Title : Proteolytic
fragmentation and sugar chains of plasma
ADAMTS13 purified by a conformation-dependent monoclonal antibody
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
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. ADAMTS13, , ADAMTS13, sugar chains, -
- 7. asialoglycoprotein, , asialoglycoprotein, -, -
- 7. glycoproteins, , glycoproteins, -, -
- 7. pADAMTS13, , pADAMTS13, N-, -
- 7. pADAMTS13, , pADAMTS13, O-linked sugar chains, -
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):