P-III hemorrhagic metalloproteinases from Russell's viper venom: cloning, characterization, phylogenetic and functional site analyses.

Two homologous P-III hemorrhagic metalloproteinases were purified from Russell's viper venoms from Myanmar and Kolkata (eastern India), and designated as daborhagin-M and daborhagin-K, respectively. They induced severe dermal hemorrhage in mice at a minimum hemorrhagic dose of 0.8-0.9 microg. Daborhagin-M specifically hydrolyzed an Aalpha-chain of fibrinogen, fibronectin, and type IV collagen in vitro. Analyses of its cleavage sites on insulin chain B and kinetic specificities toward oligopeptides suggested that daborhagin-M prefers hydrophobic residues at the P(1), P(1)', and P(2)' positions on the substrates. Of the eight Daboia geographic venom samples analyzed by Western blotting, only those from Myanmar and eastern India showed a strong positive band at 65kDa, which correlated with the high risk of systemic hemorrhagic symptoms elicited by Daboia envenoming in both regions. The full sequence of daborhagin-K was determined by cDNA cloning and sequencing, and then confirmed by peptide mass fingerprinting. Furthermore, molecular phylogenetic analyses based on 27 P-IIIs revealed the co-evolution of two major P-III classes with distinct hemorrhagic potencies, and daborhagin-K belongs to the most hemorrhagic subclass. By comparing the absolute complexity profiles between these two classes, we identified four structural motifs probably responsible for the phylogenetic subtyping and hemorrhagic potencies of P-III SVMPs.