Location of a Bombyx mori receptor binding region on a Bacillus thuringiensis delta-endotoxin.

M.K Lee,R.E Milne,A.Z Ge,D.H Dean

doi:10.1016/s0021-9258(19)50702-5

M.K Lee, R.E Milne + Show 2 more

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https://doi.org/10.1016/s0021-9258(19)50702-5

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Abstract

Receptor binding studies were performed with 125I-labeled trypsin-activated insecticidal toxins, CryIA(a) and CryIA(c), from Bacillus thuringiensis on brush-border membrane vesicles (BBMV) prepared from Bombyx mori larval midgut. Bioassays were performed by gently force feeding B. mori with diluted toxins. CryIA(a) toxin (LD50; 0.002 micrograms) was 200 times more active against B. mori larvae than CryIA(c) toxin (LD50; 0.421 micrograms) and showed high-affinity saturable binding. The Kd and the binding site concentration for CryIA(a) toxin were 3.5 nM and 7.95 pmol/mg, respectively. CryIA(c) toxin (Kd, 50.35 nM; Bmax, 2.85 pmol/mg) did not demonstrate high-affinity binding to B. mori BBMV. Control experiments with CryIA(a) and CryIA(c) toxins revealed no binding to mouse small intestine BBMV and nonspecific binding to pig kidney BBMV. These data provide evidence that binding to a specific receptor on the membrane of midgut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of insecticidal crystal proteins. To locate a B. mori receptor binding region on the CryIA(a) toxin, homologous and heterologous competition binding studies were performed with a set of mutant proteins which had previously been used to define the B. mori "specificity domain" on this toxin (Ge, A. Z., Shivarova, N. I., and Dean, D. H. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4037-4041). These mutant proteins have had regions of their genes reciprocally exchanged with the cryIA(c) gene. A B. mori receptor binding region on CryIA(a) toxin includes the amino-terminal portion of the hypervariable region, amino acids 332-450, which is identical to the previously described B. mori specificity determining region. These data provide direct evidence that delta-endotoxins contain a tract of amino acids that comprise a binding region and as a results determines the specificity of a toxin.

Highlights

The costs of publication of this article were defrayed in part by the payment of page charges
Many strainosf B. thuringiensis produce more than one type of ICP, each of which has its own insecticidal spectrum
B. thuringiensis strains may be classified into five pathotypes,depending on the insecticidal activity of their ICPs: (i) lepidopteran-active,(ii) dipteran-active, (iii) coleopteran-active, (iv) lepidopteran- and dipteran-active, and (v) no known toxic activity [2]

Summary

MATERIALS AND METHODS

Cold buffer A (300 mM mannitol, 5 mM EGTA, 17 mM Tris-HCl, pH 7.5). The isolated midgut was rinsed, blotted, and weighed. Purification and Activation of Recombinant B. thuringiensis 6Endotoxin-All cry genes were overexpressed in E. coli host strain JM103 [23] using the expression vector pKK223-3 (Pharmacia LKB BiotechnologyInc.), modified to remove the EcoRI site. Bioassays resuspended in 50 ml of buffer The crystal protein was solubilized in 50 mM sodium Na'"I (Amersham Corp.) was added to a vial which contained one carbonate buffer, pH 9.5, containing 10 mM dithiothreitol at 37 "C IODO-BEADand incubated for 5 min at room temperature. The reaction mixture was applied to a 2-ml Excellulose column (Pierce Chemical Co.) to remove free iodine and possible degradation products

NS E

Homologous Heterologous

DISCUSSION

Heterologous competition binding studies showed that all