Studies on Biosynthetic Mechanism of the Sulfated Glycosaminoglycans

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成酶的cDNA克隆及新功能的验证：我们克隆了秀丽隐杆线虫软骨素合成酶同源基因，并通过RNA介导的干扰技术使其产物表达缺失。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

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