Make Acetylcholine Great Again! Australian Skinks Evolved Multiple Neurotoxin-Proof Nicotinic Acetylcholine Receptors in Defiance of Snake Venom

Abstract

Many vertebrates have evolved resistance to snake venom as a result of coevolutionary chemical arms races. In Australian skinks (family Scincidae), who often encounter venomous elapid snakes, the frequency, diversity, and molecular basis of venom resistance have been unexplored. This study investigated the evolution of neurotoxin resistance in Australian skinks, focusing on mutations in the muscle nicotinic acetylcholine receptor (nAChR) α1 subunit's orthosteric site that prevent pathophysiological binding by α-neurotoxins. We sampled a broad taxonomic range of Australian skinks and sequenced the nAChR α1 subunit gene. Key resistance-conferring mutations at the toxin-binding site (N-glycosylation motifs, proline substitutions, arginine insertions, changes in the electrochemical state of the receptor, and novel cysteines) were identified and mapped onto the skink organismal phylogeny. Comparisons with other venom-resistant taxa (amphibians, mammals, and reptiles) were performed, and structural modelling and binding assays were used to evaluate the impact of these mutations. Multiple independent origins of α-neurotoxin resistance were found across diverse skink lineages. Thirteen lineages evolved at least one resistance motif and twelve additional motifs evolved within these lineages, for a total of twenty-five times of α-neurotoxic venoms resistance. These changes sterically or electrostatically inhibit neurotoxin binding. Convergent mutations at the orthosteric site include the introduction of N-linked glycosylation sites previously known from animals as diverse as cobras and mongooses. However, an arginine (R) substitution at position 187 was also shown to have evolved on multiple occasions in Australian skinks, a modification previously shown to be responsible for the Honey Badger's iconic resistance to cobra venom. Functional testing confirmed this mode of resistance in skinks. Our findings reveal that venom resistance has evolved extensively and convergently in Australian skinks through repeated molecular adaptations of the nAChR in response to the enormous selection pressure exerted by elapid snakes subsequent to their arrival and continent-wide dispersal in Australia. These toxicological findings highlight a remarkable example of convergent evolution across vertebrates and provide insight into the adaptive significance of toxin resistance in snake-lizard ecological interactions.

Keywords: adaptation; evolution; neurotoxin; nicotinic acetylcholine receptor; skink; venom.

Figure 1

Australian skink orthosteric site mutations linked to α-neurotoxin resistance mapped on the organismal phylogeny, with a scale bar representing millions of years of diversification. The initial tree was obtained from Timetree.org [69] and has been adapted to include data from the key literature [70,71,72,73,74,75,76,77,78,79,80,81]. Phylogenetic ambiguities at internal nodes are represented as soft polytomies. Character history was traced through Maximum Parsimony-based ancestral sequence reconstruction executed in Mesquite v.3.81 [82]. Shading shows the approximate timing (~24 MYA) of the arrival of elapid snakes in Australia [68].

Figure 2

The three key resistance mutations documented as evolving in Australian skinks: (A) N-glycosylation of arginine at position 189, with the bulky glycan chains sterically interfering with α-neurotoxin docking; (B) substitution of prolines 194 or 197, thereby changing the orthosteric site secondary structure, leading to steric hindrance of α-neurotoxin docking; or (C) changing the electrostatic state of the orthosteric site either through the introduction of a positive charge at position 187, resulting in a same-charge repulsion of the positively charged α-neurotoxins, or the loss of negative charges at the position, resulting in decreased affinity for the α-neurotoxins.

Figure 3

Biolayer interferometry assaying of native and mutant Bellatorias frerei mimotopes to test if the arginine at position 187 that confers resistance in the Honey Badger (Mellivora capensis) [53] also confers resistance in skinks. The cysteine doublet was replaced by a serine doublet during peptide synthesis to prevent uncontrolled postsynthetic thiol oxidation [83]. Venom used for binding test was Acanthophis wellsi. Values are N = 3 mean with standard deviations shown for both the line graph and the area under the curve bar graphs.