Identificação de peptídeos ligantes ao veneno de Dinoponera australis (Hymenoptera, Formicidae, Ponerinae) por phage display

Abstract

The genus Dinoponera is represented by six species, geographically distributed along South América. The species Dinoponera australis is found from Mato Grosso to Rio Grande do Sul, as well as in neighboring countries. These ants are characterized by the presence of venom glands that are responsible for the production of a toxin constituted in its majority by proteins. This investigation aimed: (a) the identification of specific ligand peptides to the D. australis venom through phage display, (b) to test the hemolytic activity inhibition by selected phages conjugated to the toxin, (c) to analyse the nucleolytic activity of the venom, and (d) to demonstrate if phage particles are inactivated by the venom. Two commercial random peptide libraries were used: Ph.D.-7 Heptapeptide Phage Display Library and Ph.D.-12 Peptide 12-mer Phage Display Library. After three rounds of selection, 96 phage clones were sequenced and translated, generating 40 valid sequences, and 21 distinct peptides. Bioinformatic analyses have identified three small frequent motifs: KVWxL, WxLL and KLxTIPM. Similarity tests have found as putative human cellular targets acting as: receptors, transcritpion factors, proteins associated with inflamation processes, calcium channel and G protein receptor. In insects, the closest identities found with putative targets were: potassium channel, cytochrome P450, ATPase, transcription factors, and proteinase. The hemolytic activity assays with selected phages conjugated with the venom were all positives, meaning that none of the peptides were able to influence the toxin activity. The nucleolytic activity assay with venom concentrations equal to 2.5 μg was shown by a smeared DNA profile in agarose gel electrophoresis, which may indicate a possible DNA degradation. The crude venom concentration of 10 μg was not able to inactivate phage particles. We have successfully identified interesting peptides that bind specifically to the D. australis venom through Phage Display, which may be important target molecules for the development of therapeutic strategies.