Sexta Brush Border Membrane Vesicles Research Articles

Rearrangement of N-Terminal α-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity.

Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II–III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.

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.

Chemical modification of Bacillus thuringiensis Cry1Aa toxin single-cysteine mutants reveals the importance of domain I structural elements in the mechanism of pore formation

Bacillus thuringiensis Cry toxins form pores in the apical membrane of insect larval midgut cells. To investigate their mechanism of membrane insertion, mutants in which cysteine replaced individual amino acids located within the pore-forming domain of Cry1Aa were chemically modified with sulfhydryl-specific reagents. The thiol group of cysteine was highly susceptible to oxidation and its reactivity was significantly increased when the toxins were purified under reducing conditions. Addition of a biotin group to the cysteine had little effect on the ability of the toxins to permeabilize Manduca sexta brush border membrane vesicles except for a slight reduction in activity for S252C and a large increase in activity for Y153C. The activity of Y153C was also significantly increased after modification by reagents that added an aromatic or a charged group to the cysteine. When permeability assays were performed in the presence of streptavidin, a large biotin-binding protein, the pore-forming activity of several mutants, including Y153C, where the altered residue is located within the hairpin comprising helices α4 and α5, or in adjacent loops, was significantly reduced. These results support the umbrella model of toxin insertion.

Membrane Insertion of the Bacillus thuringiensis Cry1Ab Toxin: Single Mutation in Domain II Block Partitioning of the Toxin into the Brush Border Membrane

The umbrella and penknife models hypothesize that insecticidal Bacillus thuringiensis Cry toxins partition into the apical membrane of the insect midgut by insertion of only two alpha-helices from domain I of the protein, alpha-helices 4 and 5 in the case of the umbrella model and alpha-helices 5 and 6 in the case of the penknife model. Neither model envisages membrane partitioning by domains II and III. In this study, we present data suggesting that mutations in the domain II residue, F371, affect insertion of the whole toxin into Manduca sexta brush border membrane vesicles (BBMVs). Using steady state fluorescence measurements combined with a proteinase K protection assay, we show that mutants of F371 have lost their ability to insert into the BBMV, even though binding to cadherin is almost unaffected. The study also identifies a difference in partitioning of toxins into artificial lipid vesicles (SUVs) as opposed to native BBMVs. While the F371 mutations block insertion of domains I and II into BBMVs, they only block domain II insertion into SUVs. Bioassay and voltage clamping of midguts also confirm the fluorescence data that the noninserting mutants are nontoxic. Our study leads us to propose that, in contrast to previous models of individual free helices inserting into the membrane, the toxin enters into the membrane as a whole molecule or oligomers of the molecule, wherein the domain II residue F371 has a vital role to play in membrane insertion.

Specific Epitopes of Domains II and III of Bacillus thuringiensis Cry1Ab Toxin Involved in the Sequential Interaction with Cadherin and Aminopeptidase-N Receptors in Manduca sexta

The Bacillus thuringiensis Cry toxins are specific to different insects. In Manduca sexta cadherin (Bt-R1) and aminopeptidase-N (APN) proteins are recognized as Cry1A receptors. Previous work showed that Cry1Ab binds to Bt-R1 promoting the formation of a pre-pore oligomer that binds to APN leading to membrane insertion. In this work we characterized the binding epitopes involved in the sequential interaction of Cry1Ab with Bt-R1 and APN. A Cry1Ab immune M13 phage repertoire was constructed using antibody gene transcripts of bone marrow or spleen from a rabbit immunized with Cry1Ab. We identified antibodies that recognize domain II loop 3 (scFvL3-3) or beta16-beta22 (scFvM22) in domain III. Enzyme-linked immunosorbent assay and toxin overlay binding competition assays in the presence of scFvL3-3, scFvM22, or synthetic peptides showed that domain II loop 3 is an important epitope for interaction with Bt-R1 receptor, whereas domain III beta16 is involved in the interaction with APN. Both scFvL3-3 and scFvM22 lowered the toxicity of Cry1Ab to M. sexta larvae indicating that interaction with both receptors is important for in vivo toxicity. scFvL3-3 and anti-loop2 scFv (scFv73) promoted the formation of the pre-pore oligomer in contrast to scFvM22. In addition, scFvL3-3 and scFv73 preferentially recognized the monomeric toxin rather than the pre-pore suggesting a conformational change in domain II loops upon oligomerization. These results indicate for the first time that both receptor molecules participate in Cry1Ab toxin action in vivo: first the monomeric toxin binds to Bt-R1 through loops 2 and 3 of domain II promoting the formation of the pre-pore inducing some structural changes, then the pre-pore interacts with APN through beta-16 of domain III promoting membrane insertion and cell death.

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K(+) permeability. A fluorometric assay performed with 3,3'-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K(+)-selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.

Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae.

Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541-544 in domain III (mutant pPB20). Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21). Toxicity assays showed a marked decrease in toxicity against S. exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity. Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S. exigua were determined for all toxins. Compared with Cry1Ca, the affinity of mutant pPB19 was slightly affected (2-fold lower), whereas the affinity of the mutants with an altered domain III (pPB20 and pPB21) was approx. 8-fold lower. Activation of Cry1Ca protoxin by incubation with S. exigua or M. sexta BBMV revealed the transient formation of an oligomeric form of Cry1Ca. The presence of this oligomeric form was tested in the activation of the different Cry1Ca mutants, and we found that those mutated in domain II (pPB19 and pPB21) could not generate the oligomeric form when activated by S. exigua BBMV. In contrast, when oligomerization was tested using BBMV prepared from M. sexta, all of the Cry1Ca mutants showed the formation of a similar oligomeric form as did the wild-type toxin. Our results show how modification of insect specificity can be achieved by manipulation of different parts of the toxin structure involved in different steps of the mode of action of B. thuringiensis toxins.

The toxicity test and hypothetical model ofBacillus thuringiensis Cry1Aa helix4

Development of targeted biological agents against agricultural insect pests is of prime importance for the elaboration and implementation of integrated pest management strategies that are environment-friendly, respectful of bio-diversity and safer to human health through reduced use of chemical pesticides. A major goal to understand how Bt toxins work is to elucidate the functions of their three domains. Domains II and III are involved in binding specificity and structural integrity, but the function of Domain I remains poorly understood. Using a Manduca sexta BBMV (brush border membrane vesicles) system, we analyzed its responses to Cry1Aa 15 single-point mutations with altered Domain I helix 4 residues. Light scattering assay showed that toxicity was almost lost in 3 mutants, and we observed significantly reduced toxicity in other 7 mutants. However, 5 mutants retained wild-type toxicity. Using computer software, we simulated the three-dimensional structures of helix 4. Both experimental and bioinformatic analysis showed that residues in Cry1Aa Domain I helix 4 were involved in the formation of ion channels that is critical for its insect toxicity.

N-terminal Activation Is an Essential Early Step in the Mechanism of Action of the Bacillus thuringiensis Cry1Ac Insecticidal Toxin

A variant form of the Bacillus thuringiensis Cry1Ac toxin that is not cleaved at the N terminus during proteolytic activation with trypsin was found to be incapable of forming pores in Manduca sexta brush border membrane vesicles in vitro and had reduced insecticidal activity in vivo. Binding studies indicated an altered binding pattern of the mutant toxin in that bound toxin could not be fully displaced by a high molar excess of fully trypsin-activated toxin. These results suggest that proteolytic removal of the N-terminal peptide of Cry1Ac is an important step in toxin activation.

Denaturation of either Manduca sexta aminopeptidase N or Bacillus thuringiensis Cry1A toxins exposes binding epitopes hidden under nondenaturing conditions.

The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with (125)I-labeled Cry1Ac, Cry1Ac mutant (509)QNR-AAA(511) (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both (125)I-Cry1Ac and (125)I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots (125)I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, (125)I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. (125)I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured (125)I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) (125)I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.

Analyses of the Pore Forming Ability of Bacillus thuringiensis Cry1A Mutant Toxins Using a Light-Scattering Technique

Abstract The pore formation properties of Bacillus thuringiensis Cry1 wild type and domain I and II mutant toxins were studied on Manduca sexta brush border membrane vesicles (BBMV) using a light-scattering technique. Wild type Cry1Ac, Cry1Ab, and Cry1Ba; Cry1Ab mutant toxins A92E, Y153D, R368A/R369A, and F371A; and Cry1Ac mutant toxin A92D were analyzed. In a direct mixing assay the mutant toxins Y153D, R368A/R369A, F371A, and Cry1Ba did not elicit a response in a 1-min signal-monitoring period. Mutant toxins A92D and A92E elicited slight responses. After preincubation of toxin with BBMV, the signal recovery response increased for all toxins. The signal recoveries caused by A92D and A92E were greater than Y153D-, F371A-, and R368A/R369A-induced signal recoveries, which were slightly greater than Cry1Ba-induced recoveries. By increasing the monitoring period to 3 min in direct mixing experiments, we observed greater pore formation by A92D. A92E had a response similar to, but lower than that of A92D. The response induced by both wild type and mutant toxins decreased when the hyperosmotic solution was changed from KCl to sucrose. However, in the presence of sucrose the responses induced by A92D and A92E were substantially reduced relative to KCl.

Role of alpha-helix seven of Bacillus thuringiensis Cry1Ab delta-endotoxin in membrane insertion, structural stability, and ion channel activity.

Domain I of the Cry1Ab insecticidal toxic protein has seven alpha-helices and is considered to be involved in the ion channel activity. While other alpha-helices, particularly alpha-4 and alpha-5, have been extensively explored, the remaining alpha-helices have been slightly studied. Site-directed mutagenesis was used to generate mutations throughout sequences encoding the alpha-helix 7 to test its role in ion channel function. Every amino acid residue in alpha-helix 7 was mutated to alanine. Most resultant proteins, e.g., D225A, W226A, Y229A, N230A, R233A, R234A, D242A, and F247A yielded no protoxin or were sensitive to degradation by trypsin or Manduca sexta midgut juice. Other mutant proteins, R224A, R228A, and E235A, were resistant to degradation to the above proteases but were 8, 30, and 12 times less toxic to M. sexta, respectively, than the wild-type Cry1Ab. Circular dichroism spectroscopy indicated a very small change in the R228A spectrum, while R224A and E235A display the same spectrum as the wild-type protein. These three mutant proteins showed little differences from Cry1Ab when analyzed by saturation binding and competition binding kinetics with (125)I-labeled toxin or by surface plasmon resonance to M. sexta brush border membrane vesicles. More conservative amino acid substitutions were introduced into alpha-helix 7 residues: R228K, F232Y, E235Q, and F247Y. In comparison with wild-type Cry1Ab, mutant proteins R228K, F232Y, E235A, and E235Q selectively discriminate between K+ and Rb+, while R224A and R228A had reduced inhibition of short-circuit current for both ions, when analyzed by voltage clamping of M. sexta midguts.

Evidence for intermolecular interaction as a necessary step for pore-formation activity and toxicity of Bacillus thuringiensis Cry1Ab toxin

Based on the observation of large conductance states formed by Bacillus thuringiensis Cry toxins in synthetic planar lipid bilayers and the estimation of a pore size of 10–20 Å, it has been proposed that the pore could be formed by an oligomer containing four to six Cry toxin monomers. However, there is a lack of information regarding the insertion of Cry toxins into the membrane and oligomer formation. Here we provide direct evidence showing that the intermolecular interaction between Cry1Ab toxin monomers is a necessary step for pore formation and toxicity. Two Cry1Ab mutant proteins affected in different steps of their mode of action (F371A in receptor binding and H168F in pore formation) were affected in toxicity against Manduca sexta larvae. Binding analysis showed that F371A protein bound more efficiently to M. sexta brush border membrane vesicles when mixed with H168F in a one to one ratio. These mutant proteins also recovered pore-formation activity, measured with a fluorescent dye with isolated brush border membrane vesicles, and toxicity against M. sexta larvae when mixed, showing that monomers affected in different steps of their mode of action can form functional hetero-oligomers.

Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis delta-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.

Two arginine residues (368-369) of Cry1Ab and Cry1Ac were mutated to alanine, glutamic acid and lysine by site-directed mutagenesis. Insecticidal activities of the mutant toxins on Manduca sexta and Lymantria dispar larvae were examined. Cry1Ac mutant toxins (c)RR-AA and (c)RR-EE and Cry1Ab mutant toxins (b)RR-AA and (b)RR-EE showed great reductions in toxicity against both insects. In contrast, conservatively changed (c)RR-KK and (b)RR-KK mutants did not alter toxicity to either insect. Binding assays with brush border membrane vesicles (BBMVs) prepared from L. dispar midguts demonstrated that (c)RR-AA, (c)RR-EE, (b)RR-AA and (b)RR-EE bound with lower affinities compared with their respective wild-type toxins. To M. sexta BBMVs, (c)RR-AA and (c)RR-EE showed great reductions in BBMV binding. However, (b)RR-AA and (b)RR-EE did not alter BBMV competition patterns, despite their reduced toxicity. Further binding assays were performed with aminopeptidase N (APN) purified from L. dispar and M. sexta BBMVs using surface plasmon resonance (BIAcore). Direct correlation between toxicity and APN binding was observed for the mutant toxins using this technique. The inconsistency between BBMV and APN binding data with Cry1Ab to M. sexta suggests the possibility of a different Cry1Ab toxin-binding mechanism or the importance of another receptor in M. sexta.

Evidence for intermolecular interaction as a necessary step for pore-formation activity and toxicity of Bacillus thuringiensis Cry1Ab toxin.

Based on the observation of large conductance states formed by Bacillus thuringiensis Cry toxins in synthetic planar lipid bilayers and the estimation of a pore size of 10-20 A, it has been proposed that the pore could be formed by an oligomer containing four to six Cry toxin monomers. However, there is a lack of information regarding the insertion of Cry toxins into the membrane and oligomer formation. Here we provide direct evidence showing that the intermolecular interaction between Cry1Ab toxin monomers is a necessary step for pore formation and toxicity. Two Cry1Ab mutant proteins affected in different steps of their mode of action (F371A in receptor binding and H168F in pore formation) were affected in toxicity against Manduca sexta larvae. Binding analysis showed that F371A protein bound more efficiently to M. sexta brush border membrane vesicles when mixed with H168F in a one to one ratio. These mutant proteins also recovered pore-formation activity, measured with a fluorescent dye with isolated brush border membrane vesicles, and toxicity against M. sexta larvae when mixed, showing that monomers affected in different steps of their mode of action can form functional hetero-oligomers.

Further characterization of BT-R 1, the cadherin-like receptor for Cry1Ab toxin in tobacco hornworm (Manduca sexta) Midguts

BT-R 1, the Manduca sexta midgut receptor for the crystal toxin CrylAb produced by Bacillus thuringiensis ssp. berliner, was partly purified by gel filtration from M. sexta brush border membrane vesicles in the presence of the detergent CHAPS. Fractions containing BT-R 1 were tested for their stability against degradation as indicated by retention of CrylAb binding on ligand blots. At 4°C and pH 7.4 in the presence of Ca 2+, BT-R 1 was stable for up to 48 h but a 65% loss of binding was observed after 100 h. Under the same conditions, no loss of binding was observed in the presence of EGTA after 100 h. CrylAb binding decreased markedly as pH increased from 6 to 10 for incubations of 24 h. at 4°C. Increasing the temperature of incubation from 4 to 37°C also decreased CrylAb binding. Neither metal ions nor free sulfhydryl groups are involved in CrylAb binding to BT-R 1. A trypsin-like, metal-ion-dependent proteolytic activity co-eluted with BT-R 1, during gel filtration. This endoproteolytic activity was unaltered by the addition of CrylAb. BT-R 1 did not co-elute with peaks of aminopeptidase, alkaline phosphatase, α-glucosidase, β-glucosidase and β-galactosidase activities. When BT-R 1 in the gel filtration fraction was further purified on a Mono Q anion exchange column, partial separation of the trypsin-like activity from BT-R 1 was observed. BT-R 1 could be removed from the appropriate Mono Q fraction by immunoprecipitation with only a slight decrease in this activity. These results demonstrate that there is no copurification of BT-R 1 and these enzymes and that BT-R 1 is unlikely to form complexes with them. Binding of CrylAa and CrylAc to BT-R 1 in gel filtration fractions is similar to that of CrylAb, indicating that BT-R 1 may be the high-affinity receptor for the CrylA toxins. Binding of CrylAb to a 120 kDa protein has not been observed in this study.

Role of Domain II, Loop 2 Residues of Bacillus thuringiensis CryIAb δ-Endotoxin in Reversible and Irreversible Binding to Manduca sexta and Heliothis virescens

Site-directed mutagenesis was used to examine the role of domain II, loop 2 residues, 368RRPFNIGI375, of Bacillus thuringiensis insecticidal protein CryIAb. Alanine substitution of residues 368RRP370, called B4, abolished potency toward Manduca sexta and Heliothis virescens, and the loss of toxicity was correlated directly to substantially reduced binding affinity to brush-border membrane vesicles (BBMV) prepared from the target insect midguts. These results indicated that these positive charges might be essential to orient the toxin to midgut receptor molecule(s). The role of residue Phe371 of CryIAb toxin to M. sexta was investigated by substituting a series of residues at this position. Irreversible binding and toxicity were affected significantly by hydrophilic, aliphatic, and smaller side-chain residues such as Cys, Val, Leu, and Ser but not by Tyr or Trp. A hydrophobic aromatic side-chain residue at position 371 was therefore essential for irreversible binding of CryIAb toxin in M. sexta. The role of residues 370PFNIGI375 of CryIAb toxin on H. virescens was also examined. Mutants D2 (deletion of residues 370-375), G374A (alanine substitution of Gly374), and I375A had reduced toxicity to H. virescens. In contrast to our findings with M. sexta, the reduction in toxicity of these mutants was correlated directly with loss of initial binding to H. virescens BBMV, indicating that these residues perform functionally distinct roles in binding and toxicity to different insects. In ligand blots, CryIAb recognized a major 210-kDa peptide in M. sexta BBMV and a 170-kDa peptide in H. virescens BBMV.

The CryIA(c) Receptor Purified from Manduca sexta Displays Multiple Specificities

The kinetic binding characteristics of four Bacillus thuringiensis CryI insecticidal crystal proteins to a Cry-binding protein, purified from Manduca sexta brush-border vesicles, were analyzed by an optical biosensor. This 120-kilodalton binding protein, previously determined to be aminopeptidase N, was converted to a 115-kilodalton water-soluble form by removing the attached glycosylphosphatidylinositol anchor with phospholipase C. The solubilized form recognized the three major subclasses of CryIA toxins but not CryIC even though all four CryI proteins are toxic to larvae of M. sexta. CryIA(a) and CryIA(b) toxins bound to a single site on the solubilized aminopeptidase N molecule whereas CryIA(c) bound to two distinct sites. Apparent kinetic rate constants were determined for each binding reaction. All three CryIA toxins exhibited moderately fast on rates (approximately 10(-5) M-1 s-1) and a slow reversible off rate (approximately 10(-3) s-1). Although the second CryIA(c)-binding site retained a moderately fast association rate, it was characterized by a rate of dissociation from the amino-peptidase an order of magnitude faster than observed for the other CryIA-binding sites. CryIA(c) binding to both sites was strongly inhibited in the presence of N-acetylgalactosamine (IC50 = 5 mM) but not N-acetylglucosamine, mannose, or glucose. CryIA(a) and CryIA(b) binding were unaffected in the presence of the same sugars. Our results serve to illustrate both the complexity and the diverse nature of toxin interactions with Cry-binding proteins.

Mutations in Domain I of Bacillus thuringiensis δ-Endotoxin CryIAb Reduce the Irreversible Binding of Toxin to Manduca sexta Brush Border Membrane Vesicles

Site-directed mutagenesis was used to generate CryIAb mutants at the selected N-terminal positions to study the function of domain I. Structurally stable mutant proteins were tested for toxicity, receptor binding kinetics, and pore function. Substitutions of tyrosine at position 153 with arginine (Y153R) or alanine (Y153A) did not affect toxicity appreciably, whereas replacing this tyrosine with aspartic acid (Y153D) resulted in a great loss of toxicity. Mutation of alanine at position 92 to glutamic acid (A92E) almost completely abolished toxicity. The initial receptor binding was unchanged as measured by competition binding assays among all mutant proteins. Reduced pore function, however, was observed for mutants A92E and Y153D as tested by voltage clamping. Further studies with specially designed association and dissociation binding assays showed that irreversible binding of these two mutant toxins to Manduca sexta brush border membrane vesicles was significantly reduced. The decrease in irreversible binding was correlated with the changes in toxicity and may reflect a severely disturbed membrane insertion process in these two mutant toxins, leading to reduced pore function and toxicity. The results support the model that domain I is involved in membrane integration and pore formation.

Chemical modification of Bacillus thuringiensis activated -endotoxin and its effect on toxicity and binding to Manduca sexta midgut membranes

The effect of chemical modification of aromatic and basic residues of the Bacillus thuringiensis insecticidal CrylA(c) toxin on toxicity and receptor binding was studied. Modification of four or more of the 43 arginine residues resulted in abolition of toxicity towards Manduca sexta and Pieris brassicae in vivo and a Choristoneura fumiferana cell line in vitro. Modification of seven or more of the 27 tyrosine residues resulted in reduction of toxicity. Upon modification of most of the 10 tryptophan residues, toxicity was reduced. Modification of histidine residues had no effect on toxicity. A quantitative binding assay was developed and optimized to compare the binding of derivatives with native toxin to M. sexta brush border membrane vesicles. The reduction or abolition of toxicity observed upon selective modification of tyrosine or arginine residues was reflected in the binding abilities of the derivatives. However a non-toxic derivative, modified at tryptophan residues, retained its ability to bind vesicles. These results suggest that tyrosine and arginine residues are involved in the binding interaction of toxin with receptor but tryptophan residues are involved in some subsequent step.