Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata (Say).
Binding and pore formation constitute key steps in the mode of action of Bacillus thuringiensis delta-endotoxins. In this work, we present a comparative analysis of toxin-binding capacities of proteolytically processed Cry3A, Cry3B and Cry3C toxins to brush border membranes (BBMV) of the Colorado potato beetle Leptinotarsa decemlineata (CPB), a major potato coleopteran-insect pest. Competition experiments showed that the three Cry3 proteolytically activated toxins share a common binding site. Also heterologous competition experiments showed that Cry3Aa and Cry3Ca toxins have an extra binding site that is not shared with Cry3Ba toxin. The pore formation activity of the three different Cry3 toxins is analysed. High pore-formation activities were observed in Cry3 toxins obtained by proteolytical activation with CPB BBMV in contrast to toxins activated with either trypsin or chymotrypsin proteases. The pore-formation activity correlated with the formation of soluble oligomeric structures. Our data support that, similarly to the Cry1A toxins, the Cry3 oligomer is formed after receptor binding and before membrane insertion, forming a pre-pore structure that is insertion-competent.
Rausell
C
C
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ap Postal 510-3, Cuernavaca 62250, Morelos, Mexico.
García-Robles
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I
Sánchez
J
J
Muñoz-Garay
C
C
Martínez-Ramírez
A C
AC
Real
M D
MD
Bravo
A
A
D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism* / Q000472:pathogenicity; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001427:Bacterial Toxins / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D001517:Coleoptera / Q000166:cytology / Q000378:metabolism*; D010450:Endopeptidases; D004731:Endotoxins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006460:Hemolysin Proteins; D008871:Microvilli / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry
D000083722:Bacillus thuringiensis Toxins; C029103:Bacillus thuringiensis protoxin; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011498:Protein Precursors; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010450:Endopeptidases