Proteolytic fragmentation and sugar chains of plasma ADAMTS13 purified by a conformation-dependent monoclonal antibody.
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.
Hiura
Hisahide
H
Department of Blood Transfusion Medicine, Nara Medical University, Kashihara, Japan.
Matsui
Taei
T
Matsumoto
Masanori
M
Hori
Yuji
Y
Isonishi
Ayami
A
Kato
Seiji
S
Iwamoto
Takaaki
T
Mori
Toshio
T
Fujimura
Yoshihiro
Y
D000017:ABO Blood-Group System; D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004795:Enzyme Stability; D000939:Epitopes / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation
D000017:ABO Blood-Group System; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007477:Ions; D008670:Metals; D010447:Peptide Hydrolases; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human