Specific epitopes of domains II and III of Bacillus thuringiensis Cry1Ab toxin involved in the sequential interaction with cadherin and aminopeptidase-N receptors in Manduca sexta
Por:
Gómez I., Arenas I., Benitez I., Miranda-Ríos J., Becerril B., Grande R., Almagro J.C., Bravo A., Soberón M.
Publicada:
1 ene 2006
Resumen:
The Bacillus thuringiensis Cry toxins are specific to different insects. In Manduca sexta cadherin (Bt-R1) and aminopeptidase-N (APN) proteins are recognized as Cry1A receptors. Previous work showed that Cry1Ab binds to Bt-R1 promoting the formation of a pre-pore oligomer that binds to APN leading to membrane insertion. In this work we characterized the binding epitopes involved in the sequential interaction of Cry1Ab with Bt-R1 and APN. A Cry1Ab immune M13 phage repertoire was constructed using antibody gene transcripts of bone marrow or spleen from a rabbit immunized with Cry1Ab. We identified antibodies that recognize domain II loop 3 (scFvL3-3) or ?16-?22 (scFvM22) in domain III. Enzyme-linked immunosorbent assay and toxin overlay binding competition assays in the presence of scFvL3-3, scFvM22, or synthetic peptides showed that domain II loop 3 is an important epitope for interaction with Bt-R1 receptor, whereas domain III ?16 is involved in the interaction with APN. Both scFvL3-3 and scFvM22 lowered the toxicity of Cry1Ab to M. sexta larvae indicating that interaction with both receptors is important for in vivo toxicity. scFvL3-3 and anti-loop2 scFv (scFv73) promoted the formation of the pre-pore oligomer in contrast to scFvM22. In addition, scFvL3-3 and scFv73 preferentially recognized the monomeric toxin rather than the pre-pore suggesting a conformational change in domain II loops upon oligomerization. These results indicate for the first time that both receptor molecules participate in Cry1Ab toxin action in vivo: first the monomeric toxin binds to Bt-R1 through loops 2 and 3 of domain II promoting the formation of the pre-pore inducing some structural changes, then the pre-pore interacts with APN through ?-16 of domain III promoting membrane insertion and cell death. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
Filiaciones:
Gómez I.:
Instituto de Biotecnología, UNAM, Departamento de Microbiología Molecular, Cuernavaca, Morelos 62250, Mexico
Arenas I.:
Instituto de Biotecnología, UNAM, Departamento de Microbiología Molecular, Cuernavaca, Morelos 62250, Mexico
Benitez I.:
Instituto de Biotecnología, UNAM, Departamento de Microbiología Molecular, Cuernavaca, Morelos 62250, Mexico
Miranda-Ríos J.:
Instituto de Biotecnología, UNAM, Departamento de Microbiología Molecular, Cuernavaca, Morelos 62250, Mexico
Becerril B.:
Instituto de Biotecnología, UNAM, Departamento de Medicina Molecular Y Bioprocesos, Cuernavaca, Morelos 62250, Mexico
Grande R.:
Instituto de Biotecnología, UNAM, Ingeniería Celular Y Biocatálisis, Apdo. Postal 510-3, Cuernavaca, Morelos 62250, Mexico
Almagro J.C.:
Instituto de Biotecnología, UNAM, Departamento de Medicina Molecular Y Bioprocesos, Cuernavaca, Morelos 62250, Mexico
Bravo A.:
Instituto de Biotecnología, UNAM, Departamento de Microbiología Molecular, Cuernavaca, Morelos 62250, Mexico
Soberón M.:
Instituto de Biotecnología, UNAM, Departamento de Microbiología Molecular, Cuernavaca, Morelos 62250, Mexico
Bronze
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