title: An engineered chymotrypsin/cathepsin G site in domain I renders Bacillus thuringiensis Cry3A active against Western corn rootworm larvae.
abstract: The western corn rootworm remains one of the most important pests of corn in the United States despite the use of many pest management tools. Cry3A, the first coleopteran-active Bacillus thuringiensis toxin isolated, has not been useful for control of the corn rootworm pest complex. Modification of Cry3A so that it contained a chymotrypsin/cathepsin G protease recognition site in the loop between alpha-helix 3 and alpha-helix 4 of domain I, however, resulted in consistent activity of the toxin ("mCry3A") against neonate western corn rootworm. In vitro chymotrypsin digests showed that there was a substantial difference between the enzyme sensitivity of mCry3A and the enzyme sensitivity of Cry3A, with mCry3A rapidly converted from a 67-kDa form to a approximately 55-kDa form. The introduced protease site was also recognized in vivo, where the approximately 55-kDa form of mCry3A toxin was rapidly generated and associated with the membrane fraction. After a point mutation in mcry3A that resulted in the elimination of the native domain I chymotrypsin site (C terminal to the introduced chymotrypsin/cathepsin G protease site of mCry3A), the in vitro and in vivo digestion patterns remained the same, demonstrating that the introduced site was required for the enhanced activity. Also, 55-kDa mCry3A generated by cleavage with chymotrypsin exhibited specific binding to western corn rootworm brush border membrane, whereas untreated 67-kDa mCry3A did not. These data indicate that the mCry3A toxicity for corn rootworm larvae was due to the introduction of a chymotrypsin/cathepsin G site, which enhanced cleavage and subsequent binding of the activated toxin to midgut cells.
authors.affiliation: Syngenta Biotechnology, Inc., Research Triangle Park, NC 27709-2257, USA. Frederick.Walters@Syngenta.com
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Walters
authors.forename: Cheryl M
authors.initials: CM
authors.lastname: Stacy
authors.forename: Mi Kyong
authors.initials: MK
authors.lastname: Lee
authors.forename: Narendra
authors.initials: N
authors.lastname: Palekar
authors.forename: Jeng S
authors.initials: JS
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D056649:Cathepsin G; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism*; D002918:Chymotrypsin / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D001517:Coleoptera / Q000254:growth & development*; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007814:Larva / Q000187:drug effects / Q000254:growth & development; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010572:Pest Control, Biological; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D003313:Zea mays / Q000469:parasitology*
chemical_list: D001426:Bacterial Proteins; D004731:Endotoxins; D002403:Cathepsins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D056649:Cathepsin G