Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin.
The mosquitocidal crystal protein, Cry19Aa, from Bacillus thuringiensis ssp. jegathesan, has high toxicity to Anopheles stephensi and Culex pipiens but is less toxic to Aedes aegypti. To study the functional role of putative domain II and surface residues in mosquito toxicity, 16 alanine substitution mutations were introduced into Cry19Aa. All mutant constructs were expressed as 65-kDa protoxins and subsequently digested by trypsin to produce further fragmented polypeptides of 40 and 25 kDa. With chymotrypsin, however, most protoxins were digested to 60 kDa and minor bands. The circular dichroism spectra of the chymotrypsin-activated toxins of Cry19Aa and muteins, Y324A, W357A, Y412A, Y414A, W416A, D418A and F485A indicated that there was no significant variation in their structure. In mosquito bioassays, Y324A, W357A, Y410A, W416A, D418A and F485A muteins showed substantial reductions in mosquitocidal activity toward A. aegypti and C. pipiens. These muteins also showed reduced competition with wild-type fluorescein 5-isothiocyanate-labeled Cry19Aa for binding to C. pipiens brush border membrane vesicles. These data suggest that the reduction of toxicity was a result of the reduced binding affinity. From these studies we have identified loop residues of domain II that are important in toxicity and receptor binding to Culex larval midgut.
Roh
Jong Yul
JY
Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Nair
Manoj S
MS
Liu
Xinyan Sylvia
XS
Dean
Donald H
DH
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D001667:Binding, Competitive; D003465:Culex / Q000254:growth & development* / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics*; D007814:Larva / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010572:Pest Control, Biological; D017434:Protein Structure, Tertiary / Q000235:genetics; D013329:Structure-Activity Relationship
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000409:Alanine