Tracing the evolution of venom phospholipases A2 in Gloydius strauchii and related pitvipers: A tale of two acidic isozymes.

Two acidic Asp49-PLA2s with Glu6 substitution and a neutral Lys49-PLA (designated Gst-K49) were cloned from G. strauchii venom glands, their full amino acid sequences were deduced. The predominant acidic PLA2 (designated Gst-E6a) contains 124 residues and the M18W30 substitutions, while the minor acidic PLA2 (designated Gst-E6b) contains 122 residues and the V18A30 substitutions. Their sequences are most similar to those of the respective orthologous PLA2s of G. intermedius venom. Gst-E6a and Gst-E6b appear to be paralogs and possibly have different predatory targets or functions. The LC-MS/MS results indicate the presence of only three PLA2 gene products in the crude venom, the relative expression levels were in the order of Gst-E6a â‰« Gst-E6b > Gst-K49, as confirmed by qPCR results. In contrast to other Gloydius, G. strauchii venom does not contain neurotoxic or basic anticoagulant Asp49-PLA2s, but Gst-K49 is the first Lys49-PLA2 identified in Gloydius venoms. However, its venom content is relatively low and its pI value 7.3 is much lower than those of other Lys49-PLA2s and. The Lys49-PLA2 genes appear to regress in the venom of most of Gloydius and related rattlesnake, and this evolutionary regression occurred before the dispersal of Asian pitvipers to the New World.

Serine protease isoforms in Gloydius intermedius venom: Full sequences, molecular phylogeny and evolutionary implications.

Nine distinct venom serine proteases (vSPs) of Gloydius intermedius were studied by transcriptomic, sub-proteomic and phylogenetic analyses. Their complete amino acid sequences were deduced after Expression Sequence Tag (EST) analyses followed by cDNA cloning and sequencing. These vSPs appear to be paralogs and contain the catalytic triads and 1-4 potential N-glycosylation sites. Their relative expression levels evaluated by qPCR were grossly consistent with their EST hit-numbers. The major vSPs were purified by HPLC and their N-terminal sequences matched well to the deduced sequences, while fragments of the minor vSPs were detected by LC-MS/MS identification. Specific amidolytic activities of the fractions from HPLC and anion exchange separation were assayed using four chromogenic substrates, respectively. Molecular phylogenetic tree based on the sequences of these vSPs and their orthologs revealed six major clusters, one of them covered four lineages of plasminogen activator like vSPs. N-glycosylation patterns and variations for the vSPs are discussed. The high sequence similarities between G. intermedius vSPs and their respective orthologs from American pitvipers suggest that most of the isoforms evolved before Asian pitvipers migrated to the New World. Our results also indicate that the neurotoxic venoms contain more kallikrein-like vSPs and hypotensive components than the hemorrhagic venoms. SIGNIFICANCE: Full sequences and expression levels of nine paralogous serine proteases (designated as GiSPs) of Gloydius intermedius venom have been studied. A kallikrein-like enzyme is most abundant and four isoforms homologous to venom plasminogen-activators are also expressed in this venom. Taken together, the present and previous data demonstrate that the neurotoxic G. intermedius venoms contain more hypotensive vSPs relative to other hemorrhagic pitviper venoms and the pitviper vSPs are highly versatile and diverse. Their structure-function relationships remain to be explored and compared. A novel, simplified phylogenetic tree based on the sequences of GiSPs and their closely related orthologs from other pitvipers reveals six major subtypes and offers a better understanding of vSP duplication and evolution in pitvipers of both the Old and New Worlds. It is well known that specific vSPs are potential therapeutic or diagnostic agents that target the plasma proteins or coagulation factors. Our results not only render deeper insights into the variation and evolution of vSPs, but may help to choose right venoms for the development of better therapeutic leads.