Abstract
Cry toxins produced by Bacillus thuringiensis have been recognized as pore-forming toxins whose primary action is to lyse midgut epithelial cells in their target insect. In the case of the Cry1A toxins, a prepore oligomeric intermediate is formed after interaction with cadherin receptor. The Cry1A oligomer then interacts with glycosylphosphatidylinositol-anchored receptors. Two Manduca sexta glycosylphosphatidylinositol-anchored proteins, aminopeptidase (APN) and alkaline phosphatase (ALP), have been shown to bind Cry1Ab, although their role in toxicity remains to be determined. Detection of Cry1Ab binding proteins by ligand blot assay revealed that ALP is preferentially expressed earlier during insect development, because it was found in the first larval instars, whereas APN is induced later after the third larval instar. The binding of Cry1Ab oligomer to pure preparations of APN and ALP showed that this toxin structure interacts with both receptors with high affinity (apparent K(d) = 0.6 nM), whereas the monomer showed weaker binding (apparent K(d) = 101.6 and 267.3 nM for APN and ALP, respectively). Several Cry1Ab nontoxic mutants located in the exposed loop 2 of domain II or in beta-16 of domain III were affected in binding to APN and ALP, depending on their oligomeric state. In particular monomers of the nontoxic domain III, the L511A mutant did not bind ALP but retained APN binding, suggesting that initial interaction with ALP is critical for toxicity. Our data suggest that APN and ALP fulfill two roles. First APN and ALP are initial receptors promoting the localization of toxin monomers in the midgut microvilli before interaction with cadherin. Then APN and ALP function as secondary receptors mediating oligomer insertion into the membrane. However, the expression pattern of these receptors and the phenotype of L511A mutant suggest that ALP may have a predominant role in toxin action because Cry toxins are highly effective against the neonate larvae that is the target for pest control programs.
Highlights
The Cry protein family is composed of more than 54 groups, among which the three-domain Cry family forms the major group, having members that show toxicity to different insect orders and to nematodes
Cry1A monomers bind to the primary receptor that has been identified in several species as a cadherin protein that is located in the microvilli of columnar midgut cells
Our data suggest that both APN and alkaline phosphatase (ALP) are functional receptor molecules that bind the oligomeric structure of Cry1Ab toxin with high affinity, facilitating membrane insertion and pore formation, and we found that ALP may a have a predominant and more important role in Cry1Ab toxicity at earlier stages of larval development
Summary
M. sexta Midgut BBMV Purification and Solubilization— Larvae of M. sexta were from a laboratory colony maintained on an artificial diet. The BBMV were centrifuged at 70,000 rpm for 40 min at 4 °C and suspended at 5 mg/ml in 20 mM Tris-HCl pH 8.5 buffer containing 100 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1% CHAPS for detergent solubilization. The BBMV proteins that were solubilized with CHAPS as stated above were concentrated by Amicon YM-50 ultrafiltration, and a 2-ml aliquot was applied to a HR 5/10 Mono Q (GE Healthcare) column equilibrated with 20 mM Tris-HCl pH 8.5 buffer containing 2 mM MgCl2, 2 mM KCl [28, 30]. CHAPS-solubilized proteins from BBMV of third instar larvae were concentrated by Amicon YM-30 ultrafiltration, and a 1-ml aliquot was loaded into the column equilibrated with 20 mM Tris-HCl pH 8.5 buffer containing 1 mM MgCl2.
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