Biochimica et biophysica acta · 2011
Cytotoxicity and inhibition of platelet aggregation caused by an l-amino acid oxidase from Bothrops leucurus venom.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Naumann Gustavo B, Silva Liliana F, Silva Luciana, Faria Gilson, Richardson Michael, Evangelista Karla, Kohlhoff Markus, Gontijo Celia M F, Navdaev Alexei, de Rezende Flavia F, Eble Johannes A, Sanchez Eladio F
Аннотация
BACKGROUND: Multifunctional l-amino acid oxidases (LAAOs) occur widely in snake venoms.
METHODS: The l-AAO from Bothrops leucurus (Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity.
RESULTS: Bl-LAAO is a 60kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination of l-amino acids with the generation of H₂O₂. The best substrates were: l-Met, l-Norleu, l-Leu, l-Phe and l-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC(50) of 0.07μM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H₂O₂ in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase.
CONCLUSION: Bl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H₂O₂ which kill the cells.
GENERAL SIGNIFICANCE: These results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism.
METHODS: The l-AAO from Bothrops leucurus (Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity.
RESULTS: Bl-LAAO is a 60kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination of l-amino acids with the generation of H₂O₂. The best substrates were: l-Met, l-Norleu, l-Leu, l-Phe and l-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC(50) of 0.07μM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H₂O₂ in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase.
CONCLUSION: Bl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H₂O₂ which kill the cells.
GENERAL SIGNIFICANCE: These results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism.
Предметные рубрики MeSH
Amino Acid SequenceAnimalsApoptosisBothropsCell LineCell Line, TumorCell SurvivalDose-Response Relationship, DrugElectrophoresis, Gel, Two-DimensionalEnzyme StabilityHumansHydrogen PeroxideHydrogen-Ion ConcentrationL-Amino Acid OxidaseLeishmania braziliensisMolecular Sequence DataPlatelet AggregationSnake VenomsSubstrate SpecificityTemperature