A depressant insect toxin with a novel analgesic effect from scorpion Buthus martensii Karsch

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Abstract

A new peptide named BmK dITAP3 from scorpion Buthus martensii Karsch (BmK) has been identified to possess a dual bioactivity, a depressant neurotoxicity on insects and an analgesic effect on mice. The bioassays also showed that the peptide was definitely devoid of the neurotoxicity on mammals, which indicated that the analgesic effect of BmK dITAP3 could not be ascribed to the syndromic effects of a mammalian neurotoxicity. BmK dITAP3 exhibited 43.0% inhibition efficiency of the analgesic effect on mice at a dose of 5 mg/kg and the FPU value of 0.5 μg/body (∼30 mg) on the fly larvae. The pI value and the molecular mass determined by MALDI-TOF MS for dITAP3 were 6.5 and 6722.7, respectively. Its first 15 N-terminal residues were determined by Edman degradation, based on which the full amino acid sequence was deduced from the cDNA sequence encoding the peptide with 3′-RACE. Circular dichroism and sequence based prediction analyses showed dITAP3 may have a similar molecular scaffold as the most scorpion toxins but with features of the more β structures and much less of α helix. The details of the purification, characterization and sequencing as well as the sequence comparison with other depressant insect toxins and the correlation between the analgesic effect and the insect toxicity will be reported and discussed, respectively.

Introduction

Scorpion venoms are well-known sources of peptide neurotoxins that bind to ionic channels at the surface of excitable cells and modify diversely their normal properties. Among others, two groups of neurotoxins, the excitatory and the depressant toxins, named according to the symptomatology developed by injected animals, have been recognized from the scorpion venoms [1]. These toxins specific for insects are characterized as single-chain polypeptides of 60–76 residues cross-linked by four disulfide bridges [2], [3], [4]. The excitatory toxins cause a fast contraction paralysis on injected animals and induce repetitive firing in insect nerves, while the depressant toxins cause a slow depressory flaccid paralysis due to depolarization of the nerve membrane and blockage of the sodium conductance in axons [1], [5], [6]. Due to their great value as tools for studying the pharmacology of insect sodium channels and bright prospect in the design of new insect selective biopesticides [7], [8], these insect toxins are widely studied by many research groups. However, it is rare to find a report to describe certain new activity other than neurotoxicity from insect toxins.

The scorpion Buthus martensii Karsch (BmK), widely distributed in East Asia, is a very mild species. Its venom is not only much less toxic and never causes death of envenomed people, but quite contrarily, is used for disease prevention and therapy in China. Probably with this background, some active peptides with diverse activities other than neurotoxicity have been identified from the BmK venom in recent years, though the mechanism remains to be clarified. For example, a peptide with anti-epilepsy effect was isolated and partially sequenced [9]; the analgesic effect was confirmed in the crude BmK venom [10]; an excitatory insect toxin with an analgesic effect was cloned and sequenced [11]; and a peptide named makatoxin with an effect on nitrergic responses has been discovered [12]. Therefore it will be no surprise if some other peptides with new biological or pharmacological effects could be found in the BmK venom.

Recently, we found a depressant insect toxin, BmK dITAP3, from the venom of scorpion BmK venom. Very interestingly, this insect toxin displays an evident analgesic effect but is devoid of any toxicity on mice, which has not been reported before for scorpion depressant insect toxins. In this paper we will report the purification and characterization of this toxin, including full sequence and secondary structure, which has established a sound basis for the further investigation on its structure and function.

Section snippets

Sample and chemicals

The venom of BmK scorpion was obtained by electrical stimulation of the post abdomen of scorpions grown in Henan Province in China. All gels used in chromatography, Sephadex G-50, Sephadex G-25 and SP-Sephadex C-25, and in electrophoresis, Ultradex, were products of Pharmacia. The acetonitrile used in reverse-phase chromatography was gradient grade (Merck). The CCA used in mass spectroscopy was from Sigma. The reagents used in N-terminal sequencing were from Perkin Elmer. Enzymes and reagents

Purification

The pure BmK dITAP3 was obtained by the successful combination of chromatography methods and preparative isoelectrofocusing electrophoresis. The second fraction eluted out from the Sephadex G-50 column (Fig. 1a) containing the anti-insect toxicity was applied to a SP-Sephadex C-25 column after being lyophilized to powder. The flow-through fraction was pooled according to its anti-insect toxicity and was then desalted on a Sephadex G-25 column, on which two protein peaks were obtained (Fig. 1b).

Sequence comparison

Sequence similarity between dITAP3 and known proteins were searched using the NCBI BLAST electronic mail servers. The results showed about 80% identity and the same cysteine motif between dITAP3 and the known depressant insect toxins, but very low identity (44.3%) between dITAP3 and the mammal-directed toxin (see Fig. 9), except a common cysteine motif. In association with the result of insect toxicity test shown in this report, dITAP3 should be classified as a depressant insect toxin. The

Note added in proof

The molecular mass of dITAP3 was more accurately determined recently by using a Finigan LCQ ion trap mass spectrometer (ThermoQuest) equipped with an electrospray ionization source. The result showed that the molecular mass of dITAP3 is 6732.0, which is in good agreement with that deduced from the sequence (6732.4).

Acknowledgements

This work was supported by the State Basic Research Program (G1990756), the National Natural Science Foundation (39970158) and a bilateral grant between China and Flinders (BIL00106). We thank Ms Shi Xiang for her help in the bioassay experiments.

References (21)

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