title: Mapping of the structural determinants of artificial and biological membrane damaging activities of a Lys49 phospholipase A2 by scanning alanine mutagenesis.
abstract: 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.
authors.affiliation: Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil.
authors.forename: Lucimara
authors.initials: L
authors.lastname: Chioato
authors.forename: Elisangela Aparecida
authors.initials: EA
authors.lastname: Aragão
authors.forename: Tatiana
authors.initials: T
authors.lastname: Lopes Ferreira
authors.forename: Alexandra Ivo de
authors.initials: AI
authors.lastname: Medeiros
authors.forename: Lúcia Helena
authors.initials: LH
authors.lastname: Faccioli
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
mesh_terms: 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
chemical_list: 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