Arg-Ser-<sup>775</sup>, <sup>792</sup> and <sup>823</sup> in Spacer Region of ADAMTS-18 Is Critical for Thrombin Cleavage — Oak Academic Publishing
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Arg-Ser-<sup>775</sup>, <sup>792</sup> and <sup>823</sup> in Spacer Region of ADAMTS-18 Is Critical for Thrombin Cleavage
Department of Pharmacology, Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai, China
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Department of Pharmacology, Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai, China
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Department of Pharmacology, Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai, China
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Key Laboratory of Brain Functional Genomics (East China Normal University), Ministry of Education, Shanghai, China
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Department of Biochemistry and Molecular Cell Biology, Institute of Medical Science, Shanghai Jiao Tong University School of Medicine, Shanghai, China
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Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, Shanghai, China
1 Department of Pharmacology, Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai, China
2 Department of Pharmacology, Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai, China
3 Department of Pharmacology, Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai, China
4 Key Laboratory of Brain Functional Genomics (East China Normal University), Ministry of Education, Shanghai, China
5 Department of Biochemistry and Molecular Cell Biology, Institute of Medical Science, Shanghai Jiao Tong University School of Medicine, Shanghai, China
6 Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, Shanghai, China
Cleavage of ADAMTS-18 by thrombin represents a new mechanism of platelet thrombus clearance via the release of active ~45-kDa C-terminal fragments that induces oxidative platelet fragmentation. The exact cleavage sites remain unclear, but Arg (R) 775 /Ser (S) 776 in spacer region of ADAMTS-18 has been shown to be one of the cleavage sites of thrombin. Here, we demonstrate that R 792 /S 793 and R 823 /S 824 are also thrombin cleavage sites by sequence analysis, amino acid mutation and mass spectrometry assay. All these cleavage sites are thrombin-specific and insensitive to other enzymes tested (e.g. cathepsin D or trypsin). Simultaneous mutation of R 775 , 792 , 823 to S 775 , 792 , 823 in ADAMTS-18 completely abrogated the cleavage by thrombin and the generation of active C-terminal 45-kDa fragments. Together with previous study, a total of three thrombin-specific cleavage sites have been identified in spacer region of ADAMTS-18.
KeywordsADAMTS-18ThrombinCleavageMass Spectrum
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