Kitagawa H, Uyama T, Sugahara K
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan.
J Biol Chem. 2001 Oct 19;276(42):38721-6. doi: 10.1074/jbc.M106871200. Epub 2001 Aug 20.
We have identified a human chondroitin synthase from the HUGE (human unidentified gene-encoded large proteins) protein data base by screening with two keywords: "one transmembrane domain" and "galactosyltransferase family." The identified protein consists of 802 amino acids with a type II transmembrane protein topology. The protein showed weak homology to the beta1,3-galactosyltransferase family on the amino-terminal side and to the beta1,4-galactosyltransferase family on the carboxyl-terminal side. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active enzyme, which transferred not only the glucuronic acid (GlcUA) from UDP-[(14)C]GlcUA but also N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to the polymer chondroitin. Identification of the reaction products demonstrated that the enzyme was chondroitin synthase, with both beta1,3-GlcUA transferase and beta1,4-GalNAc transferase activities. The coding region of the chondroitin synthase was divided into three discrete exons and localized to chromosome 15. Northern blot analysis revealed that the chondroitin synthase gene exhibited ubiquitous but markedly differential expression in the human tissues examined. Thus, we demonstrated that analogous to human heparan sulfate polymerases, the single polypeptide chondroitin synthase possesses two glycosyltransferase activities required for chain polymerization.
我们通过使用“一个跨膜结构域”和“半乳糖基转移酶家族”这两个关键词，从HUGE（人类未鉴定基因编码的大蛋白）蛋白质数据库中鉴定出一种人软骨素合酶。鉴定出的蛋白质由802个氨基酸组成，具有II型跨膜蛋白拓扑结构。该蛋白质在氨基末端与β1,3-半乳糖基转移酶家族有弱同源性，在羧基末端与β1,4-半乳糖基转移酶家族有弱同源性。该蛋白质的可溶性重组形式在COS-1细胞中的表达产生了一种活性酶，它不仅将UDP-[(14)C]葡萄糖醛酸（GlcUA）中的葡萄糖醛酸转移，还将UDP-[(3)H]N-乙酰半乳糖胺（GalNAc）中的N-乙酰半乳糖胺转移到聚合物软骨素上。反应产物的鉴定表明该酶是软骨素合酶，具有β1,3-葡萄糖醛酸转移酶和β1,4-N-乙酰半乳糖胺转移酶活性。软骨素合酶的编码区被分为三个离散的外显子，并定位于15号染色体。Northern印迹分析显示，软骨素合酶基因在所检测的人体组织中普遍存在但表达差异明显。因此，我们证明，与人类硫酸乙酰肝素聚合酶类似，单一多肽软骨素合酶具有链聚合所需的两种糖基转移酶活性。
