Primary structure and antiplatelet mechanism of a snake venom metalloproteinase, acurhagin, from Agkistrodon acutus venom.
Acurhagin has been characterized as a P-III hemorrhagic metalloproteinase. We herein report the complete sequence of acurhagin by molecular cloning. Analysis of the cDNA-predicted amino acid sequence encoding acurhagin precursor revealed that this mosaic Asn-linked glycoprotein possesses a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domains (189/205/102/114 residues), with an overall 87% identity to that of jararhagin, an integrin alpha2beta1-cleaving metalloproteinase. Acurhagin has a Ser-Glu-Cys-Asp sequence in the disintegrin-like domain instead of the typical Arg-Gly-Asp motif. In contrast to inhibiting fibrinogen-integrin alphaIIbbeta3 interaction by disintegrins, acurhagin selectively showed a dose-dependent inhibition on platelet aggregation induced by collagen, and suppression on tyrosine phosphorylation of several signaling proteins in convulxin-stimulated platelets. Although the immobilized acurhagin was shown to bind platelet GPVI and collagen in a primary structure- and steric conformation-dependent manner, respectively, the mechanism of acurhagin under short incubation is mainly through its binding to GPVI and collagen, instead of binding to alpha2beta1, or cleaving platelet membrane glycoproteins. Moreover, the molecular conformation maintained by divalent cations is required for the proteolytic activity of acurhagin toward extracellular matrix fibronectin. Taken together, these results suggest that all the three domains in mature acurhagin may cooperatively contribute to its biological function.
Wang
Wen-Jeng
WJ
Chang-Gung Institute of Technology, Kwei-Shan, Tao-Yuan, Taiwan, ROC.
Shih
Chun-Ho
CH
Huang
Tur-Fu
TF
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D045726:Metalloproteases / Q000737:chemistry; D008969:Molecular Sequence Data; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D003435:Crotalid Venoms; D017931:DNA Primers; D018076:DNA, Complementary; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; C469546:acurhagin protein, Agkistrodon acutus; D045726:Metalloproteases