Studies on Biosynthetic Mechanism of the Sulfated Glycosaminoglycans

硫酸化糖胺聚糖生物合成机制的研究

• 批准号: 14572086
• 负责人: KITAGAWA Hiroshi
• 金额: $ 2.56万
• 依托单位: Kobe Pharmaceutical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572086/
• 关键词: Glycosyltransferases; chondroitin Sulfate; Heparan Surfate; Glycosaminoglycans; Proteoglycan; tumor suppressor Gene; C.elegans; Crystal Structure; グルクロン酸; cDNAクローニング; 細胞外マトリックス

Sulfated glycosaminoglycans including heparin/heparan sulfate and chondroitin/dermatan sulfate have been implicated in numerous pathophysiological phenomena of vertebrates and invertebrates. To facilitate analyses of the functions of these glycosaminoglycans through gene manipulation in various model animals, we have cloned several genes encoding the glycosyltransferases and sulfotransferase required for the biosynthesis of the glycosaminoglycans. In this study, we cloned additional six genes encoding the glycosyltransferases and a related protein involved in glycosaminoglycan biosynthesis and found novel functions of chondroitin in Caenorhabditis elegans as follows.1)Molecular cloning of human chondroitin N-acetylgalactosaminyltransferase-2(GalNAcT-2) :We identified a novel human chondroitin GalNAcT, designated chondroitin GalNAcT-2. GalNAcT-2 transferred β1,4-N-acetylgalactosamine(GalNAc) from UDP-[^3H]GalNAc not only to a polymer chondroitin representing growing chondroitin, but als … More o to GlcUAβ1-3Galβ1-O-C2H4NHCbz, a synthetic substrate for β-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate.2)Molecular cloning of three Drosophila glucuronyltransferases :We cloned and characterized three Drosophila β1,3-glucuronytransferases. The results showed that all of them are likely involved in the synthesis of the glycosaminoglycans-protein linkage region in Drosophila.3)cDNA cloning of C.elegans chondroitin synthase and demonstration of novel functions :We cloned a chondroitin synthase homologue of C.elegans and depleted expression of its product by RNA-mediated interference. We found that blocking chondroitin synthesis results in cytokinesis defects in early embryogenesis.4)Molecular cloning of a chondroitin polymerizing factor :We previously cloned human chondroitin synthase, but chondroitin polymerization was not demonstrated in vitro using the recombinant chondroitin synthase. We reported that the chondroitin polymerizing activity requires concomitant expression of a novel protein designated chondroitin polymerizing factor with chondroitin synthase.5)In vitro heparan sulfate polymerization :We found that the core protein moieties in addition to the interaction between EXT1 and EXT2 are required for heparan sulfate polymerization. Less

硫酸化糖胺聚糖，包括肝素/硫酸乙酰肝素和软骨素/硫酸皮肤素，已经涉及脊椎动物和无脊椎动物的许多病理生理学现象。为了便于分析这些糖胺聚糖的功能，通过在各种模型动物的基因操作，我们已经克隆了几个基因编码的糖基转移酶和磺基转移酶所需的糖胺聚糖的生物合成。在这项研究中，我们克隆了另外六个基因编码的糖基转移酶和相关蛋白参与糖胺聚糖的生物合成，并发现软骨素的新功能在秀丽隐杆线虫如下。1）分子克隆的人软骨素N-乙酰半乳糖胺转移酶-2（GalNAcT-2）：我们确定了一个新的人软骨素GalNAcT，命名为软骨素GalNAcT-2。GalNAcT-2不仅将UDP-[^3H]GalNAc中的β 1，4-N-乙酰半乳糖胺（GalNAc）转移到代表生长软骨素的聚合软骨素中， ...更多信息 o为GlcUAβ1-3Galβ1-O-C2 H4 NHCbz，其为β-GalNAc转移酶I的合成底物，所述β-GalNAc转移酶I将第一个GalNAc转移到硫酸软骨素的蛋白连接区的核心四糖。结果表明，它们都可能参与果蝇糖胺聚糖-蛋白质连接区的合成。3）秀丽隐杆线虫软骨素合酶的cDNA克隆和新功能的证明：我们克隆了秀丽隐杆线虫的软骨素合酶同源物，并通过RNA介导的干扰消除了其产物的表达。我们发现，阻断软骨素合成导致早期胚胎发育中的胞质分裂缺陷。4）软骨素聚合因子的分子克隆：我们先前克隆了人软骨素合酶，但使用重组软骨素合酶在体外未证实软骨素聚合。我们报道了软骨素聚合活性需要同时表达一种新的蛋白质，称为软骨素聚合因子和软骨素脱氢酶。5）体外硫酸乙酰肝素聚合：我们发现除了EXT 1和EXT 2之间的相互作用之外，核心蛋白部分也是硫酸乙酰肝素聚合所必需的。少

项目成果

Pedersen, L.C. 他: "Crystal Structure of an 1,4-N-acetylhexosaminyltransferase (EXTL2) member of the exostosin gene family involved ・・・"J.Biol.Chem.. 278. 14420-14428 (2003)

Pedersen, L.C. 等人：“外骨蛋白基因家族的 1,4-N-乙酰己糖胺基转移酶 (EXTL2) 成员的晶体结构涉及……” J.Biol.Chem.. 278. 14420-14428 (2003)

Kim Byung-Taek他: "Identification and characterization of three Drosophila melanogaster glucuronyltransferases responsible for the Synthesis of the ・・・"J.Biol.Chem.. 278. 9116-9124 (2003)

Kim Byung-Taek 等人：“负责合成……的三种果蝇葡萄糖醛酸转移酶的鉴定和表征” J.Biol.Chem.. 278. 9116-9124 (2003)

Byung-Taek Kim, Hiroshi Kitagawa, Junko Tanaka, Jun-ichi Tamura, Kazuyuki Sugahara: "In vitro heparan sulfate polymerization : Crucial roles of core protein moieties of primer substrates in addition to the EXT1/EXT2 interaction."J.Biol.Chem.. 278(43). 416

Byung-Taek Kim、Hiroshi Kitakawa、Junko Tanaka、Jun-ichi Tamura、Kazuyuki Sugahara：“体外硫酸乙酰肝素聚合：除了 EXT1/EXT2 相互作用之外，引物底物核心蛋白部分的重要作用。”J.Biol.Chem

守山徹他: "Molecular cloning and expression of a second chondroitin N-acetylgalactosaminyltransferase involved in the initiation and elongation ・・・"J.Biol.Chem.. 278. 3072-3078 (2003)

Toru Moriyama 等人：“参与起始和延伸的第二种软骨素 N-乙酰半乳糖胺基转移酶的分子克隆和表达......” J.Biol.Chem.. 278. 3072-3078 (2003)

Kim, B.T., 北川裕之 他: "In vitro heparan sulfate polymerization : Crucial roles of core protein moieties of primer substrates in addition to the EXT1/EXT2 interaction."J.Biol.Chem.. 278・38. 36115-36127 (2003)

Kim, B.T., Hiroyuki Kitakawa 等人：“体外硫酸乙酰肝素聚合：除 EXT1/EXT2 相互作用外，引物底物核心蛋白部分的重要作用。J.Biol.Chem.. 278・36127” (2003)