Estudo comparativo do efeito leishmanicida de duas L-aminoácido oxidases isoladas de peçonhas botrópicas

Abstract

L-amino acid oxidases are oxidoreductases flavoenzymes that use L-amino acids as substrate for stereospecific catalysis in α-keto acids, releasing ammonia (NH3) and hydrogen peroxide (H2O2). SV-LAAOs (Snake Venom L-Amino Acid Oxidases) show a varied composition in venoms of different species and are considered multifunctional enzymes. They are generally homodimeric, with molecular masses ranging from 12-150 kDa, and have bactericidal, antiviral, antitumor and antiparasitic biological effects commonly attributed to the formed H2O2. The aim of this study was to compare the antiparasitic effect on two species of the Leishmania genus, caused by two isolated botropic SV-LAAOs from two different species. SV-LAAOs BjussuLAAO-II and BmooLAAO were isolated by molecular exclusion chromatographic steps on Shephacryl S-100; hydrophobic interaction in Phenyl Sepharose and affinity in Benzamidine Sepharose. Purified enzymes showed high LAAO specific activity, with 3481.17 U/mg/min for BjussuLAAO-II and 4924.77 U/mg/min for BmooLAAO. Alignment with the reference sequence ATU85535.1 (SV-LAAO of B. moojeni) showed 96.2% identity for BjussuLAAO-II and 93.4% for BmooLAAO, and 91.59% between BjussuLAAO-II and BmooLAAO. Analyzes of the post-translational modifications present in the two enzymes showed different amounts of modifications for both, being for BjussuLAAO-II and for BmooLAAO. In addition, the molecular modeling of the FAD binding site demonstrated a well conserved region in reference ATU85535.1 and SV-LAAOs studied. Cytotoxicity assays performed on Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis showed high cytotoxicity of the two SV-LAAOs tested, even at low concentrations. The SV-LAAOs studied showed lower LAAO specific activity using as substrate the complete LIT medium; but interestingly, despite the same concentration of BjussuLAAO-II and BmooLAAO producing similar amount of H2O2, different cytotoxic effects were observed in the parasite treatments. In addition, the SV-LAAOs of this work expressed antiproliferative and interference effects on mitochondrial potential (∆Ψm) of parasites. In the analysis of intracellular reactive oxygen species (ROS), surprisingly, BjussuLAAO-II increased the production of ROS only from L. (L.) amazonensis, whereas BmooLAAO acted exclusively on L. (V.) braziliensis, showing a certain specificity of the SV-LAAOs studied.