Hydrogen/deuterium exchange-LC-MS approach to characterize the action of heparan sulfate C5-epimerase
Hydrogen/deuterium exchange-LC-MS approach to characterize the action of heparan sulfate C5-epimerase
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DOI：
10.1007/s00216-011-5087-z
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发表时间：
2011-07-01
影响因子：
4.3
通讯作者：
Kuberan, Balagurunathan
中科院分区：
文献类型：
--
作者：
Babu, Ponnusamy;Victor, Xylophone V.;Kuberan, Balagurunathan
硫酸乙酰肝素（HS）蛋白聚糖在许多系统中调节许多生物学功能。HS的大部分功能归因于其独特的结构，由硫酸化和非硫酸化结构域组成，这是由于多糖链上沿着艾杜糖醛酸基和葡糖醛酸基残基的差异存在而产生的。一个单一的葡糖醛酸C5-差向异构酶作用于HS前体，将葡糖醛酸残基转化为艾杜糖醛酸残基，并调节随后的体内生物合成步骤。以前，多糖链内非硫酸化差向异构体的比例是通过使用繁琐的纸色谱技术解析放射性标记的GlcA-（A）Man（R）和IdoA-（A）Man（R）二糖来计算的。该放射性测定法基于测量H-3标记的HS前体底物的糖醛酸残基的C5碳处H-3的释放或掺入，已使用三十多年来表征HS C5-差向异构酶的作用。我们开发了一种非放射性测定法，通过在高效液相色谱上分离GlcA-（A）Man（R）和IdoA-（A）Man（R）二糖，结合差向异构化方案-液相色谱质谱法（DEEP-LC-MS）的氢/氘交换，来估计差向异构酶活性。利用这种新的，非放射性为基础的测定，深度液相色谱-质谱法，我们能够确定的程度正向和反向反应的相同底物催化的C5-差向异构酶。这项研究的结果也提供了深入了解C5-差向异构酶的行动，并提供了一个机会，描绘快照的生物合成事件发生在高尔基体中的HSPG组装。
Heparan sulfate (HS) proteoglycans regulate a number of biological functions in many systems. Most of the functions of HS are attributed to its unique structure, consisting of sulfated and non-sulfated domains, arising from the differential presence of iduronyl and glucuronyl residues along the polysaccharide chain. A single glucuronyl C5-epimerase enzyme acts on HS precursors, converts glucuronyl residues into iduronyl residues, and modulates subsequent biosynthetic steps in vivo. Previously, the ratios of non-sulfated epimers within the polysaccharide chain have been calculated by resolving radiolabeled GlcA-(A)Man(R) and IdoA-(A)Man(R) disaccharides using a tedious paper chromatography technique. This radioactive assay, based on measuring either the release or incorporation of H-3 at C5 carbon of uronyl residues of H-3-labeled HS precursor substrate, has been in use over three decades to characterize the action of HS C5-epimerase. We have developed a non-radioactive assay to estimate the epimerase activity through resolving GlcA-(A)Man(R) and IdoA-(A)Man(R) disaccharides on high-performance liquid chromatography in conjunction with hydrogen/deuterium exchange upon epimerization protocol-liquid chromatography mass spectrometry (DEEP-LC-MS). Utilizing this new, non-radioactive-based assay, DEEP-LC-MS, we were able to determine the extent of both forward and reverse reactions on the same substrate catalyzed by C5-epimerase. The results from this study also provide insights into the action of C5-epimerase and provide an opportunity to delineate snapshots of biosynthetic events that occur during the HSPG assembly in the Golgi.