Isolation and pharmacological characterisation of neurotoxins from Australo-Papuan death adder venoms (Acanthophis spp.)

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NO FULL TEXT AVAILABLE. This thesis contains 3rd party copyright material. ----- Australo-papuan death adders (Acanthophis spp.) are a cause of serious envenomations in Papua New Guinea and northern Australia, often resulting in neurotoxic paralysis. Victims occasionally present with delayed-onset neurotoxicity that sometimes responds poorly to antivenom or anticholinesterase treatment. This clinical outcome could be explained by the presence of irreversibly binding long- and short-chain postsynaptic α-neurotoxins. However, it could also be the result of potent snake presynaptic phospholipase A2 neurotoxin (SPAN) complexes and monomers, blocking neurotransmitter release and resulting in irreversible degeneration of the nerve terminal. This study attempted to determine within-genus variations in expression of high molecular mass SPAN complexes in the venoms of six major species of Acanthophis, and four geographic variants of A. antarcticus. Venoms were separated by size-exclusion liquid chromatography under non-denaturing conditions and fractions corresponding to proteins in the range of 22 to >60 kDa were subjected to pharmacological characterisation using the isolated chick biventer cervicis nerve-muscle (CBCNM) preparation. All venoms, except A. wellsi and A. pyrrhus, contained high mass fractions with phospholipase A2 activity. All high mass fractions, excluding the fraction from A. antarcticus (SA variant), inhibited twitch contractions of the CBCNM preparation. This inhibition was of slow onset, and responses to exogenous nicotinic agonists were not blocked, consistent with the presence of SPAN complexes. The results of the study indicate that clinicians may need to be aware of possible prejunctional neurotoxicity following envenomations from A. antarcticus (all geographic variants except perhaps South Australia), A. praelongus, A. rugosus and A. laevis species, and that early antivenom intervention is important in preventing further development of toxicity. Further investigations were performed on Acanthophis antarcticus (NSW variant) venom to thoroughly characterise the previously isolated presynaptic fraction. The SPAN complex P-Elapitoxin-Aa1a (P-EPTX-Aa1a) was isolated from the venom of the common death adder A. antarcticus using size-exclusion liquid chromatography methods, and subjected to biochemical and pharmacological characterisation. Using the chick biventer cervicis nerve-muscle preparation, P-EPTX-Aa1a (44,698 Da) was shown to produce inhibition of nerve-evoked twitch contractions while responses to cholinergic agonists and KCI remained unaffected. P-EPTX-Aa1a also produced significant fade in tetanic contractions and a triphasic timecourse of neuromuscular blockade under conditions of low quantal content (high [Mg2+]ₒ). These actions are consistent with the activity of other SPANs that inhibit acetylcholine release. Following RP-HPLC, P-EPTX-Aa1a was found to be a heterotrimeric complex composed of α-, β- and γ-subunits in a 1:1:1 stoichiometry with each subunit showing significant N-terminal sequence homology to taipoxin, a SPAN from the unrelated Australo-Papuan elapid Oxyuranus s. scutellatus. Like taipoxin, only the α-chain produced any signs of neurotoxicity or displayed significant PLA2 enzymatic activity. Preincubation with CSL monovalent death adder antivenom (5 U/ml) or suramin (0.3 mM), or inhibition of PLA2 activity by incubation with 4-bromophenacyl bromide, either prevented or significantly delayed the onset of toxicity. However, following complete neuromuscular blockade with P-EPTX-Aa1a, antivenom failed to restore neuromuscular function. Importantly, immediately following rapid neuromuscular blockade with whole venom, antivenom was ineffective in reversing the block of neurotransmission. This indicates the potential presence of an irreversible postsynaptic α-neurotoxin. Accordingly, fast perfusion (FPLC) and high pressure (HPLC) liquid chromatography purification techniques were used to isolate a long-chain α-neurotoxin, α-elapitoxin-Aa2a (α-EPTX-Aa2a), from A. antarcticus (NSW variant) venom. ESI-Q-TOF mass spectrometry confirmed the mass of this component to be 8850.0 Da. Therefore, α-EPTX-Aa2a is the largest long-chain α-neurotoxin isolated from elapid venom, and second only to the 'non-conventional' α-neurotoxin rufoxin from the venom of the colubrid Ramphiophis oxyrhynchus (10661 Da). This toxin displayed significant homology to a range of long-chain postsynaptic α-neurotoxins, including the presence of the characteristic fifth disulfide at the tip of loop II. In contrast to all classical long-chain α-neurotoxins possessing the critical fifth disulfide bond, the novel long-chain α-neurotoxin α-EPTX-Aa2a lacked affinity for neuronal α7-type nicotinic acetylcholine receptors (nAChRs). α-EPTX-Aa2a (0.1-1 μM) caused a concentration-dependent inhibition of indirect twitches, and blocked contractures to cholinergic agonists in the isolated CBCNM preparation, consistent with a postsynaptic curaremimetic mode of action. α-EPTX-Aa2a (1-10 nM) produced a potent pseudo-irreversible antagonism of chick muscle nAChRs, with an estimated pA2 value of 8.311 ± 0.031, which was not reversed by monovalent death adder antivenom. This is only 2.5-fold less potent than the prototypical long-chain α-neurotoxin, α-bungarotoxin. In contrast, α-EPTX-Aa2a produced complete, but weak, inhibition of 125I-α-bungarotoxin binding to rat hippocampal α7 nAChRs (pKɪ = 3.670), despite high sequence homology and similar mass to a wide range of long-chain α-neurotoxins. The mostly likely cause for the loss of α7 binding affinity is a leucine substitution, in loop II of α-EPTX-Aa2a, for the highly conserved Arg33 in long-chain α-neurotoxins. Arg33 has been shown to be critical for both neuronal and muscle activity. Despite this substitution, α-EPTX-Aa2a retains high affinity for muscle (α1)2 βγδ nAChRs. This is probably as a result of an Arg29 residue, previously shown to be critical for muscle (α1)2 βγδ nAChR affinity and highly conserved across all short-chain, but not long-chain, α-neurotoxins. α-EPTX-Aa2a therefore represents a novel atypical long-chain α-neurotoxin that includes a fifth disulfide but exhibits differential affinity for nAChR subtypes. These findings indicate that the majority of Australo-Papuan death adders contain a potent presynaptic SPAN complex and, in the case of A. antarcticus (NSW), a postsynaptic α-neurotoxin. The rapid, potent and pseudo-irreversible nature of the disruption to neuromuscular transmission by the postsynaptic α-neurotoxin, combined with the irreversible binding and destruction of the presynaptic terminal membrane by SPAN complexes, further highlights the importance of early intervention with antivenom in systemic death adder envenomation.
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