Mapping the epitope in cadherin-like receptors involved in Bacillus thuringiensis Cry1A toxin interaction using phage display.
In susceptible lepidopteran insects, aminopeptidase N and cadherin-like proteins are the putative receptors for Bacillus thuringiensis (Bt) toxins. Using phage display, we identified a key epitope that is involved in toxin-receptor interaction. Three different scFv molecules that bind Cry1Ab toxin were obtained, and these scFv proteins have different amino acid sequences in the complementary determinant region 3 (CDR3). Binding analysis of these scFv molecules to different members of the Cry1A toxin family and to Escherichia coli clones expressing different Cry1A toxin domains showed that the three selected scFv molecules recognized only domain II. Heterologous binding competition of Cry1Ab toxin to midgut membrane vesicles from susceptible Manduca sexta larvae using the selected scFv molecules showed that scFv73 competed with Cry1Ab binding to the receptor. The calculated binding affinities (K(d)) of scFv73 to Cry1Aa, Cry1Ab, and Cry1Ac toxins are in the range of 20-51 nm. Sequence analysis showed this scFv73 molecule has a CDR3 significantly homologous to a region present in the cadherin-like protein from M. sexta (Bt-R(1)), Bombyx mori (Bt-R(175)), and Lymantria dispar. We demonstrated that peptides of 8 amino acids corresponding to the CDR3 from scFv73 or to the corresponding regions of Bt-R(1) or Bt-R(175) are also able to compete with the binding of Cry1Ab and Cry1Aa toxins to the Bt-R(1) or Bt-R(175) receptors. Finally, we showed that synthetic peptides homologous to Bt-R(1) and scFv73 CDR3 and the scFv73 antibody decreased the in vivo toxicity of Cry1Ab to M. sexta larvae. These results show that we have identified the amino acid region of Bt-R(1) and Bt-R(175) involved in Cry1A toxin interaction.
Gómez
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I
Instituto de Biotecnologia, Departamento de Microbiologia Molecular, Universidad Nacional Autónoma de México, Apdo postal 510-3, Cuernavaca, Morelos 62250, México.
Oltean
D I
DI
Gill
S S
SS
Bravo
A
A
Soberón
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D015374:Biosensing Techniques; D015820:Cadherins / Q000737:chemistry*; D003001:Cloning, Molecular; D004064:Digestive System / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D000939:Epitopes / Q000032:analysis / Q000737:chemistry; D006460:Hemolysin Proteins; D007135:Immunoglobulin Variable Region; D019476:Insect Proteins; D007700:Kinetics; D007814:Larva; D018402:Manduca; D008871:Microvilli / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015820:Cadherins; C095416:Cry toxin receptors; D004731:Endotoxins; D000939:Epitopes; D006460:Hemolysin Proteins; D007135:Immunoglobulin Variable Region; D019476:Insect Proteins; D019151:Peptide Library; D010455:Peptides; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis