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Applied and Environmental Microbiology, May 2002, p. 2106-2112, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2106-2112.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Denaturation of Either Manduca sexta Aminopeptidase N or Bacillus thuringiensis Cry1A Toxins Exposes Binding Epitopes Hidden under Nondenaturing Conditions

Anu Daniel,1 Sreedhara Sangadala,2 Donald H. Dean,3 and Michael J. Adang1,2*

Department of Biochemistry and Molecular Biology,1 Department of Entomology, University of Georgia, Athens, Georgia 30602,2 Department of Molecular Genetics and Department of Biochemistry, Ohio State University, Columbus, Ohio 432103

Received 15 October 2001/ Accepted 12 February 2002

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 125I-labeled Cry1Ac, Cry1Ac mutant 509QNR-AAA511 (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 125I-Cry1Ac and 125I-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 125I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, 125I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. 125I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured 125I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) 125I-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.

* Corresponding author. Mailing address: Department of Entomology, Biosciences Building, Room 413, University of Georgia, 125 Cedar St., Athens, GA 30602-2603. Phone: (706) 542-2436. Fax: (706) 542-2279. E-mail: adang{at}uga.edu.

Applied and Environmental Microbiology, May 2002, p. 2106-2112, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2106-2112.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Pigott, C. R., Ellar, D. J. (2007). Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity. Microbiol. Mol. Biol. Rev. 71: 255-281 [Abstract] [Full Text]  
  • Sivakumar, S., Rajagopal, R., Venkatesh, G. R., Srivastava, A., Bhatnagar, R. K. (2007). Knockdown of Aminopeptidase-N from Helicoverpa armigera Larvae and in Transfected Sf21 Cells by RNA Interference Reveals Its Functional Interaction with Bacillus thuringiensis Insecticidal Protein Cry1Ac. J. Biol. Chem. 282: 7312-7319 [Abstract] [Full Text]  
  • Hua, G., Jurat-Fuentes, J. L., Adang, M. J. (2004). Bt-R1a Extracellular Cadherin Repeat 12 Mediates Bacillus thuringiensis Cry1Ab Binding and Cytotoxicity. J. Biol. Chem. 279: 28051-28056 [Abstract] [Full Text]  


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