Dynamic genetic differentiation drives the widespread structural and functional convergent evolution of snake venom proteinaceous toxins

Abstract

Background The explosive radiation and diversification of the advanced snakes (superfamily Colubroidea) was associated with changes in all aspects of the shared venom system. Morphological changes included the partitioning of the mixed ancestral glands into two discrete glands devoted for production of venom or mucous respectively, as well as changes in the location, size and structural elements of the venom-delivering teeth. Evidence also exists for homology among venom gland toxins expressed across the advanced snakes. However, despite the evolutionary novelty of snake venoms, in-depth toxin molecular evolutionary history reconstructions have been mostly limited to those types present in only two front-fanged snake families, Elapidae and Viperidae. To have a broader understanding of toxins shared among extant snakes, here we first sequenced the transcriptomes of eight taxonomically diverse rear-fanged species and four key viperid species and analysed major toxin types shared across the advanced snakes. Results Transcriptomes were constructed for the following families and species: Colubridae - Helicops leopardinus, Heterodon nasicus, Rhabdophis subminiatus; Homalopsidae – Homalopsis buccata; Lamprophiidae - Malpolon monspessulanus, Psammophis schokari, Psammophis subtaeniatus, Rhamphiophis oxyrhynchus; and Viperidae – Bitis atropos, Pseudocerastes urarachnoides, Tropidolaeumus subannulatus, Vipera transcaucasiana. These sequences were combined with those from available databases of other species in order to facilitate a robust reconstruction of the molecular evolutionary history of the key toxin classes present in the venom of the last common ancestor of the advanced snakes, and thus present across the full diversity of colubroid snake venoms. In addition to differential rates of evolution in toxin classes between the snake lineages, these analyses revealed multiple instances of previously unknown instances of structural and functional convergences. Structural convergences included: the evolution of new cysteines to form heteromeric complexes, such as within kunitz peptides (the beta-bungarotoxin trait evolving on at least two occasions) and within SVMP enzymes (the P-IIId trait evolving on at least three occasions); and the C-terminal tail evolving on two separate occasions within the C-type natriuretic peptides, to create structural and functional analogues of the ANP/BNP tailed condition. Also shown was that the de novo evolution of new post-translationally liberated toxin families within the natriuretic peptide gene propeptide region occurred on at least five occasions, with novel functions ranging from induction of hypotension to post-synaptic neurotoxicity. Functional convergences included the following: multiple occasions of SVMP neofunctionalised in procoagulant venoms into activators of the clotting factors prothrombin and Factor X; multiple instances in procoagulant venoms where kunitz peptides were neofunctionalised into inhibitors of the clot destroying enzyme plasmin, thereby prolonging the half-life of the clots formed by the clotting activating enzymatic toxins; and multiple occasions of kunitz peptides neofunctionalised into neurotoxins acting on presynaptic targets, including twice just within Bungarus venoms. Conclusions We found novel convergences in both structural and functional evolution of snake toxins. These results provide a detailed roadmap for future work to elucidate predator–prey evolutionary arms races, ascertain differential clinical pathologies, as well as documenting rich biodiscovery resources for lead compounds in the drug design and discovery pipeline.

Divergent Specialization of Simple Venom Gene Profiles among Rear-Fanged Snake Genera (Helicops and Leptodeira, Dipsadinae, Colubridae)

“…In addition, CTLs of front-fanged snakes have been shown to evolve rapidly [ 47 ]. Recently, Xie et al [ 48 ] found extensive duplication of novel dimeric CTLs genes unique to H. leopardinus . The novel CTLs found in H. leopardinus were shown to be under positive selection, but the distribution of these CTLs across the genus Helicops is currently unknown [ 48 ].…”

“…Recently, Xie et al [ 48 ] found extensive duplication of novel dimeric CTLs genes unique to H. leopardinus . The novel CTLs found in H. leopardinus were shown to be under positive selection, but the distribution of these CTLs across the genus Helicops is currently unknown [ 48 ]. A previous study of H. angulatus found its venom lacked hemorrhagic activity, which is typically associated with SVMPs and CTLs [ 36 ].…”

Differential Antivenom and Small-Molecule Inhibition of Novel Coagulotoxic Variations in Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium American Viperid Snake Venoms

“…Therefore, FX activation in P. volcanicum represents a third independent evolution of this trait within American pit-vipers. Underscoring the usefulness of this trait in prey capture, FX activation has evolved convergently in diverse Afro-Asian snake lineages such as the Daboia/Macrovipera/Montivipera viperid clade, viperid species Bitis worthingtoni , viperid genus Cerastes , viperid genus Echis , lamprophiid genus Atractaspis , and colubrid genus Rhabdophis [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. In contrast to FX activation being previously widely known as a convergent trait, Factor VII activation by snake venom has only previously been known for the Australian elapid clade of Oxyuranus + Pseudonaja [ 37 ].…”

“…Only P. dunni was observed to inhibit thrombin, producing a significant delay in clotting time within the thrombin-inhibition assay ( Figure 4 ). Future work is required to ascertain which toxin types are responsible for inhibiting the clotting factors, with characterized inhibitors of clotting enzymes in viperid-snake venoms such as kunitz peptides, the alpha–beta covalently-linked dimeric-form of lectin toxins, and Group II phospholipase A 2 toxins [ 35 , 41 , 42 , 43 ].…”

Proteomic insight into the venom composition of the largest European rear-fanged snake, Malpolon monspessulanus monspessulanus

“…Three-finger toxins exhibit a postsynaptic neurotoxic activity as antagonists of nicotinic acetylcholine receptors ( Bourne et al, 2005 ), muscarinic acetylcholine receptors ( Chung et al, 2002 ), adrenergic receptors ( Rajagopalan et al, 2007 ), and GABA receptors ( Rosso et al, 2015 ). Recently, a molecular phylogenetic study has reported the presence of a three-finger toxin from M. monspessulanus that was very similar to the related Psammophis subtaeniatus , but also to the very neurotoxic elapid species Naja kaouthia ( Xie et al, 2022 ). Therefore, an ongoing study on the venom gland transcriptomic analysis of M. monspessulanus monspessulanus is needed to clarify and identify the toxin genes produced by this species and to provide greater insight into the venom composition and mode of action of this venom.…”