Determining the involvement of two aminopeptidase Ns in the resistance of Plutella xylostella to the Bt toxin Cry1Ac: cloning and study of in vitro function

The cloning, expression in vitro, and characterization of two aminopeptidase Ns (APN5s and APN2s) isolated from the midgut of Cry1Ac-resistant (R) and susceptible (S) strains of Plutella xylostella larvae are presented in this paper. The deduced amino acid sequences of APN5s included C-terminal GPI-modification sites, the gluzincin aminopeptidase motif GATEN, and three N-glycosylated sites; those of APN2s had no GPI-modification sites, had gluzincin aminopeptidase motif GAMEN, and had four N-glycosylated sites. O-glycosylated sites were not predicted for either APN. Because APN2R and APN2S cDNAs contained the same nucleotides, only full-length cDNAs encoding APN5R and APN5S were expressed in Trichoplusia ni cells. Far-Western blotting showed that the expressed receptor APN5 bound to the Cry1Ac toxin. An enzyme-specific activity experiment also showed that APN5 genes were expressed in T. ni cells. ELISA revealed no differences in the binding of expression proteins from the resistant and susceptible strain with Cry1Ac.     

Carbohydrate analyses of Manduca sexta aminopeptidase N, co-purifying neutral lipids and their functional interactions with Bacillus thuringiensis Cry1Ac toxin.

Bacillus thuringiensis Cry1Ac insecticidal toxin binds specifically to 120kDa aminopeptidase N (APN) (EC 3.4.11.2) in the epithelial brush border membrane of Manduca sexta midguts. The isolated 120-kDa APN is a member of a functional Cry1 toxin receptor complex (FEBS Lett. 412 (1997) 270). The 120-kDa form is glycosyl-phosphatidylinositol (GPI) anchored and converted to a 115-kDa form upon membrane solubilization. The 115-kDa APN also binds Cry1A toxins and Cry1Ac binding is inhibited by N-acetylgalactosamine (GalNAc). Here we determined the monosaccharide composition of APN. APN is 4.2mol% carbohydrate and contains GalNAc, a residue involved in Cry1Ac interaction. APN remained associated with non-covalently bound lipids through anion-exchange column purification. Most associated lipids were separated from APN by hydrophobic interaction chromatography yielding a lipid aggregate. Chemical analyses of the lipid aggregate separated from APN revealed neutral lipids consisting mostly of diacylglycerol and free fatty acids. The fatty acids were long, unsaturated chains ranging from C:14 to C:22. To test the effect of APN-associated lipids on Cry1Ac function, the lipid aggregate and 115-kDa APN were reconstituted into phosphatidylcholine (PC) vesicles. The lipid aggregate increased the amount of Cry1Ac binding, but binding due to the lipid aggregate was not saturable. In contrast the lipid aggregate promoted Cry1Ac-induced release of 86Rb(+) at the lowest Cry1Ac concentration (50nM) tested. The predominant neutral lipid component extracted from the lipid aggregate promoted Cry1Ac-induced 86Rb(+) release from membrane vesicles in the presence of APN.     

Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N

Three types of binding assays were used to study the binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and the related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques. In ligand blotting experiments using SDS-PAGE-separated BBMV proteins as well as the purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for specific binding to APN. Using the surface plasmon resonance (SPR) technique with immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identified Cry1Ac binding sites, whereas the second site requires the full Cry1Ac toxin for binding. In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N-acetylgalactosamine (GalNac) was determined. Both in ligand blotting and in surface plasmon resonance experiments, as well as in binding assays using intact BBMVs, it was shown that the presence of domain III of Cry1Ac in a toxin molecule is sufficient for the inhibition of binding by GalNAc. These and other results strongly suggest that domain III of delta-endotoxins play a role in insect specificity through their involvement in specific binding to insect gut epithelial receptors.     

Structural changes of the Cry1Ac oligomeric pre-pore from bacillus thuringiensis induced by N-acetylgalactosamine facilitates toxin membrane insertion

The primary action of Cry toxins produced by Bacillus thuringiensis is to lyse midgut epithelial cells in their target insect by forming lytic pores. The toxin-receptor interaction is a complex process, involving multiple interactions with different receptor and carbohydrate molecules. It has been proposed that Cry1A toxins sequentially interact with a cadherin receptor, leading to the formation of a pre-pore oligomer structure, and that the oligomeric structure binds to glycosylphosphatidyl-inositol-anchored aminopeptidase-N (APN) receptor. The Cry1Ac toxin specifically recognizes the N-acetylgalactosamine (GalNAc) carbohydrate present in the APN receptor from Manduca sexta larvae. In this work, we show that the Cry1Ac pre-pore oligomer has a higher binding affinity with APN than the monomeric toxin. The effects of GalNAc binding on the toxin structure were studied in the monomeric Cry1Ac, in the soluble pre-pore oligomeric structure, and in its membrane inserted state by recording the fluorescence status of the tryptophan (W) residues. Our results indicate that the W residues of Cry1Ac have a different exposure to the solvent when compared with that of the closely related Cry1Ab toxin. GalNAc binding specifically affects the exposure of W545 in the pre-pore oligomer in contrast to the monomer where GalNAc binding did not affect the fluorescence of the toxin. These results indicate a subtle conformational change in the GalNAc binding pocket in the pre-pore oligomer that could explain the increased binding affinity of the Cry1Ac pre-pore to APN. Although our analysis did not reveal major structural changes in the pore-forming domain I upon GalNAc binding, it showed that sugar interaction enhanced membrane insertion of soluble pre-pore oligomeric structure. Therefore, the data presented here permits to propose a model in which the interaction of Cry1Ac pre-pore oligomer with APN receptor facilitates membrane insertion and pore formation.     

Cloning of a Heliothis virescens 110 kDa aminopeptidase N and expression in Drosophila S2 cells

We previously identified a novel Heliothis virescens 110 kDa aminopeptidase N (APN) that binds Bacillus thuringiensis (Bt) Cry1Ac and Cry1Fa delta-endotoxins, and cloned an internal region of the 110 kDa APN gene (Banks et al., 2001). Here we describe the RACE-PCR cloning and sequence of a cDNA encoding 110 kDa APN. The 110 kDa APN gene was transiently co-expressed with green fluorescent protein (GFP) in Drosophila S2 cells using the pIZT expression vector. Enrichment of total membranes purified from S2 cells transfected with the 110 kDa APN gene had 3.3 fold increased APN enzymatic activity relative to enriched total membranes purified from S2 cells transfected with vector alone. Whereas the majority of S2 cells transfected with the 110 kDa APN gene bound rhodamine-labeled Cry1Ac toxin, no S2 cells transfected with vector alone bound rhodamine-labeled Cry1Ac toxin. This indicates that toxin binding to whole cells is APN mediated. However, flow cytometry and microscopy indicated that 110 kDa APN transfected S2 cells exposed to Cry1Ac or Cry1Fa toxin did not experience an increase in membrane permeability, indicating that APN transfected cells were resistant to toxin. This suggests while the H. virescens 110 kDa APN functions as a Bt toxin binding protein, it does not mediate cytotoxicity when expressed in S2 cells.     

Disruption of Ha_BtR alters binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to midgut BBMVs of Helicoverpa armigera

Disruption of the Ha_BtR (a cadherin gene) is genetically linked to resistance to Cry1Ac delta-endotoxin of Bacillus thuringiensis in the GYBT strain of Helicoverpa armigera. Brush border membrane vesicles (BBMVs) prepared from midguts of both the Cry1Ac-resistant GYBT strain (homozygous for a deletion knockout of Ha_BtR) and the susceptible GY strain (homozygous for the wild type of Ha_BtR) possessed saturable and specific binding ability to (125)I-Cry1Ac. The binding constant (K(d)) of the GY strain was significantly lower than that of the resistant GYBT strain, whereas their binding site concentrations (B(max)) were similar. When midgut BBMVs were reacted directly with streptavidin conjugated to horseradish peroxidase, the GY strain had very clear 120- and 85-kDa protein bands, which indicated that the 120- and 85-kDa bands are endogenous biotin-containing proteins. However, the GYBT strain almost completely lost these two biotin-containing proteins. Ligand blotting with biotinylated Cry1Ac toxin showed midgut BBMVs of the GY strain contain five protein bands of 210-, 190-, 150-, 120-, and 85-kDa, respectively, while BBMVs of the GYBT strain contain only two protein bands of 150- and 120-kDa. 120-kDa bands may consist of two proteins with coincidentally the same molecular weight (putatively, an APN and a biotin-containing protein). Our results showed that the binding pattern of Cry1Ac to midgut BBMVs of H. armigera was altered quantitatively and qualitatively by knockout of Ha_BtR. There are multiple Cry1Ac-binding proteins in the midgut of susceptible H. armigera, but only the Ha_BtR can be considered as a putative functional receptor of Cry1Ac. Possible involvement of other receptor proteins in the intoxication process in vivo could not be excluded.     

Bivalent sequential binding model of a Bacillus thuringiensis toxin to gypsy moth aminopeptidase N receptor

Specificity for target insects of Bacillus thuringiensis insecticidal Cry toxins is largely determined by toxin affinity for insect midgut receptors. The mode of binding for one such toxin-receptor complex was investigated by extensive toxin mutagenesis, followed by real-time receptor binding analysis using an optical biosensor (BIAcore). Wild-type Cry1Ac, a three-domain, lepidopteran-specific toxin, bound purified gypsy moth (Lymantria dispar) aminopeptidase N (APN) biphasically. Site 1 displayed fast association and dissociation kinetics, while site 2 possessed slower kinetics, yet tighter affinity. We empirically determined that two Cry1Ac surface regions are involved in in vivo toxicity and APN binding. Mutations within domain III affected binding rates to APN site 1, whereas mutations in domain II affected binding rates to APN site 2. Furthermore, domain III contact is completely inhibited in the presence of N-acetylgalactosamine, indicating loss of domain III binding eliminates all APN binding. Based upon these observations, the following model is proposed. A cavity in lectin-like domain III initiates docking through recognition of an N-acetylgalactosamine moiety on L. dispar APN. Following primary docking, a higher affinity domain II binding mechanism occurs, which is critical for insecticidal activity.     

Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta

Bacillus thuringiensis Cry2Ab toxin has been used in combination with Cry1Ac for resistance management on the Bt-cotton that is widely planted worldwide. However, little is known regarding Cry2Ab mode of action. Particularly, there is a gap of knowledge on the identification of insect midgut proteins that bind Cry2Ab and mediate toxicity. In the case of Cry1Ab toxin, a transmembrane cadherin protein and glycosyl-phosphatidylinositol (GPI) anchored proteins like aminopeptidase-N1 (APN1) or alkaline-phosphatase (ALP) from Manduca sexta, have been shown to be important for oligomer formation and insertion into the membrane. Binding competition experiments showed that Cry2Ab toxin does not share binding sites with Cry1Ab toxin in M. sexta brush border membrane vesicles (BBMV). Also, that Cry2Ab shows reduced binding to the Cry1Ab binding molecules cadherin, APN1 or ALP. Finally, ligand blot experiments and protein sequence by LC–MS/MS identified APN2 isoform as a Cry2Ab binding protein. Cloning and expression of APN2 confirmed that APN2 is a Cry2Ab binding protein.     

Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen

Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae.     

Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxin binding to a novel 110 kDa aminopeptidase in Heliothis virescens is not N-acetylgalactosamine mediated.

We determined that Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxins recognize the same 110, 120 and 170 kDa aminopeptidase N (APN) molecules in brush border membrane vesicles (BBMV) from Heliothis virescens. The 110 kDa protein, not previously identified as an APN, contained a variant APN consensus sequence identical to that found in Helicoverpa punctigera APN 2. PCR amplification of H. virescens cDNA based on this sequence and a conserved APN motif yielded a 0.9 kb product that has 89% sequence homology with H. punctigera APN 2. Western blots revealed that the 110 kDa molecule was not recognized by soybean agglutinin, indicating the absence of GalNAc. A 125I labeled-Cry1Ac domain III mutant (509QNR(511)-AAA) that has an altered GalNAc binding pocket (Lee et al., Appl. Environ. Microbiol. 65 (1999) 4513) showed abolished binding to the 120 APN, reduced binding to the 170 kDa APN, and enhanced binding to the 110 kDa APN. Periodate treated H. virescens BBMV blots were also probed with 125I labeled-Cry1Ac and 509QNR(511)-AAA toxins. Both toxins still recognized the 110 kDa APN and a >210 kDa molecule which may be a cadherin-like protein. Additionally, 125I-(509)QNR(511)-AAA recognized periodate treated 170 kDa APN. Results indicate that the 110 kDa APN is distinct from other Cry1 toxin binding APNs and may be the first described Cry1Ac-binding APN that does not contain GalNAc.     

Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N

The Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N (APN) was analyzed, to better understand the molecular mechanism of susceptibility to the toxin and the development of resistance in insects. APN was digested with lysylendopeptidase and the ability of the resulting fragments to bind to Cry1Aa and 1Ac toxins was examined. The binding abilities of the two toxins to these fragments were different. The Cry1Aa toxin bound to the fragment containing 40-Asp to 313-Lys, suggesting that the Cry1Aa toxin-binding site is located in the region between 40-Asp and 313-Lys, while Cry1Ac toxin bound exclusively to mature APN. Next, recombinant APN of various lengths was expressed in Escherichia coli cells and its ability to bind to Cry1Aa toxin was examined. The results localized the Cry1Aa toxin binding to the region between 135-Ile and 198-Pro.     

Expression of recombinant and mosaic Cry1Ac receptors from Helicoverpa armigera and their influences on the cytotoxicity of activated Cry1Ac to Spodoptera litura Sl-HP cells

Bacillus thuringiensis (Bt) toxin receptors play important roles in the killing of pests, and investigation on characterization of the receptors is essential for utilization of Bt and management of insect resistance. Here, recombinant and mosaic receptors of Bt Cry1Ac toxin from Helicoverpa armigera were expressed in Spodoptera litura Sl-HP cells and their influences on cytotoxicity of activated Cry1Ac toxin were investigated. When H. armigera aminopeptidase N1 (APN1), alkaline phosphatase 2 (ALP2) and cadherin fused with or without GFP tag were, respectively, expressed in Sl-HP cells, live cell-immunofluorescence staining detection revealed that the quantity of the toxin binding to cadherin or cadherin-GFP was much more than that binding to ALP2 and APN1 or their fusion proteins with GFP, and only the cadherin- or cadherin-GFP-expressing cells showed aberrant cell morphology after the treatment of the toxin at low concentrations. ALP2 and APN1 fused with or without GFP tag did not significantly enhance the cadherin-mediated cytotoxicity of the toxin. The mosaic ALP-TBR-GFP-GPI was located on cell membrane, but did not bind to the toxin. The mosaic truncated cadherin-GFP-GPI was not located on cell membrane even if the signal peptide was sustained. The concentrations of the toxin resulting in swelling of 50 % cells for noncadherin-expressing Sl-HP cells and cadherin-expressing Hi5 cells were 5.08 and 9.50 µg/ml within 1 h, respectively. Taken together, our data have indicated that the binding affinity of ALP2 and APN1 to activated Cry1Ac toxin is much weaker than that of cadherin and both ALP2 and APN1 do not enhance the cytotoxicity of the toxin even though cadherin is co-expressed, and the mosaic receptor of ALP2 inserted with cadherin toxin binding domain does not mediate cytotoxicity of the toxin. In addition, the noncadherin-expressing Sl-HP cells are more susceptible to activated Cry1Ac than the cadherin-expressing Hi5 cells.     

Polydispersity of Bacillus thuringiensis Cry1 toxins in solution and its effect on receptor binding kinetics

Dynamic light scattering and surface plasmon resonance techniques were used to investigate the influence of ionic strength, buffer composition and pH on the multimerization of trypsin-activated Cry1Ac and Cry1C toxins over time and the subsequent effects of the different multimers on receptor binding models. In carbonate buffer at pH 10.5, Cry1Ac and Cry1C assumed a monomeric state. After 24 h, a complete conversion of monomeric toxin to a dimeric or trimeric form was observed only for Cry1Ac under low ionic strength condition. Cry1C and Cry1Ac in high ionic strength buffer remained monomeric. Substitution of CAPS pH 11 for carbonate buffer suppressed this Cry1Ac oligomerization effect. Once Cry1Ac toxin was in an aggregated form, increases in ionic strength failed to revert the aggregated toxin back to a monomeric form. Monomeric Cry1Ac bound to a purified 115 kDa aminopeptidase N receptor from Manduca sexta in a 2:1 molar ratio thus confirming the existence of two binding sites on this receptor. Binding rates of dimeric or higher aggregated Cry1Ac toxin forms were different from those generated using the monomeric form and could not be fitted to existing binding models. In summary, our results confirm that the M. sexta 115 kDa aminopeptidase N receptor possesses two Cry1Ac binding sites. They further suggest that although high pH and low salt conditions promote Cry1Ac aggregation, this observation cannot be applied universally to other members of the Cry family.     

Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

Phage display is an in vitro method for selecting polypeptides with desired properties from a large collection of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display single-chain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.     

A novel 96-kDa aminopeptidase localized on epithelial cell membranes of Bombyx mori midgut, which binds to Cry1Ac toxin of Bacillus thuringiensis

Proteins in the brush border membrane (BBM) of the midgut binding to the insecticidal Cry1Ac toxin from Bacillus thuringiensis were investigated to examine the lower sensitivity of Bombyx mori to Cry1Ac, and new aminopeptidase N that bound to Cry1Ac was discovered. DEAE chromatography of Triton X-100-soluble BBM proteins from the midgut revealed 96-kDa aminopeptidase that bound to Cry1Ac. The enzyme was purified to homogeneity and estimated to be a 96.4-kDa molecule on a silver-stained SDS-PAGE gel. However, the native protein was eluted as a single peak corresponding to approximately 190-kDa on gel filtration and gave a single band on native PAGE. The enzyme was determined to be an aminopeptidase N (APN96) from its substrate specificity. Antiserum to class 3 B. mori APN (BmAPN3) recognized APN96, but peptide mass fingerprinting revealed that 54% of the amino acids of matched peptides were identical to those of BmAPN3, suggesting that APN96 was a novel isoform of the APN3 family. On ligand blots, APN96 bound to Cry1Ac but not Cry1Aa or Cry1Ab, and the interaction was inhibited by GalNAc. K(D) of the APN96-Cry1Ac interaction was determined to be 1.83 +/- 0.95 microM. The lectin binding assay suggested that APN96 had an N-linked bi-antennal oligosaccharide or an O-linked mucin type one. The role of APN96 was discussed in relation to the insensitivity of B. mori to Cry1Ac.     

Cloning and complete sequence characterization of two gypsy moth aminopeptidase-N cDNAs, including the receptor for Bacillus thuringiensis Cry1Ac toxin.

The complete cDNAs corresponding to two distinct gypsy moth (Lymantria dispar) larval gut aminopeptidases, APN1 and lambda APN2, were cloned and sequenced. The 3.4 kilobasepair cDNA of APN1 which encodes a 1017 amino acid prepro-protein corresponds to the previously-identified gypsy moth APN (APN-1) that specifically binds the Cry1Ac delta-endotoxin of Bacillus thuringiensis. Analysis of the primary structure of APN1 revealed a cluster of five potential N-linked glycosylation sites near the N-terminus and a C-terminal sequence characteristic of a putative glycosylphosphatidyl-inositol (GPI) anchor signal sequence. The cDNA of APN1 encodes the N-terminal peptide sequence and nine internal sequences obtained from the purified brush border membrane vesicle Cry1Ac receptor by protein sequencing. The lambda APN2 cDNA encodes a shorter protein with 51% similarity to APN1 that also appears to have a GPI anchor signal sequence. Expression of the APN1 cDNA in a baculovirus vector was confirmed by immunoblotting.     

Binding of Phylogenetically Distant Bacillus thuringiensis Cry Toxins to a Bombyx mori Aminopeptidase N Suggests Importance of Cry Toxin's Conserved Structure in Receptor Binding

We investigated the binding proteins for three Cry toxins, Cry1Aa, Cry1Ac, and the phylogenetically distant Cry9Da, in the midgut cell membrane of the silkworm. In a ligand blot experiment, Cry1Ac and Cry9Da bound to the same 120-kDa aminopeptidase N (APN) as Cry1Aa. A competition experiment with the ligand blot indicated that the three toxins share the same binding site on several proteins. The values of the dissociation constants of the three Cry toxins and 120-kDa APN are as low as the case of other Cry toxins and receptors. These results suggest that distantly related Cry toxins bind to the same site on the same proteins, especially with APN. We propose that the conserved structure in these three toxins includes the receptor-binding site. Received: 12 January 1998 / Accepted: 17 February 1999     

Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers

The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins.     

Antisera-mediated in vivo reduction of Cry1Ac toxicity in Helicoverpa armigera

A functional assessment of Bacillus thuringiensis (Bt) toxin receptors in the midgut of lepidopteran insects will facilitate understanding of the toxin mode of action and provide effective strategies to counter the development of resistance. In this study, we produced anti-aminopeptidase (APN) and anti-cadherin sera with purified Cry1Ac toxin-binding APN or cadherin fragments from Heliocoverpa armigera. Antisera were evaluated for their effects on Cry1Ac toxicity through bioassays. Our results indicated that both the anti-APN and anti-cadherin sera reduced Cry1Ac toxicity in vivo, although cadherin antiserum reduced toxicity more than APN antiserum. These results suggest that both APN and cadherin are involved in Cry1Ac intoxication of H. armigera, evidence that the pore formation model may be representative of Cry1Ac toxin mode of action in this insect.     

N-acetylgalactosamine on the putative insect receptor aminopeptidase N is recognised by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin

Binding of the insecticidal Bacillus thuringiensis Cry1Ac toxin to the putative receptor aminopeptidase N is specifically inhibited by N-acetylgalactosamine (GalNAc), suggesting that this toxin recognises GalNAc on the receptor. A possible structural basis for involvement of domain III of the toxin in carbohydrate-mediated receptor recognition was noted in the similarity between the domain III fold of the related toxin Cry3A and a carbohydrate-binding domain in the 1,4-beta-glucanase from Cellulomonas fimi. This possibility was investigated by making selected mutations in domain III of the Cry1Ac delta-endotoxin. Mutagenesis of residues Asn506, Gln509 or Tyr513 resulted in toxins with reduced binding and a slower rate of pore formation in Manduca sexta midgut membrane vesicles compared to the wild-type Cry1Ac. These mutants also showed reduced binding to the 120 kDa Cry1Ac putative receptor aminopeptidase N. Unlike the wild-type toxin, binding of the triple mutant N506D,Q509E,Y513A (Tmut) to M. sexta midgut membrane vesicles could not be inhibited by GalNAc. These data indicate that GalNAc binding is located on domain III of Cry1Ac and therefore support a lectin-like role for this domain. A preliminary analysis of the Cry1Ac crystal structure locates Asn506, Gln509 and Tyr513 in a region on and adjacent to beta-16 in domain III, which has a unique conformation compared to the other known Cry structures. These residues are in a favourable position to interact with either soluble or protein-bound carbohydrate.