Cadherin, alkaline phosphatase, and aminopeptidase N as receptors of Cry11Ba toxin from Bacillus thuringiensis subsp. jegathesan in Aedes aegypti

Abstract

Cry11Ba is one of the most toxic proteins to mosquito larvae produced by Bacillus thuringiensis. It binds Aedes aegypti brush border membrane vesicles (BBMV) with high affinity, showing an apparent dissociation constant (K(d)) of 8.2 nM. We previously reported that an anticadherin antibody competes with Cry11Ba binding to BBMV, suggesting a possible role of cadherin as a toxin receptor. Here we provide evidence of specific cadherin repeat regions involved in this interaction. Using cadherin fragments as competitors, a C-terminal fragment which contains cadherin repeat 7 (CR7) to CR11 competed with Cry11Ba binding to BBMV. This binding was also efficiently competed by the CR9, CR10, and CR11 peptide fragments. Moreover, we show CR11 to be an important region of interaction with Cry11Ba toxin. An alkaline phosphatase (AaeALP1) and an aminopeptidase-N (AaeAPN1) also competed with Cry11Ba binding to Ae. aegypti BBMV. Finally, we found that Cry11Ba and Cry4Ba share binding sites. Synthetic peptides corresponding to loops α8, β2-β3 (loop 1), β8-β9, and β10-β11 (loop 3) of Cry4Ba compete with Cry11Ba binding to BBMV, suggesting Cry11Ba and Cry4Ba have common sites involved in binding Ae. aegypti BBMV. The data suggest that three different Ae. aegypti midgut proteins, i.e., cadherin, AaeALP1, and AaeAPN1, are involved in Cry11Ba binding to Ae. aegypti midgut brush border membranes.

FIG. 1.

Binding of Cry11Ba toxin to Ae. aegypti BBMV. (A) Specific binding of biotinylated Cry11Ba toxin to BBMV was determined in the presence of increasing concentrations of labeled Cry11Ba toxin. Specific binding was obtained from total binding minus nonspecific binding. The dissociation constant (K_d) (8.2 nM) for toxin binding affinity was determined by Origin plot analysis from two different BBMV preparations. The curve shown is from a single experiment. (B) Binding of biotinylated Cry11Ba toxin to BBMV was determined in the presence of increasing concentrations of unlabeled Cry11Ba toxin. The IC₅₀ (3.6 nM) for toxin binding affinity was determined by Origin plot analysis from three different BBMV preparations. The curve shown is from a single experiment. (C) Homologous competition in the binding of biotinylated Cry11Ba toxin to BBMV was also analyzed in solution; bound toxins to the BBMV were recovered by centrifugation, subjected to SDS-PAGE, and transferred to PVDF membranes. Biotinylated toxins were visualized with streptavidin-HRP. Different ratios of labeled to unlabeled toxins were used. At a ratio of 1:50 or 1:75, binding of biotinylated Cry11Ba was competed.

FIG. 2.

Schematic analysis of Cry11Ba toxin proteolysis. The 80-kDa Cry11Ba protoxin was processed in vitro by trypsin. The Cry11Ba toxin is cleaved between ³⁴³Lys and ³⁵⁴Gly, generating a 36-kDa N-terminal fragment and a 33-kDa C-terminal fragment. Loop region sequences in the Cry11Ba toxin are underlined in insets. Loop α8 and loop 1 were identified in the N-terminal region of Cry11Ba, whereas loop 2 and loop 3 were in the C-terminal fragment.

FIG. 3.

Cadherin is involved in binding of Cry11Ba to Ae. aegypti midgut membranes. Homologous and heterologous competition assays of binding of biotinylated Cry11Ba to Ae. aegypti BBMV were performed in the presence of different competitors, including cadherin fragments, cadherin repeats, and a cadherin synthetic peptide. Cadherin fragments G7, G10, and C13 (lanes 1 to 3, respectively) and unlabeled Cry11Ba (lane 5) were used to compete the binding of biotinylated Cry11Ba to BBMV at a 250-fold molar excess. Lane 6 shows uncompeted toxin binding. Cadherin repeats CR7 to CR11, also at 250-fold-higher concentrations, were used as competitors in lanes 8 to 12, respectively. Finally, a synthetic peptide corresponding to CR11 sequence (lanes 16 to 18) was used at molar excesses of 1:50, 1:100, and 1:250, respectively (Cry11Ba biotinylated toxin to CR11 peptide). NHE8 (lanes 4, 13, and 15), an unrelated peptide, was used as a negative control in competition experiments.

FIG. 4.

APN and ALP compete with Cry11Ba binding to Ae. aegypti membranes. Heterologous competition assays of binding of biotinylated Cry11Ba to Ae. aegypti BBMV were performed in the presence of different competitors, including AaeAPN1 and AaeALP1 to -3. Purified AaeAPN1 and AaeALP1 to -3 at a 100- or 250-fold molar excess were incubated with biotinylated Cry11Ba overnight before binding to Ae. aegypti BBMV. NHE8 (lane 6 and 12), an unrelated peptide, was used as a negative control.

FIG. 5.

Loop mutants of Cry11Ba toxin retain their ability to bind the G10 cadherin fragment. Binding of wild-type Cry11Ba (▪) and the four loop mutants α8-V256A/G257A/E258A (○), L1-R303A/E304A/N305A (▵), L3-N454A/K455A/L456A (•), and L1-H307A (□) is shown. The G10 cadherin fragment EC₅₀ for binding to Cry11Ba is 25 nM, and those for the four mutants were 38, 41, 40, and 66 nM, respectively. The EC₅₀s were determined using Origin.

FIG. 6.

Competition binding of biotinylated Cry11Ba toxin to Ae. aegypti BBMV in the presence of unlabeled Cry11Ba or Cry4Ba toxin. Results of competitive assays of binding of biotinylated Cry11Ba toxin to BBMV in the presence of Cry11Ba (•) or Cry4Ba (▪) as a competitor were plotted by using Origin. The experiment was repeated once with similar results. The concentration of Cry4Ba required to compete 50% of the binding of biotinylated Cry11Ba to BBMV was 42 nM.

FIG. 7.

Identification of loop regions of Cry4Ba that compete the binding of biotinylated Cry11Ba toxin to Ae. aegypti BBMV. Competition binding of biotinylated Cry11Ba to BBMV with 100- and 250-fold molar excesses of unlabeled Cry11Ba is shown in lanes 2 and 3, respectively. Synthetic loop peptides corresponding to Cry4Ba toxin loop regions α8, β2-3, β8-9, β6-7, β4-5, and β10-11 were used in lanes 4 to 9, respectively. Four peptides, corresponding to α8, β2-β3, β8-β9, and β10-β11 (lanes 4 to 6 and 9, respectively) were able to compete the binding of biotinylated Cry11Ba to BBMV. Numbers on the gel refer to percent Cry11Ba binding to the 33- and 36-kDa bands in comparison to uncompeted Cry11Ba (lane 1). These values are the averages from two independent experiments.