The HevCaLP protein mediates binding specificity of the Cry1A class of Bacillus thuringiensis toxins in Heliothis virescens

Retrotransposon-mediated disruption of the BtR-4 gene encoding the Heliothis virescens cadherin-like protein (HevCaLP) is linked to high levels of resistance in the YHD2 strain to Cry1Ac toxin from Bacillus thuringiensis. This suggests that HevCaLP functions as a Cry1Ac toxin receptor on the surface of midgut cells in susceptible larvae and that the BtR-4 gene disruption eliminates this protein in resistant larvae. However, Cry1Ac toxin binding to HevCaLP is yet to be reported. We used the polymerase chain reaction and immunoblotting as tools to discriminate between individual H. virescens larval midguts from susceptible (YDK) and resistant (CXC, KCBhyb, and YHD2-B) strains according to their BtR-4 gene disruption genotype and phenotype. This approach allowed us to test the correlation between BtR-4 gene disruption, lack of HevCaLP, and altered Cry1A toxin binding. Toxin-binding assays using brush border membrane vesicles revealed that a wild-type BtR-4 allele is necessary for HevCaLP production and Cry1Aa toxin binding, while most of Cry1Ab and Cry1Ac binding was independent of the BtR-4 genotype. Moreover, toxin competition experiments show that KCBhyb midguts lacking HevCaLP are more similar to midguts of the original YHD2 strain than to the current YHD2-B strain. This resolves discrepancies in published studies of Cry1A binding in YHD2 and supports our earlier suggestion that a separate genetic change occurred in YHD2 after appearance of the cadherin disruption, conferring even higher resistance in the resulting YHD2-B strain as well as a large reduction in Cry1Ab and Cry1Ac binding.     

Functional expression in insect cells of glycosylphosphatidylinositol-linked alkaline phosphatase from Aedes aegypti larval midgut: a Bacillus thuringiensis Cry4Ba toxin receptor

Bacillus thuringiensis produces insecticidal crystal (Cry) proteins which bind to cell surface receptors on the brush border membrane of susceptible midgut larvae. The toxin-receptor interaction generates pores in midgut epithelial cells resulting in cell lysis. Here, a cDNA encoding membrane-bound alkaline phosphatase from Aedes aegypti (Aa-mALP) midgut larvae, based on the sequence identity hit to Bombyx mori membrane-bound ALP, was amplified by RT-PCR and transiently expressed in Spodoptera frugiperda (Sf9) insect cells as a 58-kDa membrane-bound protein via the baculovirus expression system and confirmed by digestion with phosphatidylinositol-specific phospholipase C and LC-MS/MS analysis. Immunolocalization results showed that Cry4Ba is able to bind to only Sf9 cells-expressing Aa-mALP. Moreover, these cells were shown to undergo cell lysis in the presence of 100 ??g/ml trypsin-treated toxin. Finally, trypan blue exclusion assay also demonstrated an increase in cell death in recombinant cells treated with Cry4Ba. Overall results indicated that Aa-mALP protein was responsible for mediating Cry4Ba toxicity against Sf9 cells, suggesting its role as a receptor for Cry4Ba toxin in A. aegypti mosquito larvae.     

High level of soluble expression in Escherichia coli and characterisation of the cloned Bacillus thuringiensis Cry4Ba domain III fragment

Similar to the other known structures of Bacillus thuringiensis Cry delta-endotoxins, the crystal structure of the 65-kDa activated Cry4Ba toxin comprises three domains which are, from the N- to C-terminus, a bundle of alpha-helices, a three-beta-sheet domain, and a beta-sandwich. To investigate the properties of the C-terminal domain III in isolation from the rest of the toxin, the cloned Cry4Ba-domain III was over-expressed as a 21-kDa soluble protein in Escherichia coli, which cross-reacted with anti-Cry4Ba domain III monoclonal antibody. A highly-purified domain III was obtained in a monomeric form by ion-exchange and size-exclusion FPLC. Circular dichroism spectroscopy indicated that the isolated domain III fragment distinctly exists as a beta-sheet structure, corresponding to the domain III structure embodied in the Cry4Ba crystal structure. In vitro binding analysis via immuno-histochemical assay revealed that the Cry4Ba-domain III protein was able to bind to the apical microvilli of the susceptible Stegomyia aegypti larval midguts, albeit at lower-binding activity when compared with the full-length active toxin. These results demonstrate for the first time that the C-terminal domain III of the Cry4Ba mosquito-larvicidal protein, which can be isolated as a native folded monomer, conceivably participates in toxin-receptor recognition.     

Identification, cloning and expression of a Cry1Ab cadherin receptor from European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae)

Transgenic corn expressing the Cry1Ab toxin from Bacillus thuringiensis is highly toxic to European corn borer, Ostrinia nubilalis, larvae. A putative Cry1Ab receptor (OnBt-R(1)) molecule was cloned and sequenced from a cDNA library prepared from midgut tissue of O. nubilalis larvae. The 5.6 Kb gene is homologous with a number of cadherin genes identified as Cry1 binding proteins in other lepidopterans. Brush border membrane vesicles were prepared using dissected midguts from late instars. A 220-kDa protein was identified as a cadherin-like molecule, which bound to Cry1Ab toxin and cross-reacted with an anti-cadherin serum developed from recombinant expression of a partial O. nubilalis cadherin peptide. Two additional proteins of smaller size cross-reacted with the anti-cadherin serum indicating that Cry1Ab binds to multiple receptors or to different forms of the same protein. Spodoptera frugiperda (SF9) cells transfected with the OnBt-R(1) gene were shown to express the receptor molecule which caused functional susceptibility to Cry1Ab at concentrations as low as 0.1 microg/ml. These results in combination suggest strongly that a cadherin-like protein acts as receptor and is involved with Cry1Ab toxicity in O. nubilalis.     

Role of proteolysis in determining potency of Bacillus thuringiensis Cry1Ac delta-endotoxin

Bacillus thuringiensis protein delta-endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the B. thuringiensis Cry1Ac delta-endotoxin towards Pieris brassicae (family: Pieridae) and Mamestra brassicae (family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against P. brassicae than against M. brassicae larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from P. brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of approximately 56 kDa, whereas proteases from M. brassicae, the non-susceptible insect, generated products with molecular masses of approximately 58, approximately 40, and approximately 20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M. brassicae proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M. brassicae proteases. However, this mutation had little effect on toxicity to M. brassicae. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.     

Potency of the mosquitocidal Cry46Ab toxin produced using a 4AaCter-tag,which facilitates formation of protein inclusion bodies in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A Cry46Ab toxin derived from Bacillus thuringiensis strain TK-E6 shows mosquitocidal activity against Culex pipiens pallens Coquillett (Diptera: Culicidae) larvae as well as preferential cytotoxicity against human cancer cells. In B. thuringiensis cells, Cry46Ab is produced and accumulates as a protein crystal that is processed into the active 29-kDa toxin upon solubilization in the alkaline environment of the insect midgut. The Cry46Ab protoxin is 30 kDa, and is therefore thought to require an accessory protein such as P20 and/or ORF2 for efficient crystal formation. In the present study, the potency of the 4AaCter-tag was investigated for the production of alkali-soluble inclusion bodies of recombinant Cry46Ab in Escherichia coli. The 4AaCter-tag is a polypeptide derived from the C-terminal region of the B. thuringiensis Cry4Aa toxin and facilitates the formation of alkali-soluble protein inclusion bodies in E. coli. Fusion with the 4AaCter-tag enhanced both Cry46Ab production and the formation of Cry46Ab inclusion bodies. In addition, upon optimization of protein expression procedures, the Cry46Ab–4AaCter inclusion bodies showed mosquitocidal activity and stability in aqueous environments comparable to Cry46Ab without the 4AaCter-tag. Our study suggests that use of the 4AaCter-tag is a straightforward approach for preparing formulations of smaller-sized Cry toxins such as Cry46Ab in E. coli.     

Ion channels formed in planar lipid bilayers by the dipteran-specific Cry4B Bacillus thuringiensis toxin and its alpha1-alpha5 fragment

Trypsin activation of Cry4B, a 130-kDa Bacillus thuringiensis (Bt) protein, produces a 65-kDa toxin active against mosquito larvae. The active toxin is made of two protease resistant-products of ca. 45 kDa and ca. 20 kDa. The cloned 21-kDa fragment consisting of the N-terminal region of the toxin was previously shown to be capable of permeabilizing liposomes. The present study was designed to test the following hypotheses: (1) Cry4B, like several other Bt toxins, is a channel-forming toxin in plannar lipid bilayers; and (2) the 21-kDa N-terminal region, which maps for the first five helices (alpha1-alpha5) of domain 1 in other Cry toxins, and which putatively shares a similar tri-dimensional structure, is sufficient to account for the ion channel activity of the whole toxin. Using circular dichroism spectroscopy and planar lipid bilayers, we showed that the 21-kDa polypeptide existed as an alpha-helical structure and that both Cry4B and its alpha1-alpha5 fragment formed ion channels of 248 +/- 44 pS and 207 +/- 23 pS, respectively. The channels were cation-selective with a potassium-to-chloride permeability ratio of 6.7 for Cry4B and 4.5 for its fragment. However, contrary to the full-length toxin, the alpha1-alpha5 region formed channels at low dose; they tended to remain locked in their open state and displayed flickering activity bouts. Thus, like the full-length toxin, the alpha1-alpha5 region is a functional channel former. A pH-dependent, yet undefined region of the toxin may be involved in regulating the channel properties.     

Photorhabdus luminescens toxin-induced permeability change in Manduca sexta and Tenebrio molitor midgut brush border membrane and in unilamellar phospholipid vesicle

Photorhabdus luminescens, a Gram-negative bacterium, secretes a protein toxin (PL toxin) that is toxic to insects. In this study, the effects of the PL toxin on large receptor-free unilamellar phospholipid vesicles (LUVs) of Manduca sexta and on brush border membrane vesicles (BBMVs) of M. sexta and Tenebrio molitor were examined. Cry1Ac served as a positive control in our experiments due to its known channel-forming activity on M. sexta. Voltage clamping assays with dissected midguts of M. sexta and T. molitor clearly showed that both Cry1Ac and PL toxin caused channel formation in the midguts, although channel formation was not detected for T. molitor midguts under Cry1Ac and it was less sensitive to PL toxin than to Cry1Ac for M. sexta midguts. Calcein release experiments showed that both toxins made LUVs (unilamellar lipid vesicles) permeable, and at some concentrations of the toxins such permeabilizing effects were pH-dependent. The lowest concentrations of PL toxin were more than 600-fold and 24-fold lower to induce BBMV permeability of T. molitor and M. sexta than those to induce calcein release from LUVs of M. sexta. These further support that PL toxin is responsible for channel formation in the larvae midguts. The lower concentration to induce permeability in BBMV than in LUV is, probably, attributable to that BBMV has PL toxin receptors that facilitate the toxin to induce permeabilization. Furthermore, our results indicate that the effects of PL toxin on BBMV permeability of M. sexta were not significantly influenced by Gal Nac, but those of Cry1Ac were. This implies that PL toxin and Cry1Ac might use different molecular binding sites in BBMV to cause channel formation.     

Cytopathological effect of Bacillus thuringiensis israelensis endotoxins on the intestines of Aedes aegypti mosquito larvae

The dynamics of pathological changes in the intestine of Aedes aegypti larvae under the influence of toxins Cry11A and Cry4B produced by Bacillus thuringiensis israelensis was studied by means of electron microscope. Most significant ultrastructure changes in the intestine of the second instar larvae were observed in the midgut. The cytoplasm of cells disintegrated, and elongated lacunae appeared. The number of microvilli decreased, or they disappeared in the result of destruction. The peritrophic membrane displaced to the lumen of midgut. Any changes in epithelial cells and cuticle in time of foregut and hindgut were not observed in a comparison to control. The toxin Cry4B caused the most effective destruction of the midgut epithelium.     

In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae)

Bacillus thuringiensis subsp. medellin produces numerous proteins among which 94 kDa known as Cry11Bb, has mosquitocidal activity. The mode of action of the Cry11 proteins has been described as similar to those of the Cry1 toxins, nevertheless, the mechanism of action is still not clear. In this study we investigated the in vivo binding of the Cry11Bb toxin to the midgut of the insect species Anopheles albimanus, Aedes aegypti, and Culex quinquefasciatus by immunohistochemical analysis. Spodoptera frugiperda was included as negative control. The Cry11Bb protein was detected on the apical microvilli of the midgut epithelial cells, mostly on the posterior midgut and gastric caeca of the three mosquito species. Additionally, the toxin was detected in the Malpighian tubules of An. albimanus, Ae. aegypti, Cx. quinquefasciatus, and in the basal membrane of the epithelial cells of Ae. aegypti midgut. No toxin accumulation was observed in the peritrophic membrane of any of the mosquito species studied. These results confirm that the primary site of action of the Cry11 toxins is the apical membrane of the midgut epithelial cells of mosquito larvae.     

Production of Cry11A and Cry11Ba toxins in Bacillus sphaericus confers toxicity towards Aedes aegypti and resistant Culex populations.

Cry11A from Bacillus thuringiensis subsp. israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on the B. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegypti and Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegypti larvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE and Culex quinquefasciatus GeoR to B. sphaericus strain 2297.     

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.     

Interaction of gene-cloned and insect cell-expressed aminopeptidase N of Spodoptera litura with insecticidal crystal protein Cry1C

Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.     

Membrane proteins of Aedes aegypti larvae bind toxins Cry4B and Cry11A of Bacillus thuringiensis ssp. israelensis

Proteins of molecular weight 65 and 62 kD and having affinity for toxins Cry4B and Cry11A produced by Bacillus thuringiensis ssp. israelensis have been isolated from brush border membranes of Aedes aegypti larvae using affinity chromatography. Using a ligand blotting technique, we show that the binding of these proteins to the biotinylated toxins is reversible and that the two toxins compete for binding to the two proteins. These proteins are likely to be Cry4B and Cry11A toxin receptors in gut epithelial cells of Aedes aegypti larvae.     

Binding Properties of Bacillus thuringiensis Cry4A Toxin to the Apical Microvilli of Larval Midgut of Culex pipiens

Cry4A is a dipteran-specific δ-endotoxin produced by Bacillus thuringiensis, and toxic to Culex pipiens (mosquito) larvae. The immunohistochemical staining of the midgut sections of C. pipiens larvae revealed that Cry4A bound in vitro and in vivo to the microvilli of the epithelial cells of posterior midgut and gastric caecae. The binding of digoxigenin-labeled Cry4A (DIG-Cry4A) to the apical microvilli was almost abolished in the presence of excess unlabeled Cry4A, suggesting that the binding of Cry4A to the microvilli was specific. Several Cry4A-specific binding proteins were detected using the ligand blotting technique with DIG-Cry4A. Moreover, an insertion assay was done, where the binding of DIG-Cry4A to the BBMVs was completely irreversible and did not compete with excess unlabeled Cry4A. On the basis of these results, we propose a schematic interpretation for the binding process of Cry4A.     

Identification and characterization of Aedes aegypti aminopeptidase N as a putative receptor of Bacillus thuringiensis Cry11A toxin

Bacillus thuringiensis subsp. israelensis, which is used worldwide to control Aedes aegypti larvae, produces Cry11Aa and other toxins during sporulation. In this study, pull-down assays were performed using biotinylated Cry11Aa toxin and solubilized brush border membrane vesicles prepared from midguts of Aedes larvae. Three of the eluted proteins were identified as aminopeptidease N (APN), one of which was a 140 kDa protein, named AaeAPN1 (AAEL012778 in VectorBase). This protein localizes to the apical side of posterior midgut epithelial cells of larva. The full-length AaeAPN1 was cloned and expressed in Eschericia coli and in Sf21 cells. AaeAPN1 protein expressed in Sf21 cells was enzymatically active, had a GPI-anchor but did not bind Cry11Aa. A truncated AaeAPN1, however, binds Cry11Aa with high affinity, and also Cry11Ba but with lower affinity. BBMV but not Sf21 expressed AaeAPN1 can be detected by wheat germ agglutinin suggesting the native but Sf21 cell-expressed APN1 contains N-acetylglucosamine moieties.     

Binding properties of Bacillus thuringiensis Cry4A toxin to the apical microvilli of larval midgut of Culex pipiens.

Cry4A is a dipteran-specific delta-endotoxin produced by Bacillus thuringiensis, and toxic to Culex pipiens (mosquito) larvae. The immunohistochemical staining of the midgut sections of C. pipiens larvae revealed that Cry4A bound in vitro and in vivo to the microvilli of the epithelial cells of posterior midgut and gastric caecae. The binding of digoxigenin-labeled Cry4A (DIG-Cry4A) to the apical microvilli was almost abolished in the presence of excess unlabeled Cry4A, suggesting that the binding of Cry4A to the microvilli was specific. Several Cry4A-specific binding proteins were detected using the ligand blotting technique with DIG-Cry4A. Moreover, an insertion assay was done, where the binding of DIG-Cry4A to the BBMVs was completely irreversible and did not compete with excess unlabeled Cry4A. On the basis of these results, we propose a schematic interpretation for the binding process of Cry4A.     

Role of Proteolysis in Determining Potency of Bacillus thuringiensis Cry1Ac δ-Endotoxin

Bacillus thuringiensis protein δ-endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the B. thuringiensis Cry1Ac δ-endotoxin towards Pieris brassicae (family: Pieridae) and Mamestra brassicae (family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against P. brassicae than against M. brassicae larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from P. brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of ~56 kDa, whereas proteases from M. brassicae, the non-susceptible insect, generated products with molecular masses of ~58, ~40, and ~20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M. brassicae proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M. brassicae proteases. However, this mutation had little effect on toxicity to M. brassicae. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.     

Binding analyses of Cry1Ab and Cry1Ac with membrane vesicles from Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis

The binding properties of Bacillus thuringiensis toxins to brush border membrane vesicles of Dipel-resistant and -susceptible Ostrinia nubilalis larvae were compared using ligand-toxin immunoblot analysis, surface plasmon resonance (SPR), and radiolabeled toxin binding assays. In ligand-toxin immunoblot analysis, the number of Cry1Ab or Cry1Ac toxin binding proteins and the relative toxin binding intensity were similar in vesicles from resistant and susceptible larvae. Surface plasmon resonance with immobilized activated Cry1Ab toxin indicated that there were no significant differences in binding with fluid-phase vesicles from resistant and susceptible larvae. Homologous competition assays with radiolabeled Cry1Ab and Cry1Ac toxin and vesicles from resistant and susceptible larvae resulted in similar toxin dissociation constants and binding site concentrations. Heterologous competition binding assays indicated that Cry1Ab and Cry1Ac completely competed for binding, thus they share binding sites in the epithelium of the larval midguts of O. nubilalis. Overall, the binding analyses indicate that resistance to Cry1Ab and Cry1Ac in this Bt-resistant strain of O. nubilalis is not associated with a loss of toxin binding.     

A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11Ba toxin in the mosquito Anopheles gambiae

Bacillus thuringiensis (Bt) insecticidal toxins bind to receptors on midgut epithelial cells of susceptible insects, and binding triggers biochemical events that lead to insect mortality. Recently, a 100-kDa aminopeptidase N (APN) was isolated from brush border membrane vesicles (BBMV) of Anopheles quadrimaculatus and shown to bind Cry11Ba toxin with surface plasmon resonance (SPR) detection [Abdullah et al. (2006) BMC Biochem. 7, 16]. In our study, a 106-kDa APN, called AgAPN2, released by phosphatidylinositol-specific phospholipase C (PI-PLC) from Anopheles gambiae BBMV was extracted by Cry11Ba bound to beads. The AgAPN2 cDNA was cloned, and analysis of the predicted AgAPN2 protein revealed a zinc-binding motif (HEIAH), three potential N-glycosylation sites, and a predicted glycosylphosphatidylinositol (GPI) anchor site. Immunohistochemistry localized AgAPN2 to the microvilli of the posterior midgut. A 70-kDa fragment of the 106-kDa APN was expressed in Escherichia coli. When purified, it competitively displaced 125I-Cry11Ba binding to An. gambiae BBMV and bound Cry11Ba on dot blot and microtiter plate binding assays with a calculated K d of 6.4 nM. Notably, this truncated peptide inhibited Cry11Ba toxicity to An. gambiae larvae. These results are evidence that the 106-kDa GPI-anchored APN is a specific binding protein, and a putative midgut receptor, for Bt Cry11Ba toxin.