Specificities of three distinct human chondroitin/dermatan N-acetylgalactosamine 4-O-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor -: Implication of differential roles in dermatan sulfate biosynthesis
Specificities of three distinct human chondroitin/dermatan N-acetylgalactosamine 4-O-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor -: Implication of differential roles in dermatan sulfate biosynthesis
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DOI：
10.1074/jbc.m306044200
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发表时间：
2003-09-19
影响因子：
4.8
通讯作者：
Sugahara, K
中科院分区：
文献类型：
--
作者：
Mikami, T;Mizumoto, S;Sugahara, K
GalNAc的4-O-硫酸化是硫酸软骨素和硫酸皮肤素（DS）的高频修饰，并且已经鉴定了三种主要的GalNAc 4-O-磺基转移酶，包括皮肤素4-O-磺基转移酶-1（D4 ST-1）和软骨素4-O-磺基转移酶-1和-2（C4 ST-1和-2）。长期以来，人们一直认为DS生物合成过程中GalNAc的4-O-硫酸化是通过GlcUA的C5-差向异构化形成艾杜糖醛酸（IdoUA）的先决条件。最近基于使用微粒体的酶学研究论证了这一假设，即C5-差向异构化先于4-O-硫酸化，这进一步得到了克隆的D4 ST-1的特异性的支持，其在彻底降解的皮肤素中对侧接GlcUA-GalNAc的IdoUA-GalNAc的优势偏好超过侧接IdoUA-GalNAc的IdoUA-GalNAc。尽管反建议解释了初始反应，但显然它不能合理化成熟DS链典型的富含IdoUA-GalNAc（4-O-硫酸盐）的簇的合成机制。在这项研究中，我们研究了详细的特异性的三个重组人4-O-磺基转移酶使用部分取代DS作为受体。转移酶反应产物的酶分析表明，D4 ST-1在-IdoUA-GalNAc-IdoUA-中比在-GlcUA-GalNAc-GlcUA-序列中更有效地将硫酸根转移到GalNAc残基。相反，C4 ST-1表现出相反的偏好，C4 ST-2在两个序列中使用GalNAc残基的程度相当，这与其与D4 ST-1和C4 ST-1的系统发育关系一致。在软骨素酶AC-1消化S-35标记的转移酶反应产物后分离的寡糖的结构分析首次揭示，与C4 ST-1和C4 ST-2相比，D4 ST-1最有效地利用了不仅位于序列-IdoUA-GalNAc-IdoUA-中而且位于-GlcUA-GalNAc-IdoUA-和-IdoUA-GalNAc-GlcUA-中的GalNAc残基。分离的低聚糖结构还表明，4-O-硫酸化促进了邻近二糖单元中GalNAc的后续4-O-硫酸化。
4-O-Sulfation of GalNAc is a high frequency modification of chondroitin sulfate and dermatan sulfate (DS), and three major GalNAc 4-O-sulfotransferases including dermatan 4-O-sulfotransferase-1 (D4ST-1) and chondroitin 4-O-sulfotransferases-1 and -2 (C4ST-1 and -2) have been identified. 4-O-Sulfation of GalNAc during DS biosynthesis had long been postulated to be a prerequisite for iduronic acid (IdoUA) formation by C5-epimerization of GlcUA. This hypothesis has recently been argued based on enzymological studies using microsomes that C5-epimerization precedes 4-O-sulfation, which was further supported by the specificity of the cloned D4ST-1 with predominant preference for IdoUA-GalNAc flanked by GlcUA-GalNAc over IdoUA-GalNAc flanked by IdoUA-GalNAc in exhaustively desulfated dermatan. Whereas the counterproposal explains the initial reactions, apparently it cannot rationalize the synthetic mechanism of IdoUA-GalNAc(4-O-sulfate)-rich clusters typical of mature DS chains. In this study, we examined detailed specificities of the three recombinant human 4-O-sulfotransferases using partially desulfated DS as an acceptor. Enzymatic analysis of the transferase reaction products showed that D4ST-1 far more efficiently transferred sulfate to GalNAc residues in -IdoUA-GalNAc-IdoUA- than in -GlcUA-GalNAc-GlcUA-sequences. In contrast, C4ST-1 showed the opposite preference, and C4ST-2 used GalNAc residues in both sequences to comparable degrees, being consistent with its phylogenetic relations to D4ST-1 and C4ST-1. Structural analysis of the oligosaccharides, which were isolated after chondroitinase AC-I digestion of the S-35-labeled transferase reaction products, revealed for the first time that D4ST-1, as compared with C4ST-1 and C4ST-2, most efficiently utilized GalNAc residues located not only in the sequence -IdoUA-GalNAc-IdoUA- but also in -GlcUA-GalNAc-IdoUA- and -IdoUA-GalNAc-GlcUA-. The isolated oligosaccharide structures also suggest that 4-O-sulfation promotes subsequent 4-O-sulfation of GalNAc in the neighboring disaccharide unit.