Biochemical characterization and in vitro evaluation of the fibroblast activation and the leishmanicide potential of an L-amino acid oxidase (LAAO) from Crotalus durissus terrificus venom

Author(s)

Gisele Adriano Wiezel

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2016-09-02

Examining board members

Eliane Candiani Arantes Braga; Heloisa Sobreiro Selistre de Araújo; Ana Patricia Yatsuda Natsui

• Advisor: Eliane Candiani Arantes Braga

Abstract

Acidents caused by venomous animals represent a serious publich health problem, mainly in remote areas where the acess to the health service is difficult. In Brazil, the genus Crotalus is the most lethal genus among the Brazilian snakes. The L-amino acid oxidases (LAAOs) are present in the venom from this genus and they are flavoenzymes that catalyze the oxidation of L-amino acids, producing hydrogen peroxide and ammonia concomitantly. LAAOs have been demonstrating many activities, including cytotoxic, antimicrobial, antitumor, antiparasitic and action on platelet aggregation. The main objectives of this study included the isolation and biochemical characterization of a LAAO from C. d. terrificus, and the evaluation of its leishmanicide potential and the fibroblasts activation. Two protocols were developed to the LAAO isolation from C. d. terrificus venom. First one consists in ionic exchange, gel filtration and hydrophobic interaction chromatographies. The second protocol has a modification in the third step which is affinity chromatography. Reverse-phase chromatography of the isolated LAAO showed high purity degree and the separation of FAD from the enzyme. The LAAO molecular mass was determined by MALDI-TOF mass spectrometry (58,702.196 Da). The structural characterization also included the deduction of primary sequence and the glycosylation site and disulfide bonds determinations through LCMS/ MS with different fragmentation modes (HCD, ETD, EThcD). The primary sequence (498 amino acid residues) was obtained after the LAAO digestion using different proteases. The glycosylation site was located in the Asn361. A SDS-PAGE analysis of reduced LAAO and reduced/deglycosylated LAAO showed that about 5% of the LAAO mass is due to the sugar presence. The disulfide bonds were determined after LAAO digestion at low pH and LC-MS/MS analysis. It showed bonds between Cys10-Cys171 and Cys331-Cys412. The qualitative evaluation of substrate specificity revealed preference for hydrophobic L-amino acids. The specificity order, determined through the kinetics evaluation, is L-Phe>L-Leu>LMet> L-Trp>L-Ile. The LAAO stability was evaluated at different temperatures, timecourse and storage conditions. The enzyme lost its activity over time, and lyophilization and freezing at -20 °C completely inhibited its activity. The thermal stability, evaluated by the Termofluor method, demonstrated that the best LAAO structural stability is achieved at acid pH, different substrate concentrations and at absence of NaCl. Leishmania amazonensis promastigotes were stimulated with LAAO (55 mEAU) and the parasites death was about 30%. The fibroblasts cell line L929 was also stimulated with LAAO, and at low concentration (1.83 mEAU) the cellular viability was close to zero. At lower concentrations, without significative cellular death, the fibroblast activation was evaluated through the nitric oxide e cytokines production, but none of the compounds were released. Therefore, in this study, a LAAO from C. d. terrificus venom was isolated and characterized. Moreover, this enzyme presented leishmanicide against L. amazonensis promastigotes and high cytotoxicity to fibroblasts, without the activation of these cells. (AU)

• FAPESP's process: 14/06170-8 - Biochemical and in vitro evaluation of fibroblast activation and leishmanicidal potential of an L-amino acid oxidase (LAAO) from Crotalus durissus terrificus venom
• Grantee: Gisele Adriano Wiezel
• Support Opportunities: Scholarships in Brazil - Master