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

被引:14
作者
Hiura, Hisahide [1 ]
Matsui, Taei [2 ]
Matsumoto, Masanori [1 ]
Hori, Yuji [1 ]
Isonishi, Ayami [1 ]
Kato, Seiji [1 ]
Iwamoto, Takaaki [3 ]
Mori, Toshio [3 ]
Fujimura, Yoshihiro [1 ]
机构
[1] Nara Med Univ, Dept Blood Transfus Med, Nara 6348522, Japan
[2] Fujita Hlth Univ, Sch Hlth Sci, Dept Biol, Fac Med Technol, Aichi 4701192, Japan
[3] Nara Med Univ, Radioisotope Res Ctr, Nara 6348521, Japan
关键词
enzyme digestion; lectin blotting; plasma ADAMTS13; purification; VON-WILLEBRAND-FACTOR; FACTOR-CLEAVING PROTEASE; THROMBOTIC THROMBOCYTOPENIC PURPURA; LIVER; MICROANGIOPATHIES; TRANSPLANTATION; INACTIVATION; PURIFICATION; SECRETION; SEQUENCE;
D O I
10.1093/jb/mvq075
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 alpha 2-6 and alpha 2-3-linked sialic acid residues with penultimate beta-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.
引用
收藏
页码:403 / 411
页数:9
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