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A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to target insects.

ABSTRACT: BACKGROUND: Despite evidence suggesting a role in plant defence, the use of plant lectins in crop protection has been hindered by their low and species-specific insecticidal activity. Snowdrop lectin (Galanthus nivalis agglutinin; GNA) is transported to the haemolymph of insects after oral ingestion, and can be used as a basis for novel insecticides. Recombinant proteins containing GNA expressed as a fusion with a peptide or protein, normally only toxic when injected into the insect haemolymph, have the potential to show oral toxicity as a result of GNA-mediated uptake. RESULTS: A gene encoding a toxin, ButaIT, from the red scorpion (Mesobuthus tamulus) was synthesised and assembled into expression constructs. One construct contained ButaIT alone, whereas the other contained ButaIT fused N-terminally to a GNA polypeptide (ButaIT/GNA). Both recombinant proteins were produced using the yeast Pichia pastoris as an expression host, and purified. Recombinant ButaIT and ButaIT/GNA were acutely toxic when injected into larvae of tomato moth (Lacanobia oleracea), causing slow paralysis, leading to mortality or decreased growth. ButaIT/GNA was chronically toxic when fed to L. oleracea larvae, causing decreased survival and weight gain under conditions where GNA alone was effectively non-toxic. Intact ButaIT/GNA was detected in larval haemolymph from insects fed the fusion protein orally, demonstrating transport of the linked polypeptide across the gut. Proteolysis of the fusion protein was also observed. ButaIT/GNA was significantly more toxic that GNA alone when fed to the homopteran Nilaparvata lugens (rice brown planthopper) in liquid artificial diet. CONCLUSION: The ButaIT/GNA recombinant fusion protein is toxic to lepidopteran larvae both when injected and when fed orally, showing the utility of GNA as a carrier to transport potentially toxic peptides and proteins across the insect gut. Although ButaIT has been claimed to be lepidopteran-specific, the fusion protein has more wide-ranging insecticidal activity. Fusion proteins based on plant lectins have potential applications in crop protection, both as exogenously applied treatments and as endogenous products in transgenic plants.

SUBMITTER: Pham Trung N

PROVIDER: S-EPMC1459149 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to target insects.

Pham Trung Nghia N Fitches Elaine E Gatehouse John A JA

BMC biotechnology 20060316

<h4>Background</h4>Despite evidence suggesting a role in plant defence, the use of plant lectins in crop protection has been hindered by their low and species-specific insecticidal activity. Snowdrop lectin (Galanthus nivalis agglutinin; GNA) is transported to the haemolymph of insects after oral ingestion, and can be used as a basis for novel insecticides. Recombinant proteins containing GNA expressed as a fusion with a peptide or protein, normally only toxic when injected into the insect haemo ...[more]

PMID: 16542451

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Project description:?-Scorpion toxins constitute a family of peptide modulators that induce a prolongation of the action potential of excitable cells by inhibiting voltage-gated sodium channel inactivation. Although they all adopt a conserved structural scaffold, the potency and phylogentic preference of these toxins largely vary, which render them an intriguing model for studying evolutionary diversification among family members. Here, we report molecular characterization of a new multigene family of ?-toxins comprising 13 members (named MeuNaTx?-1 to MeuNaTx?-13) from the scorpion Mesobuthus eupeus. Of them, five native toxins (MeuNaTx?-1 to -5) were purified to homogeneity from the venom and the solution structure of MeuNaTx?-5 was solved by nuclear magnetic resonance. A systematic functional evaluation of MeuNaTx?-1, -2, -4, and -5 was conducted by two-electrode voltage-clamp recordings on seven cloned mammalian voltage-gated sodium channels (Na(v)1.2 to Na(v)1.8) and the insect counterpart DmNa(v)1 expressed in Xenopus oocytes. Results show that all these four peptides slow inactivation of DmNa(v)1 and are inactive on Na(v)1.8 at micromolar concentrations. However, they exhibit differential specificity for the other six channel isoforms (Na(v)1.2 to Na(v)1.7), in which MeuNaTx?-4 shows no activity on these isoforms and thus represents the first Mesobuthus-derived insect-selective ?-toxin identified so far with a half maximal effective concentration of 130 ± 2 nm on DmNa(v)1 and a half maximal lethal dose of about 200 pmol g(-1) on the insect Musca domestica; MeuNaTx?-2 only affects Na(v)1.4; MeuNaTx?-1 and MeuNaTx?-5 have a wider range of channel spectrum, the former active on Na(v)1.2, Na(v)1.3, Na(v)1.6, and Na(v)1.7, whereas the latter acting on Na(v)1.3-Na(v)1.7. Remarkably, MeuNaTx?-4 and MeuNaTx?-5 are two nearly identical peptides differing by only one point mutation at site 50 (A50V) but exhibit rather different channel subtype selectivity, highlighting a switch role of this site in altering the target specificity. By the maximum likelihood models of codon substitution, we detected nine positively selected sites (PSSs) that could be involved in functional diversification of Mesobuthus ?-toxins. The PSSs include site 50 and other seven sites located in functional surfaces of ?-toxins. This work represents the first thorough investigation of evolutionary diversification of ?-toxins derived from a specific scorpion lineage from the perspectives of sequence, structure, function, and evolution.

Dataset Information

A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to target insects.

Publications

A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to target insects.

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