The cooperative activity between the carboxyl-terminal TSP1 repeats and the CUB domains of ADAMTS13 is crucial for recognition of von Willebrand factor under flow
The cooperative activity between the carboxyl-terminal TSP1 repeats and the CUB domains of ADAMTS13 is crucial for recognition of von Willebrand factor under flow
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DOI：
10.1182/blood-2007-04-083329
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发表时间：
2007-09-15
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：
Zhang, Ping;Pan, Weilan;Zheng, X. Long
ADAMTS13在A2亚基中心的Tyr（1605）和Met（1606）残基之间切割von Willebrand因子（VWF）。ADAMTS13蛋白酶的氨基端似乎足以在静态和变性条件下结合和切割VWF。然而，ADAMTS13的羧基末端在底物识别中的作用仍然存在争议。目前的研究表明，ADAMTS13在一个小型涡流器上以旋转速度和蛋白酶浓度依赖的方式切割VWF。在相同条件下，去除CUB结构域（delCUB）或截断间隔结构域（MDTCS）会显著削弱其切割VWF的能力。ADAMTS13和delCUB（而不是MDTCS）在流动中与VWF结合，解离常数（K-D）分别约为50 nM和274 nM。分离的CUB结构域既不足以检测到结合VWF，也不能抑制ADAMTS13在流动下对VWF的蛋白水解裂解。在CUB结构域添加TSP1 5-8 （T5-8CUB）或TSP1 2-8重复序列（T2-8CUB）恢复了对VWF的结合亲和力，并抑制了ADAMTS13在流动条件下对VWF的切割作用。这些数据直接和定量地证明了中间羧基末端TSP1重复序列与末端羧基末端CUB结构域之间的协同活性可能对流动条件下VWF的识别和切割至关重要。
ADAMTS13 cleaves von Willebrand factor (VWF) between Tyr(1605) and Met(1606) residues at the central A2 subunit. The aminoterminus of ADAMTS13 protease appears to be sufficient to bind and cleave VWF under static and denatured condition. However, the role of the carboxyl-terminus of ADAMTS13 in substrate recognition remains controversial. Present study demonstrates that ADAMTS13 cleaves VWF in a rotation speed- and protease concentration-dependent manner on a mini vortexer. Removal of the CUB domains (delCUB) or truncation after the spacer domain (MDTCS) significantly impairs its ability to cleave VWF under the same condition. ADAMTS13 and delCUB (but not MDTCS) bind VWF under flow with dissociation constants (K-D) of about 50 nM and about 274 nM, respectively. The isolated CUB domains are neither sufficient to bind VWF detectably nor capable of inhibiting proteolytic cleavage of VWF by ADAMTS13 under flow. Addition of the TSP1 5-8 (T5-8CUB) or TSP1 2-8 repeats (T2-8CUB) to the CUB domains restores the binding affinity toward VWF and the inhibitory effect on cleavage of VWF by ADAMTS13 under flow. These data demonstrate directly and quantitatively that the cooperative activity between the middle carboxyl-terminal TSP1 repeats and the distal carboxyl-terminal CUB domains may be crucial for recognition and cleavage of VWF under flow.