Manduca sexta cadherin-like protein (Bt-R1) is a receptor for Bacillus thuringiensis Cry1A toxins. Bt-R1a has an ectodomain that contains 12 tandemly repeated units referred to as cadherin (cad) repeats. The goal of this study was to identify regions on Bt-R1a that function as a Cry1Ab toxin receptor. Our approach was to express truncated derivatives of Bt-R1a on the surface of Drosophila melanogaster S2 cells and then analyze toxin interactions using binding and cytotoxicity assays. On ligand blots, Cry1Ab bound denatured full-length cadherin, plus peptides fragments containing cad7 to the end (called cad7E), cad10E and cad11E. Expressed fragments cad7, cad11, cad12E did not bind Cry1Ab on ligand blots. Since proteins on blots are denatured and denaturation is known to effect toxin binding, we analyzed toxin binding to non-denatured Bt-R1a and fragments on dot blot. Cry1Ab bound to the same fragments as detected by ligand blot experiments; additionally Cry1Ab bound the cad12E fragment. Using a flow cytometry assay, we show that Cry1Ab killed S2 cells expressing BtR1a cadherin, cad7E, cad10E, cad11E and cad12E. These results demonstrate that cad12 of Bt-R1a is sufficient to bind Cry1Ab with high-affinity and induce cytotoxicity.
Species 1: Lepidoptera Sphingidae Manduca sexta (tobacco hornworm)
Keywords: cadherin, Bt-toxin
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