title: 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.
abstract: 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 beta16-beta22 (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 beta16 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 beta-16 of domain III promoting membrane insertion and cell death.
authors.affiliation: Instituto de Biotecnología, UNAM, Departamento de Microbiología Molecular, Apdo. Postal 510-3, Cuernavaca, Morelos 62250, México.
authors.forename: Isabel
authors.initials: I
authors.lastname: Gómez
authors.forename: Iván
authors.initials: I
authors.lastname: Arenas
authors.forename: Itzel
authors.initials: I
authors.lastname: Benitez
authors.forename: Juan
authors.initials: J
authors.lastname: Miranda-Ríos
authors.forename: Baltazar
authors.initials: B
authors.lastname: Becerril
authors.forename: Ricardo
authors.initials: R
authors.lastname: Grande
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Almagro
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D018826:CD13 Antigens / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism*; D004731:Endotoxins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D000939:Epitopes / Q000032:analysis / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D007114:Immunization; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000378:metabolism; D007306:Insecticides / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D018402:Manduca / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D010572:Pest Control, Biological; D011485:Protein Binding; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015820:Cadherins; D004731:Endotoxins; D000939:Epitopes; D006460:Hemolysin Proteins; D007135:Immunoglobulin Variable Region; D007306:Insecticides; D019151:Peptide Library; D010455:Peptides; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens