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Amino acid residues Arg(659), Arg(660), and Tyr(661) in the spacer domain of ADAMTS13 are critical for cleavage of von Willebrand factor.

Abstract
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 the ADAMTS13 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.
AuthorsSheng-Yu Jin, Christopher G Skipwith, X Long Zheng
JournalBlood (Blood) Vol. 115 Issue 11 Pg. 2300-10 (Mar 18 2010) ISSN: 1528-0020 [Electronic] United States
PMID20075158 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
Chemical References
  • Immobilized Proteins
  • Mutant Proteins
  • Peptides
  • Recombinant Fusion Proteins
  • von Willebrand Factor
  • Tyrosine
  • Arginine
  • ADAM Proteins
  • ADAMTS13 Protein
  • ADAMTS13 protein, human
Topics
  • ADAM Proteins (chemistry, metabolism)
  • ADAMTS13 Protein
  • Amino Acid Sequence
  • Arginine (metabolism)
  • Cells, Cultured
  • Humans
  • Immobilized Proteins (metabolism)
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutant Proteins (chemistry, isolation & purification, metabolism)
  • Peptides (metabolism)
  • Protein Binding
  • Protein Multimerization
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins (chemistry, metabolism)
  • Rheology
  • Stress, Mechanical
  • Structure-Activity Relationship
  • Tyrosine (metabolism)
  • von Willebrand Factor (metabolism)

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