title: Evidence of caspase-mediated apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom.
abstract: The aim of this work was to investigate the involvement of caspases in apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom. The isolation of LAAO involved three chromatographic steps: molecular exclusion on a G-75 column; ion exchange column by HPLC and affinity chromatography on a Lentil Lectin column. SDS-PAGE was used to confirm the expected high purity level of BatroxLAAO. It is a glycoprotein with 12% sugar and an acidic character, as confirmed by its amino acid composition, rich in "Asp and Glu" residues. It displays high specificity toward hydrophobic l-amino acids. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom LAAOs. This enzyme induces in vitro platelet aggregation, which may be due to H2O2 production by LAAOs, since the addition of catalase completely inhibited the aggregation effect. It also showed cytotoxicity towards several cancer cell lines: HL60, Jurkat, B16F10 and PC12. The cytotoxicity activity was abolished by catalase. A fluorescence microscopy evaluation revealed a significant increase in the apoptotic index of these cells after BatroxLAAO treatment. This observation was confirmed by phosphatidyl serine exposure and activation of caspases. BatroxLAAO is a protein with various biological functions that can be involved in envenomation. Further investigations of its function will contribute to toxicology advances.
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, FCFRP, Universidade de São Paulo, USP, Ribeirão Preto-USP, Brazil.
authors.forename: Raquel Melo
authors.initials: RM
authors.lastname: Alves
authors.forename: Gilmara Ausech
authors.initials: GA
authors.lastname: Antonucci
authors.forename: Helder Henrique
authors.initials: HH
authors.lastname: Paiva
authors.forename: Adélia Cristina Oliveira
authors.initials: AC
authors.lastname: Cintra
authors.forename: João José
authors.initials: JJ
authors.lastname: Franco
authors.forename: Elaine Paula
authors.initials: EP
authors.lastname: Mendonça-Franqueiro
authors.forename: Daniel Junqueira
authors.initials: DJ
authors.lastname: Dorta
authors.forename: José Roberto
authors.initials: JR
authors.lastname: Giglio
authors.forename: José César
authors.initials: JC
authors.lastname: Rosa
authors.forename: André Lopes
authors.initials: AL
authors.lastname: Fuly
authors.forename: Marcelo
authors.initials: M
authors.lastname: Dias-Baruffi
authors.forename: Andreimar Martins
authors.initials: AM
authors.lastname: Soares
authors.forename: Suely Vilela
authors.initials: SV
authors.lastname: Sampaio
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D017837:Bothrops / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D018922:HL-60 Cells; D006801:Humans; D066298:In Vitro Techniques; D050830:L-Amino Acid Oxidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity*; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010446:Peptide Fragments / Q000235:genetics; D010449:Peptide Mapping; D010974:Platelet Aggregation / Q000187:drug effects; D011817:Rabbits; D051381:Rats; D012910:Snake Venoms / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000633:toxicity*; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D012910:Snake Venoms; D050830:L-Amino Acid Oxidase; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases