Ai Jihui, Smith Paula, Wang Shuwei, Zhang Ping, Zheng X Long
Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia and The University of Pennsylvania School of Medicine, Philadelphia, 19104, USA.
J Biol Chem. 2005 Aug 19;280(33):29428-34. doi: 10.1074/jbc.M505513200. Epub 2005 Jun 23.
ADAMTS13 limits platelet-rich thrombosis by cleaving von Willebrand factor at the Tyr(1605)-Met(1606) bond. Previous studies showed that ADAMTS13 truncated after spacer domain remains proteolytically active or hyperactive. However, the relative contribution of each domain within the proximal carboxyl terminus of ADAMTS13 in substrate recognition and specificity is not known. We showed that a metalloprotease domain alone was unable to cleave the Tyr-Met bond of glutathione S-transferase (GST)-VWF73-H substrate in 3 h, but it did cleave the substrate at a site other than the Tyr-Met bond after 16-24 h of incubation. Remarkably, the addition of even one or several proximal carboxyl-terminal domains of ADAMTS13 restored substrate specificity. Full proteolytic activity, however, was not achieved until all of the proximal carboxyl-terminal domains were added. The addition of TSP1 2-8 repeats and two CUB domains did not further increase proteolytic activity. Furthermore, ADAMTS13 truncated after the spacer domain with or without metalloprotease domain bound GST-VWF73-H with a K(d) of approximately 7.0 or 13 nm, comparable with full-length ADAMTS13 (K(d) = 4.6 nm). Metalloprotease domain did not bind GST-VWF73-H detectably, but the disintegrin domain, first TSP1 repeat, Cys-rich domain, and spacer domain bound GST-VWF73-H with K(d) values of 489, 136, 121, and 108 nm, respectively. These proximal carboxyl-terminal domains dose-dependently inhibited cleavage of fluorescent resonance energy transfer (FRETS)-VWF73 by full-length ADAMTS13 and ADAMTS13 truncated after the spacer domain. These data demonstrated that the proximal carboxyl-terminal domains of ADAMTS13 determine substrate specificity and are all required for recognition and cleavage of von Willebrand factor between amino acid residues Asp(1595) and Arg(1668).
ADAMTS13 通过在Tyr(1605)-Met(1606)键处切割血管性血友病因子来限制富含血小板的血栓形成。先前的研究表明，在间隔域后截断的ADAMTS13 仍具有蛋白水解活性或高活性。然而，ADAMTS13 近端羧基末端内每个结构域在底物识别和特异性中的相对贡献尚不清楚。我们发现单独的金属蛋白酶结构域在3小时内无法切割谷胱甘肽S-转移酶(GST)-VWF73-H底物的Tyr-Met键，但在孵育16-24小时后，它确实在Tyr-Met键以外的位点切割了底物。值得注意的是，即使添加一个或几个ADAMTS13的近端羧基末端结构域也能恢复底物特异性。然而，直到添加了所有近端羧基末端结构域才实现完全的蛋白水解活性。添加TSP1 2-8重复序列和两个CUB结构域并没有进一步提高蛋白水解活性。此外，在间隔域后截断的ADAMTS13，无论有无金属蛋白酶结构域，与GST-VWF73-H的结合解离常数(K(d))约为7.0或13 nM，与全长ADAMTS13相当(K(d)=4.6 nM)。金属蛋白酶结构域未检测到与GST-VWF73-H结合，但解整合素结构域、第一个TSP1重复序列、富含半胱氨酸结构域和间隔域与GST-VWF73-H的结合解离常数(K(d))分别为489、136、121和108 nM。这些近端羧基末端结构域以剂量依赖的方式抑制全长ADAMTS13和在间隔域后截断的ADAMTS13对荧光共振能量转移(FRETS)-VWF73的切割。这些数据表明，ADAMTS13的近端羧基末端结构域决定底物特异性，并且是识别和切割血管性血友病因子在氨基酸残基Asp(1595)和Arg(1668)之间所必需的。
