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P-III hemorrhagic metalloproteinases from Russell's viper venom: cloning, characterization, phylogenetic and
Hong-Sen Chen1, Hsin-Yu Tsai, Ying-Ming Wang
1Graduate Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.
Abstract:
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.
Insights
Two Russell's viper venom metalloproteinases, daborhagin-M and daborhagin-K, cause severe hemorrhage. Phylogenetic analysis reveals structural motifs linked to hemorrhagic potency in P-III SVMPs.
Area of Science:
- Biochemistry
- Toxicology
- Molecular Biology
Background:
- Russell's viper venom contains potent hemorrhagic metalloproteinases.
- Geographic variations in venom composition can influence toxicity.
Purpose of the Study:
- To purify and characterize two homologous P-III hemorrhagic metalloproteinases from Russell's viper venoms.
- To investigate the structural basis of hemorrhagic potency in these enzymes.
Main Methods:
- Purification of metalloproteinases from Russell's viper venom.
- Hemorrhagic dose determination in mice.
- Substrate specificity analysis (fibrinogen, fibronectin, collagen, insulin B chain).
- cDNA cloning, sequencing, and peptide mass fingerprinting for daborhagin-K.
- Western blotting of geographic venom samples.
- Molecular phylogenetic analysis of P-III metalloproteinases.
Main Results:
- Daborhagin-M and daborhagin-K were purified and shown to induce severe dermal hemorrhage.
- Daborhagin-M hydrolyzed fibrinogen, fibronectin, and type IV collagen, with a preference for hydrophobic residues.
- Venom samples from Myanmar and eastern India showed a 65kDa band, correlating with high hemorrhagic risk.
- Phylogenetic analysis placed daborhagin-K in the most hemorrhagic subclass of P-III SVMPs.
- Four structural motifs were identified as potentially responsible for phylogenetic subtyping and hemorrhagic potency.
Conclusions:
- Daborhagin-M and daborhagin-K are potent hemorrhagic metalloproteinases from Russell's viper venom.
- Enzyme activity and geographic distribution correlate with hemorrhagic symptoms.
- Structural motifs within P-III SVMPs are key determinants of their hemorrhagic potency and evolutionary divergence.
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