Bryan G. Fry

The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms.

Throughout evolution, numerous proteins have been convergently recruited into the venoms of various animals, including centipedes, cephalopods, cone snails, fish, insects (several independent venom systems), platypus, scorpions, shrews, spiders, toxicoferan reptiles (lizards and snakes), and sea anemones. The protein scaffolds utilized convergently have included AVIT/colipase/prokineticin, CAP, chitinase, cystatin, defensins, hyaluronidase, Kunitz, lectin, lipocalin, natriuretic peptide, peptidase S1, phospholipase A(2), sphingomyelinase D, and SPRY. Many of these same venom protein types have also been convergently recruited for use in the hematophagous gland secretions of invertebrates (e.g., fleas, leeches, kissing bugs, mosquitoes, and ticks) and vertebrates (e.g., vampire bats). Here, we discuss a number of overarching structural, functional, and evolutionary generalities of the protein families from which these toxins have been frequently recruited and propose a revised and expanded working definition for venom. Given the large number of striking similarities between the protein compositions of conventional venoms and hematophagous secretions, we argue that the latter should also fall under the same definition.

Molecular Evolution and Phylogeny of Elapid Snake Venom Three-Finger Toxins

Animal venom components are of considerable interest to researchers across a wide variety of disciplines, including molecular biology, biochemistry, medicine, and evolutionary genetics. The three-finger family of snake venom peptides is a particularly interesting and biochemically complex group of venom peptides, because they are encoded by a large multigene family and display a diverse array of functional activities. In addition, understanding how this complex and highly varied multigene family evolved is an interesting question to researchers investigating the biochemical diversity of these peptides and their impact on human health. Therefore, the purpose of our study was to investigate the long-term evolutionary patterns exhibited by these snake venom toxins to understand the mechanisms by which they diversified into a large, biochemically diverse, multigene family. Our results show a much greater diversity of family members than was previously known, including a number of subfamilies that did not fall within any previously identified groups with characterized activities. In addition, we found that the long-term evolutionary processes that gave rise to the diversity of three-finger toxins are consistent with the birth-and-death model of multigene family evolution. It is anticipated that this “three-finger toxin toolkit” will prove to be useful in providing a clearer picture of the diversity of investigational ligands or potential therapeutics available within this important family.

Evolution of an Arsenal Structural and Functional Diversification of the Venom System in the Advanced Snakes (Caenophidia)

Venom is a key innovation underlying the evolution of advanced snakes (Caenophidia). Despite this, very little is known about venom system structural diversification, toxin recruitment event timings, or toxin molecular evolution. A multidisciplinary approach was used to examine the diversification of the venom system and associated toxins across the full range of the ∼100 million-year-old advanced snake clade with a particular emphasis upon families that have not secondarily evolved a front-fanged venom system (∼80% of the 2500 species). Analysis of cDNA libraries revealed complex venom transcriptomes containing multiple toxin types including three finger toxins, cobra venom factor, cysteine-rich secretory protein, hyaluronidase, kallikrein, kunitz, lectin, matrix metalloprotease, phospholipase A2, snake venom metalloprotease/a disintegrin and metalloprotease, and waprin. High levels of sequence diversity were observed, including mutations in structural and functional residues, changes in cysteine spacing, and major deletions/truncations. Morphological analysis comprising gross dissection, histology, and magnetic resonance imaging also demonstrated extensive modification of the venom system architecture in non-front-fanged snakes in contrast to the conserved structure of the venom system within the independently evolved front-fanged elapid or viperid snakes. Further, a reduction in the size and complexity of the venom system was observed in species in which constriction has been secondarily evolved as the preferred method of prey capture or dietary preference has switched from live prey to eggs or to slugs/snails. Investigation of the timing of toxin recruitment events across the entire advanced snake radiation indicates that the evolution of advanced venom systems in three front-fanged lineages is associated with recruitment of new toxin types or explosive diversification of existing toxin types. These results support the role of venom as a key evolutionary innovation in the diversification of advanced snakes and identify a potential role for non-front-fanged venom toxins as a rich source for lead compounds for drug design and development.

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity.

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle α1βγδ-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 Å by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snakes diet.

Isolation of a Neurotoxin (α-colubritoxin) from a Nonvenomous Colubrid: Evidence for Early Origin of Venom in Snakes

The evolution of venom in advanced snakes has been a focus of long-standing interest. Here we provide the first complete amino acid sequence of a colubrid toxin, which we have called α-colubritoxin, isolated from the Asian ratsnake Coelognathusradiatus (formerly known as Elapheradiata), an archetypal nonvenomous snake as sold in pet stores. This potent postsynaptic neurotoxin displays readily reversible, competitive antagonism at the nicotinic receptor. The toxin is homologous with, and phylogenetically rooted within, the three-finger toxins, previously thought unique to elapids, suggesting that this toxin family was recruited into the chemical arsenal of advanced snakes early in their evolutionary history. LC-MS analysis of venoms from most other advanced snake lineages revealed the widespread presence of components of the same molecular weight class, suggesting the ubiquity of three-finger toxins across advanced snakes, with the exclusion of Viperidae. These results support the role of venom as a key evolutionary innovation in the early diversification of advanced snakes and provide evidence that forces a fundamental rethink of the very concept of nonvenomous snake.

Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails.

Venomous animals are thought to inject the same combination of toxins for both predation and defence, presumably exploiting conserved target pharmacology across prey and predators. Remarkably, cone snails can rapidly switch between distinct venoms in response to predatory or defensive stimuli. Here, we show that the defence-evoked venom of Conus geographus contains high levels of paralytic toxins that potently block neuromuscular receptors, consistent with its lethal effects on humans. In contrast, C. geographus predation-evoked venom contains prey-specific toxins mostly inactive at human targets. Predation- and defence-evoked venoms originate from the distal and proximal regions of the venom duct, respectively, explaining how different stimuli can generate two distinct venoms. A specialized defensive envenomation strategy is widely evolved across worm, mollusk and fish-hunting cone snails. We propose that defensive toxins, originally evolved in ancestral worm-hunting cone snails to protect against cephalopod and fish predation, have been repurposed in predatory venoms to facilitate diversification to fish and mollusk diets.

Antimicrobial activity of omwaprin, a new member of the waprin family of snake venom proteins

We have isolated and characterized omwaprin, a 50-amino-acid cationic protein from the venom of inland taipan (Oxyuranus microlepidotus). It is a new member of the waprin family of snake venom proteins. A synthetic gene was designed and constructed for expressing the recombinant protein in Escherichia coli. Recombinant omwaprin was used for carrying out functional analyses. The protein is non-toxic to Swiss albino mice at doses of up to 10 mg/kg when administered intraperitoneally. However, it shows selective and dose-dependant antibacterial activity against Gram-positive bacteria. The minimum inhibitory doses were in the range 2-10 microg for selected species of bacteria in radial diffusion assays. The antibacterial activity is salt-tolerant up to 350 mM NaCl. However, omwaprin lost its antibacterial activity upon reduction and alkylation of its cysteine residues, or upon deletion of six N-terminal amino acid residues, four of which are positively charged. These observations indicate that the three-dimensional structure constrained by four disulfide bonds and the N-terminal residues are essential for its activity. The mechanism of action is via membrane disruption, as shown by scanning electron microscopy. Importantly, omwaprin lacks haemolytic activity on human erythrocytes. This demonstrates the specificity of omwaprin for bacterial membranes. Unlike other reported WAP (whey acidic protein) domain-containing antibacterial proteins, including elafin, EPPIN (epididymal proteinase inhibitor), SWAM1 and SWAM2 [single WAP (whey acidic protein) motif proteins 1 and 2] and SLPI (secretory leucocyte proteinase inhibitor), omwaprin shows species-specific activity on the Gram-positive bacteria tested.

Evolutionary origin and development of snake fangs

Many advanced snakes use fangs—specialized teeth associated with a venom gland—to introduce venom into prey or attacker. Various front- and rear-fanged groups are recognized, according to whether their fangs are positioned anterior (for example cobras and vipers) or posterior (for example grass snakes) in the upper jaw. A fundamental controversy in snake evolution is whether or not front and rear fangs share the same evolutionary and developmental origin. Resolving this controversy could identify a major evolutionary transition underlying the massive radiation of advanced snakes, and the associated developmental events. Here we examine this issue by visualizing the tooth-forming epithelium in the upper jaw of 96 snake embryos, covering eight species. We use the sonic hedgehog gene as a marker, and three-dimensionally reconstruct the development in 41 of the embryos. We show that front fangs develop from the posterior end of the upper jaw, and are strikingly similar in morphogenesis to rear fangs. This is consistent with their being homologous. In front-fanged snakes, the anterior part of the upper jaw lacks sonic hedgehog expression, and ontogenetic allometry displaces the fang from its posterior developmental origin to its adult front position—consistent with an ancestral posterior position of the front fang. In rear-fanged snakes, the fangs develop from an independent posterior dental lamina and retain their posterior position. In light of our findings, we put forward a new model for the evolution of snake fangs: a posterior subregion of the tooth-forming epithelium became developmentally uncoupled from the remaining dentition, which allowed the posterior teeth to evolve independently and in close association with the venom gland, becoming highly modified in different lineages. This developmental event could have facilitated the massive radiation of advanced snakes in the Cenozoic era, resulting in the spectacular diversity of snakes seen today.

Eggs-Only Diet: Its Implications for the Toxin Profile Changes and Ecology of the Marbled Sea Snake (Aipysurus eydouxii)

Studies so far have correlated the variation in the composition of snake venoms with the target prey population and snake’s diet. Here we present the first example of an alternative evolutionary link between venom composition and dietary adaptation of snakes. We describe a dinucleotide deletion in the only three finger toxin gene expressed in the sea snake Aipysurus eydouxii (Marbled Sea Snake) venom and how it may have been the result of a significant change in dietary habits. The deletion leads to a frame shift and truncation with an accompanying loss of neurotoxicity. Due to the remarkable streamlining of sea snake venoms, a mutation of a single toxin can have dramatic effects on the whole venom, in this case likely explaining the 50- to 100-fold decrease in venom toxicity in comparison to that of other species in the same genus. This is a secondary result of the adaptation of A. eydouxii to a new dietary habit — feeding exclusively on fish eggs and, thus, the snake no longer using its venom for prey capture. This was parallel to greatly atrophied venom glands and loss of effective fangs. It is interesting to note that a potent venom was not maintained for use in defense, thus reinforcing that the primary use of snake venom is for prey capture.

Structure–function properties of venom components from Australian elapids

A comprehensive review of venom components isolated thus far from Australian elapids. Illustrated is that a tremendous structural homology exists among the components but this homology is not representative of the functional diversity. Further, the review illuminates the overlooked species and areas of research.