Rational attenuation of elapid long-chain neurotoxin by targeted residue substitution and disulfide architecture simplification: implications for receptor interaction and immune recognition.

Elapid long-chain neurotoxins (LNTxs) exert their toxicity through high-affinity binding to nicotinic acetylcholine receptors (nAChRs). Specific residues mediate this process, including basic (Lys23, Arg33, Arg36, Lys49), structural (Cys26, Cys30), and aromatic (Phe29) amino acids. To investigate the functional and immunogenic implications of modifying these residues, we designed two recombinant variants of a consensus LNTx. The first variant, rLcNTx V1, contains charge-reversal mutations at four key basic positions, while the second, rLcNTx V2, lacks the segment corresponding to the fifth disulfide bond. We expressed both variants in Escherichia coli Origami cells. rLcNTx V1 was recovered in a soluble form, but rLcNTx V2 was present only in inclusion bodies. We assessed the binding affinity of both proteins to muscle and neuronal nicotinic acetylcholine receptors (nAChRs) and observed no channel blockade for either variant, as expected. While this lack of activity in rLcNTx V2 points to the structural importance of the fifth disulfide region, it also suggests that its deletion disrupts the structure of the tip of loop II. Immunizing rabbits with these proteins elicited antibodies capable of recognizing α-bungarotoxin and various elapid venoms, but they failed to neutralize venom toxicity in vivo. These results emphasize that preserving the native electrostatic and conformational integrity of the basic core is essential for maintaining neutralizing epitopes in LNTxs.