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September 3rd, 2025
Version: 2
University of Bristol
biochemistry
bioRxiv

Purified Zymogens Reveal Mechanisms of Snake Venom Metalloproteinase Auto-Activation

Hall, S.Open in Google Scholar•Cardoso, I. A.Open in Google Scholar•Wilkinson, M. C.Open in Google Scholar•Carretero, M. M.Open in Google Scholar•Lingappa, S.Open in Google Scholar•Rand, B.Open in Google Scholar•Shen, D.Open in Google Scholar•Boldrini-Franca, J.Open in Google Scholar•Stenner, R.Open in Google Scholar•Menzies, S. K.Open in Google Scholaret al.

Snake venoms contain diverse mixtures of toxins that evolved to incapacitate prey, but in humans they cause extensive pathology following snakebite envenomation. In viper venom, the most potent toxins are the haemorrhagic and coagulopathic snake venom metalloproteinases (SVMPs). Because venoms contain a SVMP cocktail, and due to their cytotoxicity, SVMP characterizations have been hampered by the lack of purified enzymes. By incorporating their prodomain, which blocks the active SVMP site, we overcame their cytotoxicity and enabled recombinant production of zymogens from all three structurally variable SVMP classes (PI, PII and PIII) using our baculovirus/insect cell expression system. Zymogens were auto-activated by incubation with Zn2+ ions, resulting in prodomain cleavage, PII disintegrin cleavage and PIII prodomain proteolysis. Auto-activated SVMPs were characterized using protein substrate degradation, platelet aggregation and blood coagulation assays, benchmarked to native venom-purified SVMP. Our recombinant zymogen production protocol is generically applicable for the expression of SVMPs, unlocking biomedical use in haematology, and discovery of novel snakebite therapeutics.

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