Chinese hamster ovary cell mutants defective in glycosaminoglycan assembly and glucuronosyltransferase I
Chinese hamster ovary cell mutants defective in glycosaminoglycan assembly and glucuronosyltransferase I
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DOI：
10.1074/jbc.274.19.13017
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发表时间：
1999-05-07
影响因子：
4.8
通讯作者：
Esko, JD
中科院分区：
文献类型：
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作者：
Bai, XM;Wei, G;Esko, JD
动物细胞的蛋白聚糖通常含有一种或多种硫酸乙酰肝素或硫酸软骨素链。这些糖胺聚糖在四糖引物 -GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta-O- 上组装，附着在核心蛋白中的特定丝氨酸残基上。对途径第一种或第二种酶（木糖基转移酶和半乳糖基转移酶 I）有缺陷的中国仓鼠卵巢细胞突变体的研究表明，引物的组装是通过将单个单糖残基从相应的高能核苷酸糖供体顺序转移到生长链的非还原端来进行的。为了研究连接四糖组装中涉及的其他反应，我们设计了一种基于诱导对由碱性成纤维细胞生长因子皂草素组成的丝裂毒素的抗性的强大选择方法。一类突变体不将 (SO4)-S-35 和 [6-H-3]GlcN 掺入糖胺聚糖链中。这些细胞与萘酚-β-D-木糖苷 (Xyl beta-O-Np) 一起孵育，导致含有 1 或 2 mol 半乳糖（Gal beta 1、4Xyl beta-O-Np 和 Gal beta 1、3Gal beta 1、4Xyl beta-O-Np）和唾液酸（Sia α 2,3Gal beta 1、3Gal）的连接区中间体积累beta 1, 4Xyl beta-O-Np)，但不包括任何含 GlcA 的寡糖。突变体的提取物完全缺乏 UDP-葡萄糖醛酸：Gal beta 1,3Gal-R 葡萄糖醛酸基转移酶 (GlcAT-I) 活性，通过 GlcA 从 UDP-GlcA 转移到 Gal beta 1,3Gal beta-O-萘甲醇来测量（
The proteoglycans of animal cells typically contain one or more heparan sulfate or chondroitin sulfate chains. These glycosaminoglycans assemble on a tetrasaccharide primer, -GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta-O-, attached to specific serine residues in the core protein. Studies of Chinese hamster ovary cell mutants defective in the first or second enzymes of the pathway (xylosyl-transferase and galactosyltransferase I) show that the assembly of the primer occurs by sequential transfer of single monosaccharide residues from the corresponding high energy nucleotide sugar donor to the non-reducing end of the growing chain. In order to study the other reactions involved in linkage tetrasaccharide assembly, we have devised a powerful selection method based on induced resistance to a mitotoxin composed of basic fibroblast growth factor-saporin. One class of mutants does not incorporate (SO4)-S-35 and [6-H-3]GlcN into glycosaminoglycan chains. Incubation of these cells with naphthol-beta-D-xyloside (Xyl beta-O-Np) resulted in accumulation of linkage region intermediates containing 1 or 2 mol of galactose (Gal beta 1, 4Xyl beta-O-Np and Gal beta 1, 3Gal beta 1, 4Xyl beta-O-Np) and sialic acid (Sia alpha 2,3Gal beta 1, 3Gal beta 1, 4Xyl beta-O-Np) but not any GlcA-containing oligosaccharides. Extracts of the mutants completely lacked UDP-glucluronic acid:Gal beta 1,3Gal-R glucuronosyltransferase (GlcAT-I) activity, as measured by the transfer of GlcA from UDP-GlcA to Gal beta 1,3Gal beta-O- naphthalenemethanol (