Structural and functional characterisation of isolated puff adder (Bitis arietans) venom serine proteases

Serine proteases (SVSPs) are known to play a major role in the haemotoxic actions of viper venom, but compared with those from other medically important vipers, the serine proteases of puff adder venoms have not been extensively characterised. To address this, we isolated, identified and characterised the bioactivity of the serine proteases within the venom of the Nigerian puff adder, which we had previously shown to be especially rich in this class of toxin. Two distinct groups were identified, each with different protein substrate specificities. Both had similar molecular weights of 52-62 kDa, with 4-6 N-glycans, but one group consisted of trypsin-like acidic SVSPs and the other of non-trypsin-like basic SVSPs with a specificity for aliphatic amino acids at the P₁ position. Each acted differently on fibrinogen: the acidic SVSPs showed thrombin-like alpha/beta-fibrinogenase activity, whereas the basic forms were shown to be alpha-fibrinogenases. The acidic SVSPs acted on a chromogenic substrate commonly used to determine kallikrein-like activity but also possess strong gelatinase activity - a novel activity for SVSPs and the first example of an SVSP acting on proteins other than those of the haemostatic system. The basic SVSPs were able to specifically convert angiotensin I into the vasoconstrictor angiotensin 1-8. Analysis of the transcripts of each set of SVSPs revealed structural details of the substrate-binding sites that supported the experimental findings. The activity and sequences of the basic SVSPs show that they are very like the alpha-fibrinogenase ML-AF of M. lebetina, which until now was considered to be a unique SVSP. Thus, this basic SVSP and the acidic SVSP with its gelatinase activity can be considered to be atypical viper serine proteases. The levels of these SVSPs in venom were found to vary geographically and this, alongside the regional variation in the SVMP activities that we observed in a previous study, is discussed with reference to the potential implications on pathology of snakebite envenoming and the development of therapeutic interventions.