Biochemical characterisation and substrate-specific proteolytic diversity of venom metalloproteinases in African puff...

The puff adder (Bitis arietans) is a highly venomous viper responsible for many snakebite fatalities in Africa, yet there have been few geographically comprehensive analyses of its venom proteins, particularly of the proteases that play a key role in pathology of envenoming. To address this, we have isolated, identified and characterised the bioactivity of the venom metalloproteases of puff adders obtained from a range of localities. Prominent in all venoms was a PI snake venom metalloproteinase (SVMP), derived from a larger PII precursor. This protein existed as either non-glycosylated (21 kDa) or glycosylated, the latter containing either one (26 kDa) or two N-glycans (30 kDa). All the venoms we tested contained either one or the other form: none had both. The 21 kDa form proved to be highly bioactive, with alpha-, beta- and in some cases gamma-fibrinogenase activities and was very destructive towards laminin. Prothrombin and factor X were also extensively degraded by the 21 kDa SVMP, but in neither case did this result in generation of the respective active forms of these clotting factors. This protein was also shown to specifically and efficiently convert angiotensin I to the vasodilatory product, angiotensin 1-7. In contrast, the two-glycan forms were markedly less active against all of these substrates. The one-glycan form isolated from a Kenyan venom possessed activities that were intermediate between the non- and two-glycan forms. Using insulin B chain as a substrate, it was shown that these SVMPs cleave at the same peptide bonds as those observed for similar proteases found in other snake venoms, but the higher potency of the 21 kDa form was due to its ability to cleave peptide bonds C-terminal to glycine residues. The PIII SVMP content of the puff adder venoms was quite low, atypically for African vipers. In some Kenyan venoms, however, there was an abundant PIII SVMP with strong gelatinase activity. This protein possesses an unusual oligomeric structure, being a 140 kDa homodimer (c.f. PIII-c SVMP) but without the disulphide bonds that normally hold the monomers together in this class of SVMP. This diversity in venom metalloprotease activities is discussed with reference to the potential implications on the pathology of envenoming and the development of therapeutic interventions.