Molecular characterization of a new excitatory insect neurotoxin with an analgesic effect on mice from the scorpion Buthus martensi Karsch

Besides the neurotoxins active on mammals, a new excitatory insect selectiv e toxin with a mice analgesic activity was found and purified from the veno m of the scorpion Buthus martensi Karsch (BmK) (Ji, Y.H., Mansuelle, P., Te rakawa, S., Kopeyan, C., Yanaihara, N., Hsu, K., Rochat. H., 1996. Toxicon 34, 987; Luo, M.J., Xiong, Y.M., Wang, M., Wang, D.C., Chi, C.W., 1997. Tox icon 35, 723.). This peptide (designated as BmK IT-AP) is composed of 72 am ino acid residues, Its primary structure was determined by automated Edman degradation of the N-terminal part of the reduced and S-carboxamidemethylat ed protein and its lysylendopeptidase degraded fragments. Based on the dete rmined sequence, the gene specific primers were designed and synthesized fo r 3' and 5' RACE (rapid amplification of cDNA ends). Their partial cDNA fra gments obtained by 3' and 5' RACE were cloned and sequenced and the full le ngth cDNA sequence of BmK IT-AP was then completed by overlapping their two partial cDNA sequences. It encodes a precursor of 90 amino acid residues: a signal peptide of 18 residues and a mature peptide of 72 residues which a re consistent with the determined protein sequence of BmK IT-AP. The genomi c DNA of the peptide was also amplified by PCR from the scorpion genomic DN A and sequenced, which is a first report on the genomic structure of a scor pion toxin specific for insects. Its sequence revealed an intron of 590 bp inserted in the end part of the signal peptide. The peptide caused a fast e xcitatory contraction paralysis on house fly larvae. Furthermore. the pepti de also showed an obvious analgesic effect on mice, as assayed by using a t wisting test model. This effect of BmK; IT-AP well characterized at molecul ar level is first reported among the known scorpion insect neurotoxins. (C) 1999 Elsevier Science Ltd. All rights reserved.