Amino acid residues Arg659, Arg660, and Tyr661 in the spacer domain of ADAMTS13 are critical for cleavage of von Willebrand factor
Amino acid residues Arg659, Arg660, and Tyr661 in the spacer domain of ADAMTS13 are critical for cleavage of von Willebrand factor
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DOI：
10.1182/blood-2009-07-235101
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发表时间：
2010-03-18
期刊：
影响因子：
20.3
通讯作者：
Zheng, X. Long
中科院分区：
文献类型：
--
作者：
Jin, Sheng-Yu;Skipwith, Christopher G.;Zheng, X. Long
先前的研究表明，ADAMTS13 间隔区是切割血管性血友病因子 (VWF) 所必需的。然而，该结构域内对于底物识别至关重要的确切氨基酸残基尚不清楚。来自血栓性血小板减少性紫癜患者的抗 ADAMTS13 免疫球蛋白 G 的表位作图以及人类、小鼠和斑马鱼的 ADAMTS13 间隔区与人类和小鼠 ADAMTS1（ADAMTS 家族密切相关成员）的序列比对，为研究 ADAMTS13 间隔区的 Arg (659) 和 Glu (664) 之间的氨基酸残基在底物中的作用提供了线索。认可。从 ADAMTS13 间隔结构域中删除所有这 6 个氨基酸残基（即 Arg(659)-Glu(664)）会导致 VWF73 肽、盐酸胍变性 VWF 和流体剪切应力下的天然 VWF 以及内皮细胞上的超大 VWF 的蛋白水解活性显着降低。定点诱变、动力学分析和肽抑制测定进一步确定了氨基酸残基 Arg(659)、Arg(660) 和 Tyr(661) 在静态和流体剪切应力条件下各种底物的蛋白水解裂解中的作用。这些发现可能为 ADAMTS13 的结构与功能关系提供新的见解，并帮助我们了解血栓性血小板减少性紫癜和其他与 VWF 蛋白水解受损相关的动脉血栓的发病机制。 （血。2010；115：2300-2310）
Previous studies have shown that ADAMTS13 spacer domain is required for cleavage of von Willebrand factor (VWF). However, the exact amino acid residues within this domain critical for substrate recognition are not known. Epitope mapping of anti-ADAMTS13 immunoglobulin G from patients with thrombotic thrombocytopenic purpura and sequence alignment of the ADAMTS13 spacer domains of human, mouse, and zebrafish with these of human and murine ADAMTS1, a closely related member of ADAMTS family, have provided hints to investigate the role of the amino acid residues between Arg(659) and Glu(664) of theADAMTS13 spacer domain in substrate recognition. A deletion of all these 6 amino acid residues (ie, Arg(659)-Glu(664)) from the ADAMTS13 spacer domain resulted in dramatically reduced proteolytic activity toward VWF73 peptides, guanidine-HCl denatured VWF, and native VWF under fluid shear stress, as well as ultralarge VWF on endothelial cells. Site-directed mutagenesis, kinetic analyses, and peptide inhibition assays have further identified a role for amino acid residues Arg(659), Arg(660), and Tyr(661) in proteolytic cleavage of various substrates under static and fluid shear stress conditions. These findings may provide novel insight into the structural-function relationship of ADAMTS13 and help us to understand pathogenesis of thrombotic thrombocytopenic purpura and other arterial thromboses associated with compromised VWF proteolysis. (Blood. 2010; 115: 2300-2310)