Bt-R1a extracellular cadherin repeat 12 mediates Bacillus thuringiensis Cry1Ab binding and cytotoxicity.
The cadherin protein Bt-R(1a) is a receptor for Bacillus thuringiensis Cry1A toxins in Manduca sexta. Cry1Ab toxin is reported to bind specific epitopes located in extracellular cadherin repeat (CR) 7 and CR11 on Bt-R(1) (Gomez, B., Miranda-Rios, J., Riudino-Pinera, E., Oltean, D. I., Gill, S. S., Bravo, A., and Soberon, M. (2002) J. Biol. Chem. 277, 30137-30143; Dorsch, J. A., Candas, M., Griko, N., Maaty, W., Midboe, E., Vadlamudi, R., and Bulla, L. (2002) Insect Biochem. Mol. Biol. 32, 1025-1036). We transiently expressed CR domains of Bt-R(1a) in Drosophila melanogaster Schneider 2 (S2) cells as fusion peptides between a signal peptide and a terminal region that included membrane-proximal, membrane-spanning, and cytoplasmic domains. A domain consisting of CR11 and 12 was the minimal (125)I-Cry1Ab binding region detected under denaturing conditions. Only CR12 was essential for Cry1Ab binding and cytotoxicity to S2 cells when tested under native conditions. Under these conditions expressed CR12 bound (125)I-Cry1Ab with high affinity (K(com) = 2.9 nm). Flow cytometry assays showed that expression of CR12 conferred susceptibility to Cry1Ab in S2 cells. Derivatives of Bt-R(1a) with separate deletions of CR7, 11, and 12 were expressed in S2 cells. Only deletion of CR12 caused loss of Cry1Ab binding and cytotoxicity. These results demonstrate that CR12 is the essential Cry1Ab binding component on Bt-R(1) that mediates Cry1Ab-induced cytotoxicity.
Hua
Gang
G
Department of Entomology, University of Georgia, Athens, GA 30602-2603, USA.
Jurat-Fuentes
Juan Luis
JL
Adang
Michael J
MJ
D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry*; D015820:Cadherins / Q000737:chemistry*; D002460:Cell Line; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000737:chemistry*; D005434:Flow Cytometry; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006460:Hemolysin Proteins; D015151:Immunoblotting; D019476:Insect Proteins / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D020449:Repetitive Sequences, Amino Acid; D015398:Signal Transduction
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015820:Cadherins; C095416:Cry toxin receptors; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D008024:Ligands; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D049452:Green Fluorescent Proteins