Dataset Information

Three-fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of snake venom toxins.

ABSTRACT: Three-finger toxins (3FTx) represent one of the most abundantly secreted and potently toxic components of colubrid (Colubridae), elapid (Elapidae) and psammophid (Psammophiinae subfamily of the Lamprophidae) snake venom arsenal. Despite their conserved structural similarity, they perform a diversity of biological functions. Although they are theorised to undergo adaptive evolution, the underlying diversification mechanisms remain elusive. Here, we report the molecular evolution of different 3FTx functional forms and show that positively selected point mutations have driven the rapid evolution and diversification of 3FTx. These diversification events not only correlate with the evolution of advanced venom delivery systems (VDS) in Caenophidia, but in particular the explosive diversification of the clade subsequent to the evolution of a high pressure, hollow-fanged VDS in elapids, highlighting the significant role of these toxins in the evolution of advanced snakes. We show that Type I, II and III ?-neurotoxins have evolved with extreme rapidity under the influence of positive selection. We also show that novel Oxyuranus/Pseudonaja Type II forms lacking the apotypic loop-2 stabilising cysteine doublet characteristic of Type II forms are not phylogenetically basal in relation to other Type IIs as previously thought, but are the result of secondary loss of these apotypic cysteines on at least three separate occasions. Not all 3FTxs have evolved rapidly: ?-neurotoxins, which form non-covalently associated heterodimers, have experienced a relatively weaker influence of diversifying selection; while cytotoxic 3FTx, with their functional sites, dispersed over 40% of the molecular surface, have been extremely constrained by negative selection. We show that the a previous theory of 3FTx molecular evolution (termed ASSET) is evolutionarily implausible and cannot account for the considerable variation observed in very short segments of 3FTx. Instead, we propose a theory of Rapid Accumulation of Variations in Exposed Residues (RAVER) to illustrate the significance of point mutations, guided by focal mutagenesis and positive selection in the evolution and diversification of 3FTx.

SUBMITTER: Sunagar K

PROVIDER: S-EPMC3847720 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Three-fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of snake venom toxins.

Sunagar Kartik K Jackson Timothy N W TN Undheim Eivind A B EA Ali Syed A SA Antunes Agostinho A Fry Bryan G BG

Toxins 20131118 11

Three-finger toxins (3FTx) represent one of the most abundantly secreted and potently toxic components of colubrid (Colubridae), elapid (Elapidae) and psammophid (Psammophiinae subfamily of the Lamprophidae) snake venom arsenal. Despite their conserved structural similarity, they perform a diversity of biological functions. Although they are theorised to undergo adaptive evolution, the underlying diversification mechanisms remain elusive. Here, we report the molecular evolution of different 3FTx ...[more]

PMID: 24253238

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Project description:Snakebite is classified as a priority Neglected Tropical Disease by the World Health Organization. Understanding the pathology of individual snake venom toxins is of great importance when developing more effective snakebite therapies. Snake venoms may induce a range of pathologies, including haemolytic activity. Although snake venom-induced erythrocyte lysis is not the primary cause of mortality, haemolytic activity can greatly debilitate victims and contributes to systemic haemotoxicity. Current assays designed for studying haemolytic activity are not suitable for rapid screening of large numbers of toxic compounds. Consequently, in this study, a high-throughput haemolytic assay was developed that allows profiling of erythrocyte lysis, and was validated using venom from a number of medically important snake species (Calloselasma rhodostoma, Daboia russelii, Naja mossambica, Naja nigricollis and Naja pallida). The assay was developed in a format enabling direct integration into nanofractionation analytics, which involves liquid chromatographic separation of venom followed by high-resolution fractionation and subsequent bioassaying (and optional proteomics analysis), and parallel mass spectrometric detection. Analysis of the five snake venoms via this nanofractionation approach involving haemolytic assaying provided venom-cytotoxicity profiles and enabled identification of the toxins responsible for haemolytic activity. Our results show that the elapid snake venoms (Naja spp.) contained both direct and indirect lytic toxins, while the viperid venoms (C. rhodostoma and D. russelii) only showed indirect lytic activities, which required the addition of phospholipids to exert cytotoxicity on erythrocytes. The haemolytic venom toxins identified were mainly phospholipase A2s and cytotoxic three finger toxins. Finally, the applicability of this new analytical method was demonstrated using a conventional snakebite antivenom treatment and a small-molecule drug candidate to assess neutralisation of venom cytotoxins.

Project description:Despite the unparalleled diversity of venomous snakes in Australia, research has concentrated on a handful of medically significant species and even of these very few toxins have been fully sequenced. In this study, venom gland transcriptomes were sequenced from eleven species of small Australian elapid snakes, from eleven genera, spanning a broad phylogenetic range. The particularly large number of sequences obtained for three-finger toxin (3FTx) peptides allowed for robust reconstructions of their dynamic molecular evolutionary histories. We demonstrated that each species preferentially favoured different types of ?-neurotoxic 3FTx, probably as a result of differing feeding ecologies. The three forms of ?-neurotoxin [Type I (also known as (aka): short-chain), Type II (aka: long-chain) and Type III] not only adopted differential rates of evolution, but have also conserved a diversity of residues, presumably to potentiate prey-specific toxicity. Despite these differences, the different ?-neurotoxin types were shown to accumulate mutations in similar regions of the protein, largely in the loops and structurally unimportant regions, highlighting the significant role of focal mutagenesis. We theorize that this phenomenon not only affects toxin potency or specificity, but also generates necessary variation for preventing/delaying prey animals from acquiring venom-resistance. This study also recovered the first full-length sequences for multimeric phospholipase A2 (PLA2) 'taipoxin/paradoxin' subunits from non-Oxyuranus species, confirming the early recruitment of this extremely potent neurotoxin complex to the venom arsenal of Australian elapid snakes. We also recovered the first natriuretic peptides from an elapid that lack the derived C-terminal tail and resemble the plesiotypic form (ancestral character state) found in viper venoms. This provides supporting evidence for a single early recruitment of natriuretic peptides into snake venoms. Novel forms of kunitz and waprin peptides were recovered, including dual domain kunitz-kunitz precursors and the first kunitz-waprin hybrid precursors from elapid snakes. The novel sequences recovered in this study reveal that the huge diversity of unstudied venomous Australian snakes are of considerable interest not only for the investigation of venom and whole organism evolution but also represent an untapped bioresource in the search for novel compounds for use in drug design and development.

Project description:BackgroundBothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults.ResultsOptimized conditions for two-dimensional gel electrophoresis (2-DE) of pooled venom samples were achieved using immobilized pH gradient (IPG) gels of non-linear 3-10 pH range during the isoelectric focusing step and 10-20% gradient polyacrylamide gels in the second dimension. Software-assisted analysis of the 2-DE gels images demonstrated differences in the number and intensity of spots in juvenile, sub-adult and adult venoms. Although peptide mass fingerprinting (PMF) failed to identify even a minor fraction of spots, it allowed us to group spots that displayed similar peptide maps. The spots were subjected to a combination of tandem mass spectrometry and Mascot and MS BLAST database searches that identified several classes of proteins, including metalloproteinases, serine proteinases, lectins, phospholipases A2, L-amino oxidases, nerve growth factors, vascular endothelial growth factors and cysteine-rich secretory proteins.ConclusionThe analysis of B. atrox samples from specimens of different ages by 2-DE and mass spectrometry suggested that venom proteome alters upon ontogenetic development. We identified stage specific and differentially expressed polypeptides that may be responsible for the activities of the venom in each developmental stage. The results provide insight into the molecular basis of the relation between symptomatology of snakebite accidents in humans and the venom composition. Our findings underscore the importance of the use of venoms from individual specimen at various stages of maturation for the production of antivenoms.

Dataset Information

Three-fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of snake venom toxins.

Publications

Three-fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of snake venom toxins.

Similar Datasets

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