Molecular Characterization of a Cytotoxic L-Amino Acid Oxidase Isolated from Bothrops moojeni Snake Venom: Antimicrobial, Antitumoral and Apoptosis-Inducing Properties
Stábeli R.G , Ticli, F.K, Malta-Neto, N.R. Sant´Ana C.D., Ribeiro P.H., Pires. M.G., Nomizo, A., Albuquerque S., Sampaio, S.V., Soares. A.M.
Laboratório de Bioquímica do Instituto de Pesquisas em Patologias Tropicais (IPEPATRO) e Universidade Federal de Rondônia, UNIR, Porto Velho-RO;
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, FCFRP, Universidade de São Paulo, USP, Ribeirão Preto-SP, Brazil;
Unidade de Biotecnologia, Universidade de Ribeirão Preto, UNAERP, Ribeirão Preto-SP, Brazil.
L-amino acid oxidases are widely found in snake venoms and are thought to contribute to the toxicity upon envenomation. The mechanism of these toxic effects and whether they result from the enzymatic activity are still uncertain although many papers describing the biological and pharmacological effects of L-amino acid oxidases have appeared recently, which provide more information about their action on platelets, induction of apoptosis, haemorrhagic effects, and cytotoxicity. Here, an L-amino acid oxidase (BmooLAAO-I) from Bothrops moojeni snake venom was purified to a high degree and functionally characterized. This enzyme was purified by sequential CM-Sepharose ion-exchange and Phenyl-Sepharose chromatography. The purified BmooLAAO-I presented a molecular weight of 64,889.05 and about 137,789.76, in denatured and non-denatured conditions, respectively, when analyzed for mass spectrometry. The BmooLAAO-I is a homeodimeric, acidic
glycoprotein, pI~4.7 and its sequence show close structural homology with other snake venom LAAOs. This enzyme was inactivated by freezing or low pH and, the secondary structure analysis by circular dichroism, revealed 48% α-helix, 20% β-sheet, 12% β-turn, and 20% random coil structure. BmooLAAO-I exhibited bactericidal, antitumor, trypanocidal, edematogenic and platelet aggregation activities, all these effects were inhibited by catalase, suggesting that these biological effects are mediated by the H2O2 production. This enzyme induced a typical DNA fragmentation apoptotic pattern in HL-60 cells, which was inhibited by catalase.
Keywords: L-amino acid oxidase, snake venom, Bothrops moojeni, platelet aggregation,
antimicrobial and cytotoxic effects, structural analysis.
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