New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors.
New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors.
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DOI：
10.1042/bcj20180265
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发表时间：
2018-08-14
期刊：
影响因子：
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通讯作者：
Eyers PA
中科院分区：
文献类型：
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作者：
Byrne DP;Li Y;Ramakrishnan K;Barsukov IL;Yates EA;Eyers CE;Papy-Garcia D;Chantepie S;Pagadala V;Liu J;Wells C;Drewry DH;Zuercher WJ;Berry NG;Fernig DG;Eyers PA
碳水化合物残基的硫酸化发生在用于分泌的各种聚糖上，并且这种修饰对于有效的基于基质的信号转导是必不可少的。硫酸乙酰肝素（HS）糖胺聚糖控制从血液凝固到细胞增殖的生理功能。HS生物合成涉及膜结合高尔基体磺基转移酶，包括HS 2-O-磺基转移酶（HS 2ST），其将硫酸盐从辅因子PAPS（3′-磷酸腺苷5′-磷酸硫酸盐）转移到成熟多糖链中α-l-艾杜糖醛酸的2-O位置。目前缺乏可用于量化碳水化合物硫酸化水平的简单的非放射性酶测定，这阻碍了该过程的动力学分析和HS 2ST抑制剂的发现。在本文中，我们描述了一个新的程序纯化HS 2ST的热位移分析。使用这种方法，我们使用一种新的合成荧光底物定量HS 2ST催化的寡糖硫酸化，并筛选已发表的激酶抑制剂集，以评估抑制催化的化合物。我们报告了HS 2ST对多种细胞渗透性化合物的体外敏感性，包括聚阴离子极性分子、蛋白激酶抑制剂rottlerin和基于羟吲哚的RAF激酶抑制剂。在与本研究背靠背发表的一项相关研究中，我们证明了酪氨酰蛋白磺基转移酶也被各种蛋白激酶抑制剂抑制。我们建议，经过适当验证的小分子化合物可以成为快速抑制细胞中聚糖（和蛋白质）硫酸化的新工具，并且蛋白激酶抑制剂可以重新利用或重新设计以特异性抑制HS 2ST。
Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.