The proximal carboxyl-terminal domains of ADAMTS13 determine substrate specificity and are all required for cleavage of von Willebrand factor

Academic Article

Abstract

  • ADAMTS13 limits platelet-rich thrombosis by cleaving von Willebrand factor at the Tyr1605-Met1606 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 Kd of ≈7.0 or 13 nM, comparable with full-length ADAMTS13 (Kd = 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 Kd 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 Asp1595 and Arg1668. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
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    Digital Object Identifier (doi)

    Author List

  • Ai J; Smith P; Wang S; Zhang P; Zheng XL
  • Start Page

  • 29428
  • End Page

  • 29434
  • Volume

  • 280
  • Issue

  • 33