Functional Expression and Genetic Alteration of an Alpha Scorpion Neurotoxin

Abstract

The alpha neurotoxin Lqh alpha IT is toxic to both insects and mammals but exhibits a bioactivity ratio favoring insects (insect/mammal approximately 2). With the objective of increasing this ratio by genetic manipulation of the amino acid sequence, a cDNA clone encoding Lqh alpha IT was used to produce recombinant variants of the toxin in a high efficiency bacterial expression system. The unmodified recombinant toxin, isolated from inclusion bodies and renatured in vitro, exhibited chemical and biological properties indistinguishable from those of the authentic native toxin. Alteration of the toxin by site-directed mutagenesis led to a substantial reduction in anti-mammalian toxicity (mouse LD50 reduced 6.4-fold) but only a slight reduction (x 1.5) in the insect ED50 value for paralysis. The reduction in anti-mammalian toxicity was correlated with a approximately 2-fold reduction of its potency for slowing of sodium channel inactivation in mammalian neurons, while no change in mutant toxin binding affinity to insect neuronal receptors was registered. These results demonstrate for the first time expression of a recombinant sodium channel neurotoxin in Escherichia coli and the use of site-directed mutagenesis to improve phylogenetic selectivity. This recombinant approach provides a promising strategy for optimizing the selective toxicity of peptide neurotoxins.

Evolutionary Diversification of Mesobuthus α-Scorpion Toxins Affecting Sodium Channels

“…MeuNaTx␣-2 specifically inhibits the inactivation process of Na v 1.4 that is the only one VGSC subtype expressed in skeletal muscles, whereas MeuNaTx␣-4 shows a prospect in developing a new pest control agent because of its selective on DmNa v 1 and its killing activity on insects. Among all scorpion ␣-toxins previously identified, Lqh␣IT from Leiurus quinquestriatus is considered as the best candidate for developing an agent for pest control because of its strong insecticidal activity (46). However, this has been challenged by its absence of selectivity on mammalian VGSCs.…”

Common Features in the Functional Surface of Scorpion β-Toxins and Elements That Confer Specificity for Insect and Mammalian Voltage-gated Sodium Channels

“…DNA Manipulations-A synthetic Css4 gene was constructed using eight overlapping oligonucleotide primers designed with a codon usage based on the nucleotide sequences of long-chain scorpion toxin genes that have been successfully expressed in E. coli (Table I) (9,17,18). A mixture of 1 M of each of primers 1-8 (Table I) heated to 95°C was cooled stepwise to 4°C for annealing.…”

“…Css4 -Protocols that were previously shown to be useful for the production and extensive mutagenesis of a number of long-chain scorpion toxins using the expression vector pET-11c (9,10,17,18,26) were unsuccessful in attempts to overexpress Css4. However, large quantities of recombinant toxin, His-Css4, were obtained using the expression vector pET-14b (see "Materials and Methods").…”

Medical Defense Against Protein Toxin Weapons

“…157). Zilberberg et al applied site-directed mutagenesis to modify the binding specificity of a scorpion neurotoxin (Lqh a IT) (158). Additionally, the potency of one long-chain scorpion toxin (BotlX) could be enhanced by the transferring select residues from a scorpion alpha toxin (Lqh a IT) (159).…”