PMID: 20624874

 

    Legend: Sugar

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

Abstract :

1. ADAMTS13 is a metalloproteinase that specifically cleaves unusually large von Willbrand factor multimers under high-shear stress
2. Deficiency of ADAMTS13 activity induces a life-threatening generalized disease, thrombotic thrombocytopenic purpura
3. 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
4. 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
5. Purified pADAMTS13 had a specific activity of 300 U/mg (25,057-fold purification) and the pI was 5.1-5.5
6. 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
7. 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