Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca2+-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117—122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys122, these same substitutions did not significantly alter the Ca2+-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca2+-independent membrane-damaging activity but, with the exception of Lys122, had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115—119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A2, and support a model of a Ca2+-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.
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September 2002
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Research Article|
September 15 2002
Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2
Lucimara CHIOATO;
Lucimara CHIOATO
∗Departamento de Bioquímica e Imunologia, FMRP-USP, Universidade de São Paulo, Brazil
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Arthur H.C. de OLIVEIRA;
Arthur H.C. de OLIVEIRA
†Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, FMRP-USP, Universidade de São Paulo, Brazil,
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Roberto RULLER;
Roberto RULLER
∗Departamento de Bioquímica e Imunologia, FMRP-USP, Universidade de São Paulo, Brazil
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Juliana M. SÁ;
Juliana M. SÁ
∗Departamento de Bioquímica e Imunologia, FMRP-USP, Universidade de São Paulo, Brazil
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Richard J. WARD
Richard J. WARD
1
‡Departamento de Química, FFCLRP-USP, Brazil
1To whom correspondence should be addressed (e-mail rjward@fmrp.usp.br).
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Publisher: Portland Press Ltd
Received:
January 15 2002
Revision Received:
March 22 2002
Accepted:
June 21 2002
Accepted Manuscript online:
June 21 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (3): 971–976.
Article history
Received:
January 15 2002
Revision Received:
March 22 2002
Accepted:
June 21 2002
Accepted Manuscript online:
June 21 2002
Citation
Lucimara CHIOATO, Arthur H.C. de OLIVEIRA, Roberto RULLER, Juliana M. SÁ, Richard J. WARD; Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2. Biochem J 15 September 2002; 366 (3): 971–976. doi: https://doi.org/10.1042/bj20020092
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