Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von Willebrand factor-induced platelet agglutination
Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von Willebrand factor-induced platelet agglutination
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Botrocetin-2（一种蛇毒辅助因子，用于冯维勒布兰德因子诱导的血小板凝集）的鉴定和重组分析
DOI：
10.1021/bi300442c
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发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
Matsui T
中科院分区：
文献类型：
--
作者：
Yamamoto-Suzuki Y;Sakurai Y;Fujimura Y;Matsumoto M;Hamako J;Kokubo T;Kitagawa H;Kawsar SMA;Fujii Y;Ozeki Y;Matsushita F;Matsui T
Botrocetin 是一种异二聚体蛇毒蛋白，可在体外诱导血管性血友病因子 (VWF) 和血小板糖蛋白 Ib (GPIb) 依赖性血小板凝集。我们从哈拉卡角蚁毒腺cDNA文库中克隆了与botrocetin亚基高度相似的botrocetin-2 cDNA。在 293T 细胞中表达的重组 botrocetin-2 表现出与天然 botrocetin 相当的辅因子活性。在单亚基表达实验中，获得了β亚基的二聚体，它表现出降低但明显的血小板凝集活性。 Ala 扫描诱变显示，β 亚基中 Asp62、Asp70、Arg115 或 Lys117 的取代降低了血小板凝集活性。 Botrocetin-2与VWF A1结构域和GPIbα复合的3D同源模型表明，β亚基的Asp62、Arg115和Lys117分别位于GPIbα的Arg218和Asp222附近，并且Aspβ70靠近A1结构域的Gln1391。我们的结果表明，β 亚基中的这些带电氨基酸残基在 botrocetin-2 的活性中具有优先作用。由于从天然毒液中获得均质的 Botrocetin 既费时又困难，因此重组 Botrocetin-2 作为标准试剂对止血和血栓形成的临床和基础研究具有潜在的益处。
Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutinationin vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland ofBothrops jararacahaving a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the β subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the β subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the β subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspβ70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these charged amino acid residues in the β subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.