Binding of the CryIVD Toxin of Bacillus thuringiensis subsp. israelensis to Larval Dipteran Midgut Proteins

Ligand-blotting experiments on dipteran brush border membrane vesicles (BBMVs) showed binding of CryIVD toxin of Bacillus thuringiensis subsp. israelensis to proteins of 148 kDa in Anopheles stephensi and of 78 kDa in Tipula oleracea, both species being susceptible to CryIVD. Binding of CryIVD with BBMVs of A. stephensi resulted in a stronger signal than with BBMVs of T. oleracea. Likewise, larvae of A. stephensi are 10,000-fold more susceptible to the CryIVD toxin than are larvae of T. oleracea. Binding was also found with six proteins ranging in size from 48 to 110 kDa in BBMVs from the lepidopteran species Manduca sexta, but CryIVD was not toxic for M. sexta larvae. No binding of trypsinated CryIVD to BBMV proteins was observed. With the lepidopteran-specific toxin CryIA(b), no binding to dipteran BBMVs was found. Binding of CryIA(b) to nine different BBMV proteins ranging in size from 71 to 240 kDa was observed in M. sexta. The major binding signal was observed with a protein of 240 kDa for CryIA(b).     

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.     

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.     

Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots.

We investigated the role of the constituent domains of the CryIA(b) and CryIA(c) delta-endotoxins in binding to midgut epithelial cell membrane proteins of Spodoptera exigua and Manduca sexta on ligand blots. A collection of wild-type and CryIC-CryIA hybrid toxins was used for this purpose. As demonstrated elsewhere (R. A. de Maagd, M. S. G. Kwa, H. van der Klei, T. Yamamoto, B. Schipper, J. M. Vlak, W. J. Stiekema, and D. Bosch, Appl. Environ. Microbiol. 62:1537-1543, 1996), CryIA(b) domain III recognized a 205-kDa protein on S. exigua blots, while no specific binding by domain I or II could be detected. In contrast, on ligand blots of M. sexta proteins CryIA(b) domain II recognized a 210-kDa protein and CryIA(b) domain III recognized a 250-kDa protein. Domain III is responsible for the interaction of CryIA(c) with 120-kDa major binding proteins of both S. exigua and M. sexta. In addition, in M. sexta CryIA(c) also reacts with a 210-kDa binding protein through its domain I and/or domain II. These results show that besides domain II, domain III of delta-endotoxins plays a major role in binding to putative receptors on ligand blots. However, for S. exigua there was no clear correlation between binding of toxins on ligand blots and the in vivo toxicity of the toxins. These and previous results suggest that interactions of insect membrane proteins with both domain II and domain III can occur and that detection of these interactions depends on the type of binding assay used.     

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.     

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.     

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.     

Selective antagonism to the cadherin BT-R1 interferes with calcium-induced adhesion of epithelial membrane vesicles

BT-R(1) is a member of the cadherin superfamily of proteins and is expressed in the midgut epithelium of Manduca sexta during larval development. Previously, we showed that calcium ions influence the structure and stability of BT-R(1) on brush border membrane vesicles (BBMVs) prepared from M. sexta midgut epithelium. In the present study, the effects of calcium and Cry1Ab toxin, produced by Bacillus thuringiensis, on the adhesive properties of BBMVs were investigated. Addition of calcium to a suspension of BBMVs promoted adhesion and aggregation of the vesicles. Treatment of BBMVs with trypsin or lowering the pH (pH 4.0) of the BBMV suspension abolished calcium-induced vesicle aggregation, whereas treatment with deglycosylating enzymes did not affect the aggregation of vesicles, indicating that adhesion and clustering of BBMVs involves protein-protein interactions. Preincubation of BBMVs with Cry1Ab toxin, which specifically binds to BT-R(1) with high affinity and disrupts the midgut epithelium of M. sexta, caused a 50% decrease in calcium-induced vesicle aggregation. The inhibitory effects of the Cry1Ab toxin on BBMV aggregation was blocked completely when the toxin was preincubated with a peptide containing the toxin-binding site of BT-R(1). Cry3A toxin, which is similar in molecular structure to Cry1Ab but does not bind to BT-R(1) and is not toxic to M. sexta larvae, did not affect BBMV aggregation. The results of this study demonstrate that the adhesive function of BT-R(1) is compromised by the Cry1Ab toxin, which acts as a selective antagonist, and supports the notion that BT-R(1) is critical in preserving the integrity of larval midgut epithelium in M. sexta.     

Study of the irreversible binding of Bacillus thuringiensis Cry1Aa to brush border membrane vesicles from Bombyx mori midgut

The binding of Bacillus thuringiensis δ-endotoxin to brush border membrane vesicles (BBMVs) from the target insect larval midgut comprises with not only a reversible but also an irreversible component. The irreversible binding of δ-endotoxin is thought to be a pathologically important factor. Here, we studied the irreversible binding of Cry1Aa to the BBMVs of Bombyx mori. The ¹²⁵I-labeled Cry1Aa bound to the solubilized brush border membrane (BBM) through rapid dissociation only, unlike the binding to BBMVs, indicating that the toxin bound to the solubilized BBM through only a reversible process. Low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the toxin bound irreversibly to BBMVs formed an oligomer of 220 kDa, whereas that bound reversibly to the solubilized BBM did not oligomeraize. When the ¹²⁵I-labeled Cry1Aa bound irreversibly to the BBMVs was digested by proteinase K, approximately 40% of the toxin observed to be resistant to proteinase K. The molecular mass of the toxin resistant to proteinase K was 60 kDa, suggesting that the irreversible binding comprise two forms. These results support the notion that the irreversible binding of the toxin to BBMVs is due to the insertion of the toxin into the lipid bilayers and oligomerization to form channels.     

Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in Heliothis virescens midgut.

The binding proteins, or receptors, for insecticidal Bacillus thuringiensis subsp. kurstaki delta-endotoxins are located in the brush border membranes of susceptible insect midguts. The interaction of one of these toxins, CryIA(c), with proteins isolated from Heliothis virescens larval midguts was investigated. To facilitate the identification of solubilized putative toxin-binding proteins, a solid-phase binding assay was developed and compared with toxin overlay assays. The overlay assays demonstrated that a number of proteins of 170, 140, 120, 90, 75, 60, and 50 kDa bound the radiolabeled CryIA(c) toxin. Anion-exchange fractionation allowed the separation of these proteins into three toxin binding fractions, or pools. Toxin overlay assays demonstrated that although the three pools had distinct protein profiles, similar-size proteins could be detected in these three pools. However, determination of toxin affinity by using the solid-phase binding assay showed that only one of the three pools contained high-affinity binding proteins. The Kd obtained, 0.65 nM, is similar to that of the unsolubilized brush border membrane vesicles. Thus, the solid-phase binding assay in combination with the toxin overlay assay facilitates the identification and purification of high-affinity B. thuringiensis toxin-binding proteins from the insect midgut.     

Identification and characterization of Heliothis virescens midgut membrane proteins binding Bacillus thuringiensis delta-endotoxins.

To investigate the specificity of Bacillus thuringiensis var. kurstaki strain HD1 insecticidal crystal proteins (ICP), we used membrane preparations obtained from the midgut of Heliothis virescens larvae to perform separate ligand-blot experiments with the three activated CryIA toxins. The CryIA(a) and the CryIA(b) toxins bind the same 170-kDa protein, but most likely at two different binding sites. The CryIA(c) toxin binds two proteins of molecular masses 140 kDa and 120 kDa. We also demonstrate that the binding proteins for each of the B. thuringiensis toxins are not part of a covalent complex. Although the 170-kDa protein is a glycoprotein, endoglycosidase treatment does not prevent the binding of the CryIA(a) or CryIA(b) toxin. This indicates that the sugars are not important for the binding of these toxins. A model for a protein complex binding the B. thuringiensis HD1 ICPs is presented. Our results support the idea that binding proteins on membranes of the gut epithelial cells of H. virescens larvea are important for the specificity of the bacterial toxins.     

Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects

To study the molecular basis of differences in the insecticidal spectrum of Bacillus thuringienesis delta-endotoxins, we have performed binding studies with three delta-endotoxins on membrane preparations from larval insect mid-gut. Conditions for a standard binding assay were established through a detailed study of the binding of 125I-labeled Bt2 toxin, a recombinant B. thuringiensis delta-endotoxin, to brush border membrane vesicles of Manduca sexta. The toxins tested (Bt2, Bt3 and Bt73 toxins) are about equally toxic to M. sexta but differ in their toxicity against Heliothis virescens. Equilibrium binding studies revealed saturable, high-affinity binding sites on brush border membrane vesicles of M. sexta and H. virescens. While the affinity of the three toxins was not significantly different on H. virescens vesicles, marked differences in binding site concentration were measured which reflected the differences in in vivo toxicity. Competition experiments revealed heterogeneity in binding sites. For H. virescens, a three-site model was proposed. In M. sexta, one population of binding sites is shared by all three toxins, while another is only recognized by Bt3 toxin. Several other toxins, non-toxic or much less toxic to M. sexta than Bt2 toxin, did not or only marginally displace binding of 125I-labeled Bt2 toxin in this insect. No saturable binding of this toxin was observed to membrane preparations from tissues of several non-susceptible organisms. Together, these data provide new evidence that binding to a specific receptor on the membrane of gut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of B. thuringiensis insecticidal crystal proteins.     

Isolation and partial characterization of binding proteins for immobilized delta endotoxin from solubilized brush border membrane vesicles of the silkworm, Bombyx mori, and the common cutworm, Spodoptera litura.

1. Brush border membrane vesicles (BBMV) were prepared from the Bombyx mori, and Spodoptera litura, midguts. BBMV was solubilized. 2. Activated delta endotoxin from Bacillus thuringiensis were immobilized. 3. Solubilized BBMV proteins were applied to the toxin column and the proteins bound were analyzed by SDS-PAGE. 4. In the case of B. mori M(r) = 220,000, 150,000 and 130,000 and in the case of S. litura 160,000 bands were detected. 5. The bindings were inhibited by N-acetyl galactosamine and mannose. 6. The binding proteins applied onto a Con A column and eluted by 0.1 M methyl-alpha-glucose, suggesting that hybrid type sugar sidechain may involve in the interaction.     

Detection of bacterial toxins with monosaccharide arrays

A large number of bacterial toxins, viruses and bacteria target carbohydrate derivatives on the cell surface to attach and gain entry into the cell. We report here the use of a monosaccharide-based array to detect protein toxins. The array-based technique provides the capability to perform simultaneous multianalyte analyses. Arrays of N-acetyl galactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) derivatives were immobilized on the surface of a planar waveguide and were used as receptors for protein toxins. These arrays were probed with fluorescently labeled bacterial cells and protein toxins. While Salmonella typhimurium, Listeria monocytogenes, Escherichia coli and staphylococcal enterotoxin B (SEB) did not bind to either of the monosaccharides, both cholera toxin and tetanus toxin bound to GalNAc and Neu5Ac. The results show that the binding of the toxins to the carbohydrates is density dependent and semi-selective. Both toxins were detectable at 100 ng/ml.     

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.     

Clostridium botulinum serotype D neurotoxin and toxin complex bind to bovine aortic endothelial cells via sialic acid

Botulinum neurotoxin (BoNT) is produced as a large toxin complex (L-TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, -33 and -17). The binding properties of BoNT to neurons and L-TC to intestinal epithelial cells are well documented, while those to other tissues are largely unknown. Here, to obtain novel insights into the pathogenesis of foodborne botulism, we examine whether botulinum toxins bind to vascular endothelial cells. BoNT and 750 kDa L-TC (a complex of BoNT, NTNHA and HAs) of Clostridium botulinum serotype D were incubated with bovine aortic endothelial cells (BAECs), and binding to the cells was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Both BoNT and L-TC bound to BAECs, with L-TC showing stronger binding. Binding of BoNT and L-TC to BAECs was significantly inhibited by N-acetyl neuraminic acid in the cell culture medium or by treatment of the cells with neuraminidase. However, galactose, lactose or N-acetyl galactosamine did not significantly inhibit toxin binding to the cells. This is the first report demonstrating that BoNT and L-TC bind to BAECs via sialic acid, and this mechanism may be important in the trafficking pathway of BoNT in foodborne botulism.     

Binding of the delta endotoxin from Bacillus thuringiensis to brush-border membrane vesicles of the cabbage butterfly (Pieris brassicae)

The insecticidal delta endotoxin of Bacillus thuringiensis was labeled with iodine-125. Brush-border membrane vesicles, prepared from the midgut epithelium of Pieris brassicae larvae, known to be highly susceptible to the toxin, and from a non-target tissue: the small intestine of rat, were examined for binding of 125I-toxin. The toxin was bound specifically only to insect vesicles. Its binding to the insect membrane system was competitively inhibited by 127I-toxin and non-iodinated toxin, whereas the binding of the 125I-toxin to the mammalian membrane system was not affected by unlabeled toxin. Vesicles of P. brassicae possess two individual binding-site populations for iodinated toxin with dissociation constants of 46 nM and 490 nM. The Hill coefficients of both sites were approximately 1 and the binding capacities were 0.2 pmol and 30 pmol/mg vesicle protein for the high and the low-affinity sites respectively. The estimation of the dissociation constant for non-iodinated toxin, using a competition experiment, revealed only one binding-site population which possessed a dissociation constant of 235 nM. It is concluded that this is the binding site for the native toxin. This site was sensitive towards treatment with proteases or mixed glycosidases. It is suggested that it is a protein or a glycoprotein.     

Expression in Spodoptera frugiperda (Sf21) insect cells of BT-R(1), a cadherin-related receptor from Manduca sexta for Bacillus thuringiensis Cry1Ab toxin

The cadherin-related receptor of Manduca sexta, BT-R(1), for the Cry1A family of Bacillus thuringiensis insecticidal toxins, was expressed in cultured Spodoptera frugiperda (Sf21) insect cells utilizing the expression vector deltaOp-gp64. Recombinant BT-R(1) was released by the Sf21 cells in soluble form into the culture medium and represents approximately 58% of total BT-R(1) produced by the cells. The soluble protein was purified by affinity chromatography using Cry1Ab toxin coupled to Sepharose 4B. The apparent molecular mass of purified soluble recombinant BT-R(1) is 195 kDa. Radiolabeled toxin bound to purified soluble BT-R(1) with a K(d) value of 1.1 nM, which is similar to that of both membrane-bound BT-R(1) in Sf21 cells and natural BT-R(1) from M. sexta larval midgut tissue. Binding of radiolabeled toxin to soluble BT-R(1) was competitively inhibited by unlabeled Cry1Ab toxin but not by other Cry toxins as was observed also for membrane-bound BT-R(1). The recombinant soluble protein was stable in culture medium for at least 3 days at 27 degrees C and for 7 days at 4 degrees C and exhibited toxin-binding properties similar to the natural protein. Apparently, neither membrane association nor the extent of glycosylation influences the binding affinity and specificity of BT-R(1). Approximately 1 mg of purified BT-R(1) was obtained per liter of insect cell culture supernatant, representing approximately 2 x 10(9) Sf21 cells.     

Brush border membrane aminopeptidase-n in the midgut of the gypsy moth serves as the receptor for the CryIA(c) δ-endotoxin of Bacillus thuringiensis

Aminopeptidase-N (AP-N) was purified from gypsy moth (Lymantria dispar, L.) brush border membrane vesicles (BBMV) proteins by mono-Q chromatography and Superdex-75 gel filtration in the presence of the zwitterionic detergent, CHAPS, using FPLC. The purified AP-N, identified by its enzymatic activity, had an apparent size of 100 kDa, and was identified as the unique Bacillus thuringiensis insecticidal toxin, CryIA(c), binding protein. AP-N clearly displayed strong binding to CryIA(c), exhibiting little or no binding to CryIA(a) or CryIA(b), and showing no binding for the coleopteran-specific toxin, CryIIIA. Protein blots of the BBMV proteins probed with biotin-labeled and ¹²⁵I-labeled insecticidal proteins revealed that CryIAc binds only to 120 kDa protein which is a slightly larger size in comparison to purified AP-N. Antibodies raised against the gypsy moth AP-N demonstrated that the purified AP-N and the 120 kDa CryIA(c) binding protein of total BBMV proteins are antigenically identical.     

Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush-border membranes from mosquito larvae.

The presence of specific receptors for Bacillus sphaericus binary toxin on brush-border membrane fractions (BBMF) from Culex pipiens larvae midgut cells was demonstrated by an in vitro binding assay. Both activated and radiolabelled polypeptides from the 51-kDa and 42-kDa binary toxin of B. sphaericus 1593 specifically bound to BBMF. Direct binding and homologous competition experiments indicated a single class of B. sphaericus toxin receptors, with a dissociation constant (Kd) of approximately 20 nM and a maximum binding capacity (Bmax) of approximately 7 pmol/mg BBMF protein. The sugars GalNAc, GlcNAc and N-acetyl neuraminic acid had no detectable inhibitory effect on toxin binding to C. pipiens BBMF. Binding experiments with the non-susceptible mosquito species Aedes aegypti failed to detect significant binding of B. sphaericus binary toxin to A. aegypti BBMF.