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Studies of the Manduca sexta cadherin-like receptor binding epitopes of Bacillus thuringiensis Cry1Aa toxin and protein engineering of mosquitocidal activity

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2005, Doctor of Philosophy, Ohio State University, Ohio State Biochemistry Program.

Bacillus thuringiensis, an aerobic, gram-positive spore-forming bacterium commonly found in soil, produces parasporal crystal (Cry) proteins with insecticidal activity against a wide range of pests. Cry toxin binding to receptors on the brush border membrane in insect midgut is required, among other factors, to exert the toxic effect. The binding epitopes on Cry1Aa toxin to the cadherin-like receptor in Manduca sexta (tobacco hornworm) was mapped by a combined approach of molecular modeling, site-directed mutagenesis, bioassay on insect larvae, and kinetic analysis.

CAD-D, a truncated fragment (CR11 and 12) of Bt-R1a (63), the cadherin-like receptor from Manduca sexta for Bacillus thuringiensis Cry1A toxins, was expressed and purified as a soluble MBP (maltose binding protein) fusion protein. Binding affinity of Cry1Aa to CAD-D measured by real time SPR was at the 10nM level. Some of the CAD-D binding epitopes on Cry1Aa toxin were mapped by alanine scanning mutagenesis. Designing of the area targeted for mutagenesis was based on structural information derived from topology prediction and computational docking of the toxin with the receptor. Loop 2 residues in domain II and three clusters of surface residues in domains II and III were demonstrated to be involved in binding to CAD-D. The interaction surface was defined by the loss of binding for mutants on the predicted face of the toxin and no effects on another set of substitutions located on the opposite face of domain III.

The Bacillus thuringiensis crystal protein Cry1Aa is naturally selectively active to caterpillar larvae. In further manipulation of receptor binding epitopes through rational design, toxicity (µg/ml) to the mosquito Culex pipiens was introduced by selected deletions and substitutions of the loop residues of domain II. Toxicity to its natural target Manduca sexta was concomitantly abolished. The successful grafting of the alternate mosquito toxicity onto the original lepidopteran Cry1Aa toxin by exchanging the specificity-determining loop regions demonstrates the possibility of designing and engineering a desired toxicity into any toxin of a common scaffold by reshaping the receptor binding region with desired specificities.

Taken together, these studies provided promising evidence that epitope-mapping and protein-engineering under the guidance of molecular modeling can serve as a rational and useful tool in understanding the mode of action of Cry toxins, and ultimately in producing better toxins.

Donald Dean (Advisor)

Recommended Citations

Citations

  • Liu, X. (2005). Studies of the Manduca sexta cadherin-like receptor binding epitopes of Bacillus thuringiensis Cry1Aa toxin and protein engineering of mosquitocidal activity [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1117655918

    APA Style (7th edition)

  • Liu, Xinyan. Studies of the Manduca sexta cadherin-like receptor binding epitopes of Bacillus thuringiensis Cry1Aa toxin and protein engineering of mosquitocidal activity. 2005. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1117655918.

    MLA Style (8th edition)

  • Liu, Xinyan. "Studies of the Manduca sexta cadherin-like receptor binding epitopes of Bacillus thuringiensis Cry1Aa toxin and protein engineering of mosquitocidal activity." Doctoral dissertation, Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1117655918

    Chicago Manual of Style (17th edition)