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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 Coelognathus radiatus (formerly known as Elaphe radiata), 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.

In this study, venoms from species in the Colubrinae, Homalopsinae, Natricinae, Pseudoxyrhophiinae and Psammophiinae snake families were assayed for activity in the chick biventer cervicis skeletal nerve muscle preparation. Boiga dendrophila , Boiga cynodon , Boiga dendrophila gemincincta , Boiga drapiezii , Boiga irregularis , Boiga nigriceps and Telescopus dhara venoms (10 μg/ml) displayed postsynaptic neuromuscular activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches. Neostigmine (5 μM) reversed the inhibition caused by B. cynodon venom (10 μg/ml) while the inhibitory effects of Psammophis mossambicus venom (10 μg/ml) spontaneously reversed, indicating a reversible mode of action for both venoms. Trimorphodon biscutatus (10 μg/ml) displayed irreversible presynaptic neurotoxic activity. Detectable levels of phospholipase A 2 activity were found only in T. biscutatus , T. dhara and P. mossambicus venoms. The results demonstrate a hitherto unsuspected diversity of pharmacological actions in all lineages which may have implications ranging from clinical management of envenomings to venom evolution.

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.

The evolution of the venomous function of snakes and the diversification of the toxins has been of tremendous research interest and considerable debate. It has become recently evident that the evolution of the toxins in the advanced snakes (Colubroidea) predated the evolution of the advanced, front-fanged delivery mechanisms. Historically, the venoms of snakes lacking front-fanged venom-delivery systems (conventionally grouped into the paraphyletic family Colubridae) have been largely neglected. In this study we used liquid chromatography with mass spectrometry (LC/MS) to analyze a large number of venoms from a wide array of species representing the major advanced snake clades Atractaspididae, Colubrinae, Elapidae, Homalopsinae, Natricinae, Psammophiinae, Pseudoxyrhophiinae, Xenodontinae, and Viperidae. We also present the first sequences of toxins from Azemiops feae as well as additional toxin sequences from the Colubrinae. The large body of data on molecular masses and retention times thus assembled demonstrates a hitherto unsuspected diversity of toxins in all lineages, having implications ranging from clinical management of envenomings to venom evolution to the use of isolated toxins as leads for drug design and development. Although definitive assignment of a toxin to a protein family can only be done through demonstrated structural studies such as N-terminal sequencing, the molecular mass data complemented by LC retention information, presented here, do permit formulation of reasonable hypotheses concerning snake venom evolution and potential clinical effects to a degree not possible till now, and some hypotheses of this kind are proposed here. The data will also be useful in biodiscovery.

1. Tiger snake antivenom, raised against Notechis scutatus venom, is indicated not only for the treatment of envenomation by this snake, but also that of the copperhead (Austrelaps superbus) and Stephen's banded snake (Hoplocephalus stephensi). The present study compared the neuromuscular pharmacology of venom from these snakes and the in vitro efficacy of tiger snake antivenom. 2. In chick biventer cervicis muscle and mouse phrenic nerve diaphragm preparations, all venoms (3-10 microg/mL) produced inhibition of indirect twitches. In the biventer muscle, venoms (10 micro g/mL) inhibited responses to acetylcholine (1 mmol/L) and carbachol (20 micromol/L), but not KCl (40 mmol/L). The prior (10 min) administration of 1 unit/mL antivenom markedly attenuated the neurotoxic effects of A. superbus and N. scutatus venoms (10 microg/mL), but was less effective against H. stephensi venom (10 microg/mL); 5 units/mL antivenom attenuated the neurotoxic activity of all venoms. 3. Administration of 5 units/mL antivenom at t90 partially reversed, over a period of 3 h, the inhibition of twitches produced by N. scutatus (10 microg/mL; 41% recovery), A. superbus (10 microg/mL; 25% recovery) and H. stephensi (10 microg/mL; 50% recovery) venoms. All venoms (10-100 microg/mL) also displayed signs of in vitro myotoxicity. 4. The results of the present study indicate that all three venoms contain neurotoxic activity that is effectively attenuated by tiger snake antivenom.

Death adders (genus Acanthophis) are unique among elapid snakes in both morphology and venom composition. Despite this genus being among the most divergent of all elapids, the venom has been historically regarded as relatively quite simple. In this study, liquid chromatography/mass spectrometry (LC/MS) analysis has revealed a much greater diversity in venom composition, including the presence of molecules of novel molecular weights that may represent a new class of venom component. Furthermore, significant variation exists between species and populations, which allow for the LC/MS fingerprinting of each species. Mass profiling of Acanthophis venoms clearly demonstrates the effectiveness of this technique which underpins fundamental studies ranging from chemotaxonomy to drug design.

Background: Spiders have evolved pharmacologically complex venoms that serve to rapidly subdue prey and deter predators. The major toxic factors in most spider venoms are small, disulfide-rich peptides. While there is abundant evidence that snake venoms evolved by recruitment of genes encoding normal body proteins followed by extensive gene duplication accompanied by explosive structural and functional diversification, the evolutionary trajectory of spider-venom peptides is less clear. Results: Here we present evidence of a spider-toxin superfamily encoding a high degree of sequence and functional diversity that has evolved via accelerated duplication and diversification of a single ancestral gene. The peptides within this toxin superfamily are translated as prepropeptides that are posttranslationally processed to yield the mature toxin. The N-terminal signal sequence, as well as the protease recognition site at the junction of the propeptide and mature toxin are conserved, whereas the remainder of the propeptide and mature toxin sequences are variable. All toxin transcripts within this superfamily exhibit a striking cysteine codon bias. We show that different pharmacological classes of toxins within this peptide superfamily evolved under different evolutionary selection pressures. Conclusions: Overall, this study reinforces the hypothesis that spiders use a combinatorial peptide library strategy to evolve a complex cocktail of peptide toxins that target neuronal receptors and ion channels in prey and predators. We show that the ω-hexatoxins that target insect voltage-gated calcium channels evolved under the influence of positive Darwinian selection in an episodic fashion, whereas the κ-hexatoxins that target insect calcium-activated potassium channels appear to be under negative selection. A majority of the diversifying sites in the ω-hexatoxins are concentrated on the molecular surface of the toxins, thereby facilitating neofunctionalisation leading to new toxin pharmacology. © 2014 Pineda et al.; licensee BioMed Central Ltd.

Although viperlike in appearance and habit, death adders belong to the Elapidae family of snakes. Systemic envenomation represents a serious medical problem with antivenom, which is raised against Acanthophis antarcticus venom, representing the primary treatment. This study focused on the major Acanthophis variants from Australia and islands in the Indo-Pacific region. Venoms were profiled using liquid chromatography-mass spectrometry, and analyzed for in vitro neurotoxicity (0.3-10 mug/ml), as well as the effectiveness of antivenom. (1-5 units/ml; 10 min prior to the addition of 10 mug/ml venom). The following death adder venoms were examined: A. antarcticus (from separate populations in New South Wales, Queensland, South Australia, and Western Australia), A. hawkei, A. praelongus, A. pyrrhus, A. rugosus, A. wellsi, and venom from an unnamed species from the Indonesian island of Seram. All venoms abolished indirect twitches of the chick isolated biventer cervicis nerve-muscle preparation in a dose-dependent manner. In addition, all venoms blocked responses to exogenous acetylcholine (1 m-M) and carbachol (20 muM), but not KCl (40 mM), suggesting postsynaptic neurotoxicity. Death adder antivenom (1 unit/ml) prevented the neurotoxic effects of A. pyrrhus, A. praelongus, and A. hawkei venoms, although it was markedly less effective against venoms from A. antarcticus (NSW, SA, WA), A. rugosus, A. wellsi, and A. sp. Scram. However, at 5 units/ml, antivenom was effective against all venoms tested. Death adder venoms, including those from A. antarcticus geographic variants, differed not only in their venom composition but also in their neurotoxic activity and susceptibility to antivenom. For the first time toxicological aspects of A. hawkei, A. wellsi, A. rugosus, and A. sp. Seram venoms were studied. (C) 2001 Academic Press.

Nat. Chem. Biol. 9, 826–833 (2013); published online 13 October 2013; corrected after print 11 December 2013 In the version of this article initially published, the US Department of Energy Office of Basic Energy Sciences, Energy Frontier Research Center grant number was incorrect. The correct numberis DE-SC 0001035.

High efficiencies and catalytic activities of natural oxido-reductases keep inspiring development of new enzymes with the goal to produce better catalysts, novel drug therapies, bioelectronics devices, bioremediation and alternative energy sources. However, the immense complexity of natural proteins and their very evident resistance and fragility to modification prevents scientists from deciphering what part of the protein structure is essential for reproducing the catalytic function itself apart from the many other demands made on a natural protein within a cell. To overcome this obstacle, we are designing general structural platforms (maquettes) that accommodate variety of oxido-reductase functions, including light energy harvesting, photochemical charge separation, oxygen transport, and oxidative metabolism. They also serve as models for understanding the fundamental properties of enzyme activity, stability, and folding. Here we will present a transmembrane maquette designed to form electron transfer chain across a lipid bilayer. This artificial membrane maquette contains four membrane spanning α-helices that are linked into a single chain. It has been expressed in high yields in inclusion bodies using E. Coli, purified and refolded in charged and uncharged detergent micelles, as well as in lipid vesicles. Circular dichroism studies revealed 70% α-helical nature in SDS and no melting in high temperatures and common denaturants, indicating its strong structural stability. We will present maquette assembly, stability, cofactor binding and redox characteristics in different membrane environments and how they compare to natural proteins.

In our earlier analysis of Varanus body shape, size was a dominating factor with some qualitative phylogenetic patterns and grouping of species into ecological categories. With a phylogeny and an improved capacity to account for the effects of size, we have reanalysed our morphometric data for male Australian goannas (Varanus spp.) using an increased number of specimens and species to examine whether variations in body shape can be accounted for by retreat choice, as it can for Western Australian Ctenophorus dragon lizards. After accounting for body size in the current analysis, four ecotypes based on retreat choice (i.e. those that retreat to oblique crevices between large rocks or rock faces, those that retreat to burrows dug into the ground, those that retreat to spaces under rocks or in tree hollows, and those that retreat to trees but not tree hollows) accounted for much of the variation in body shape. There is a phylogenetic pattern to the ecotypes, but accounting for phylogenetic effects did not weaken the link between body shape and ecotype based on retreat choice. This suggests that there are large differences in body shape among ecotypes, and shape is relatively independent of phylogeny. The strong link between shape and choice of retreat site in Varanus spp. is consistent with that for Ctenophorus spp. We speculate on why there might be a strong link between retreat choice and body shape for both Varanus and Ctenophorus.

We analyzed the origin and evolution of snake venom toxin families represented in both viperid and elapid snakes by means of phylogenetic analysis of the amino acid sequences of the toxins and related nonvenom proteins. Out of eight toxin families analyzed, five provided clear evidence of recruitment into the snake venom proteome before the diversification of the advanced snakes (Kunitz-type protease inhibitors, CRISP toxins, galactose-binding lectins, M12B peptidases, nerve growth factor toxins), and one was equivocal (cystatin toxins). In two others (phospholipase A(2) and natriuretic toxins), the nonmonophyly of venom toxins demonstrates that presence of these proteins in elapids and viperids results from independent recruitment events. The ANP/BNP natriuretic toxins are likely to be basal, whereas the CNP/BPP toxins are Viperidae only. Similarly, the lectins were recruited twice. In contrast to the basal recruitment of the galactose-binding lectins, the C-type lectins were shown to be Viperidae only, with the alpha-chains and beta-chains resulting from an early duplication event. These results provide strong additional evidence that venom evolved once, at the base of the advanced snake radiation, rather than multiple times in different lineages, with these toxins also present in the venoms of the "colubrid" snake families. Moreover, they provide a first insight into the composition of the earliest ophidian venoms and point the way toward a research program that could elucidate the functional context of the evolution of the snake venom proteome.

A new species of viviparous sea snake, Hydrophis donaldi sp. nov. (Hydrophiinae), is described from the Gulf of Carpen-taria, northern Australia. Molecular analyses reveal this species as a deeply divergent lineage within the Hydrophis sub-group, and separate it from all other sampled taxa by fixed nucleotide substitutions at three independent mitochondrial andnuclear loci. The new species is assigned to Hydrophis based on the current morphological diagnosis of this large but pa-raphyletic genus, and is distinguished from all other Hydrophis species and closely allied genera by a combination of mor-phological characters relating to scalation, colour pattern and osteology. Using current keys for sea snakes, H. donaldi sp. nov. might be mistaken for H. coggeri, H. sibauensis or H. torquatus diadema but it is readily distinguished from thesespecies by a higher number of bands on the body and tail, lower ventral count, strongly spinous body scales, and a wider,more rounded head. Sea snakes have been sampled intensively in the Gulf of Carpentaria due to their vulnerability to by-catch in the region’s commercial prawn-trawl fisheries. That this highly distinctive new species has evaded discovery inthe region until now is surprising, but might be explained by its habitat preferences. All known specimens of H. donaldi sp. nov. were found in estuarine habitats that are relatively poorly surveyed and are not targeted by commercial fisheries.

The Australian snake fauna is unique in harbouring more venomous species than non-venomous ones. Although taxonomic research on the elapid snakes of Australia goes back to the late 18th century, in stark contrast to other developed regions of the world (e.g. the continental USA), Australian snake taxonomy is very much in its infancy. Despite this, or perhaps because of this, the taxonomy of Australian snakes has been extraordinarily controversial, and many of the taxonomists involved correspondingly colourful. In this review, we explore the sometimes-tortured history of the taxonomic exploration of the venomous snake fauna of Australia, looking at some of the more colourful and notable contributors and highlighting systematic pitfalls that persist even today.

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.

Despite the staggering diversity of venomous animals, there seems to be remarkable convergence in regard to the types of proteins used as toxin scaffolds. However, our understanding of this fascinating area of evolution has been hampered by the narrow taxonomical range studied, with entire groups of venomous animals remaining almost completely unstudied. One such group is centipedes, class Chilopoda, which emerged about 440 Ma and may represent the oldest terrestrial venomous lineage next to scorpions. Here, we provide the first comprehensive insight into the chilopod “venome” and its evolution, which has revealed novel and convergent toxin recruitments as well as entirely new toxin families among both high- and low molecular weight venom components. The ancient evolutionary history of centipedes is also apparent from the differences between the Scolopendromorpha and Scutigeromorpha venoms, which diverged over 430 Ma, and appear to employ substantially different venom strategies. The presence of a wide range of novel proteins and peptides in centipede venoms highlights these animals as a rich source of novel bioactive molecules. Understanding the evolutionary processes behind these ancient venom systems will not only broaden our understanding of which traits make proteins and peptides amenable to neofunctionalization but it may also aid in directing bioprospecting efforts.

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 μg 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.

Electron transport chains are fundamental to both photosynthesis and oxidative phosphorylation. Protein-based electron transport chains transfer electrons from high-energy donors to lower-energy acceptors and are commonly coupled to the translocation of protons across a membrane, producing a transmembrane electrochemical potential gradient. Electron transfer rates within these chains are governed primarily by the distance between redox centers and by the driving force that originates from the redox mid-point potentials or coupled catalytic reactions. The complexity of natural redox protein structures contrasts the relatively simple rules of cofactor placement that, in principle, govern the electron transfer behavior. Rather than focusing on the structural details of a specific natural protein, we are designing general protein structural scaffolds (“maquettes”) to accommodate a variety of functions. Here we will present transmembrane electron transfer via AP6, an amphiphilic tetra-helical maquette that binds up to 6 hemes. We demonstrate that AP6 self-assembles with phospholipids into vesicles. Our stop flow experiments confirm that the AP6 maquette significantly increases the electron transfer rates between oxidizing interior and an external redox mediator dye, as shown below.View Large Image | View Hi-Res Image | Download PowerPoint Slide

We have designed a simple protein, monomeric four-α-helix bundle, which can serve as a platform for diverse set of oxidoreductase related functions. We describe the progressive design steps taken from the simplest α-helical peptide toward functionalized proteins. This platform has considerable latitude for external charge patterning and internal control of stability, both globally and locally. We show how ligation of photoactive and redox cofactors can induce the stabilization and structuring of helices. We demonstrate that this platform can accomplish not one, but variety of functions, including electron transfer to natural proteins, O2 binding sustained for seconds, CO and NO sensing, millisecond superoxide bursts and triplet-excited state-driven nanosecond charge-separation followed by micro to millisecond electron tunneling reactions. We will present our strategies for optimizing bacterial expression, assembly of natural cofactors such as hemes, flavins, iron-sulfur clusters, quinone, nicotinamide and light active cofactors (porphyrins, chlorins) and many more synthetic analogues with a control over the distance and orientation that allow tuning for specific selected function. These maquette proteins can be altered to assemble in water, membranes and on a range of surfaces including titania. Such versatility makes this protein platform suitable for further iterative designs for light energy harvesting, photochemical charge separation, oxygen transport, oxidative metabolism as well as understanding of fundamental properties of enzyme activity, stability, and folding.

Protein-driven electron transfers are essential steps in a wide range of biochemical processes. Electron transfer rates are governed primarily by the distance between redox centers and by the driving force originating from midpoint potentials and coupled catalytic reactions. The structural complexity of natural proteins contrasts with the relatively simple rules of cofactor selection and placement that, in principle, govern the electron transfer behavior. Here we present two examples of minimal 4-α-helical bundle proteins ("maquettes") that leverage these basic design principles to illustrate functional electron transfer. Both proteins support bis-histidine ligation of natural heme cofactors within a tetrahelical bundle. The first of these maquettes is BT6, a water-soluble monomeric protein with a net charge of −14. Reduced heme-bound BT6 transfers electrons to cytochrome c at 2x107 M/s, a rate similar to the electron transfer between the natural b-heme protein neuroglobin and cytochrome c during apoptosis. As in the neuroglobin-cyt c system, we believe that the rapid electron transfer from glutamate-rich BT6 to lysine-rich cytochrome c is enhanced through electrostatic interaction between the two proteins. The second maquette discussed here is AM1, an amphiphilic 4-α-helical monomer. AM1 incorporates three bis-histidine binding sites to ligate a chain of heme cofactors spanning a phospholipid bilayer. We performed stopped flow experiments to probe transmembrane electron transfer, mixing natural cytochrome c with AM1 liposomes encapsulating photoreduced flavin mononucleotide. In the presence of protein and heme, transmembrane electron transfer rates are significantly faster than in the absence of either.

Venom research and technology has advanced greatly, rapidly transforming the area of reptile venom. Research developments-like the development of molecular systematics-provide the framework necessary to reconstruct the evolutionary history of glands and fangs. Such research developments result in a fundamental shift in scientists' understanding of venom's evolution and usefulness in therapeutic development. Startling evolutionary expansion, including the development of new protein functions, enable the development of novel drug therapies and impact the effectiveness of anti-venoms available for the treatment of humans.Venomous Reptiles And Their Toxins is a comprehensive study of the entire scope of reptile venom, from its evolution to drug design and development. This book devotes a chapter to each toxin class found in reptile venom, detailing the full trajectory of research on the toxin in question. The comprehensive synthesis of research deals with the impact that venom has had on biomedical applications and snake evolution and ecology.