Timothy N. W. Jackson

Three-Fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of Snake Venom Toxins

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 α-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: κ-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.

Differential Evolution and Neofunctionalization of Snake Venom Metalloprotease Domains

Snake venom metalloproteases (SVMP) are composed of five domains: signal peptide, propeptide, metalloprotease, disintegrin, and cysteine-rich. Secreted toxins are typically combinatorial variations of the latter three domains. The SVMP-encoding genes of Psammophis mossambicus venom are unique in containing only the signal and propeptide domains. We show that the Psammophis SVMP propeptide evolves rapidly and is subject to a high degree of positive selection. Unlike Psammophis, some species of Echis express both the typical multidomain and the unusual monodomain (propeptide only) SVMP, with the result that a lower level of variation is exerted upon the latter. We showed that most mutations in the multidomain Echis SVMP occurred in the protease domain responsible for proteolytic and hemorrhagic activities. The cysteine-rich and disintegrin-like domains, which are putatively responsible for making the P-III SVMPs more potent than the P-I and P-II forms, accumulate the remaining variation. Thus, the binding sites on the molecules surface are evolving rapidly whereas the core remains relatively conserved. Bioassays conducted on two post-translationally cleaved novel proline-rich peptides from the P. mossambicus propeptide domain showed them to have been neofunctionalized for specific inhibition of mammalian a7 neuronal nicotinic acetylcholine receptors. We show that the proline rich postsynaptic specific neurotoxic peptides from Azemiops feae are the result of convergent evolution within the precursor region of the C-type natriuretic peptide instead of the SVMP. The results of this study reinforce the value of studying obscure venoms for biodiscovery of novel investigational ligands.

Dracula's children: molecular evolution of vampire bat venom

UNLABELLED While vampire bat oral secretions have been the subject of intense research, efforts have concentrated only on two components: DSPA (Desmodus rotundus salivary plasminogen activator) and Draculin. The molecular evolutionary history of DSPA has been elucidated, while conversely draculin has long been known from only a very small fragment and thus even the basic protein class was not even established. Despite the fact that vampire bat venom has a multitude of effects unaccounted by the documented bioactivities of DSPA and draculin, efforts have not been made to establish what other bioactive proteins are secreted by their submaxillary gland. In addition, it has remained unclear whether the anatomically distinct anterior and posterior lobes of the submaxillary gland are evolving on separate gene expression trajectories or if they remain under the shared genetic control. Using a combined proteomic and transcriptomic approach, we show that identical proteins are simultaneously expressed in both lobes. In addition to recovering the known structural classes of DSPA, we recovered a novel DSPA isoform as well as obtained a very large sequence stretch of draculin and thus established that it is a mutated version of the lactotransferrin scaffold. This study reveals a much more complex secretion profile than previously recognised. In addition to obtaining novel versions of scaffolds convergently recruited into other venoms (allergen-like, CRiSP, kallikrein, Kunitz, lysozyme), we also documented novel expression of small peptides related to calcitonin, PACAP, and statherin. Other overexpressed protein types included BPI-fold, lacritin, and secretoglobin. Further, we investigate the molecular evolution of various vampire bat venom-components and highlight the dominant role of positive selection in the evolution of these proteins. Conspicuously many of the proteins identified in the proteome were found to be homologous to proteins with known activities affecting vasodilation and platelet aggregation. We show that vampire bat venom proteins possibly evade host immune response by the mutation of the surface chemistry through focal mutagenesis under the guidance of positive Darwinian selection. These results not only contribute to the body of knowledge regarding haematophagous venoms but also provide a rich resource for novel lead compounds for use in drug design and development. BIOLOGICAL SIGNIFICANCE These results have direct implications in understanding the molecular evolutionary history of vampire bat venom. The unusual peptides discovered reinforce the value of studying such neglected taxon for biodiscovery.

Venom proteomic characterization and relative antivenom neutralization of two medically important Pakistani elapid snakes (Bungarus sindanus and Naja naja)

UNLABELLED Intra- and interspecific variation in venom composition has been shown to have a major effect upon the efficacy of antivenoms. Due to the absence of domestically produced antivenoms, Pakistan is wholly reliant upon antivenoms produced in other countries, such as India. However, the efficacy of these antivenoms in neutralising the venoms of Pakistani snakes has not been ascertained. This is symptomatic of the general state of toxicological research in this country, which has a myriad of highly toxic and medically important venomous animals. Thus, there is a dire need for knowledge regarding the fundamental proteomics of these venoms and applied knowledge of the relative efficacy of foreign antivenoms. Here we present the results of our proteomic research on two medically important snakes of Pakistan: Bungarus sindanus and Naja naja. Indian Polyvalent Antivenom (Bharat Serums and Vaccines Ltd), which is currently marketed for use in Pakistan, was completely ineffective against either Pakistani species. In addition to the expected pre- and post-synaptic neurotoxic activity, the venom of the Pakistan population of N. naja was shown to be quite divergent from other populations of this species in being potently myotoxic. These results highlight the importance of studying divergent species and isolated populations, where the same data not only elucidates clinical problems in need of immediate attention, but also uncovers sources for novel toxins with potentially useful activities. BIOLOGICAL SIGNIFICANCE Pakistan Bungarus sindanus and Naja naja venoms are differentially complex. Naja naja is potently myotoxic. Neither venom is neutralized by Indian antivenom. These results have direct implications for the treatment of envenomed patients in Pakistan. The unusually myotoxic effects of Naja naja demonstrates the value of studying remote populations for biodiscovery.

Molecular Evolution of Vertebrate Neurotrophins: Co-Option of the Highly Conserved Nerve Growth Factor Gene into the Advanced Snake Venom Arsenalf

Neurotrophins are a diverse class of structurally related proteins, essential for neuronal development, survival, plasticity and regeneration. They are characterized by major family members, such as the nerve growth factors (NGF), brain-derived neurotrophic factors (BDNF) and neurotrophin-3 (NT-3), which have been demonstrated here to lack coding sequence variations and follow the regime of negative selection, highlighting their extremely important conserved role in vertebrate homeostasis. However, in stark contrast, venom NGF secreted as part of the chemical arsenal of the venomous advanced snake family Elapidae (and to a lesser extent Viperidae) have characteristics consistent with the typical accelerated molecular evolution of venom components. This includes a rapid rate of diversification under the significant influence of positive-selection, with the majority of positively-selected sites found in the secreted β-polypeptide chain (74%) and on the molecular surface of the protein (92%), while the core structural and functional residues remain highly constrained. Such focal mutagenesis generates active residues on the toxin molecular surface, which are capable of interacting with novel biological targets in prey to induce a myriad of pharmacological effects. We propose that caenophidian NGFs could participate in prey-envenoming by causing a massive release of chemical mediators from mast cells to mount inflammatory reactions and increase vascular permeability, thereby aiding the spread of other toxins and/or by acting as proapoptotic factors. Despite their presence in reptilian venom having been known for over 60 years, this is the first evidence that venom-secreted NGF follows the molecular evolutionary pattern of other venom components, and thus likely participates in prey-envenomation.

Venom Down Under: Dynamic Evolution of Australian Elapid Snake Toxins

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 α-neurotoxic 3FTx, probably as a result of differing feeding ecologies. The three forms of α-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 α-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.

Squeezers and leaf-cutters: differential diversification and degeneration of the venom system in toxicoferan reptiles

Although it has been established that all toxicoferan squamates share a common venomous ancestor, it has remained unclear whether the maxillary and mandibular venom glands are evolving on separate gene expression trajectories or if they remain under shared genetic control. We show that identical transcripts are simultaneously expressed not only in the mandibular and maxillary glands, but also in the enigmatic snake rictal gland. Toxin molecular frameworks recovered in this study were three-finger toxin (3FTx), CRiSP, crotamine (beta-defensin), cobra venom factor, cystatin, epididymal secretory protein, kunitz, l-amino acid oxidase, lectin, renin aspartate protease, veficolin, and vespryn. We also discovered a novel low-molecular weight disulfide bridged peptide class in pythonid snake glands. In the iguanian lizards, the most highly expressed are potentially antimicrobial in nature (crotamine (beta-defensin) and cystatin), with crotamine (beta-defensin) also the most diverse. However, a number of proteins characterized from anguimorph lizards and caenophidian snakes with hemotoxic or neurotoxic activities were recruited in the common toxicoferan ancestor and remain expressed, albeit in low levels, even in the iguanian lizards. In contrast, the henophidian snakes express 3FTx and lectin toxins as the dominant transcripts. Even in the constricting pythonid and boid snakes, where the glands are predominantly mucous-secreting, low-levels of toxin transcripts can be detected. Venom thus appears to play little role in feeding behavior of most iguanian lizards or the powerful constricting snakes, and the low levels of expression argue against a defensive role. However, clearly the incipient or secondarily atrophied venom systems of these taxa may be a source of novel compounds useful in drug design and discovery.

A proteomics and transcriptomics investigation of the venom from the Barychelid spider Trittame loki (brush-foot trapdoor)

Although known for their potent venom and ability to prey upon both invertebrate and vertebrate species, the Barychelidae spider family has been entirely neglected by toxinologists. In striking contrast, the sister family Theraphosidae (commonly known as tarantulas), which last shared a most recent common ancestor with Barychelidae over 200 million years ago, has received much attention, accounting for 25% of all the described spider toxins while representing only 2% of all spider species. In this study, we evaluated for the first time the venom arsenal of a barychelid spider, Trittame loki, using transcriptomic, proteomic, and bioinformatic methods. The venom was revealed to be dominated by extremely diverse inhibitor cystine knot (ICK)/knottin peptides, accounting for 42 of the 46 full-length toxin precursors recovered in the transcriptomic sequencing. In addition to documenting differential rates of evolution adopted by different ICK/knottin toxin lineages, we discovered homologues with completely novel cysteine skeletal architecture. Moreover, acetylcholinesterase and neprilysin were revealed for the first time as part of the spider-venom arsenal and CAP (CRiSP/Allergen/PR-1) were identified for the first time in mygalomorph spider venoms. These results not only highlight the extent of venom diversification in this neglected ancient spider lineage, but also reinforce the idea that unique venomous lineages are rich pools of novel biomolecules that may have significant applied uses as therapeutics and/or insecticides.

Firing the Sting: Chemically Induced Discharge of Cnidae Reveals Novel Proteins and Peptides from Box Jellyfish (Chironex fleckeri) Venom

Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which has a notable impact on public health. By utilizing scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or “venom” recovery, we utilized both top-down and bottom-up transcriptomics–proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts.

Rapid Radiations and the Race to Redundancy: An Investigation of the Evolution of Australian Elapid Snake Venoms.

Australia is the stronghold of the front-fanged venomous snake family Elapidae. The Australasian elapid snake radiation, which includes approximately 100 terrestrial species in Australia, as well as Melanesian species and all the world’s true sea snakes, may be less than 12 million years old. The incredible phenotypic and ecological diversity of the clade is matched by considerable diversity in venom composition. The clade’s evolutionary youth and dynamic evolution should make it of particular interest to toxinologists, however, the majority of species, which are small, typically inoffensive, and seldom encountered by non-herpetologists, have been almost completely neglected by researchers. The present study investigates the venom composition of 28 species proteomically, revealing several interesting trends in venom composition, and reports, for the first time in elapid snakes, the existence of an ontogenetic shift in the venom composition and activity of brown snakes (Pseudonaja sp.). Trends in venom composition are compared to the snakes’ feeding ecology and the paper concludes with an extended discussion of the selection pressures shaping the evolution of snake venom.