Institute for Molecular Science of Medicine, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan.
J Biol Chem. 2012 Dec 21;287(52):43390-400. doi: 10.1074/jbc.M112.412676. Epub 2012 Nov 5.
Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-D-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ∼90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ∼29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions.
硫酸软骨素（CS）是一种线性酸性多糖，由重复的二糖单位葡萄糖醛酸和 N-乙酰-D-半乳糖胺组成，并在不同位置修饰硫酸根基团，在发育和疾病中发挥各种作用。在这里，我们使用细菌软骨素聚合酶和重组 CS 硫酸转移酶，包括软骨素-4-硫酸转移酶 1（C4ST-1）、软骨素-6-硫酸转移酶 1（C6ST-1）、N-乙酰半乳糖胺 4-硫酸 6-硫酸转移酶（GalNAc4S-6ST）和尿苷酰基 2-硫酸转移酶（UA2ST），从六胺连接在还原端的六糖硫酸软骨素开始，化学酶法合成了具有不同长度和不同硫酸化组成的各种 CS 物质。连续修饰 CS 与一系列 CS 硫酸转移酶揭示了它们的独特特征，包括它们的底物特异性。与软骨素聚合酶的反应生成非硫酸化的软骨素，而与 C4ST-1 和 C6ST-1 的反应分别生成含有>95%4S 和 6S 单位的均匀硫酸化的 CS。GalNAc4S-6ST 和 UA2ST 生成高度硫酸化的 CS，具有约 90%相应的二硫酸化二糖单位。与 UA2ST 和 GalNAc4S-6ST 的连续反应生成进一步高度硫酸化的 CS，含有混合的二硫酸化单元结构。令人惊讶的是，与 GalNAc4S-6ST 和 UA2ST 的连续反应生成了一种含有约 29%三硫酸化二糖单位的新型 CS 分子。使用 CS 文库和末端用生物素修饰的天然 CS 产物进行酶联免疫吸附试验和表面等离子体共振分析，揭示了 CS 物质与抗 CS 抗体以及与 CS 结合分子（如中期因子和多效蛋白）相互作用的细节。化学酶法合成能够生成具有所需长度、组成和独特结构的 CS 链，所得文库将是研究 CS 功能的有用工具。
