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 chroma...

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Published in:Comparative biochemistry and physiology. Part A, Molecular & integrative physiology Molecular & integrative physiology, 2008-12, Vol.151 (4), p.542-550
Main Authors: Alves, Raquel Melo, Antonucci, Gilmara Ausech, Paiva, Helder Henrique, Cintra, Adélia Cristina Oliveira, Franco, João José, Mendonça-Franqueiro, Elaine Paula, Dorta, Daniel Junqueira, Giglio, José Roberto, Rosa, José César, Fuly, André Lopes, Dias-Baruffi, Marcelo, Soares, Andreimar Martins, Sampaio, Suely Vilela
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Apoptosis
Apoptosis - drug effects
Apoptosis - physiology
Bothrops
Bothrops - genetics
Bothrops - metabolism
Bothrops atrox
Caspase 3 - metabolism
Caspase 9 - metabolism
Caspases
Caspases - metabolism
Cell Line, Tumor
Cell lines culture
HL-60 Cells
Humans
In Vitro Techniques
L-Amino Acid Oxidase - genetics
L-Amino Acid Oxidase - isolation & purification
L-Amino Acid Oxidase - metabolism
L-Amino Acid Oxidase - toxicity
LAAO
Molecular Sequence Data
PC12 Cells
Peptide Fragments - genetics
Peptide Mapping
Platelet Aggregation - drug effects
Rabbits
Rats
Snake Venoms - chemistry
Snake Venoms - enzymology
Snake Venoms - genetics
Snake Venoms - toxicity
Substrate Specificity
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Summary: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 H 2O 2 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.
ISSN:1095-6433
1531-4332
DOI:10.1016/j.cbpa.2008.07.007