Characterization of the transcript for a depressant insect selective neurotoxin gene with an isolated cDNA clone from the scorpion Buthotus judaicus

AbstractThe poly(A)+ mRNA isolated from the venomous terminal segments of the scorpion Buthotus judaicus was reversed transcribed into cDNA. PCR amplification of the cDNA in presence of oligonucleotide primers prepared on basis of the known amino acid sequence of the depressant insect toxin II yielded a 125 bp long product. This fragment was cloned and its sequence determined. The deduced amino acid sequence has revealed a complete homology with the amino acid sequence of the toxin. This clone was used to probe a Northern blot resolving the poly(A)+ and poly(A)− fractions derived from the scorpion. An organ specific 360 nucleotide transcript which might be the processed product of a 4̃.0 kb precursor was elucidated. This cDNA clone may pave the way for a molecular genetic approach to study the structure-function relationship of scorpion selective insect toxins

Isolation of the first toxin from the scorpion Buthus occitanus israelis showing preference for Shaker potassium channels

AbstractWe have purified BoiTx1, the first toxin from the venom of the Israeli scorpion, Buthus occitanus israelis, and studied its activity and genomic organization. BoiTx1 is a 37 amino acid-long peptide contained six conserved cysteines, and is classified as an α-KTx3.10 toxin. The pharmacological effects of BoiTx1 were studied on various cloned K+ channels expressed in Xenopus laevis oocytes. BoiTx1 inhibited currents through Drosophila Shaker channels with an IC50 value of 3.5±0.5nM, yet had much lesser effect on its mammalian orthologs. Thus, BoiTx1 is the first member of the α-KTx3 family that preferentially affects insect potassium channels

Forward transport. 14-3-3 binding overcomes retention in endoplasmic reticulum by dibasic signals.

Proteins with dibasic retention motifs are subject to retrograde transport to endoplasmic reticulum (ER) by COPI-coated vesicles. As forward transport requires escape from ER retention, general release mechanisms have been expected. Here, KCNK3 potassium channels are shown to bear two cytoplasmic trafficking motifs: an N-terminal dibasic site that binds beta-COP to hold channels in ER and a C-terminal "release" site that binds the ubiquitous intracellular regulator 14-3-3beta on a nonclassical motif in a phosphorylation-dependent fashion to suppress beta-COP binding and allow forward transport. The strategy appears to be common. The major histocompatibility antigen class II-associated invariant chain Iip35 exhibits dibasic retention, carries a release motif, and shows mutually exclusive binding of beta-COP and 14-3-3beta on adjacent N-terminal sites. Other retained proteins are demonstrated to carry functional 14-3-3beta release motifs

Assembling an arsenal, the scorpion way

<p>Abstract</p> <p>Background</p> <p>For survival, scorpions depend on a wide array of short neurotoxic polypeptides. The venoms of scorpions from the most studied group, the Buthida, are a rich source of small, 23–78 amino acid-long peptides, well packed by either three or four disulfide bridges that affect ion channel function in excitable and non-excitable cells.</p> <p>Results</p> <p>In this work, by constructing a toxin transcripts data set from the venom gland of the scorpion <it>Buthus occitanus israelis</it>, we were able to follow the evolutionary path leading to mature toxin diversification and suggest a mechanism for leader peptide hyper-conservation. Toxins from each family were more closely related to one another than to toxins from other species, implying that fixation of duplicated genes followed speciation, suggesting early gene conversion events. Upon fixation, the mature toxin-coding domain was subjected to diversifying selection resulting in a significantly higher substitution rate that can be explained solely by diversifying selection. In contrast to the mature peptide, the leader peptide sequence was hyper-conserved and characterized by an atypical sub-neutral synonymous substitution rate. We interpret this as resulting from purifying selection acting on both the peptide and, as reported here for the first time, the DNA sequence, to create a toxin family-specific codon bias.</p> <p>Conclusion</p> <p>We thus propose that scorpion toxin genes were shaped by selective forces acting at three levels, namely (1) diversifying the mature toxin, (2) conserving the leader peptide amino acid sequence and intriguingly, (3) conserving the leader DNA sequences.</p