Denaturation of either Manduca sexta aminopeptidase N or Bacillus thuringiensis Cry1A toxins exposes binding epitopes hidden under nondenaturing conditions.
The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with (125)I-labeled Cry1Ac, Cry1Ac mutant (509)QNR-AAA(511) (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both (125)I-Cry1Ac and (125)I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots (125)I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, (125)I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. (125)I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured (125)I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) (125)I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.
Daniel
Anu
A
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Sangadala
Sreedhara
S
Dean
Donald H
DH
Adang
Michael J
MJ
D000626:Aminopeptidases / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D001413:Bacillus thuringiensis / Q000737:chemistry*; D001426:Bacterial Proteins; D000939:Epitopes; D015151:Immunoblotting; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism; D007457:Iodine Radioisotopes; D008024:Ligands; D018402:Manduca / Q000201:enzymology*; D011489:Protein Denaturation; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; C095416:Cry toxin receptors; D000939:Epitopes; D019476:Insect Proteins; D007457:Iodine Radioisotopes; D008024:Ligands; D011956:Receptors, Cell Surface; D000626:Aminopeptidases