The Snake with the Scorpion’s Sting: Novel Three-Finger Toxin Sodium Channel Activators from the Venom of the Long-Glanded Blue Coral Snake (Calliophis bivirgatus)

Abstract

Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake’s body length and nestle within the rib cavity. Despite the snake’s notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of NaV1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (NaV) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that NaV modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera re...

Multifunctional Toxins in Snake Venoms and Therapeutic Implications: From Pain to Hemorrhage and Necrosis

“…Pre-and post-synaptic toxin, block of neuromuscular transmission through modulation of nAChR, AChE, Na V 1.4, and L-type calcium channels (Grant and Chiappinelli, 1985;Changeux, 1990;De Weille et al, 1991;Garcia et al, 2001;Marquer et al, 2011;Yang et al, 2016) Their functional properties associated to pain, inflammation, hemorrhage, necrosis and paralysis are described.…”

“…For example, αneurotoxins inhibit muscle acetylcholine receptors (nAChR) (Changeux, 1990), κ-neurotoxins inhibits neuronal AChR (Grant and Chiappinelli, 1985), muscarinic toxins inhibit muscarinic receptors (Marquer et al, 2011), fasciculins inhibit acetylcholinesterase (AChE) (Marchot et al, 1998), calciseptine modulates L-type calcium channels (De Weille et al, 1991;Garcia et al, 2001), cardiotoxins interact non-specifically with phospholipids (Konshina et al, 2017), or induce insulin secretion (Nguyen et al, 2012), mambin interacts with platelet receptors (McDowell et al, 1992), exactin inhibits Factor X (Girish and Kini, 2016), β-cardiotoxins inhibit β-adrenoreceptors (Rajagopalan et al, 2007), MTα inhibits α-adrenoreceptors (Koivula et al, 2010), mambalgins inhibit ASIC channels (Diochot et al, 2012), Tx7335 that activates potassium channels (Rivera- Torres et al, 2016) and calliotoxin activates voltagegated sodium channels (Na V ) (Yang et al, 2016). Their toxic biological effects include flaccid or spastic paralysis due to the inhibition of AChE and ACh receptors (Grant and Chiappinelli, 1985;Changeux, 1990;Marchot et al, 1998;Marquer et al, 2011), and activation of Na V 1.4 (Yang et al, 2016) and L-type calcium channels (Garcia et al, 2001) in the periphery, necrosis through the action of cardiotoxins (cytotoxins) (Konshina et al, 2017), alteration of the cardiac rate through modulation of αand β-adrenoreceptors (Rajagopalan et al, 2007;Koivula et al, 2010), and altered homeostasis through inhibition of platelet aggregation (McDowell et al, 1992) and Factor X (Girish and Kini, 2016). In addition to their multitude of bio-activities, 3FTXs can remarkably display toxicities that target distinct classes of organisms as demonstrated in non-front fanged snake venoms that produce 3FTX isoforms which are non-toxic to mice but highly toxic to lizards, and vice-versa (Modahl et al, 2018b).…”

Scorpion toxins interact with nicotinic acetylcholine receptors

“…These toxins belong to the Kunitz inhibitor structural type. Recently, a new toxin called calliotoxin has been identified in the venom of the blue coral snake Calliophis bivirgatus, which enhances the peak inward current and delays the inactivation of Nav1.4 channel [35]. It is the first identified Nav activator from snake venom belonging to the large family of three-finger toxins.…”

A TetR-family protein activates transcription from a new promoter motif associated with essential genes for autotrophic growth in acetogens

“…Detection of proteins in both the digestion products of gel band excision and the whole captured material was performed using a QTOF Sciex 5600 or a Thermo Orbitrap Elite mass spectrometer (depending on instrument availability) with details described elsewhere (Kappler and Nouwens, 2013) (Yang et al 2016) with a modified liquid chromatographic (LC) gradient. Protein identification was performed using the software ProteinPilot v5.0 (ABSciex) with the Paragon Algorithm against the NC_022592.1 and CP006763 genome annotations with the following search parameters: Trypsin+LysC digestion; IAA as cysteine alkylation; Thorough search effort; FDR analysis.…”