title: ADAMTS-13 cysteine-rich/spacer domains are functionally essential for von Willebrand factor cleavage.
abstract: A severe lack of von Willebrand factor-cleaving protease (VWF-CP) activity can cause thrombotic thrombocytopenic purpura (TTP). This protease was recently identified as a member of the ADAMTS family, ADAMTS-13. It consists of a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, additional Tsp1 repeats, and CUB domains. To explore the structural and functional relationships of ADAMTS-13, we prepared here 13 sequential COOH-terminal truncated mutants and a single-point mutant (ArgGlyAsp [RGD] to ArgGlyGlu [RGE] in the cysteine-rich domain) and compared the activity of each mutant with that of the wild-type protein. The results revealed that the truncation of the cysteine-rich/spacer domains caused a remarkable reduction in VWF-CP activity. We also prepared immunoglobulin G (IgG) fractions containing inhibitory autoantibodies against ADAMTS-13 from plasma from 3 patients with acquired TTP, and we performed mapping of their epitopes using the aforementioned mutants. The major epitopes of these antibodies were found to reside within the cysteine-rich/spacer domains. These results suggest that the ADAMTS-13 cysteine-rich/spacer domains are essential for VWF-CP activity.
authors.affiliation: First Research Department, The Chemo-Sero-Therapeutic Research Institute, Kawabe, Kyokushi, Kikuchi, Kumamoto 869-1298, Japan. soejima@kaketsuken.or.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Soejima
authors.forename: Masanori
authors.initials: M
authors.lastname: Matsumoto
authors.forename: Koichi
authors.initials: K
authors.lastname: Kokame
authors.forename: Hideo
authors.initials: H
authors.lastname: Yagi
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Ishizashi
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Maeda
authors.forename: Chikateru
authors.initials: C
authors.lastname: Nozaki
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Miyata
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Fujimura
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Nakagaki
mesh_terms: D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D001323:Autoantibodies / Q000097:blood; D003545:Cysteine; D018604:Epitope Mapping; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000502:physiology; D011697:Purpura, Thrombotic Thrombocytopenic / Q000276:immunology; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D001323:Autoantibodies; D007074:Immunoglobulin G; D014841:von Willebrand Factor; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D003545:Cysteine