Isolation and Structural and Pharmacological Characterization of α-Elapitoxin-Dpp2d, an Amidated Three Finger Toxin from Black Mamba Venom

Abstract

We isolated a novel, atypical long-chain three-finger toxin (TFT), α-elapitoxin-Dpp2d (α-EPTX-Dpp2d), from black mamba (Dendroaspis polylepis polylepis) venom. Proteolytic digestion with trypsin and V8 protease, together with MS/MS de novo sequencing, indicated that the mature toxin has an amidated C-terminal arginine, a posttranslational modification rarely observed for snake TFTs. α-EPTX-Dpp2d was found to potently inhibit α7 neuronal nicotinic acetylcholine receptors (nAChR; IC₅₀, 58 ± 24 nM) and muscle-type nAChR (IC₅₀, 114 ± 37 nM) but did not affect α3β2 and α3β4 nAChR isoforms at 1 μM concentrations. Competitive radioligand binding assays demonstrated that α-EPTX-Dpp2d competes with epibatidine binding to the Lymnea stagnalis acetylcholine-binding protein (Ls-AChBP; IC₅₀, 4.9 ± 2.3 nM). The activity profile and binding data are reminiscent of classical long-chain TFTs with a free carboxyl termini, suggesting that amidation does not significantly affect toxin selectivity. The crystal structure of α-EPTX-Dpp2d was determined at 1.7 Å resolution and displayed a dimeric toxin assembly with each monomer positioned in an antiparallel orientation. The dimeric structure is stabilized by extensive intermolecular hydrogen bonds and electrostatic interactions, which raised the possibility that the toxin may exist as a noncovalent homodimer in solution. However, chemical cross-linking and size-exclusion chromatography coupled with multiangle laser light scattering (MALLS) data indicated that the toxin is predominantly monomeric under physiological conditions. Because of its high potency and selectivity, we expect this toxin to be a valuable pharmacological tool for studying the structure and function of nAChRs.

Revered but Poorly Understood: A Case Report of Dendroaspis polylepis (Black Mamba) Envenomation in Watamu, Malindi Kenya, and a Review of the Literature

“…Proteomic characterization of the venom has revealed 41 different proteins and one nucleoside [ 4 ]. Major proteins reported include dendrotoxins, α-neurotoxins, muscarinic toxins, fasciculins, calciseptine, mamba intestinal toxins, and mambalgin [ 4 , 19 , 20 , 21 , 22 , 23 ]. Minor proteins include metalloproteinases, hyaluronidase, prokinecitin, nerve growth factor, phospholipase A 2 , 5’-nucleotidase, and phosphodiesterase [ 4 ].…”

“…Dendrotoxins primarily act on the voltage-dependent potassium channels where they potentiate effects of acetylcholine by facilitating its release at pre-synaptic nerve terminals, thereby eliciting excitatory effects [ 11 , 24 , 25 ]. α-Neurotoxins bind to the nicotinic cholinergic receptors at the motor end-plates of muscle fibers and have the capacity to cause flaccid paralysis that leads to labored breathing and death [ 19 ]. Muscarinic toxins bind to muscarinic cholinergic receptors [ 26 ], and fasciculins inhibit anticholinesterase; thus, there is an increase in the levels of acetylcholine at neuromuscular junctions, which manifests as generalized, long-lasting fasciculations [ 20 ].…”

Snake Venom Peptides: Tools of Biodiscovery

“…This post-translational modification is rarely observed for snake venom three-finger toxins. Assay binding studies showed that α-elapitoxin strongly inhibits α 7 nAChR; however, it was suggested that the C-terminal modification does not significantly affect toxin selectivity [ 73 ]. The three-finger toxin peptides have served as an excellent molecular probe in deciphering the structural and functional details of nAChR since the discovery of the iconic peptide α-bungarotoxin from the venom of a krait, Bungarus multicinctus [ 74 , 75 ].…”

High-throughput immuno-profiling of mamba (Dendroaspis) venom toxin epitopes using high-density peptide microarrays

“…Structure built upon35; (G,H) Alfa-elapitoxin-Dpp2c (P01397) from D. polylepis as an example of a type 2 α-neurotoxin. Structure built upon56. (A,C,E,G) Residues coloured according to alanine substitution effect in log 2 fold-change, where magenta indicates that a residue is of particular importance for antibody recognition.…”