Proteolytic fragmentation and sugar chains of plasma ADAMTS13 purified by a conformation-dependent monoclonal antibody

Hisahide Hiura, Taei Matsui, Masanori Matsumoto, Yuji Hori, Ayami Isonishi, Seiji Kato, Takaaki Iwamoto, Toshio Mori, Yoshihiro Fujimura

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

ADAMTS13 is a metalloproteinase that specifically cleaves unusually large von Willbrand factor multimers under high-shear stress. Deficiency of ADAMTS13 activity induces a life-threatening generalized disease, thrombotic thrombocytopenic purpura. We established a simple and efficient method to purify plasma ADAMTS13 (pADAMTS13) from cryosupernatant using an anti-ADAMTS13 monoclonal antibody (A10) that recognizes a conformational epitope within the disintegrin-like domain. Using the purified pADAMTS13, the amino acid residues involved in cleavage by thrombin, plasmin and leucocyte elastase were determined, and the carbohydrate moieties of this enzyme was analysed by lectin blots. Purified pADAMTS13 had a specific activity of 300 U/mg (25,057-fold purification) and the pI was 5.1-5.5. Cleavage sites of the purified pADAMTS13 by three proteases were identified; thrombin cleaved the four peptidyl bonds between Arg257-Ala258, Arg459-Ser460, Arg888-Thr889 and Arg1176-Arg1177, plasmin cleaved the three peptidyl bonds between Arg257-Ala258, Arg888-Thr889 and Arg1176-Arg1177, and elastase cleaved the two peptidyl bonds between Ile380-Ala381 and Thr874-Ser875. Lectin blot analysis indicated the presence of non-reducing terminal α2-6 and α2-3-linked sialic acid residues with penultimate β-galactose residues on the N- and O-linked sugar chains of pADAMTS13, suggesting that pADAMTS13 is cleared from the circulation via the hepatic asialoglycoprotein receptor like other plasma glycoproteins.

Original languageEnglish
Pages (from-to)403-411
Number of pages9
JournalJournal of Biochemistry
Volume148
Issue number4
DOIs
Publication statusPublished - 01-10-2010

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'Proteolytic fragmentation and sugar chains of plasma ADAMTS13 purified by a conformation-dependent monoclonal antibody'. Together they form a unique fingerprint.

  • Cite this

    Hiura, H., Matsui, T., Matsumoto, M., Hori, Y., Isonishi, A., Kato, S., Iwamoto, T., Mori, T., & Fujimura, Y. (2010). Proteolytic fragmentation and sugar chains of plasma ADAMTS13 purified by a conformation-dependent monoclonal antibody. Journal of Biochemistry, 148(4), 403-411. https://doi.org/10.1093/jb/mvq075