Purification and functional characterization of AAV1, a novel P-III metalloproteinase, from Formosan Agkistrodon acutus venom.
AAV1, an alkaline glycoprotein (GP), was purified from Agkistrodon acutus venom by two chromatographic steps on successive DEAE-Sephadex A-50 and Superdex 75 FPLC columns. AAV1 on SDS-PAGE under non-reducing conditions migrated as a monomeric and a polymeric forms with apparent molecular mass of 57 and 180 kDa, respectively. Upon reduction, it appeared as a single broad band with a mass of 50.3 kDa corresponding to the size of a typical P-III metalloproteinase acurhagin. The N-terminal sequence of an autoproteolytical 30 kDa-fragment of AAV1 showed a high homology to that of venom proteins with Metalloproteinase, Disintegrin-like, and Cysteine-rich (MDC) domains. Although it was devoid of cleaving activity toward gelatin, fibronectin and prothrombin, AAV1 preferentially digested the Aalpha chain of fibrinogen and followed by the Bbeta chain, leading to the inhibition of fibrinogen-induced platelet aggregation in elastase-treated human platelets. However, the proteolytic activity of AAV1 was completely inactivated by the chelating agent but not serine proteinase inhibitor. Furthermore, AAV1 could concentration-dependently inhibit platelet aggregation and suppress tyrosine phosphorylation of intracellular proteins in collagen- and convulxin-stimulated platelets, respectively. The interaction of MDC domains in AAV1 molecule with platelet GPVI was responsible for the inhibitory effect of AAV1 on collagen- and convulxin-induced platelet aggregation. Taken together, these pieces of evidence suggest that AAV1 from Formosan viper venom belongs to a new member of high-molecular mass metalloproteinase family and functions as a GPVI antagonist.
Wang
Wen-Jeng
WJ
Chang-Gung Institute of Technology, Room A810, No. 261 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 33303, Taiwan. wjwang@gw.cgit.edu.tw
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D045726:Metalloproteases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors; D013624:Taiwan
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D045726:Metalloproteases