Sulfation of the galactose residues in the glycosaminoglycan-protein linkage region by recombinant human chondroitin 6-O-sulfotransferase-1
Sulfation of the galactose residues in the glycosaminoglycan-protein linkage region by recombinant human chondroitin 6-O-sulfotransferase-1
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DOI：
10.1074/jbc.m803279200
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发表时间：
2008-10-10
影响因子：
4.8
通讯作者：
Sugahara, Kazuyuki
中科院分区：
文献类型：
--
作者：
Kitagawa, Hiroshi;Tsutsumi, Kae;Sugahara, Kazuyuki
从鲨鱼软骨6-硫酸软骨素中分离出的糖胺聚糖-蛋白连接区GlcUA β 1-3Gal β 1-3Gal- 4xyl β 1-O-Ser （Sugahara， K., Ohi， Y., Harada， T., de ward， P., and vliegenhart， J. F. G. (1992) J. Biol.）。化学，267,6027-6035)。在这项研究中，我们研究了重组人软骨素6-亚砜转移酶-1 （C6ST-1）是否使用结构上确定的受体底物催化C6在连接区域的两个半乳糖残基上的硫酸化。C6ST-1以可溶性蛋白a嵌合形式在COS-1细胞中表达，并使用IgG-Sepharose纯化。纯化的C6ST-1利用了三、四、五、六糖丝氨酸和六糖醛醇的连锁，包括GlcUA β 1-3GalNAc（4- o -硫酸盐）β 1-4GlcUA β 1-3Gal（4- o -硫酸盐）β 1-3Gal β 1-4Xy1 -o -ser和Delta GlcUA β 1-3Gal- nac （6- o -硫酸盐）β 1-4GlcUA β 1-3Gal β 1-3Gal（6- o -硫酸盐）β 1- 4xygal β 1-3Gal（6- o -硫酸盐）β 1- 4xygal β 1- 4xygal （6- o -硫酸盐）β 1-4Xyl-ol。通过对四糖、五糖和六糖的连锁反应产物的鉴定，发现C6ST-1催化了C6在连锁区域的两个半乳糖残基上的磺化反应。值得注意的是，链接四糖肽GlcUA β 1-3Gal β 1-3Gal β 1-4Xyl β 1-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能发生在第一个n -乙酰己糖残基转移到链接四糖之前。相比之下，δ GlcUA β 1- 3GalNAc(4-O-sulfate) β 1- 4glcua β 1- 3gal (4-O-sulfate) β 1- 3gal β 1- 4xyl -ol中未观察到掺入，这表明硫酸盐从还原端转移到第二个糖渣Gal中需要完整的木糖。这些结果清楚地表明，重组C6ST-1在体外催化C6在连接区域的两个半乳糖残基上的硫酸化。这是第一次鉴定负责在糖胺聚糖-蛋白连接区域的半乳糖残基磺化的硫代转移酶。
6-O-Sulfated galactose residues have been demonstrated in the glycosaminoglycan-protein linkage region GlcUA beta 1-3Gal beta 1-3Gal-4Xyl beta 1-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 GlcUA beta 1-3GalNAc(4-O-sulfate)beta 1-4GlcUA beta 1-3Gal(4-O-sulfate) beta 1-3Gal beta 1-4Xy1 beta 1-O-Ser and Delta GlcUA beta 1-3Gal-NAc(6-O-sulfate)beta 1-4GlcUA beta 1-3Gal beta 1-3Gal(6-O-sulfate)beta 1-4Xyl-ol. Identification of the reaction products obtained with the linkage tetra-, penta-, and hexasaccharideserines revealed that the C6ST-1 catalyzed the sulfation of C6 on both galactose residues in the linkage region. Notably, the linkage tetrasaccharide-peptide GlcUA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-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 Delta GlcUA beta 1 3GalNAc(4-O-sulfate)beta 1-4GlcUA beta 1-3Gal(4-O-sulfate)beta 1-3Gal beta 1-4Xyl-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.