Role of alkaline phosphatase from Manduca sexta in the mechanism of action of Bacillus thuringiensis Cry1Ab toxin

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.

FIGURE 1.

Expression of aminopeptidase N and alkaline phosphatase during larval development. A, ligand blot analysis of biotin-labeled Cry1Ab toxin to BBMV proteins from M. sexta larvae isolated from each instar larvae (first through fifth indicated). B, specific enzymatic activities of APN (black bars) and ALP (gray bars) determined in BBMV from each larval instar.

FIGURE 2.

Purification of M. sexta APN and ALP proteins. Silver stain 10% SDS-PAGE of APN (lane 1) and ALP (lane 2) pure fractions from Mono Q (Table 1) and affinity chromatography (Table 2), respectively, are shown. Ligand blots with biotin-labeled Cry1Ab toxin of APN (lane 3) and ALP (lane 4) fractions are shown. Molecular mass markers are shown at the left of the figure.

FIGURE 3.

Cry1Ab domain II loop 2 and domain III mutants are structurally stable. A, SDS-PAGE electrophoresis pattern of trypsin activated Cry1Ab (lane 1), L511A (lane 2), R368A/R369A (lane 3), and F371A (lane 4) toxins. B, Western blot of pure oligomer samples obtained after size exclusion chromatography of toxin samples activated in the presence of cadherin fragment CR7-CR12. Cry1Ab (lane 1), L511A (lane 2), R368A/R369A (lane 3), and F371A (lane 4) toxins are shown. Molecular mass markers are shown at the left of the figure.

FIGURE 4.

Binding of Cry1Ab oligomer and monomer structure to APN and ALP proteins. The purified APN (Table 1) and ALP (Table 2) were immobilized on ELISA plates and incubated with Cry1Ab oligomer (A, 0–2 nm) or monomer (B, 0–1000 nm). K_d values obtained by Scatchard analysis are indicated inside the graphs.

FIGURE 5.

Binding analysis of Cry1Ab and loop 2 and domain III mutants to APN or ALP. A, ELISA binding assays of 25 nm of monomeric structures of Cry1Ab and mutant toxins to APN (black bars) or ALP (gray bars). B, ELISA binding assays of 0.1 nm of oligomeric structures of Cry1Ab and mutant toxins to APN (black bars) or ALP (gray bars). Standard deviations from three replica plates were obtained.