Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, USA.
J Biol Chem. 2010 Oct 29;285(44):34155-67. doi: 10.1074/jbc.M110.109553. Epub 2010 Aug 21.
Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.
硫酸软骨素 (CS) 是一种由 N-乙酰-D-半乳糖胺和 d-葡萄糖醛酸残基重复二糖单位组成的多糖，在各种位置带有硫酸化残基修饰。迄今为止，已经鉴定出六种用于软骨素合成的糖基转移酶，并且假定硫酸软骨素合酶-1 (CSS1)/软骨素合酶-1 (ChSy-1) 和硫酸软骨素合酶-2 (CSS2)/软骨素聚合因子复合物在 CS 生物合成中起主要作用。我们在数据库中发现了一种小鼠 CSS2 的替代剪接变体，该变体缺乏 N 端跨膜结构域，与原始 CSS2 形成对比。在这里，我们研究了 CSS2 变体的作用。在组织中，原始酶和剪接变体（分别命名为 CSS2A 和 CSS2B）以不同的水平和比例表达。对培养的小鼠胚胎成纤维细胞的 Western blot 分析证实，这两种酶实际上都作为蛋白质合成，并定位于内质网和高尔基体中。下拉测定表明，CSS2A、CSS2B 和 CSS1/ChSy-1 中的任何一种都可以异质和同质相互作用，表明它们形成多聚体复合物。体外糖基转移酶测定表明，CSS2B 的葡萄糖醛酸基转移酶活性降低，并且与 CSS1 共表达时没有聚合活性，而与 CSS1 共表达的 CSS2B 则没有聚合活性。用放射性标记分析过表达每种变体的 COS-7 细胞表明，CSS2A 促进 CS 生物合成，而 CSS2B 抑制 CS 生物合成。CSS2A 和 CSS2B 的分子建模为它们的特性提供了支持。这些发现表明 CSS2 变体调节 CS 链聚合，为阐明 CS 生物合成的机制提供了线索。
