Kitagawa Hiroshi, Tsutsumi Kae, Ikegami-Kuzuhara Akemi, Nadanaka Satomi, Goto Fumitaka, Ogawa Tomoya, Sugahara Kazuyuki
Department of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan.
Department of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan.
J Biol Chem. 2008 Oct 10;283(41):27438-27443. doi: 10.1074/jbc.M803279200. Epub 2008 Aug 11.
6-O-Sulfated galactose residues have been demonstrated in the glycosaminoglycan-protein linkage region GlcUAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser isolated from shark cartilage chondroitin 6-sulfate (Sugahara, K., Ohi, Y., Harada, T., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 6027-6035). In this study, we investigated whether a recombinant human chondroitin 6-sulfotransferase-1 (C6ST-1) catalyzes the sulfation of C6 on both galactose residues in the linkage region using structurally defined acceptor substrates. The C6ST-1 was expressed as a soluble protein A chimeric form in COS-1 cells and purified using IgG-Sepharose. The purified C6ST-1 utilized the linkage tri-, tetra-, penta-, and hexasaccharide-serines and hexasaccharide alditols, including GlcUAbeta1-3GalNAc(4-O-sulfate)beta1-4GlcUAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xylbeta1-O-Ser and DeltaGlcUAbeta1-3GalNAc(6-O-sulfate)beta1-4GlcUAbeta1-3Galbeta1-3Gal(6-O-sulfate)beta1-4Xyl-ol. Identification of the reaction products obtained with the linkage tetra-, penta-, and hexasaccharide-serines revealed that the C6ST-1 catalyzed the sulfation of C6 on both galactose residues in the linkage region. Notably, the linkage tetrasaccharide-peptide GlcUAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-(Gly)Ser-(Gly-Glu) was a good acceptor substrate for the C6ST-1, suggesting that the sulfation of the galactose residues can occur before the transfer of the first N-acetylhexosamine residue to the linkage tetrasaccharide. In contrast, no incorporation was observed into DeltaGlcUAbeta1-3GalNAc(4-O-sulfate)beta1-4GlcUAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xyl-ol, indicating that an intact xylose is necessary for the transfer of a sulfate to the second sugar residue Gal from the reducing end. These findings clearly demonstrated that the recombinant C6ST-1 catalyzes the sulfation of C6 on both galactose residues in the linkage region in vitro. This is the first identification of the sulfotransferase responsible for the sulfation of galactose residues in the glycosaminoglycan-protein linkage region.
在从鲨鱼软骨硫酸软骨素6 - 硫酸酯中分离得到的糖胺聚糖 - 蛋白质连接区域GlcUAbeta1 - 3Galbeta1 - 3Galbeta1 - 4Xylbeta1 - O - Ser中已证实存在6 - O - 硫酸化半乳糖残基（菅原健、大日义、原田彻、德瓦尔德、弗利根哈特，《生物化学杂志》，1992年，第267卷，第6027 - 6035页）。在本研究中，我们使用结构明确的受体底物，研究了重组人软骨素6 - 硫酸转移酶 - 1（C6ST - 1）是否催化连接区域中两个半乳糖残基上C6位的硫酸化。C6ST - 1在COS - 1细胞中表达为可溶性蛋白A嵌合形式，并使用IgG - 琼脂糖进行纯化。纯化后的C6ST - 1利用连接三糖、四糖、五糖和六糖 - 丝氨酸以及六糖醇，包括GlcUAbeta1 - 3GalNAc(4 - O - 硫酸酯)beta1 - 4GlcUAbeta1 - 3Gal(4 - O - 硫酸酯)beta1 - 3Galbeta1 - 4Xylbeta1 - O - Ser和DeltaGlcUAbeta1 - 3GalNAc(6 - O - 硫酸酯)beta1 - 4GlcUAbeta1 - 3Galbeta1 - 3Gal(6 - O - 硫酸酯)beta1 - 4Xyl - ol。对连接四糖、五糖和六糖 - 丝氨酸反应产物的鉴定表明，C6ST - 1催化连接区域中两个半乳糖残基上C6位的硫酸化。值得注意的是，连接四糖 - 肽GlcUAbeta1 - 3Galbeta1 - 3Galbeta1 - 4Xylbeta1 - O - (Gly)Ser - (Gly - Glu)是C6ST - 1的良好受体底物，这表明半乳糖残基的硫酸化可以在第一个N - 乙酰己糖胺残基转移到连接四糖之前发生。相比之下，未观察到DeltaGlcUAbeta1 - 3GalNAc(4 - O - 硫酸酯)beta1 - 4GlcUAbeta1 - 3Gal(4 - O - 硫酸酯)beta1 - 3Galbeta1 - 4Xyl - ol中有掺入现象，这表明完整的木糖对于从还原端向第二个糖残基半乳糖转移硫酸是必需的。这些发现清楚地表明，重组C6ST - 1在体外催化连接区域中两个半乳糖残基上C6位的硫酸化。这是首次鉴定出负责糖胺聚糖 - 蛋白质连接区域中半乳糖残基硫酸化的硫酸转移酶。
