Isolation, characterization, and peptide synthesis of fragments from the first phospholipase A2 identified in Rhinella diptycha toad poison

Abstract

Toad poison is composed of a range of molecules, such as alkaloids, bufotoxin, proteins, and peptides, including phospholipases A₂ (PLA₂). PLA₂s are responsible for catalyzing the hydrolysis of the ester bonds of phospholipids, which are one of the main compounds of the cellular membrane. PLA₂s may have hemolytic, myotoxic, and antimicrobial functions. However, the function of this protein in Rhinella's poison is still unknown. In this study, Rhinella diptycha mucous poison was fractionated using reversed-phase chromatography, and fraction 16, which contained a PLA₂-sized protein, was subjected to a phospholipase assay, N-terminal sequencing, and an agar diffusion antimicrobial assay against Escherichia coli. Molecular modeling and in-silico analysis of PLA₂ were performed to identify potential antimicrobial peptides encoded in its sequence. Selected peptides were subjected to solid-phase peptide synthesis and analyzed using minimum inhibitory concentration and minimum bactericidal concentration assays against Staphylococcus aureus and E. coli. PLA₂ was successfully isolated, showing enzymatic activity. N-terminal sequencing confirmed its identity as RsPLA₂ from the R. diptycha skin transcriptome. The antimicrobial assay revealed no growth inhibition of E. coli at the RsPLA₂ tested concentrations. In-silico analysis identified three potential antimicrobial peptides, two of which were synthesized and tested against E. coli and S. aureus, showing limited antimicrobial activity. Further investigations are required to assess its potential against other microorganisms. This study enhances our understanding of toad poisons and their potential biotechnological applications.