Isolation and characterisation of serine proteases and metalloproteases from the venom of African puff adders

Abstract

The puff adder (Bitis arietans) is a highly venomous viper responsible for many fatalities in Africa, yet there have been few 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 wide range of sources. Prominent in all venoms was an SVMP PI, derived from a PII precursor. This protein existed in either of two forms: non-glycosylated (21 kDa) or glycosylated with either one (26 kDa) or two N-glycans (30 kDa). All the venoms we tested here were found to contain either one or the other form: none had both. The 21 kDa form proved to be highly potent, with alpha-, beta- and in some cases gamma-fibrinogenase activities and were 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. 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 was intermediate between the non- and two-glycan forms. Because of the predominance and ubiquity of these SVMPs in puff adders, and their undoubted clinical significance, we propose to name them the arilysins. The SVMP PIII content of the puff adder venoms was, atypically for African vipers, quite low. In some Kenyan venoms, however, there was an abundant SVMP PIII, with strong gelatinase activity. This protein possesses an unusual oligomeric structure, being a 140 kDa homodimer (c.f. SVMPIII-c) 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 envenomation and the development of therapeutic interventions.