Identification and synthesis of palmitoylated and branched peptides to produce Tityus antivenom

J. A. RODRIGUEZ; L.C. IGLESIAS GARCÍA; G. R. BARREDO VACCHELLI; G. ACOSTA; F. ALBERICIO; S. A. CAMPERI

Sitges, España

Simposio; 36th European and 12th International Peptide Symposium (EPS 2022).; 2022

European peptide society

Tityus trivittatus is one of the main scorpions of medical importance in South America. Antivenoms to treat envenoming are produced immunizing horses repeatedly with scorpion venom which is collected by a very dangerous, arduous and low-yielding electrostimulation process. Hence, new approaches are needed for more efficient production to meet the demands. Moreover, peptide neurotoxins, responsable for envenoming symptoms, are low immunogenic Cys-rich peptides. Their high stability hindered their digestion in the antigen-presenting cells, necessary to trigger the adaptive immune response.Beta-mammal Tt1g neurotoxin is the main responsible of T. trivittatus envenoming symptoms in humans. In this work, the epitopes from Tt1g were identified and immunogenic peptides were designed to develop synthetic immunogens. The tool “MHC-II Binding Predictions” from the “Immune Epitope Database Analysis Resource” was used to identify the epitopes in the toxin. Its most immunogenic fragment corresponding to its C-terminus, LPNWVKVWERATNRC, was synthesized. Cys was replaced by α-aminobutyric acid to avoid Cys bonds formation. To increase the immunogenicity, branched peptides were synthesized using Fmoc-Lys(Fmoc)-OH. Also, linear, and branched lipopeptides were developed incorporating palmitic acid in their N-terminal. All peptides were analyzed by ESI-MS and RP-HPLC. All the peaks obtained in the mass spectrum corresponded to the synthesized immunogens , demonstrating the high purity of the synthesis. The RP-HPLC analysis of the linear and the palmitoylated epitopes demonstrated a purity higher than 85%. The branched peptide chromatogram showed a broad peak, while the branched palmitoylated peptide showed two peaks. Probably, the branched palmitoylated peptide exists in two forms that convert very slowly between both conformations. Similar results were obtained with a branched palmitoylated peptide of the spider´s Tx2-6 neurotoxin, also synthesized in our laboratory. All the immunogens developed will be assayed to produce T. trivittatus antivenom.