engleski

Ammodytagin, the first heterodimeric P-IIIc metalloproteinase from Vipera ammodytes ammodytes venom with strong hemorrhagic activity

The most pronounced pathophysiological manifestations following the viperine snakes’ bites are hemorrhage and necrosis. Identification of the molecules involved in such pathologies is prerequisite for improvements in diagnosis and therapy of envenomations, as well as in antivenom production. In this study we report the properties of the novel Zn2+-dependent metalloproteinase with strong hemorrhagic activity, named ammodytagin, in the Vipera ammodytes ammodytes (Vaa) venom. It is a glycosylated heterodimer of 108 kDa, as determined by MALDI mass spectrometry, and highly basic molecule, according to its chromatographic behaviour. Partial amino acid sequencing by Edman degradation and MS/MS analysis identified sequences belonging to metalloproteinase, disintegrin-like and cysteine-rich domains, which in addition to its heterodimeric nature allows classification into the P-IIIc group of snake venom metalloproteinases (SVMPs). Only few members of that group have been described so far. Ammodytagin possesses potent azocaseinolytic activity which can be inhibited by Na2EDTA, Zn2+ and DTT. Also, it cleaves insulin B-chain, hydrolysing it at positions Gln4-His5, His10-Leu11 and Tyr16-Leu17. Furthermore, ammodytagin acts as a strong hemorrhagin in both rats and mice. Investigation of substrate specificity revealed that the hemorrhagicity of the novel SVMP might be the result of its involvement in cleavage of basal membrane components and depletion of fibrinogen, prothrombin and factor X in blood circulation. Finally, antiserum raised against ammodytagin was able to completely neutralise the hemorrhagic activity of the whole venom, suggesting it might be one of the key molecules towards which effective antivenoms should be directed.

Snake venom metalloproteinase; Hemorrhagin; Substrate specificity; Vipera ammodytes; MS/MS

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