Heparan sulfate is synthesized by most animal cells and interacts with numerous proteins via specific sulfation motifs to regulate various physiological processes. Various 3-O-sulfated motifs are considered to be key in controlling the binding specificities to the functional proteins. One such motif, synthesized by 3-O-sulfotransferase-1 (3OST-1) serves as a binding site for antithrombin (AT), and has been thoroughly studied because of its pharmacological importance. However, the physiological roles of 3-O-sulfates produced by other 3OST isoforms, which do not bind AT, remain obscure, in part due to the lack of a standard method to analyze this rare modification. This study aims to establish a method for quantifying 3-O-sulfated components of heparan sulfate, focusing on non-AT-binding units. We previously examined the reaction products of human 3OST isoforms and identified five 3-O-sulfated components, including three non-AT-binding disaccharides and two AT-binding tetrasaccharides, as digestion products of heparin lyases. In this study, we prepared these five components as a standard saccharide for HPLC analysis. Together with eight non-3-O-sulfated disaccharides, a standard mixture of thirteen units was prepared. Using reverse-phase ion-pair HPLC with a post-column fluorescent labeling system, the separation conditions were optimized to quantify the thirteen units. Finally, we analyzed the compositional changes of 3-O-sulfated units in heparan sulfate from P19 cells before and after neuronal differentiation. We successfully detected the 3-O-sulfated units specifically expressed in the differentiated neurons. This is the first report that shows the quantification of three non-AT-binding 3-O-sulfated units, and establishes a new approach to explore the physiological functions of 3-O-sulfate.

中文翻译：

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硫酸乙酰肝素中非抗凝血酶结合 3-O-硫酸化单元的定量方法

硫酸乙酰肝素由大多数动物细胞合成，并通过特定的硫酸化基序与多种蛋白质相互作用，以调节各种生理过程。各种 3-O-硫酸化基序被认为是控制功能蛋白结合特异性的关键。其中一个基序由 3-O-磺基转移酶-1 (3OST-1) 合成，可作为抗凝血酶 (AT) 的结合位点，并且由于其药理学重要性而已得到彻底研究。然而，其他不结合 AT 的 3OST 亚型产生的 3-O-硫酸盐的生理作用仍然不清楚，部分原因是缺乏分析这种罕见修饰的标准方法。本研究旨在建立一种定量硫酸乙酰肝素 3-O-硫酸化成分的方法，重点关注非 AT 结合单元。我们之前检查了人 3OST 亚型的反应产物，并鉴定了五种 3-O-硫酸化成分，包括三种非 AT 结合二糖和两种 AT 结合四糖，作为肝素裂解酶的消化产物。在本研究中，我们制备了这五种成分作为 HPLC 分析的标准糖。与八种非 3-O-硫酸化二糖一起，制备了十三个单位的标准混合物。使用带有柱后荧光标记系统的反相离子对 HPLC，优化分离条件以定量 13 个单位。最后，我们分析了神经元分化前后 P19 细胞硫酸乙酰肝素中 3-O-硫酸化单元的组成变化。我们成功检测到了分化神经元中特异表达的3-O-硫酸化单元。这是第一份对三个非AT结合的3-O-硫酸盐单元进行定量的报告，并建立了探索3-O-硫酸盐生理功能的新方法。