Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxin binding to a novel 110 kDa aminopeptidase in Heliothis virescens is not N-acetylgalactosamine mediated.
We determined that Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxins recognize the same 110, 120 and 170 kDa aminopeptidase N (APN) molecules in brush border membrane vesicles (BBMV) from Heliothis virescens. The 110 kDa protein, not previously identified as an APN, contained a variant APN consensus sequence identical to that found in Helicoverpa punctigera APN 2. PCR amplification of H. virescens cDNA based on this sequence and a conserved APN motif yielded a 0.9 kb product that has 89% sequence homology with H. punctigera APN 2. Western blots revealed that the 110 kDa molecule was not recognized by soybean agglutinin, indicating the absence of GalNAc. A 125I labeled-Cry1Ac domain III mutant (509QNR(511)-AAA) that has an altered GalNAc binding pocket (Lee et al., Appl. Environ. Microbiol. 65 (1999) 4513) showed abolished binding to the 120 APN, reduced binding to the 170 kDa APN, and enhanced binding to the 110 kDa APN. Periodate treated H. virescens BBMV blots were also probed with 125I labeled-Cry1Ac and 509QNR(511)-AAA toxins. Both toxins still recognized the 110 kDa APN and a >210 kDa molecule which may be a cadherin-like protein. Additionally, 125I-(509)QNR(511)-AAA recognized periodate treated 170 kDa APN. Results indicate that the 110 kDa APN is distinct from other Cry1 toxin binding APNs and may be the first described Cry1Ac-binding APN that does not contain GalNAc.
Banks
D J
DJ
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
Jurat-Fuentes
J L
JL
Dean
D H
DH
Adang
M J
MJ
D000116:Acetylgalactosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism*; D001427:Bacterial Toxins; D001710:Biotin; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000276:immunology / Q000378:metabolism*; D006460:Hemolysin Proteins; D019476:Insect Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007457:Iodine Radioisotopes; D037102:Lectins / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D009036:Moths; D010504:Periodic Acid; D037121:Plant Lectins; D016133:Polymerase Chain Reaction / Q000379:methods; D012967:Sodium Dodecyl Sulfate; D030262:Soybean Proteins; D013194:Staining and Labeling / Q000379:methods; D010835:Phytohemagglutinins
D000116:Acetylgalactosamine; D000626:Aminopeptidases; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001710:Biotin; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D007457:Iodine Radioisotopes; D037102:Lectins; D008024:Ligands; D010504:Periodic Acid; D037121:Plant Lectins; D012967:Sodium Dodecyl Sulfate; D030262:Soybean Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C028824:soybean lectin; D010835:Phytohemagglutinins; C009288:metaperiodate