Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis delta-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.
Two arginine residues (368-369) of Cry1Ab and Cry1Ac were mutated to alanine, glutamic acid and lysine by site-directed mutagenesis. Insecticidal activities of the mutant toxins on Manduca sexta and Lymantria dispar larvae were examined. Cry1Ac mutant toxins (c)RR-AA and (c)RR-EE and Cry1Ab mutant toxins (b)RR-AA and (b)RR-EE showed great reductions in toxicity against both insects. In contrast, conservatively changed (c)RR-KK and (b)RR-KK mutants did not alter toxicity to either insect. Binding assays with brush border membrane vesicles (BBMVs) prepared from L. dispar midguts demonstrated that (c)RR-AA, (c)RR-EE, (b)RR-AA and (b)RR-EE bound with lower affinities compared with their respective wild-type toxins. To M. sexta BBMVs, (c)RR-AA and (c)RR-EE showed great reductions in BBMV binding. However, (b)RR-AA and (b)RR-EE did not alter BBMV competition patterns, despite their reduced toxicity. Further binding assays were performed with aminopeptidase N (APN) purified from L. dispar and M. sexta BBMVs using surface plasmon resonance (BIAcore). Direct correlation between toxicity and APN binding was observed for the mutant toxins using this technique. The inconsistency between BBMV and APN binding data with Cry1Ab to M. sexta suggests the possibility of a different Cry1Ab toxin-binding mechanism or the importance of another receptor in M. sexta.
Lee
M K
MK
Departments of Biochemistry and Molecular Genetics, The Ohio State University, Biological Sciences, 484 West 12th Avenue, Columbus, OH 43210, USA.
Rajamohan
F
F
Jenkins
J L
JL
Curtiss
A S
AS
Dean
D H
DH
D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001413:Bacillus thuringiensis / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018826:CD13 Antigens / Q000378:metabolism*; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006460:Hemolysin Proteins; D018402:Manduca / Q000201:enzymology*; D009036:Moths / Q000201:enzymology*; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D001120:Arginine; D018826:CD13 Antigens