IsCT2

Seven analogs of the natural, α-helix peptides IsCT1 and IsCT2-found in the venom of scorpion Opithancatus Madagascariensis-have been synthesized and tested to compare their antibacterial and hemolytic activity against natural peptides. In general, results show that increasing hydrophobicity by substituting positions 5 and 9 of the sequences with alanine, valine, and leucine, enhances antibacterial activity. However, this also increases hemolytic activity. The analog with an increased net positive charge from +1 to +3 produces moderate bacterial growth inhibition but also has high hemolytic activity. On the other hand, the analog with a negative net charge (-1) has low antibacterial properties but also no cytotoxicity under the tested conditions, a similar result was found for five of the seven studied analogs.

In the previous report [Biochem. Biophys. Res. Commun. 286 (2001) 820], we described a novel short linear peptide, IsCT, with cytolytic activity isolated from the venom of scorpion Opisthacanthus madagascariensis. From the same scorpion venom, we further purified and characterized three short linear peptides named IsCT2, IsCTf, and IsCT2f that shared high homology with IsCT, while with different C-terminal areas between IsCT/IsCT2 and IsCTf/IsCT2f. Structure-activity relationship was analyzed by performing vivo and vitro assays and circular dichroism spectroscopy. Like IsCT, IsCT2 showed broad activity spectra against microbes (Gram positive and negative bacteria as well as fungi) and relatively weak hemolytic activity against sheep red blood cells. It adopts an amphipathic alpha-helical structure in aqueous TFE and is able to disrupt the artificial membrane. However, the other two peptides IsCTf and IsCT2f showed no activity in antimicrobial or hemolytic assay. Furthermore, IsCTf and IsCT2f cannot form amphipathic alpha-helix while demonstrating random coil structure in aqueous TFE, which might result in their lost cytolytic activity. IsCTf and IsCT2f both exist in the crude venom and are proved to be enzymatic products from IsCT and IsCT2. Whether they have some other biological activity is still unclear. In addition, we got the cDNAs encoding the precursors of IsCT and IsCT2. Besides the signal peptide, they still contain an unusual acidic pro-peptide at the C-terminal that was quite different from other known precursors of scorpion venom peptides. The novel structure and biological activity of these peptides proposed them to be a new class in scorpion venom.

Scorpion venoms have been studied for over fifty years; however, the majority of research has focussed primarily on medically important Buthidae species. Additionally, venoms of the estimated 200 species of scorpion native to Australia have received very little attention. The first venom mass profiles of six non-buthid and one buthid scorpion species are presented herein, four of which are endemic to Australia. While masses under 5 kDa dominated the venoms of all species, the buthid venom contained considerably more masses between 7 and 8 kDa than those of the non-buthids, corroborating the emergent trend that buthids are richer in long-chain neurotoxins than non-buthids. The Australian scorpion venom fractions were also analysed with the relatively new MALDI-ToF matrix 1,5-DAN. Over forty partial sequences were obtained, the majority of which are homologous to scorpion antimicrobials such as opistoporin and IsCT2. Overall, this study is the single most comprehensive mass spectrometric analysis of scorpion venom landscapes to date and provides an insight into untapped Australian species.