Mapping of the structural determinants of artificial and biological membrane damaging activities of a Lys49 phospholipase A2 by scanning alanine mutagenesis.
Scanning alanine mutagenesis has been used to study the structural determinants of several activities of bothropstoxin-I (BthTx-I), a lysine 49 Phospholipases A(2) from the venom of Bothrops jararacussu. A total of 31 mutants were generated in the interfacial recognition site and C-terminal loop regions of the protein. The effects of mutagenesis on the in vivo myotoxic activity, the cytolytic activity against cultured C2C12 myoblasts, the bactericidal activity, and the Ca(2+)-independent membrane damaging activity against liposome membranes were compared. Residues 116-119 and 122-125 in the C-terminal loop region are structural determinants for these activities, indicating that membrane permeabilization by the BthTx-I is an important general property in all the measured effects. The structural determinants of myotoxicity and myoblast membrane permeabilization are highly correlated, demonstrating that cultured C2C12 myoblasts are a good model for the myotoxic effect. However, comparison of the structural determinants for all activities revealed several differences in the structural determinants between the effects against myoblast and bacterial membranes, and further differences when compared to the liposome membrane damaging effect. These membrane dependent effects are interpreted to be the consequence of differences in the activation of the membrane bound form of the protein on biological and artificial membranes.
Chioato
Lucimara
L
Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil.
Aragão
Elisangela Aparecida
EA
Lopes Ferreira
Tatiana
T
Medeiros
Alexandra Ivo de
AI
Faccioli
Lúcia Helena
LH
Ward
Richard J
RJ
D000409:Alanine / Q000378:metabolism*; D000818:Animals; D017837:Bothrops / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000097:blood; D004926:Escherichia coli / Q000187:drug effects; D008081:Liposomes; D008239:Lysine / Q000378:metabolism*; D008297:Male; D008567:Membranes, Artificial; D051379:Mice; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D016296:Mutagenesis; D050505:Mutant Proteins / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D054467:Phospholipases A2; D017433:Protein Structure, Secondary; D012910:Snake Venoms / Q000201:enzymology / Q000494:pharmacology; D013329:Structure-Activity Relationship
D008081:Liposomes; D008567:Membranes, Artificial; D050505:Mutant Proteins; D012910:Snake Venoms; D003402:Creatine Kinase; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine; D000409:Alanine; D002118:Calcium