New tools for carbohydrate sulphation analysis: Heparan Sulphate 2- O -sulphotranserase (HS2ST) is a target for small molecule protein kinase inhibitors
New tools for carbohydrate sulphation analysis: Heparan Sulphate 2- O -sulphotranserase (HS2ST) is a target for small molecule protein kinase inhibitors
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碳水化合物硫酸化分析的新工具：硫酸乙酰肝素 2-O-磺基转移酶 (HS2ST) 是小分子蛋白激酶抑制剂的靶标
DOI：
10.1101/296533
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Byrne D
中科院分区：
文献类型：
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作者：
Byrne D
碳水化合物残基的磺化发生在多种聚糖上，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的生物合成涉及到膜结合的高尔基硫转移酶，包括硫酸乙酰肝素2- o-硫转移酶（HS2ST），它将硫酸盐从辅助因子PAPS（3 ' -磷酸腺苷5 ' -硫酸磷酸）转移到成熟寡糖链中α- l -伊杜酸的2-对偶体上。目前缺乏可用于量化碳水化合物硫酸化水平的简单非放射性酶测定方法，这阻碍了对这一过程的动力学分析和HS2ST抑制剂的发现。本文描述了纯化HS2ST热移分析的新方法。使用这种方法，我们使用一种新的合成荧光底物来量化hs2st催化的寡糖硫酸化，并筛选已发表的激酶抑制剂集（PKIS），以评估抑制催化的化合物。我们在体外报道了HS2ST对多种细胞渗透性化合物的敏感性，包括聚阴离子极性分子、蛋白激酶抑制剂rottlerin和基于氧吲哚的RAF激酶抑制剂。在与本文一起发表的一项相关研究中，我们证明了酪氨酸蛋白硫转移酶（TPSTs）也受到多种蛋白激酶抑制剂的抑制。我们提出，适当验证的小分子化合物可能成为快速抑制细胞中聚糖（和蛋白质）磺化的新工具，并且蛋白激酶抑制剂可能被重新利用或重新设计用于特异性抑制HS2ST。我们报道，HS2ST是一种依赖于ppps的聚糖亚硫酸盐转移酶，可以使用多种新的生化方法进行检测，包括一种基于非放射性酶的实时检测聚糖底物硫酸化的方法。HS2ST活性可被不同种类的化合物（包括已知的蛋白激酶抑制剂）抑制，这为评估HS2ST依赖的小分子硫酸化在细胞中的作用提供了新的方法。
Sulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2-O-sulphotransferase (HS2ST), which transfers sulphate from the co-factor PAPS (3’-phosphoadenosine 5’-phosphosulphate) to the 2-Oposition of α-L-iduronate in the maturing oligosaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compoundsin 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 this article, we demonstrate that Tyrosyl Protein Sulpho Tranferases (TPSTs) 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) sulphation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.SUMMARY STATEMENTWe report that HS2ST, which is a PAPS-dependent glycan sulphotransferase, can be assayed using a variety of novel biochemical procedures, including a non-radioactive enzyme-based assay that detects glycan substrate sulphation in real time. HS2ST activity can be inhibited by different classes of compounds, including known protein kinase inhibitors, suggesting new approaches to evaluate the roles of HS2ST-dependent sulphation with small molecules in cells.