Evidence of caspase-mediated apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom.
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.
Alves
Raquel Melo
RM
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, FCFRP, Universidade de São Paulo, USP, Ribeirão Preto-USP, Brazil.
Antonucci
Gilmara Ausech
GA
Paiva
Helder Henrique
HH
Cintra
Adélia Cristina Oliveira
AC
Franco
João José
JJ
Mendonça-Franqueiro
Elaine Paula
EP
Dorta
Daniel Junqueira
DJ
Giglio
José Roberto
JR
Rosa
José César
JC
Fuly
André Lopes
AL
Dias-Baruffi
Marcelo
M
Soares
Andreimar Martins
AM
Sampaio
Suely Vilela
SV
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
D010446:Peptide Fragments; D012910:Snake Venoms; D050830:L-Amino Acid Oxidase; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases