title: Single amino acid mutations in the cadherin receptor from Heliothis virescens affect its toxin binding ability to Cry1A toxins.
abstract: Bacillus thuringiensis Cry protein exerts its toxic effect through a receptor-mediated process. Both aminopeptidases and cadherin proteins were identified as putative Cry1A receptors from Heliothis virescens and Manduca sexta. The importance of cadherin was implied by its correlation with a Cry1Ac resistant H. virescens strain (Gahan, L. J., Gould, F., and Heckel, D. G. (2001) Science 293, 857-860). In this study, the Cry1Ac toxin-binding region in H. virescens cadherin was mapped to a 40-amino-acid fragment, from amino acids 1422 to 1440. This site overlaps with a Cry1Ab toxin-binding site, amino acids 1363-1464 recently reported in M. sexta (Hua, G., Jurat-Fuentes, J. L., and Adang, M. J. (2004) J. Biol. Chem. 279, 28051-28056). Further, feeding of the anti-H. virescens cadherin antiserum or the partial cadherins, which contain the toxin-binding region, in combination with Cry1Ab/Cry1Ac reduced insect mortality by 25.5-55.6% to first instar H. virescens and M. sexta larvae, suggesting a critical function for this cadherin domain in insect toxicity. Mutations in this region, to which the Cry1Ac binds through its loop 3, resulted in the loss of toxin binding. For the first time, we show that the cadherin amino acids Leu(1425) and Phe(1429) are critical for Cry1Ac toxin interaction, and if substituted with charged amino acids, result in the loss of toxin binding, with a K(D) of < 10(-5) m. Mutation of Gln(1430) to an alanine, however, increased the Cry1Ac affinity 10-fold primarily due to an increase on rate. The L1425R mutant can result from a single nucleotide mutation, CTG --> CGG, suggesting that these mutants, which have decreased toxin binding, may lead to Cry1A resistance in insects.
authors.affiliation: Environmental Toxicology Graduate Program, Department of Cell Biology and Neuroscience, University of California, Riverside 92521, USA.
authors.forename: Ruiyu
authors.initials: R
authors.lastname: Xie
authors.forename: Meibao
authors.initials: M
authors.lastname: Zhuang
authors.forename: Linda S
authors.initials: LS
authors.lastname: Ross
authors.forename: Isabel
authors.initials: I
authors.lastname: Gomez
authors.forename: Daniela I
authors.initials: DI
authors.lastname: Oltean
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
authors.forename: Mario
authors.initials: M
authors.lastname: Soberon
authors.forename: Sarjeet S
authors.initials: SS
authors.lastname: Gill
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001681:Biological Assay; D015820:Cadherins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry* / Q000378:metabolism; D006460:Hemolysin Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D016296:Mutagenesis; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014118:Toxins, Biological / Q000737:chemistry
chemical_list: D000596:Amino Acids; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015820:Cadherins; D018076:DNA, Complementary; D004731:Endotoxins; D006460:Hemolysin Proteins; D010455:Peptides; D014118:Toxins, Biological; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D012313:RNA; D000409:Alanine