Wayne C. Hodgson

Early evolution of the venom system in lizards and snakes

Among extant reptiles only two lineages are known to have evolved venom delivery systems, the advanced snakes and helodermatid lizards (Gila Monster and Beaded Lizard). Evolution of the venom system is thought to underlie the impressive radiation of the advanced snakes (2,500 of 3,000 snake species). In contrast, the lizard venom system is thought to be restricted to just two species and to have evolved independently from the snake venom system. Here we report the presence of venom toxins in two additional lizard lineages (Monitor Lizards and Iguania) and show that all lineages possessing toxin-secreting oral glands form a clade, demonstrating a single early origin of the venom system in lizards and snakes. Construction of gland complementary-DNA libraries and phylogenetic analysis of transcripts revealed that nine toxin types are shared between lizards and snakes. Toxinological analyses of venom components from the Lace Monitor Varanus varius showed potent effects on blood pressure and clotting ability, bioactivities associated with a rapid loss of consciousness and extensive bleeding in prey. The iguanian lizard Pogona barbata retains characteristics of the ancestral venom system, namely serial, lobular non-compound venom-secreting glands on both the upper and lower jaws, whereas the advanced snakes and anguimorph lizards (including Monitor Lizards, Gila Monster and Beaded Lizard) have more derived venom systems characterized by the loss of the mandibular (lower) or maxillary (upper) glands. Demonstration that the snakes, iguanians and anguimorphs form a single clade provides overwhelming support for a single, early origin of the venom system in lizards and snakes. These results provide new insights into the evolution of the venom system in squamate reptiles and open new avenues for biomedical research and drug design using hitherto unexplored venom proteins.

Pharmacology and biochemistry of spider venoms.

Spider venoms represent an incredible source of biologically active substances which selectively target a variety of vital physiological functions in both insects and mammals. Many toxins isolated from spider venoms have been invaluable in helping to determine the role and diversity of neuronal ion channels and the process of exocytosis. In addition, there is enormous potential for the use of insect specific toxins from animal sources in agriculture. For these reasons, the past 15-20 years has seen a dramatic increase in studies on the venoms of many animals, particularly scorpions and spiders. This review covers the pharmacological and biochemical activities of spider venoms and the nature of the active components. In particular, it focuses on the wide variety of ion channel toxins, novel non-neurotoxic peptide toxins, enzymes and low molecular weight compounds that have been isolated. It also discusses the intraspecific sex differences in given species of spiders.

A central role for venom in predation by Varanus komodoensis (Komodo Dragon) and the extinct giant Varanus (Megalania) priscus

The predatory ecology of Varanus komodoensis (Komodo Dragon) has been a subject of long-standing interest and considerable conjecture. Here, we investigate the roles and potential interplay between cranial mechanics, toxic bacteria, and venom. Our analyses point to the presence of a sophisticated combined-arsenal killing apparatus. We find that the lightweight skull is relatively poorly adapted to generate high bite forces but better adapted to resist high pulling loads. We reject the popular notion regarding toxic bacteria utilization. Instead, we demonstrate that the effects of deep wounds inflicted are potentiated through venom with toxic activities including anticoagulation and shock induction. Anatomical comparisons of V. komodoensis with V. (Megalania) priscus fossils suggest that the closely related extinct giant was the largest venomous animal to have ever lived.

Ohanin, a novel protein from king cobra venom, induces hypolocomotion and hyperalgesia in mice.

We have identified, purified, and determined the complete amino acid sequence of a novel protein, ohanin from Ophiophagus hannah (king cobra) venom. It is a small protein containing 107 amino acid residues with a molecular mass of 11951.47 ± 0.67 Da as assessed by electrospray ionization-mass spectrometry. It does not show similarity to any known families of snake venom proteins and hence is the first member of a new family of snake venom proteins. It shows similarity to PRY and SPRY domain proteins. It is nontoxic up to 10 mg/kg when injected intraperitoneally in mice. Ohanin produced statistically significant and dose-dependent hypolocomotion in mice. In a pain threshold assay, it showed dose-dependent hyperalgesic effect. The ability of the protein to elicit a response at greatly reduced doses when injected intracerebroventricularly as compared with intraperitoneal administration in both the locomotion and hot plate experiments strongly suggests that ohanin acts on the central nervous system. Since the natural abundance of the protein in the venom is low (∼1 mg/g), a synthetic gene was constructed and expressed. The recombinant protein, which was obtained in the insoluble fraction in Escherichia coli, was purified under denaturing condition and was refolded. Recombinant ohanin is structurally and functionally similar to native protein as determined by circular dichroism and hot plate assay, suggesting that it will be useful in future structure-function relationship studies.

Development of a sensitive enzyme immunoassay for measuring taipan venom in serum.

The detection and measurement of snake venom in blood is important for confirming snake identification, determining when sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays (EIA) have had persistent problems with poor sensitivity and high background absorbance leading to false positive results. This is particularly problematic with Australasian snakes where small amounts of highly potent venom are injected, resulting in low concentrations being associated with severe clinical effects. We aimed to develop a venom EIA with a limit of detection (LoD) sufficient to accurately distinguish mild envenoming from background absorbance at picogram concentrations of venom in blood. Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before and after antivenom, and from rats given known venom doses. A sandwich EIA was developed using biotinylated rabbit anti-snake venom antibodies for detection. For low venom concentrations (i.e. <1 ng/mL) the assay was done before and after addition of antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the standard assay and 0.1 ng/mL for the antivenom difference method. In 11 pre-antivenom samples the median venom concentration was 10 ng/mL (Range: 0.3-3212 ng/mL). In four patients with incomplete venom-induced consumption coagulopathy the median venom concentration was 2.4 ng/mL compared to 30 ng/mL in seven patients with complete venom-induced consumption coagulopathy. No venom was detected in any post-antivenom sample and the median antivenom dose prior to this first post-antivenom sample was 1.5 vials (1-3 vials), including 7 patients administered only 1 vial. In rats the assay distinguished a 3-fold difference in venom dose administered and there was small inter-individual variability. There was small but measurable cross-reactivity with black snake (Pseudechis), tiger snake (Notechis) and rough-scale snake (Tropidechis carinatus) venoms with the assay for low venom concentrations (<1 ng/mL). The use of biotinylation and the antivenom difference method in venom EIA produces a highly sensitive assay that will be useful for determining antivenom dose, forensic and clinical diagnosis.

Novel Venom Proteins Produced by Differential Domain-Expression Strategies in Beaded Lizards and Gila Monsters (genus Heloderma)

The origin and evolution of venom proteins in helodermatid lizards were investigated by multidisciplinary techniques. Our analyses elucidated novel toxin types resultant from three unique domain-expression processes: 1) The first full-length sequences of lethal toxin isoforms (helofensins) revealed this toxin type to be constructed by an ancestral monodomain, monoproduct gene (beta-defensin) that underwent three tandem domain duplications to encode a tetradomain, monoproduct with a possible novel protein fold; 2) an ancestral monodomain gene (encoding a natriuretic peptide) was medially extended to become a pentadomain, pentaproduct through the additional encoding of four tandemly repeated proline-rich peptides (helokinestatins), with the five discrete peptides liberated from each other by posttranslational proteolysis; and 3) an ancestral multidomain, multiproduct gene belonging to the vasoactive intestinal peptide (VIP)/glucagon family being mutated to encode for a monodomain, monoproduct (exendins) followed by duplication and diversification into two variant classes (exendins 1 and 2 and exendins 3 and 4). Bioactivity characterization of exendin and helokinestatin elucidated variable cardioactivity between isoforms within each class. These results highlight the importance of utilizing evolutionary-based search strategies for biodiscovery and the virtually unexplored potential of lizard venoms in drug design and discovery.

Effectiveness of snake antivenom: Species and regional venom variation and its clinical impact.

The ubiquity of venom variation in snakes poses special problems for the manufacture of antivenom and has undermined the commercial attractiveness of this class of therapeutic agent. In particular, it has been amply documented that both interspecific and intraspecific variation in venom composition can affect the neutralisation capacity of antivenoms. This may be exacerbated by the selective use of tests of venom toxicity and antivenom efficacy, such as the lethal dose and ED50, resulting in inadequate neutralisation of time, rather than dose, dependent toxins, particularly enzymes involved in defibrinogenating, haemorrhagic and necrotising venom activities. The clinical consequences can be reduced efficacy against some important venom activities or even complete treatment failure in critical envenomations. All these factors, combined with the ongoing reduction in the number of antivenom manufacturers world‐wide, and concomitant contraction in the range of available antivenoms, present significant challenges for the treatment of snakebite in the 21st century.

Functional and Structural Diversification of the Anguimorpha Lizard Venom System

Venom has only been recently discovered to be a basal trait of the Anguimorpha lizards. Consequently, very little is known about the timings of toxin recruitment events, venom protein molecular evolution, or even the relative physical diversifications of the venom system itself. A multidisciplinary approach was used to examine the evolution across the full taxonomical range of this ∼130 million-year-old clade. Analysis of cDNA libraries revealed complex venom transcriptomes. Most notably, three new cardioactive peptide toxin types were discovered (celestoxin, cholecystokinin, and YY peptides). The latter two represent additional examples of convergent use of genes in toxic arsenals, both having previously been documented as components of frog skin defensive chemical secretions. Two other novel venom gland-overexpressed modified versions of other protein frameworks were also recovered from the libraries (epididymal secretory protein and ribonuclease). Lectin, hyaluronidase, and veficolin toxin types were sequenced for the first time from lizard venoms and shown to be homologous to the snake venom forms. In contrast, phylogenetic analyses demonstrated that the lizard natriuretic peptide toxins were recruited independently of the form in snake venoms. The de novo evolution of helokinestatin peptide toxin encoding domains within the lizard venom natriuretic gene was revealed to be exclusive to the helodermatid/anguid subclade. New isoforms were sequenced for cysteine-rich secretory protein, kallikrein, and phospholipase A2 toxins. Venom gland morphological analysis revealed extensive evolutionary tinkering. Anguid glands are characterized by thin capsules and mixed glands, serous at the bottom of the lobule and mucous toward the apex. Twice, independently this arrangement was segregated into specialized serous protein-secreting glands with thick capsules with the mucous lobules now distinct (Heloderma and the Lanthanotus/Varanus clade). The results obtained highlight the importance of utilizing evolution-based search strategies for biodiscovery and emphasize the largely untapped drug design and development potential of lizard venoms.

Hypotensive Agents from Snake Venoms

Many snake venoms contain toxins which produce profound cardiovascular effects. The site of action of these toxins includes cardiac muscle, vascular smooth muscle and the capillary vascular bed. Some snake venoms, for example, contain peptides that inhibit angiotensin converting enzyme and potentiate the biological actions of bradykinin. Other snake venoms contain structural and functional equivalents of mammalian natriuretic peptides. Sarafotoxins are short peptide toxins found in the venoms of snakes from Atractaspis spp. which display potent vasoconstriction properties. These peptides, which share a high degree of sequence identity with endothelins, recognize and bind to endothelin receptors. Snakes have also evolved toxins which block L-type Ca(2+) currents (eg. calciseptine, FS2 toxins, C(10)S(2)C(2) and S(4)C(8)). Snake venom proteins have also been shown to increase vascular permeability. One such protein, increasing capillary permeability protein (ICPP) has recently been isolated from the venom of Vipera lebetina. ICPP is an extremely potent permeability factor with a structure similar to vascular endothelial growth factor (VEGF). Thus there is a vast array of snake toxins with potent cardiovascular activity. Some of these proteins and peptides have proven to be highly selective tools in the study of physiological processes. Others have been used as probes of potential therapeutic targets or as lead compounds in the development of therapeutic agents. Therefore these and other related snake venom proteins hold great promise in the future understanding and treatment of cardiovascular diseases.

The effects of antivenom on the in vitro neurotoxicity of venoms from the taipans Oxyuranus scutellatus, Oxyuranus microlepidotus and Oxyuranus scutellatus canni.

The venoms of the inland (Oxyuranus microlepidotus), coastal (O. scutellatus) and Papuan (O. s. canni) taipans are among the most potent in the world. The present study compared the in vitro neurotoxic effects of these venoms and the protective effects of taipan antivenom. Venom (10 microg/ml) from all three snakes abolished nerve-mediated twitches of the chick biventer cervicis muscle preparation with the following rank order of potency (based on the time taken to inhibit 90% of the twitch response; t90): O. microlepidotus (27+/-3 min) > O. scutellatus (42+/-3 min) = O. S. canni (48+/-5 min). This inhibitory effect of all three venoms was primarily postsynaptic in origin as evidenced by the inhibition of responses to exogenous acetylcholine (ACh; 1 mM) and carbachol (CCh; 20 microM), but not potassium chloride (40 mM). In contrast, the presynaptic neurotoxins taipoxin (3 microg/ml) and paradoxin (3 microg/ml) abolished nerve-mediated twitches without producing a significant effect on contractile responses to exogenous agonists. Prior incubation of the tissue with taipan antivenom (1 unit/ml for 10 min) markedly attenuated the inhibitory effects of taipoxin (3 microg/ml) and paradoxin (3 microg/ml), as well as O. scutellatus (10 microg/ml) and O. s. canni (10 microg/ml) venom. However, in the presence of antivenom, O. microlepidotus venom (10 microg/ml) still abolished nerve-mediated twitches and responses to ACh and CCh. The results of the current study indicate that taipan antivenom, raised against O. scutellatus venom, is effective, in vitro, against the neurotoxic effects of venom from the Papuan and coastal taipans, as well as the presynaptic effects of venom from the inland taipan. However, the antivenom appears less effective against the postsynaptic effects of the latter. It is possible that inland taipan venom contains a component not neutralised by the antivenom which may contribute to the extreme potency of this venom.