａｇｍｅｎｔをＥｃｏＲＩとＢａｍＨＩで消化し、ｐｃＤＮＡ３（Ｉｎｓ．ｌｅａｄｅｒ＋６×Ｈｉｓ）ベクターに組込み発現プラスミドとした｛ｐｃＤＮＡ３（Ｉｎｓ．ｌｅａｄｅｒ＋６×Ｈｉｓ）−ＣｈＰＦ｝。
ｂ）ＣｈＳｙ：５’−Ｐｒｉｍｅｒは５’末端にＸｂａＩ ｓｉｔｅ（下線）を含む５’−ＧＣＴＣＴＡＧＡＧＧＣＴＧＣＣＧＧＴＣＣＧＧＧＣＡＧ−３’を、３’−ｐｒｉｍｅｒは５’末端にＸｂａＩ ｓｉｔｅ（下線）を含む５’−ＧＣＴＣＴＡＧＡＣＡＡＴＣＴＴＡＡＡＧＧＡＧＴＣＣＴＡＴＧＴＡ−３’を用い、上記方法（１）と同条件でＰＣＲを行った。増幅したｆｒａｇｍｅｎｔをＸｂａＩで消化し、ｐｃＤＮＡ３（Ｉｎｓ．ｌｅａｄｅｒ＋６×Ｈｉｓ）に組込み発現プラスミドとした｛ｐｃＤＮＡ３（Ｉｎｓ．ｌｅａｄｅｒ＋６×Ｈｉｓ）−ＣｈＳｙ｝。
（５−２）培養細胞への遺伝子の導入と組換えタンパク質の精製
上記ａ）およびｂ）で調製した発現プラスミド６．０μｇをＦｕＧＥＮＥ^ＴＭ６ｔｒａｎｓｆｅｃｔｉｏｎ ｒｅａｇｅｎｔを用いてＣＯＳ−１細胞に遺伝子導入した。共発現の場合には、ｐｃＤＮＡ３（Ｉｎｓ．ｌｅａｄｅｒ＋６×Ｈｉｓ）−ＣｈＳｙとｐＥＦ−ＢＯＳ／ＩＰ−ＣｈＰＦあるいはｐｃＤＮＡ３（Ｉｎｓ．ｌｅａｄｅｒ＋６×Ｈｉｓ）−ＣｈＰＦとｐＥＦ−ＢＯＳ／ＩＰ−ＣｈＳｙを３．０μｇずつ同様にＦｕＧＥＮＥ^ＴＭ６ｔｒａｎｓｆｅｃｔｉｏｎ ｒｅａｇｅｎｔを用いて、ＣＯＳ−１細胞に両方のプラスミドを導入した。Ｔｒａｎｓｆｅｃｔｉｏｎ後、ＤＭＥＭ／１０％ ＦＢＳ培地で２日間、３７℃／５％ ＣＯ_２の条件でｉｎｃｕｂａｔｉｏｎした後、培養上清をＮｉ−ＮＴＡ ａｇａｒｏｓｅと４℃で１時間混合し、Ｈｉｓ−ｔａｇ融合タンパク質を結合させた。その後、１，０００ｒｐｍで２ｍｉｎ遠心し上清を除き、沈殿した樹脂を０．１５Ｍ ＮａＣｌ／０．０２％ Ｔｗｅｅｎ−２０／５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）で洗浄した。
（５−３）ＳＤＳ−ＰＡＧＥおよびｗｅｓｔｅｒｎ ｂｌｏｔ分析によるタンパク質相互作用の確認
上記方法で得たＨｉｓ−ｔａｇ融合タンパク質／Ｎｉ−ＮＴＡ ａｇａｒｏｓｅを１．３ｍＭ ｄｉｔｈｉｏｔｈｒｅｉｔｏｌ（ＤＴＴ）の存在下で９４℃、５分間ｉｎｃｕｂａｔｉｏｎした。その後、５，０００ｒｐｍで５ｍｉｎ遠心し、上清をサンプルとした。そのサンプルをＳＤＳ−ＰＡＧＥによって分離し、分離されたタンパク質を電気的にＰＶＤＦ膜に移行させた。１次抗体にはｐｒｏｔｅｉｎ Ａと高親和性を持つマウスモノクローナル抗体ＩｇＧ_３（ＭＳＷ１１３）、２次抗体にはｈｏｒｓｅｒａｄｉｓｈ ｐｅｒｏｘｉｄａｓｅ（ＨＲＰ）が結合したａｎｔｉ−ｍｏｕｓｅ ＩｇＧを用い、ＥＣＬ Ａｄｖａｎｃｅ Ｄｅｔｅｃｔｉｏｎ Ｒｅａｇｅｎｔｓ ｋｉｔのプロトコールに従い、目的タンパク質を検出した。
（６）ＣｈＰＦのｎｏｒｔｈｅｒｎ ｂｌｏｔ解析
メンブランはＨｕｍａｎ１２−Ｌａｎｅ ＭＴＮ^ＴＭ ｂｌｏｔ用い、ｐｒｏｂｅは、ＣｈＰＦが組込まれたプラスミドを鋳型として、ＰＣＲ法により増幅し、増幅したフラグメントをＢｇｌＩＩ消化することにより、ＣｈＰＦに特異的な８４０ｂｐのフラグメントを精製したものを用いた。上記方法（１）と同条件でＰＣＲを行い、５’−ｐｒｉｍｅｒは５’−ＣＧＧＧＡＴＣＣＡＣＴＣＣＴＣＴＧＧＣＴＧＣＴＣＴＧＧ−３’を、３’−ｐｒｉｍｅｒは５’−ＣＧＧＧＡＴＣＣＧＣＴＣＴＧＧＴＴＴＴＧＧＧＧＧＡＧＡＡＧ−３’を用いた。Ｐｒｏｂｅの標識は［α−^３２Ｐ］ｄＣＴＰを使用し、ランダムプライム法に基づくｏｌｉｇｏｌａｂｅｌｌｉｎｇ ｋｉｔを用いて行った。次に、メンブランをプレハイブリダイゼーションし、^３２Ｐ標識ｐｒｏｂｅを加え、６８℃で２４時間ハイブリダイゼーションを行った。メンブランを洗浄した後、ＢＩＯＭＡＸ^ＴＭ ＭＡ（Ｋｏｄａｋ社）に１週間露光し、検出した。
（７）酵素活性の測定方法
（７−１）Ｓｕｐｅｒｄｅｘ Ｐｅｐｔｉｄｅ、Ｓｕｐｒｅｄｅｘ ７５、Ｓｕｐｅｒｄｅｘ ２００の各種カラムを用いたゲルろ過クロマトグラフィー
酵素反応後のサンプルを遠心する事によって夾雑物を取り除き、予め０．２Ｍ ＮＨ_４ＨＣＯ_３で平衡化しておいたカラムにアプライした。０．４ｍｌ／ｍｉｎの流速でゲルろ過クロマトグラフィーを行い、０．４ｍｌずつ分画した画分の放射活性を、液体シンチレーションカウンターで測定した。
（７−２）シリンジカラムアッセイ
１ｍｌのシリンジ（０．４５ｘ６．８ｃｍ）に０．２Ｍ ＮＨ_４ＨＣＯ_３で平衡化したＳｅｐｈａｄｅｘ Ｇ−２５（ｓｕｐｅｒｆｉｎｅ）を充填し、１５０ｇｘ５ｍｉｎ、続いて９００ｇｘ５ｍｉｎ遠心して、ゲル内の溶媒を除去した。０．２Ｍ ＮＨ_４ＨＣＯ_３を３０μｌ加え、全量５０μｌとしたサンプルをこの樹脂に添加して９００ｇｘ５ｍｉｎで遠心し、さらに０．２Ｍ ＮＨ_４ＨＣＯ_３を５０μｌ加えてもう１度、９００ｇｘ５ｍｉｎで遠心し、未反応のＵＤＰ−糖を取り除き、その溶出液の放射活性を液体シンチレーションカウンターで測定した。
〔実験結果の考察〕
ＣｈＰＦとＣｈＳｙを共発現させることにより、ｉｎ ｖｉｔｒｏでＣｈｎ鎖の重合化が可能になることを明らかにした。ＣｈＰＦ自身はＧａｌＮＡｃＴ−ＩＩおよびＧｌｃＡＴ−ＩＩ活性をほとんど保持していないにも関わらず、ＣｈＰＦは今までに同定されている他の５つのコンドロイチン硫酸（ＣＳ）の生合成に関与している糖転移酵素と高い相同性を示す。それゆえ、ＣｈＰＦはＣＳの生合成に関与する６番目の遺伝子ファミリー（ＣｈＳｙ ｆａｍｉｌｙ）のｍｅｍｂｅｒであると考えられる。
以前、ｉｎ ｖｉｔｒｏでヒトの悪性黒色腫Ｇ３６１細胞培養上清を酵素源に用い、α−ＴＭ上にＣｈｎ鎖の重合化がおこることが報告されている。さらに基質阻害実験により、この推定されるｃｈｏｎｄｒｏｉｔｉｎ ｐｏｌｙｍｅｒａｓｅは３つの糖転移活性を保持していることが予想された。１つはＣＳ合成の開始に関与する四糖結合領域に最初のＧａｌＮＡｃを転移するＧａｌＮＡｃＴ−Ｉ活性、２つ目および３つ目はＣＳ鎖を伸長させるＧａｌＮＡｃＴ−ＩＩおよびＧｌｃＡＴ−ＩＩ活性である。今回、ＣｈＰＦとＣｈＳｙを共発現した組換えタンパク質を酵素源とした際に、α−ＴＭの四糖結合領域上にＣｈｎ鎖の重合化が起こったことより、Ｃｈｎの重合化は単独のタンパク質により担われるのではなく、ＣｈＰＦとＣｈＳｙの複合体により担われていることが示唆された。Ｐｕｌｌ−ｄｏｗｎ法による相互作用解析からも、ＣｈＰＦとＣｈＳｙが同一細胞内で複合体を形成していることが強く示唆された（図６）。さらに、Ｇ３６１細胞の培養上清から部分精製した酵素活性の分子量は１６０ｋＤａであり、今回クローニングしたＣｈＰＦおよびＣｈＳｙの分泌型のタンパク質の分子量は、それぞれ約８０ｋＤａであり、両者が１：１で複合体を形成していることが示唆された。
以前の報告と同様に、ＣｈＳｙはＣｈｎポリマーやＣｈｎ由来のオリゴ糖に対して低い糖転移活性しか示さなかった（表１）。しかし、ＣｈＳｙとＣｈＰＦを可溶性の融合タンパク質として共発現させると、ＣｈＳｙ単独時よりもＧａｌＮＡｃＴ−ＩＩおよびＧｌｃＡＴ−ＩＩ両活性は２０倍以上に上昇した（表１）。このことから、ＣｈＳｙが十分な活性を発揮するためにはＣｈＰＦが必要であると考えられる。
さらに、Ｃｈｎ鎖の重合化にはコアタンパク質が重要な役割を果たしていることが示唆された。ＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−Ｓｅｒ、ＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−（Ｇｌｙ）Ｓｅｒ−Ｇｌｙ−Ｇｌｕ、ＧｌｃＡβ１−３Ｇａｌβ１−３Ｇａｌβ１−４Ｘｙｌβ１−Ｏ−Ｓｅｒ−Ｇｌｙ−Ｔｒｐ−Ｐｒｏ−Ａｓｐ−Ｇｌｙ、ＧｌｃＡβ１−３Ｇａｌβ１−Ｏ−Ｃ_２Ｈ_４ＮＨ−ｂｅｎｚｙｌｏｘｙｃａｒｂｏｎｙｌ、（ＧｌｃＡβ１−３ＧａｌＮＡｃ）_３、およびＣｈｎポリマーを受容体基質に用いたときと比較し、α−ＴＭを受容体基質に用いたときの方がより長く効率よくＣｈｎ鎖の重合化が起った（図３）。これに関連して、以前、ＰＧであるｄｅｃｏｒｉｎのコアタンパク質の様々な欠失変異体では短いＧＡＧ鎖しか合成されず、その理由としてコアタンパク質と糖転移酵素間の親和性が低下したためであるとの報告がある。それ故、今回の結果はＣｈｎ鎖の長さが、受容体となるコアタンパク質によって影響を受け、ＣｈＳｙとＣｈＰＦの複合体がコアタンパク質と相互作用し、ＧＡＧ鎖の長さを調節していることが示唆された。
動物細胞においてβ−Ｄ−ｘｙｌｏｓｉｄｅをプライマーとしてＧＡＧが合成されることが知られており、このことより疎水的な性質を保持しているｘｙｌｏｓｉｄｅのアグリコン部分はコアタンパク質を模倣することが示唆されている。実際、ＣｈＰＦとＣｈＳｙの組換え型タンパク質を同一細胞内で共発現させたものを酵素源とし、ＧｌｃＡβ１−３Ｇａｌβ１−Ｏ−Ｃ_２Ｈ_４ＮＨ−ｂｅｎｚｙｌｏｘｙｃａｒｂｏｎｙｌを受容体とした際、Ｃｈｎ鎖の重合反応が起った（図３）。しかし、α−ＴＭを受容体に用いたときよりも短いＣｈｎ鎖しか重合しなかった（図３）。このことは疎水的なアグリコンがＧＡＧ鎖の付加するコアタンパク質の最小限の性質を保持しており、ＣｈＳｙとＣｈＰＦの複合体と相互作用することが可能であることを示唆している。
これまでの研究では、通常、重合化反応と硫酸化修飾は同じＧｏｌｇｉ区画で起り、硫酸化修飾と糖鎖の伸長および停止との間には密接な相関があると考えられている。伸長しているＣＳ鎖の硫酸化は糖鎖の重合化に影響を与えるといわれているが、今回の結果よりｉｎ ｖｉｔｒｏではＣｈｎ鎖の重合化には硫酸化修飾は必須ではないことが示された。
Ｎｏｒｔｈｅｒｎ ｂｌｏｔ解析により、ＣｈＰＦは末梢白血球を除いて調べた組織ほとんど全てに発現が認められた。また、ＣｈＰＦはＣｈＳｙのｍＲＮＡの発現パターンと非常に類似していたことより、両者が共同してＣＳの生合成に関与していることが強く示唆された（図７）。
今回、組換え型タンパク質として共発現させたＣｈＳｙとＣｈＰＦの複合体を用いてｉｎ ｖｉｔｒｏでＣｈｎ鎖の重合化を起こすことができた。この知見は、ＣＳの生合成機構の解明およびその機能解析にも役立つと考えられる。
尚、発明を実施するための最良の形態の項においてなした具体的な実施態様または実施例は、あくまでも、本発明の技術内容を明らかにするものであって、そのような具体例にのみ限定して狭義に解釈されるべきものではなく、次に記載する特許請求の範囲内で、様々に変更して実施することができる。  The present invention, in collaboration with chondroitin synthase (ChSy), identifies a polymerization factor that promotes the polymerization of chondroitin chains and a gene that encodes the same, and analyzes the function in detail. We propose a method for synthesizing chondroitin chains. Therefore, in the following, the sequence and structure of this chondroitin polymerization factor and its gene (polynucleotide) will be described, and then its function, usage method, etc. will be described.
(1) Chondroitin polymerization factor and gene sequence and structure according to the present invention
  A BLAST search was performed based on the amino acid sequence of human ChSy. As a result, gene information (accession number: BC021223) having high homology with ChSy was found. However, the deduced amino acid sequence disclosed in this information was a short one consisting of 522 residues. As a result of proceeding with the analysis based on the obtained data, the present inventor has a novel protein comprising 775 amino acids, showing 23% homology with human ChSy and 57% homology with human Chondroitin GlcAT ( The presence of ChPF) was identified (see FIG. 1).
  As will be described later, this novel protein did not have a DXD motif which is a binding motif of UDP-sugar which is a sugar donor. This suggests that this gene product is involved in the biosynthesis of chondroitin sulfate, but does not retain glycosyl transfer activity. The results of functional analysis also revealed that this gene product is not a glycosyltransferase but a factor that polymerizes chondroitin in cooperation with ChSy. Therefore, the present inventor has identified this novel protein as a chondroitin polymerization factor ( It was named chondroitin polymerizing factor (ChPF)).
  SEQ ID NO: 1 in the sequence listing shows the cDNA sequence of the human ChPF gene, and SEQ ID NO: 2 shows the amino acid sequence of human ChPF encoded by the gene. SEQ ID NO: 4 shows the amino acid sequence of a soluble ChPF recombinant protein obtained by recombining 62 amino acids from the N-terminal side containing the predicted transmembrane domain of ChPF with other sequences. SEQ ID NO: 3 Shows the cDNA sequence. As will be described later, functional analysis of ChPF was performed by expressing this soluble ChPF.
  As a result of searching and analyzing the genome information, as shown in FIG. 2, ChPF was present on the second chromosome in a spread of about 5 kb, and the gene in the translation region was composed of four exons. This genomic structure was similar to the Chondroitin GlcAT gene.
  The protein according to the present invention, that is, chondroitin polymerization factor is (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, (b) a soluble protein consisting of the amino acid sequence shown in SEQ ID NO: 4, (c) SEQ ID NO: 2 Or a protein consisting of an amino acid sequence comprising the amino acid sequence shown in 4 (for example, a fusion protein), (d) in the amino acid sequence of any one of (a) to (c) above, one or several amino acids are substituted or missing It may be any of proteins consisting of lost, inserted and / or added amino acid sequences (functioning as a chondroitin polymerization factor). Moreover, not only human origin but chondroitin polymerization factor derived from other organisms may be sufficient.
  The above “one or several amino acids are substituted, deleted, inserted, and / or added” is a degree that can be replaced, deleted, inserted, and / or added by a known mutant protein production method such as a point mutagenesis method. Means that a certain number of amino acids are substituted, deleted, inserted, and / or added. Thus, in other words, the protein of (d) above is the mutant protein of (a) to (c) above, and the “mutation” mentioned here is artificially introduced mainly by a known mutant protein production method. The same mutant protein that exists in nature may be isolated and purified.
  The protein according to the present invention may contain a known additional sequence such as HA or Flag at the end or may be a fusion protein in order to easily purify and detect the protein. Moreover, you may give various well-known modifications and may form a multimer more than a dimer.
  The “polynucleotide (gene)” of the present invention only needs to have a sequence encoding the protein of any one of the above (a) to (d), and further includes a sequence of an untranslated region (UTR). It may be.
  The “polynucleotide (gene)” of the present invention includes RNA and DNA. RNA includes mRNA, siRNA, and the like, and DNA includes cDNA, genomic DNA, and the like. The DNA may be double-stranded or single-stranded, and the single-stranded DNA may be a coding DNA serving as a sense strand or an anti-coding strand serving as an antisense strand. The antisense strand can be used as a probe or as an antisense drug. Moreover, siRNA can be utilized for gene expression suppression etc. as RNAi.
(2) Function of chondroitin polymerization factor and its utilization method
  As a result of functional analysis, the following functions have been found in the chondroitin polymerization factor (ChPF) (for details, refer to Examples described later). 1) When ChSy and ChPF retaining GalNAcT-II and GlcAT-II activities were co-expressed in the same cell, the activities of GalNAcT-II and GlcAT-II were increased by 20 times or more compared to the ChSy single expression. . 2) As a result of enzymatic reaction in the presence of UDP-GalNAc and UDP-GlcA using α-TM as a receptor substrate and secreted protein coexpressed with ChSy and ChPF as an enzyme source, the result is the same as that of natural chondroitin chain We succeeded in causing polymerization to a certain length. 3) In the polymerization reaction of chondroitin chains using ChPF, it is preferable to use a core protein such as α-TM as a receptor substrate. 4) It is considered that ChSy co-expressed as a secretory protein and ChPF form a complex and interact with each other. 5) ChPF was expressed in most tissues examined, and its expression pattern was similar to ChSy. From this, it is considered that both of them are involved in the biosynthesis of chondroitin sulfate in vivo.
  Therefore, the chondroitin polymerization factor (ChPF) and its gene can be used for artificial synthesis of a sugar chain skeleton of a sulfated glycosaminoglycan (chondroitin sulfate, dermatan sulfate) having physiological activity. For example, chondroitin has been prepared by chemically desulfating chondroitin sulfate prepared from whale cartilage and the like, but ChSy and ChPF are co-expressed and used as an enzyme source. It becomes possible to stably and easily mass-produce chondroitin sugar chains in vitro.
  In the sugar chain synthesis using the chondroitin polymerization factor (ChPF), it is preferable to synthesize a chondroitin sugar chain on a core protein such as α-thrombomodulin (α-TM).
  Furthermore, by using various sulfated transferases having different specificities, it can be used for the synthesis of various chondroitin sulfates having physiological activity and dermatan sulfate, which is an isomer thereof.
  In addition, the chondroitin polymerization factor and gene thereof of the present invention can be used as a research reagent and a test reagent, as well as diseases caused by abnormal chondroitin polymerization factor, sulfates such as chondroitin sulfate and dermatan sulfate. The present invention can be used for pathological analysis of diseases related to glycated glycosaminoglycan and development of therapeutic agents, therapeutic methods, diagnostic agents, diagnostic methods and the like for the diseases.
(3) Protein and gene acquisition method according to the present invention
  The method for obtaining the ChPF gene according to the present invention is not particularly limited, and DNA fragments containing the gene sequence can be isolated and cloned by various methods based on the above-described disclosed sequence information and the like. it can.
  For example, a probe that specifically hybridizes with a partial sequence of the cDNA of the ChPF gene may be prepared, and a human genomic DNA library or cDNA library may be screened. As such a probe, any probe of any sequence / length may be used as long as it specifically hybridizes to at least part of the cDNA sequence of the ChPF gene or its complementary sequence. In silico screening may also be performed.
  The clone obtained by the above screening can be analyzed in more detail by preparing a restriction enzyme map and determining its nucleotide sequence (sequencing). From these analyses, it can be easily confirmed whether a DNA fragment containing the gene sequence according to the present invention has been obtained.
  Further, if the sequence of the probe is selected from the highly specific region of the ChPF gene and a genomic DNA (or cDNA) library of human, mouse or other organisms is screened, the same sequence as the ChPF protein is obtained. There is a high possibility that a gene encoding a homologous molecule or a related molecule having a function can be isolated and cloned.
  The method for obtaining the gene (polynucleotide) according to the present invention includes a method using an amplification means such as PCR in addition to the above screening method. For example, among the above-mentioned cDNA sequences of ChPF, primers are prepared from sequences of untranslated regions on the 5 ′ side and 3 ′ side (or their complementary sequences), and human genomic DNA (or cDNA) is prepared using these primers. ) And the like as a template, and amplifying a DNA region sandwiched between both primers, a large amount of DNA fragments containing the polynucleotide of the present invention can be obtained.
  The method for obtaining the ChPF protein or the like according to the present invention is not particularly limited. For example, the gene obtained as described above (cDNA or the like encoding the ChPF protein or a homologous molecule thereof) is used. According to the present invention, the introduced recombinant expression vector is prepared and incorporated into microorganisms such as Escherichia coli and yeast or animal cells by a well-known method, and the protein encoded by the cDNA is expressed and purified as a transformant. Protein can be easily obtained.
  The method for producing the mutant protein of the ChPF protein is not particularly limited. For example, the base sequence of the gene obtained as described above using a known mutant protein production method such as a point mutation introduction method. In which one or more bases can be modified by substitutions, deletions, insertions and / or additions. Moreover, you may utilize a commercially available kit for preparation of a mutein.
(4) ChPF knockout animal of the present invention
  The ChPF knockout animal of the present invention, that is, a non-human animal obtained by modifying all or part of the ChPF gene sequence in the genome and having one or both ChPF genes on the homologous chromosome disrupted, is a gene targeting method. Can be used.
  Here, the phrase “ChPF gene has been destroyed” means that a protein having the original function of ChPF is not expressed. Therefore, a partial fragment (polypeptide) of the ChPF protein having no ChPF intrinsic activity may be expressed.
  The gene targeting method may be performed according to a normal method. For example, first, a targeting vector (targeting construct) for homologous recombination is constructed. The targeting vector can be prepared by a known method. Generally, a ChPF genomic DNA clone, a commercially available plasmid, a marker for positive selection (eg, PGK-neo cassette), and a marker for negative selection (DTA gene, HSV- Each fragment such as a tk gene) can be appropriately ligated. At this time, the targeting vector may be designed so that the target restriction enzyme cleavage site is located at an appropriate position. Moreover, since the efficiency of targeting depends on the length of the homologous region, it is preferable that the homologous region is as long as possible. Furthermore, since the targeting vector is preferably linear rather than circular, an appropriate restriction enzyme cleavage site may be provided in a portion other than the homologous region for linearization.
  The ChPF gene targeting vector prepared by the above method is introduced into a totipotent cell derived from a target animal such as an ES cell by electroporation or the like, and then a cell in which the desired homologous recombination has occurred is selected. The screening can be efficiently performed using a drug by a positive-negative selection method. After selection, the cells in which the desired homologous recombination has occurred are confirmed by Southern blotting or PCR. The totipotent cells finally confirmed to have homologous recombination are introduced into blastocysts (blast cysts) collected from the uterus during pregnancy. The introduction of cells into the blastocyst can be performed by a microinjection method or the like, but is not limited thereto.
  The blastocyst is transplanted to a temporary parent according to a conventional method. By crossing a germline chimeric animal (preferably male) born from a foster parent with a wild-type animal (preferably female) having a wild-type ChPF gene homozygously, as a first generation (F1), A heterozygote in which one ChPF gene is disrupted by homologous recombination can be obtained. Furthermore, by mating these heterozygotes, a homozygote in which both ChPF genes on the homologous chromosome are disrupted can be obtained as the second generation (F2). The homozygote can be identified by cutting a part of the body (eg, tail), extracting the DNA, and examining the genotype by Southern blotting or PCR. Further, as a second generation (F2), a wild-type animal (having a wild-type gene homozygous) that is the same as a ChPF knockout animal can be obtained, and this wild-type animal can be suitably used for a control experiment.
  The gene targeting method described above is merely an example, and it goes without saying that various known modifications can be made.
  The animal that is the subject of the ChPF knockout animal is not particularly limited, and examples thereof include mammals such as cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, and rats. Of these, rabbits, dogs, cats, guinea pigs, hamsters, mice, and rats are preferred for use as experimental animals, of which rodents are more preferred, and many inbred strains have been produced. Particularly preferred are mice that are equipped with techniques such as in vitro fertilization. As totipotent cells, in addition to fertilized eggs and early embryos, pluripotent ES cells and cultured cells such as somatic stem cells can be targeted.
  The ChPF knockout animal of the present invention is useful for functional analysis of ChPF.
  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples. Detailed experimental methods in each example will be described together in the last section.
[Example 1: Cloning of ChPF]
  As a result of a BLAST search using the amino acid sequence of human ChSy, it was found that a novel gene having homology with ChSy exists. This gene was composed of a 236 bp 5 'untranslated region, a 2325 bp translated region encoding 775 amino acids (Figure 1 and SEQ ID NOs: 1 and 2), and an approximately 500 bp 3' untranslated region. The sequence predicted to be the translation initiation region followed the Kozak consensus sequence. Moreover, the protein translated from this gene is a characteristic II of a glycosyltransferase localized in the Golgi apparatus having one transmembrane domain consisting of 23 amino acids on the N-terminal side from Kyte-Doolittle hydropathy analysis. It was identified as a type membrane protein and further retained three N-glycosylation sites. This gene showed 23% homology with ChSy at the amino acid sequence level and 57% homology with human Chondroitin GlcAT (FIG. 1). However, there was no DXD motif, which is a binding motif of UDP-sugar, which is a sugar donor. From this, it was predicted that this gene product is involved in the biosynthesis of chondroitin sulfate but does not retain transglycosylation activity. The results of functional analysis also revealed that this gene product is not a glycosyltransferase but a factor that polymerizes chondroitin (hereinafter referred to as “Chn” for short) in cooperation with ChSy. Named this novel protein a chondroitin polymerizing factor (ChPF).
  As a result of the analysis, it was found that the above-mentioned ChPF ortholog was also present in Caenorhabditis elegans and Drosophila melanogaster, with 27 and 25% homology, respectively.
[Example 2: ChPF gene structure and chromosome mapping]
  As shown in FIG. 2, ChPF was present on the second chromosome extending over about 5 kb, and the gene in the translation region was composed of four exons. Each intron / exon boundary was GT / AG rule compatible.
[Example 3: ChPF expression and functional analysis]
  In order to analyze the function of ChPF, 62 amino acids from the N-terminal side including the transmembrane domain of ChPF were recombined with a sequence of the IgG-binding domain of insulin leader and protein A, which are extracellular secretion signals. -1 cells expressed secreted protein fused with protein A. This ChPF recombinant protein was used as an enzyme source, and transglycosylation activity was measured using Chn as a sugar acceptor substrate and UDP-GalNAc or UDP-GlcA as a sugar donor. Table 1).
  Therefore, ChPF was considered to be a polymerization factor rather than a glycosyltransferase, and ChSy having glycosyltransferase activity that synthesizes the disaccharide repeat region of the Chn chain was coexpressed with ChPF in the same cell. As a result, when the co-expression of ChPF with ChSy was used as the enzyme source, the GalNAcT-II and GlcAT-II activities increased more than 20 times compared to ChSy alone (Table 1). In addition, no increase in transglycosylation activity was observed in a mixture of recombinant proteins in which ChSy and ChPF were individually expressed. This suggested that ChSy and ChPF form a complex in the cell. Furthermore, it was considered that the complex was not formed when expressed alone, but was formed by co-expressing ChSy and ChPF in the same cell.

[Example 4: Polymerization reaction using various sugar acceptor substrates]
  As described above, ChSy expressed as a recombinant protein has both GalNAcT-II and GlcAT-II activities that transfer GalNAc and GlcA with monosaccharides, but is not present on α-TM. The activity of polymerizing the Chn chain was not retained.
  Therefore, since the increase in transglycosylation activity was observed in the co-expressed ChPF and ChSy, the co-expressed ChPF and ChSy may have the activity of polymerizing the Chn chain. It was. In order to analyze this point, α-TM, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, and α-TM derived from the tetrasaccharide-binding region were obtained using the recombinant protein coexpressed with ChSy and ChPF as the enzyme source. GlcAβ1-3Galβ1-3Galβ1-GlcAβ1-3Galβ1-3Galβ1-Peptide-bound GlcAβ1-3Galβ1-3Galβ1-O- (Gly) Ser-Gly-Glu; 4Xylβ1-O-Ser-Gly-Trp-Pro-Asp-Gly and the artificial substrate GlcAβ1-3Galβ1-OC₂H₄Polymerization activity was measured using NH-benzoxycarbonyl as a sugar acceptor substrate. The results are shown in graphs A and B in FIG.
  Explanation of FIG. GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser (●), GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O- (Gly) Ser-Gly-Glu (×), GlcAβ1-3Galβ1-3Gal1 -4Xylβ1-O-Ser-Gly-Trp-Pro-Asp-Gly (Δ), GlcAβ1-3Galβ1-OC₂H₄NH-benzoxycarbonyl (（) and α-TM (◯) were used. [³H] -labeled polymerization reaction products were analyzed by gel filtration chromatography using a Superdex peptide column. When α-TM is used as a receptor substrate, [³The H] labeled polymerization reaction product was subjected to alkali reduction treatment and then used for analysis. The arrow indicates the elution position of Chn-derived octasaccharide to decasaccharide. Vt is around the fraction (Fr.) 60. B. The sugar chain (◯) synthesized on α-TM by the polymerization reaction was analyzed using a Superdex 75 column (1.6 × 60 cm). The radioactivity of the eluate after gel filtration chromatography was measured. Chn polymer was also analyzed as a molecular weight standard, and ultraviolet absorption at 210 nm of the eluate was measured (●).
  As shown in FIG. 3, polymerization of the Chn chain occurred on the various substrates used, but the Chn chain was elongated most efficiently on α-TM.
  In addition, Chn-derived hexasaccharide (GlcAβ1-3GalNAc) is used to confirm whether the Chn chain is extended by the fusion protein in which ChSy and ChPF are co-expressed in the already extended Chn chain.₃Then, a Chn chain polymerization reaction was carried out using the Chn polymer as an acceptor substrate. The results are shown in graphs AC in FIG.
  Explanation of FIG. Chn-derived oligosaccharide (GlcAβ1-3GalNAc) as an acceptor substrate for polymerization reaction of Chn chain₃Was used. [³H] The labeled polymerization reaction product was separated by gel filtration chromatography using a Superdex peptide column, and the radioactivity of the eluate was measured (●). The arrow indicates the elution position of Chn-derived octasaccharide to decasaccharide. Vt is Fr. Around 60. B. Polymerization reaction was carried out using Chn polymer as an acceptor substrate and co-expression of ChPF and ChSy (◯) and ChSy alone (●) as an enzyme source, [³H] The product of the polymerization reaction was isolated by gel filtration chromatography. Isolated [³H] The labeled polymerization reaction product was subjected to gel filtration chromatography using a Superdex 200 column (1.6 × 60 cm), and the radioactivity of the eluate was measured. Arrows indicate the elution positions at the respective molecular weights determined by dextran used as molecular weight markers. C. The reaction product isolated in B above was digested with CSaseACII, and after digestion, the reaction product was separated by gel filtration chromatography using a Superdex Peptide column, and measured with a liquid scintillation counter. After CSase digestion of reaction product when using ChPF and ChSy co-expressed as enzyme source (○) and after CSase digestion of reaction product when using ChSy alone expressed as enzyme source The chromatogram of (●) is shown. The arrow indicates the elution position of Chn-derived disaccharide and GalNAc.
  As shown in FIG. 4, Chn-derived hexasaccharide (GlcAβ1-3GalNAc)₃When was used as a receptor substrate, it was predicted that the sugar chain was elongated from about disaccharide to tetrasaccharide because the peak was detected at the position of octadecasaccharide (Fig. A). When Chn polymer was used as the acceptor substrate, the length of the extended sugar chain was not significantly different from the product obtained by using only ChSy as the control as the enzyme source and analyzed by gel filtration chromatography. (Fig. B). However, as a result of CSaseACII digestion, a radioactive peak was detected only at the position of the monosaccharide from the reaction product prepared using ChSy, whereas the enzyme source was obtained by co-expressing ChPF and ChSy. From the reaction product used in the above, a prominent peak was also detected at the position of the disaccharide (Fig. C). From this, it was clarified that a sugar chain elongation reaction of at least several sugars occurred on the Chn polymer substrate compared with ChSy alone.
[Example 5: Analysis of Chn chain polymerized to tetrasaccharide binding region of α-TM]
  Next, it was confirmed whether the elongation reaction of the Chn chain occurred using the tetrasaccharide binding region of α-TM as a primer. The results are shown in graphs A and B in FIG.
  Explanation of FIG. Perform polymerization reaction using α-TM as the acceptor substrate, [³The H] labeled polymerization reaction product was isolated by gel filtration. Isolated [³H] The labeled polymerization reaction product was alkali-treated and then fluorescently labeled with 2-aminobenzamide (AB). The fluorescently labeled sugar chain is completely digested with CSaseABC, ΔDi-0S is added as an unsaturated disaccharide standard, fractionated by gel filtration chromatography using a Superdex peptide column, and the radioactivity of the eluate (●) Was measured. The arrow indicates the elution position of unsaturated disaccharide (ΔHex α 1-3GalNAc). B. The radiolabeled fraction containing the binding region (Fr. 1 in graph A) was collected and analyzed by HPLC using an anion exchange column. Fr. 1 shows chromatograms before digestion (◯) and after digestion (●) with 1 chondro-glycuronidase. The radioactivity of the eluate was measured. The arrows indicate the elution positions of the binding region pentasaccharide and hexasaccharide labeled with 2-AB. The wavy line is NaH₂PO₄The concentration gradient is shown.
  As shown in FIG. 5A, the radiolabeled sugar chain was digested with CSaseABC, and Fr. Fr. containing 1 and disaccharides. 2 was obtained. Fr. 1 and Fr. Since the ratio of the radioactivity of 2 was 1:47, the synthesized Chn chain was expected to consist of an average of about a hundred sugars.
  Thereafter, Fr. When 1 was analyzed by anion exchange HPLC, one radioactive peak (arrow 2 in FIG. 5B) was detected, and the elution position was determined as the binding region hexasaccharide standard product ΔHexA α 1-3GalNAcβ1-4GlcAβ1- It was consistent with that of 3Galβ1-3Galβ1-4Xyl-2-AB. Further, this peak was digested with chondro-glycuronidase to digest the non-reducing end ΔHexA and moved to the elution position of GalNAcβ1-4GlcAβ1-3Galβ1-3Galβ1-4Xyl-2-AB (arrow 1 in FIG. 5B). These results indicated that sugar chain elongation occurred on the α-TM binding domain tetrasaccharide.
  Interestingly, it was revealed that the complex of ChSy and ChPF retains not only GalNAcT-II and GlcAT-II activity but also GalNAcT-I activity that first transfers GalNAc to the binding region. This result was similar to the previously reported polymerization activity found in the culture supernatant of human malignant melanoma G361 cells.
[Example 6: Analysis of interaction between ChPF and ChSy]
  From the above results, it has been clarified that when ChSy and ChPF are co-expressed in the same cell, an increase in sugar transfer activity and polymerization of the Chn chain occur. Furthermore, no increase in transglycosylation activity was observed in a mixture of fusion proteins in which each was expressed alone. This suggests that no complex was formed when expressed alone, but a complex was formed by co-expressing ChSy and ChPF in the same cell. Therefore, in order to confirm whether ChSy and ChPF actually form a complex, ChPF and ChSy are co-expressed as a secreted fusion protein with 6 × His or protein A in the same cell, and the pull-down method is used. The interaction between ChPF and ChSy was examined. The result is shown in FIG.
  Explanation of FIG. 6: First, ChPF and ChSy were expressed alone and in the same cell as a secreted fusion protein with 6 × His or protein A, and then pull-down assay using Ni-NTA agarose was performed. Then, it isolate | separated by SDS-PAGE and the fusion protein with protein A was detected by Western blotting using IgG antibody. In Lane 1, a band of about 110 kDa, which is expected to be the size of protein A-ChPF, was detected. In Lane 4, a band of about 120 kDa, which was expected to be protein A-ChSy, was detected. Samples for each lane are as follows. Lane1: protein A-ChPF / His-ChSy, lane2: His-ChSy, lane3: protein A-ChSy + His-ChPF (mixed proteins expressed alone), lane4: protein A-ChSy / His-ChPF : His-ChPF.
  Thus, protein A-ChPF (about 110 kDa) and protein A-ChSy (about 110 kDa) and protein A-ChPF (about 110 kDa) and protein A-ChPF / His-ChSy and protein A-ChSy / His-ChPF co-expressed with ChPF and ChSy, respectively, A band expected to be about 120 kDa) was detected. However, no band was detected in those expressed alone or in the case where each protein was expressed alone and mixed with each protein. This indicates that ChPF and ChSy interact in the same cell.
[Example 7: Expression pattern analysis of ChPF by Northern blot]
  In order to examine the expression pattern of ChPF, a northern blot analysis was performed. The result is shown in FIG.
  Explanation of FIG. 7: Expression of ChPF (upper) and ChSy (lower) was examined using mRNA extracted from various human tissues. Samples for each lane are as follows.
Lane 3, heart; lane 3, skeleton; lane 4, colon; lane 5, thymus; lane 6, splene; lane 7, kidney; lane 8, lancer; lane 9, phne lunar; leukocytes.
  As shown in FIG. 7, the size of mRNA was about 3.4 kb, and expression was observed in all the tissues examined except for peripheral leukocytes. This was consistent with the result that chondroitin sulfate was expressed in almost all tissues. However, expression levels differed depending on the tissue, with the strongest expression in the placenta and heart followed by strong expression in the brain, skeletal muscle, kidney and liver. Furthermore, ChPF was very similar to the expression pattern of ChSy mRNA. From this, it was shown that both were jointly involved in the biosynthesis of chondroitin sulfate.
〔experimental method〕
(1) Preparation of a soluble form expression plasmid for functional analysis of ChPF
  In order to avoid contamination with endogenous glycosyltransferases, an expression plasmid for expressing ChPF as a soluble recombinant protein in the culture supernatant was prepared. A sequence excluding the first 62 amino acids expected to be a transmembrane domain from the N-terminal side of ChPF was amplified by PCR. 5'-Primer contains 5'-CG containing a BamHI site (underlined) at the 5 'endGGATCCAACTCGGTGCAGC-3 ', 3'-primer contains 5'-CG containing BamHI site (underlined) at the 5' endGGATCCGCTCTGGTTTTGGGGGAGAAG-3 'was used. For PCR, KOD DNA polymerase was used and 50 ng of cDNA library derived from human malignant melanoma cell line G361 (ATCC CRL-1424) was used as a template. The reaction was carried out in the presence of 5% DMSO for 32 cycles at 94 ° C., 30 sec; 55 ° C., 30 sec; 68 ° C., 180 sec. The amplified fragment was digested with BamHI and incorporated into a pEF-BOS / IP vector to form an expression plasmid (pEF-BOS / IP-ChPF).
(2) Introduction of genes into cultured cells and purification of recombinant proteins
  6.0 μg of the expression plasmid pEF-BOS / IP-ChPF was added to FuGENE.^TMThe gene was introduced into COS-1 cells derived from African green monkey kidney using 6-translation reagent. In the case of co-expression, FuGENE is similarly used for 3.0 μg each of pEF-BOS / IP-ChPF and pEF-BOS / IP-ChSy.^TMBoth plasmids were introduced into COS-1 cells using 6 transfection reagent. After Transfection, DMEM / 10% FBS medium for 2 days at 37 ° C / 5% CO₂After incubation under the conditions described above, the culture supernatant was mixed with IgG-Sepharose at 4 ° C. for 1 hour to bind the protein A fusion protein. Thereafter, the mixture was centrifuged at 1,000 rpm for 2 minutes, the supernatant was removed, and the precipitated resin was washed with 0.15 M NaCl / 0.02% Tween-20 / 50 mM Tris-HCl (pH 7.5). This was repeated three times, and purified fusion protein / IgG-Sepharose was used as the enzyme source.
(3) Glycosyltransferase activity measurement conditions
  (3-1) Measurement of GalNAcT-II activity
  As final concentration, 50 mM MES-NaOH (pH 6.5), 10 mM MnCl₂8.57 μM UDP- [³H] GalNAc (3.60x10⁵171 μg of Chn was added as a sugar receptor substrate under the condition of adding dpm). The enzyme source used was purified by the above method (2). This reaction solution was incubated at 37 ° C. for 2 hours. The free isotope was removed from the reaction product using a syringe column, and the radioactivity of the eluate was measured with a liquid scintillation counter.
  (3-2) Measurement of GlcAT-II activity
  As final concentration, 50 mM sodium acetate buffer (pH 5.6), 10 mM MnCl₂, 14.3 μM UDP- [¹⁴C] GlcA (1.46 × 10⁵171 μg of Chn was added as a sugar receptor substrate under the condition of adding dpm). The enzyme source used was purified by the above method (2). This reaction solution was incubated at 37 ° C. for 2 hours. The free isotope was removed from the reaction product using a syringe column, and the radioactivity of the eluate was measured with a liquid scintillation counter.
  (3-3) Measurement of Polymerization activity
  Final concentration of 100 mM MES-NaOH (pH 6.5), 10 mM MnCl₂, 250 μM UDP- [³H] GalNAc (50.0x10⁵dpm), 1 nmol α-TM, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O- (Gly) Ser-Gly-GluGlu, with 250 μM UDP-GlcA added -3Galβ1-3Galβ1-4Xylβ1-O-Ser-Gly-Trp-Pro-Asp-Gly, 100 nmol GlcAβ1-3Galβ1-O-C₂H₄NH-benzoxycarbonyl, 10 nmol (GlcAβ1-3GalNAc)₃Alternatively, 171 μg Chn was added as a sugar acceptor substrate. The enzyme source used was purified by the above method (2). This reaction solution was incubated at 37 ° C. for 12 hours.
(4) Identification of Polymerization reaction product
  (4-1) Identification of product when α-TM is used as a sugar receptor substrate
  The purified product after the polymerization reaction of α-TM was isolated by removing a free isotope using a syringe column. On α-TM³H] GalNAc-labeled sugar chain was excised from the core protein by alkali treatment with 0.5M LiOH, and a quarter was subjected to gel filtration chromatography using a Superdex 75 column, and the radioactivity of the fractionated fraction was determined. Measured with a liquid scintillation counter. The remaining three quarters treated with alkali were fluorescently labeled with 2-aminobenzamide (AB). The resulting fluorescently labeled sugar chain was completely digested with 50 mIU CSaseABC for 1 hour. The digested product of CSaseABC was subjected to gel filtration chromatography using a Superdex Peptide column, and a fraction considered to be a binding region hexasaccharide labeled with 2-AB was fractionated, and a polyamine-bound silica column (PA-03) was used. Was analyzed by HPLC. In addition, digestion with chondro-glycuronidase was performed and analyzed by HPLC. The reaction product was identified by co-chromatography with a binding region hexasaccharide standard and a binding region pentasaccharide standard labeled with 2-AB.
  (4-2) Identification of product when sugar chain or glycopeptide is used as receptor substrate
  The product after the polymerization reaction was separated by gel filtration chromatography using a Superdex Peptide column, and the radioactivity of the fractionated eluate was measured with a liquid scintillation counter.
  (4-3) Identification of product when Chn polymer is used as receptor substrate
  The product after the polymerization reaction was isolated by removing free isotopes using a syringe column. Thereafter, the purified product was directly analyzed by gel filtration chromatography using a Superdex 200 column. In addition, the isolated purified product was digested with CSaseACII, separated by gel filtration chromatography using a Superdex Peptide column after digestion, and the radioactivity of the fractionated fraction was measured with a liquid scintillation counter.
(5) Analysis of interaction between ChPF and ChSy
  (5-1) Preparation of His-tag fusion protein expression plasmid for interaction analysis
  When confirming the interaction between ChPF and ChSy, in order to remove the influence of endogenous glycosyltransferase, expression for expressing HisPF-labeled ChPF and ChSy as secretory recombinant proteins in the culture supernatant A plasmid was prepared. Sequences after the transmembrane domain of ChPF and ChSy were amplified by PCR as follows.
a) ChPF: 5'-Primer contains an EcoRI site (underlined) at the 5 'end 5'-CGGAATTCAACTCGGTGCAGCCCGGAGC-3 ', 3'-primer contains 5'-CG containing BamHI site (underlined) at the 5' endGGATCCPCR was performed using GCTCTGGTTTGGGGGAGAAG-3 'under the same conditions as in the above method (1). The amplified fragment was digested with EcoRI and BamHI, and incorporated into a pcDNA3 (Ins.leader + 6 × His) vector as an expression plasmid {pcDNA3 (Ins.leader + 6 × His) -ChPF}.
b) ChSy: 5'-Primer contains 5'-GC containing an XbaI site (underlined) at the 5 'endTCTAGAGCGTCGCCGTCGCGGGCAG-3 ', 3'-primer contains 5'-GC containing XbaI site (underlined) at the 5' endTCTAGAPCR was performed using CAATCTTAAAGGAGCTCTATGTA-3 'under the same conditions as in the above method (1). The amplified fragment was digested with XbaI and incorporated into pcDNA3 (Ins.leader + 6 × His) as an expression plasmid {pcDNA3 (Ins.leader + 6 × His) -ChSy}.
  (5-2) Introduction of genes into cultured cells and purification of recombinant proteins
  6.0 μg of the expression plasmid prepared in the above a) and b) was added to FuGENE.^TMThe gene was introduced into COS-1 cells using 6-transfecting reagent. In the case of co-expression, pcDNA3 (Ins.leader + 6 × His) -ChSy and pEF-BOS / IP-ChPF or pcDNA3 (Ins.leader + 6 × His) -ChPF and pEF-BOS / IP-ChSy in the same amount of 3.0 μg FuGENE^TMBoth plasmids were introduced into COS-1 cells using 6 transfection reagent. After Transfection, DMEM / 10% FBS medium for 2 days at 37 ° C / 5% CO₂After incubation under the conditions, the culture supernatant was mixed with Ni-NTA agarose at 4 ° C. for 1 hour to bind the His-tag fusion protein. Thereafter, the mixture was centrifuged at 1,000 rpm for 2 minutes, the supernatant was removed, and the precipitated resin was washed with 0.15 M NaCl / 0.02% Tween-20 / 50 mM Tris-HCl (pH 7.5).
  (5-3) Confirmation of protein interaction by SDS-PAGE and Western blot analysis
  The His-tag fusion protein / Ni-NTA agarose obtained by the above method was incubated at 94 ° C. for 5 minutes in the presence of 1.3 mM dithiothreitol (DTT). Thereafter, the mixture was centrifuged at 5,000 rpm for 5 minutes, and the supernatant was used as a sample. The sample was separated by SDS-PAGE, and the separated protein was electrically transferred to a PVDF membrane. Mouse monoclonal antibody IgG with high affinity for protein A as the primary antibody₃(MSW113) Anti-mouse IgG conjugated with horseradish peroxidase (HRP) was used as the secondary antibody, and the target protein was detected according to the protocol of ECL Advance Detection Reagents kit.
(6) Northern blot analysis of ChPF
  The membrane is Human12-Lane MTN^TM  Blot was used and probe was obtained by amplifying by PCR using a plasmid incorporating ChPF as a template, and digesting the amplified fragment with BglII to purify a 840 bp fragment specific for ChPF. PCR was performed under the same conditions as in the above method (1), and 5'-primer was 5'-CGGGATCCACTCCCTCTGGCTGCCTCTGG-3 ', and 3'-primer was 5'-CGGGATCCGCTCTGGTTTTGGGGGGAGAAG-3'. The label of Probe is [α-³²P] dCTP was used, and an oligobolating kit based on the random prime method was used. Next, prehybridize the membrane,³²P-labeled probe was added and hybridization was performed at 68 ° C. for 24 hours. After washing the membrane, BIOMAX^TM  MA (Kodak) was exposed for 1 week and detected.
(7) Measuring method of enzyme activity
  (7-1) Gel filtration chromatography using various columns of Superdex Peptide, Superdex 75, Superdex 200
  The sample after the enzyme reaction is centrifuged to remove impurities, and 0.2M NH in advance.₄HCO₃And applied to the column that had been equilibrated with. Gel filtration chromatography was performed at a flow rate of 0.4 ml / min, and the radioactivity of fractions fractioned by 0.4 ml was measured with a liquid scintillation counter.
  (7-2) Syringe column assay
  0.2 ml NH in a 1 ml syringe (0.45 x 6.8 cm)₄HCO₃Sephadex G-25 (superfine) equilibrated in (1) was filled, followed by centrifugation at 150 g × 5 min and then 900 g × 5 min to remove the solvent in the gel. 0.2M NH₄HCO₃Was added to this resin, centrifuged at 900 g × 5 min, and further 0.2 M NH₄HCO₃Was added, and centrifuged again at 900 g × 5 min to remove unreacted UDP-sugar, and the radioactivity of the eluate was measured with a liquid scintillation counter.
[Consideration of experimental results]
  It was clarified that Chn chains can be polymerized in vitro by co-expressing ChPF and ChSy. Although ChPF itself retains little GalNAcT-II and GlcAT-II activity, ChPF is involved in the biosynthesis of the other five chondroitin sulfates (CS) that have been identified so far. High homology with the enzyme. Therefore, ChPF is considered to be a member of the sixth gene family (ChSy family) involved in CS biosynthesis.
  Previously, it has been reported that Chn chain polymerization occurs on α-TM using human malignant melanoma G361 cell culture supernatant in vitro as an enzyme source. Furthermore, it was predicted from the substrate inhibition experiment that this estimated chondroitin polymerase retained three glycosyltransferase activities. One is GalNAcT-I activity that transfers the first GalNAc to the tetrasaccharide binding region involved in the initiation of CS synthesis, and the second and third are GalNAcT-II and GlcAT-II activities that extend the CS chain. In this study, when a recombinant protein coexpressed with ChPF and ChSy was used as the enzyme source, the polymerization of Chn occurred on the tetrasaccharide binding region of α-TM. It was suggested that it was carried by a complex of ChPF and ChSy rather than being carried. The interaction analysis by the Pull-down method also strongly suggested that ChPF and ChSy formed a complex in the same cell (FIG. 6). Furthermore, the molecular weight of the enzyme activity partially purified from the culture supernatant of G361 cells is 160 kDa, and the molecular weights of the secreted proteins of ChPF and ChSy cloned this time are about 80 kDa, both of which are 1: 1 and complex. It was suggested that
  Similar to previous reports, ChSy showed only low transglycosylation activity for Chn polymers and Chn-derived oligosaccharides (Table 1). However, when ChSy and ChPF were co-expressed as a soluble fusion protein, both GalNAcT-II and GlcAT-II activities increased more than 20 times compared to ChSy alone (Table 1). From this, it is considered that ChPF is necessary for ChSy to exhibit sufficient activity.
  Furthermore, it was suggested that the core protein plays an important role in the polymerization of the Chn chain. GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O- (Gly) Ser-Gly-Glu, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Srp-Gly-β -Gly, GlcAβ1-3Galβ1-OC₂H₄NH-benzoxycarbonyl, (GlcAβ1-3GalNAc)₃As compared with the case where the Chn polymer was used as the receptor substrate, polymerization of the Chn chain occurred longer and more efficiently when α-TM was used as the receptor substrate (FIG. 3). In this connection, various deletion mutants of the core protein of decorin, PG, previously synthesized only a short GAG chain because the affinity between the core protein and glycosyltransferase was reduced. There is a report. Therefore, the result of this time is that the length of the Chn chain is affected by the core protein as a receptor, and the complex of ChSy and ChPF interacts with the core protein and regulates the length of the GAG chain. Was suggested.
  It is known that GAG is synthesized by using β-D-xyloside as a primer in animal cells. This suggests that the aglycon part of xyloside that retains hydrophobic properties mimics the core protein. Yes. In fact, the enzyme source is a co-expressed recombinant protein of ChPF and ChSy in the same cell, and GlcAβ1-3Galβ1-OC₂H₄When NH-benzoylcarbonyl was used as an acceptor, a Chn chain polymerization reaction occurred (FIG. 3). However, only a shorter Chn chain polymerized than when α-TM was used for the receptor (FIG. 3). This suggests that the hydrophobic aglycon retains the minimal properties of the core protein to which the GAG chain is added and can interact with the complex of ChSy and ChPF.
  In previous studies, polymerization reaction and sulfation modification usually occur in the same Golgi compartment, and it is considered that there is a close correlation between sulfation modification and sugar chain elongation and termination. Although it is said that the sulfation of the extending CS chain affects the polymerization of sugar chains, the present results indicate that sulfation modification is not essential for polymerization of Chn chains in vitro. It was.
  According to the Northern blot analysis, ChPF was found to be expressed in almost all tissues examined except for peripheral leukocytes. Moreover, since ChPF was very similar to the expression pattern of mRNA of ChSy, it was strongly suggested that both were involved in the biosynthesis of CS (FIG. 7).
  This time, the Chn chain could be polymerized in vitro using a complex of ChSy and ChPF co-expressed as a recombinant protein. This finding may be useful for elucidating the biosynthetic mechanism of CS and analyzing its function.
  It should be noted that the specific embodiments or examples made in the best mode for carrying out the invention are merely to clarify the technical contents of the present invention, and are limited to such specific examples. Therefore, the present invention should not be interpreted in a narrow sense, and various modifications can be made within the scope of the following claims.

Industrial applicability

  As described above, the chondroitin polymerization factor (ChPF) of the present invention forms a complex with chondroitin synthase (ChSy) and promotes the polymerization of chondroitin sugar chains. It can be used for artificial synthesis of the sugar chain skeleton of sulfated glycosaminoglycans having useful physiological activities such as sulfa and dermatan sulfate, and has various usefulness.

Claims (3)

A complex obtained by co-expressing a protein comprising any one of the following amino acid sequences (a) to (d) and chondroitin synthase :
(A) the amino acid sequence shown in SEQ ID NO: 2,
(B) the amino acid sequence shown in SEQ ID NO: 4,
(C) an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 or 4,
(D) Amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in (a) or (b) above
(However, (c) or (d) has a polymerization effect of chondroitin chains in cooperation with chondroitin synthase). The chondroitin chain polymerization method as described in the above, wherein a complex obtained by co-expressing a protein comprising any one of the following amino acid sequences (a) to (d) and chondroitin synthase :
(A) the amino acid sequence shown in SEQ ID NO: 2,
(B) the amino acid sequence shown in SEQ ID NO: 4,
(C) an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 or 4,
(D) In the amino acid sequence shown in (a) or (b) above, an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added is included.
(However, (c) or (d) has a polymerization effect of chondroitin chains in cooperation with chondroitin synthase). The chondroitin chain polymerization method according to claim 2 , wherein a chondroitin chain is synthesized on a core protein of α-thrombomodulin to which a tetrasaccharide is bound.

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