©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutations in Domain I of Bacillus thuringiensis -Endotoxin CryIAb Reduce the Irreversible Binding of Toxin to Manduca sexta Brush Border Membrane Vesicles (*)

(Received for publication, October 11, 1994; and in revised form, December 8, 1994)

Xue Jun Chen April Curtiss Edwin Alcantara Donald H. Dean (§)

From the Department of Biochemistry, Ohio State University, Columbus, Ohio 43210

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

During sporulation, Bacillus thuringiensis can produce crystal proteins known as -endotoxins that have been shown to be toxic to a number of insect larvae in the orders Lepidoptera, Diptera, and Coleoptera(1) . Recently, toxins with activity against nematodes have also been reported(2) . It is believed that after ingestion and solubilization, protoxins are converted to active toxins that pass through the peritrophic membrane and bind to receptors on the surface of midgut epithelium cells of susceptible insects before insertion into the membrane. The inserted toxins disturb the osmotic balance by generating pores in the cell membrane, leading to cell lysis and insect death. However, the precise mode of action is not completely understood.

The correlation of specificity with the presence of high affinity receptors was demonstrated by Hofmann et al.(3, 4) and Van Rie et al.(5) . These studies indicated that binding is a crucial step in toxicity. This conclusion is supported by the isolation of a specific receptor for CryIAc toxin from Manduca sexta BBMV(^1)(6, 7) . However, it has been observed that CryI toxins bind specifically to the midgut membrane preparation from nonsusceptible insects(8, 9) . Wolfersberger (10) has shown that toxicities of CryIAb and CryIAc are inversely related to their binding affinity to Lymantria dispar BBMV. Additionally, Gould et al.(11) have observed that a CryIAc-resistant Heliothis virescens strain binds to the toxin as well as the sensitive strain does. Toxin binding on BBMV has been shown to become irreversible within a short incubation time, implying a membrane insertion step following the initial binding(5) . Thus, it appears that post-binding events, such as membrane insertion and channel formation and function, together with binding, contribute to the insecticidal activity of -endotoxins.

Because of the presence of five highly conserved amino acid blocks among B. thuringiensis toxins(1) , it is believed that -endotoxins may adopt a similar tertiary structure composed of three domains, as elucidated in CryIIIA by Li et al.(12) . Several identified insect specificity regions are located in domain II (13, 14, 15, 16) , including one that has been proved to be the Bombyx mori-specific binding region for CryIAa(17, 18) , indicating that domain II is a receptor-binding domain. Domain III has been shown to be involved in structural stability and channel function as well as specificity(14, 15, 19, 20) . Domain I, a seven-alpha-helix bundle covering the most conserved N-terminal region, is predicted to be the membrane insertion and pore formation domain. Ahmad and Ellar (21) have found that mutations A165P, L167M, F50N, and V51N in the N-terminal hydrophobic regions of CryIAa do not affect binding to tissue culture cells, but lower cytotoxicity. They concluded that this region is involved in formation of the transmembrane pore rather than in receptor recognition. To the contrary, Wu and Aronson (22) have observed that the domain I mutation A92D in CryIAc affects both binding and toxicity in M. sexta. More studies on domain I, which could be the key factor in post-binding events, are important to fully understand the mode of action of -endotoxins.

To further investigate the function of domain I and to gain more insights into events subsequent to binding, selected amino acid residues, alanine at position 92, phenylalanine at position 148, and tyrosine at position 153, in domain I of CryIAb were mutagenized. Based on the structural alignment with CryIIIA, all these amino acids should be located on the side facing the cell membrane and could directly participate in the domain I-membrane interaction(12, 23) . Mutant toxins were tested for structural stability, toxicity, receptor binding, and pore function. We found that the dramatic decrease in toxicity, resulting from mutation, is correlated with a significant decrease in irreversible binding or insertion, which may lead to poor channel formation and function. These results support the prediction that domain I is important for membrane insertion and pore function.


MATERIALS AND METHODS

Bacterial Strains, Plasmids, and Mutagenesis

The plasmid pSB033, constructed by cloning a cryIAb gene from B. thuringiensis subsp. kurstaki 133 into a pBluescript vector, was kindly provided by T. Yamamoto (Sandoz Agro Inc.). A cryIC gene 5`-region and a cryIAc gene 3`-region were used as a promoter and a terminator, respectively, in this plasmid for expressing the cloned cryIAb gene. pSB033 is capable of both generating single-strand DNA templates for mutagenesis and expressing the mutant gene. In vitro site-directed mutagenesis was performed using a Bio-Rad Muta-Gene phagemid in vitro mutagenesis kit following the instructions from the manufacturer. Uracil-containing DNA templates were made in Escherichia coli strain CJ236, and mutant protoxins were expressed in E. coli strain MV1190. All mutations were confirmed by DNA sequencing. Oligonucleotides directing mutations were as follows: 1) 5`-AGAATAGAAGAGTTCGAGAGGAACCAAGCC-3` for A92E, 2) 5`-CTTTTTGCAGTGCAGAATGCTCAAGTTCCTCTT-3` for Y153A, 3) 5`-CTTTTTGCAGTGCAGAATGATCAAG TTCCTCTT-3` for Y153D, 4) 5`-CTTTTTGCAGTGCAGAATCGTCAAGTTCCTCTT-3` for Y153A, 5) 5`-GCTATTCCTCTTGATGCAGTTCAAAAT-3` for F148D, and 6) 5`-GCTATTCCTCTTCGTGCAGTTCAAAAT-3` for F148R. All oligonucleotides were synthesized at Sandoz Agro Inc.

Purification and Protease Digestion of Protoxin

Wild type and mutant genes were expressed in E. coli strain MV1190 (Bio-Rad). Cells were grown for 48 h in 400 ml of LB medium containing 50 µg of ampicillin/ml. Crystal extracts were prepared and solubilized in sodium carbonate buffer (50 mM Na(2)CO(3)/NaHCO(3), 10 mM dithiothreitol, pH 9.5) as described by Chen et al.(20) . Protoxin was digested to the active toxin with trypsin at a trypsin/protoxin ratio of 1:50 (w/w) for 1 h at 37 °C, followed by an equal dose of trypsin for another hour. The concentration of protoxin and toxin was determined by Coomassie Blue protein assay reagent (Pierce). Protoxin and toxin were examined by SDS-12% polyacrylamide gel electrophoresis according to Laemmli(24) . For iodination and CD spectrum study, Sephadex G-100 column-purified toxins were used.

Toxicity Assay

Bioassays on M. sexta were performed by the surface contamination method with wild type or mutant toxins. Artificial diet (from BioServ Inc., Frenchtown, New Jersey) was prepared by following the manufacturer's instruction. About 100 µl of the diet was added to a well with a diameter of 0.75 cm in a Falcon dish. After the diet was solidified, a 20-µl drop of diluted toxin solution was evenly spotted on the surface of the diet. The toxin solution was air-dried, and a newly hatched larva was placed on the surface of the diet. Five or six toxin concentrations were used to calculate the medium lethal concentration (LC) value with at least 20 larvae at each concentration. LC values and 95% fiducial limits were obtained with the PROBIT.SAS program(25) . Solubilization buffer was used as a nontoxic control for the bioassay.

Isolation of Midguts and Preparation of Brush Border Membrane Vesicles

M. sexta eggs were purchased from Carolina Biological Supply. The eggs were hatched, and larvae were raised to the fifth instar on the artificial diet. The fifth instar larvae, weighing an average of 7.5 g, were chilled on ice for 10-15 min and transected after the third appendage from the head, and the integument was opened with a longitudinal cut. The midgut was cut open, and the peritrophic membrane and gut contents were discarded. The isolated midgut was rinsed twice with buffer A (300 mM mannitol, 5 mM EDTA, 17 nM Tris-HCl, pH 7.5), blotted, weighed, and stored in liquid nitrogen vapor.

BBMV were prepared from the isolated midgut following the method described by Wolfersberger et al.(26) . The final BBMV pellet was resuspended in binding buffer (150 mM NaCl, 8 mM Na(2)HPO(4), 2 mM KH(2)PO(4), pH 7.4), and the concentration of BBMV proteins was assessed by Coomassie Blue protein assay reagent using bovine serum albumin as a standard. The final BBMV suspension contained 0.1% bovine serum albumin.

Iodination of Toxins

Trypsin-digested toxin was dialyzed against labeling buffer (0.05 mM Na(2)CO(3)/NaHCO(3), pH 9.5) overnight and then further purified by passage through a Sephadex G-100 column. Iodination of toxins was conducted with IODO-BEADS (Pierce). One IODO-BEAD was rinsed with the labeling buffer, dried, and then placed in a 1.5-ml plastic cup. One mCi of NaI (Amersham Corp.) was first added to the cup and incubated with the IODO-BEAD for 5 min at room temperature. Then 25 µg of toxin was added to the reaction cup, and the incubation was continued for another 15 min at room temperature. The iodination reaction was terminated by removing the reaction solution from the IODO-BEAD. The reaction solution was loaded onto and passed through a 2.0-ml Excellulose GF-5 column (Pierce) to separate labeled toxin from free iodine.

Immunoblotting Assay

Forty µg of M. sexta BBMV proteins was electrophoresed on a 7.5% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Immobilon-P, Bio-Rad). The membrane was cut into strips, which were incubated for 2 h at room temperature in TTBS (50 mM Tris, 0.9% NaCl, 0.1% Triton X-100, 0.05% Tween 20, pH 7.5) containing 5% nonfat dry milk powder to block nonspecific binding. After blocking, membrane strips were incubated first with 30 µg of CryIAb or mutant toxins or with M. sexta nontoxic CryIIIA toxin (CryIIIA control) or without toxin (no toxin control) in 6 ml of TTBS for 2 h, then with primary antibody for 1 h, and finally with secondary antibody for 1 h. Each incubation was followed by three 10-min washes with TTBS. Primary antibody was raised in rabbit against purified wild type CryIAb toxin. Secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) purchased from Bio-Rad. Two controls were included to examine potential nonspecific binding of primary antibodies to M. sexta BBMV: the first one was a no toxin control (see Fig. 3, lane2), and the second one used M. sexta nontoxic CryIIIA toxin (lane3). Visualization of toxin-binding bands was accomplished by treating the strips with a solution made from a Sigma Fast(TM) DAB peroxidase substrate tablet set (Sigma).


Figure 3: Immunoblotting of M. sexta BBMV proteins with wild type and mutant toxins. 40 µg of M. sexta BBMV proteins was blotted and incubated as described under ``Materials and Methods.'' Detection of the binding of wild type or mutant toxins to BBMV-binding proteins was carried out using polyclonal antisera to CryIAb toxin. Lane1, Coomassie Blue-stained M. sexta BBMV proteins only; lane2, control (no toxin); lane3, M. sexta nonspecific CryIIIA toxin control; lane4, IAb; lane5, A92E; lane6, Y153A; lane7, Y153D; lane8, Y153R. Positions of protein markers (in kilodaltons) are shown on the left.



Ligand Binding Assay

Binding assays were performed as described by Lee et al.(17) . For the competition binding assay, 20 µg of BBMV was incubated with 0.5 nMI-labeled toxin in 100 µl of binding buffer in the presence of varying concentrations of unlabeled toxins. Labeled and unlabeled toxins were mixed together before adding them to the BBMV. More than 12 concentrations of unlabeled toxins were used in the homologous or heterologous competition binding studies. Data from competition binding studies were analyzed using the LIGAND computer program(27) .

Dissociation and Association Binding Assays

For the dissociation experiment, 0.5 nMI-labeled toxin was first incubated with 20 µg of BBMV in 100 µl of binding buffer at room temperature. After 1 h of incubation, a 10-µl unlabeled toxin (1000 nM) solution was added to the I-labeled toxin and BBMV mixture to start chasing. Chasing was terminated at different times by spinning down the incubation mixture. The resulting pellets were washed twice with 300 µl of binding buffer and then counted in a -counter (Beckman Instruments). Nonspecific binding was determined by testing the binding of labeled toxin to BBMV in the presence of 1000 nM unlabeled toxin. Nonspecific binding was subtracted in the final data analysis.

To measure irreversible and reversible binding at different incubation times, the association experiment was designed as follows. Two sample sets of a mixture of 0.5 nMI-labeled toxin and 20 µg of BBMV in a total volume of 100 µl were incubated at room temperature. At the selected time, the BBMV bound by labeled toxin in the first set was quickly spun down at 16,500 rpm for 10 min to measure total binding. At the same time, 10 µl of unlabeled toxin (1000 nM) was added to the second set. Incubation was continued for 60 min at room temperature to chase off the reversible binding at the selected time. The labeled toxin-bound BBMV pellets were always washed twice with binding buffer before counting in a -counter. Nonspecific binding was obtained by adding 0.5 nMI-labeled toxin and 1000 nM unlabeled toxin together into BBMV at the beginning of the incubation. The counts of the first set were the total specific binding plus the nonspecific binding, whereas the counts of the second set were the irreversible binding plus the nonspecific binding. Reversible binding counts were obtained by subtracting the counts of the second set from the counts of the first set. Irreversible binding counts were calculated by subtracting the nonspecific binding from the counts of the second set.

Voltage Clamping Assay

Inhibition of mutant toxins on the short circuit current (I) across the M. sexta midgut membrane was tested using the voltage clamping assay as described by Chen et al.(20) . The anterior part of the midgut of fifth instar M. sexta larvae was mounted on an aperture (diameter = 0.5 cm) that was assembled on the voltage clamping chamber. The same amount (final concentration = 50 ng/ml) of wild type or mutant toxins was added to the luminal side of the chamber. The I was traced with a Kipp & Zonen recorder, and the slope of falling I was measured after addition of toxins.


RESULTS

Effect of Mutations on Expression of Protoxin and Stability of Toxin

The domain I mutants constructed in this study are listed in Fig. 1. Mutants F148D and F148R, which replaced phenylalanine at position 148 with aspartic acid and arginine, respectively, yielded no detectable protoxins in E. coli. Other mutants, A92E, Y153A, Y153D, and Y153R, expressed protoxins as well as wild type CryIAb did. Toxins were prepared by trypsin digestion of protoxin inclusions. The stability of trypsin-resistant cores of these mutants was further examined by incubation with M. sexta midgut juice and CD spectrum analysis. All these toxins were as stable as the wild type in the presence of the gut juice and showed essentially identical CD spectra as compared with the wild type. (^2)


Figure 1: Mutations in domain I of CryIAb and their effect on protoxin expression and toxin stability. Amino acid sequences between positions 96 and 145 are not shown. +, expression of protoxin or stable toxin; -, no detectable expression. WT, wild type; mut, mutant.



Effect of Mutations on Toxicity

Insecticidal activities of wild type and mutant toxins were tested against M. sexta larvae and are reported in Table 1. No mortality was observed with mutant A92E toxin even at a concentration up to 1.14 times 10^5 ng/cm^2. Y153D toxin (LC = 3584 ng/cm^2) is much less toxic to M. sexta relative to the wild type (154 ng/cm), whereas Y153A and Y153R are 2-3-fold less toxic than wild type toxin.



Effect of Mutations on Receptor Binding

Competition binding assays were performed on M. sexta BBMV to analyze the effect on receptor binding. Three types of competition binding studies were conducted: 1) I-labeled wild type IAb toxin was competed by unlabeled mutant toxins (Fig. 2A); 2) I-labeled mutant toxins were competed by the unlabeled wild type toxin (Fig. 2B); and 3) I-labeled mutant toxins were competed with the same unlabeled mutant toxins (Fig. 2C). All these toxins displayed high affinity binding to M. sexta BBMV. As reported in Table 1, the K(d) values of A92E and Y153D, from both heterologous and homologous competition assays, are similar to that of the wild type. However, the K(d) values of Y153A and Y153R tested by heterologous displacement are 3-4-fold higher than the ones tested by homologous displacement. The K(d) values of Y153A and Y153R from heterologous displacement are 2-3-fold higher than that of the wild type, while the ones from homologous displacement are very close to that of the wild type. If the K(d) values calculated from heterologous competition data are more valid, they might account for the 2-fold loss of toxicity, but we would generally consider the K(d) values calculated from homologous competition binding to be a more representative value of binding of the mutant toxins to the receptor. At this point, we are not sure what causes this difference, except that it indicates the complexity of binding of this toxin to its receptor.


Figure 2: Competition binding studies of wild type and mutant toxins to M. sexta BBMV. Binding is expressed as percentages of the amount bound with labeled toxin alone. Unlabeled competing toxins are shown as follows: IAb, bullet; A92E, circle; Y153A, times; Y153D, ; and Y153R, . A, labeled wild type toxins competed by increasing amounts of unlabeled mutant toxins; B, labeled mutant toxin competed by increasing amounts of corresponding unlabeled mutant toxin; C, labeled mutant toxin competed by increasing amounts of unlabeled wild type IAb toxin.



All mutant toxins exhibited almost two times higher B(max) values than the wild type did. This minor difference could be caused by some errors in the determination of the specific activity of I-labeled toxins or by other unknown reasons, but the increases in the B(max) for mutants cannot account for their loss in toxicity.

Effect of Mutations on Immunoblotting Assay

BBMV proteins of M. sexta were blotted onto polyvinylidene difluoride membranes and incubated with either wild type or mutant toxins. The potential toxin and binding protein complexes were examined. Two major protein bands, 210 and 123 kDa, were visualized for wild type IAb and all mutant toxins (Fig. 3, lanes 4-8), whereas no bands were detected in the incubations without toxin or with M.sexta nontoxic CryIIIA toxin, indicating that the binding detected here is specific. Under the same experimental conditions, no apparent differences in binding were observed among wild type IAb and mutant toxins.

Effect of Mutations on Inhibition of I

Voltage clamp analysis was carried out with isolated M. sexta midgut membrane to study the effect of mutations on channel function. At a toxin concentration of 50 ng/ml, wild type toxin caused a sharp reduction in I with a slope of -3.4 ± 0.1 (Fig. 4). Under the same conditions, A92E toxin did not affect I, while Y153D toxin caused a slow decline in I (-0.3 ± 0.03). Mutant Y153A and Y153R toxins did affect I, but not as strongly as wild type toxin did.


Figure 4: Typical response curves of M. sexta midgut membrane to CryIAb and mutant toxins in voltage clamping assays. The same amounts of wild type IAb and mutant toxins in the same volume were respectively applied to the luminal side of the chamber. The final concentration for each toxin was 50 ng/ml. A control experiment was carried out with the addition of the same volume of solubilization buffer without toxin. Time 0 indicates the time of addition of the toxins. Curves were reproduced based on the recorded data. bullet, IAb; circle, A92E; times, Y153A; , Y153D; , Y153R; box, control.



Effect of Mutations on Irreversible Binding

To examine the effect of mutations on irreversible binding, the association binding and the dissociation binding of labeled wild type and mutant toxins to M. sexta BBMV were studied. In the presence of excess amounts of unlabeled toxin, I-labeled A92E and Y153D toxins dissociated from BBMV much more rapidly than did wild type toxin, as shown in Fig. 5. After a 20-min chase, wild type toxin dissociation was complete, while A92E and Y153D continued to dissociate for >40 min. After a 60-min chase with unlabeled toxin, 42% of Y153D toxin and 20% of A92E toxin remained in the BBMV, whereas >60% of wild type toxin was still bound to the BBMV. Mutants Y153A and Y153R had dissociation curves similar to that of the wild type.


Figure 5: Assay of dissociation binding of labeled wild type and mutant toxins to M. sexta BBMV. Binding is expressed as the percentage of the amount without addition of unlabeled toxin. Nonspecific binding was obtained by incubating the labeled toxin with BBMV in the presence of a 1000-fold excessive amount of unlabeled toxin and was subtracted from the total binding. bullet, IAb; circle, A92E; times, Y153A; , Y153D; , Y153R.



Association studies are shown in Fig. 6and summarized in Table 2. In this study, total specific binding, reversible binding, and irreversible binding at different incubation times were tested for each toxin. For wild type toxin, more than half of the total specific binding was irreversible at all tested time points. For mutant A92E, after 5 min of incubation, no irreversible binding was detected, and after 80 min of incubation, only 22% of the total specific binding was irreversible. Similarly, for Y153D toxin, irreversible binding accounted for only 40% of the total specific binding. In the case of mutants Y153A and Y153R, their association binding behaved similarly to that of the wild type.


Figure 6: Assay of association binding of wild type and mutant toxins to M. sexta BBMV. The curves show the total binding (circle), reversible binding (bullet), and irreversible binding () expressed as counts/minute at selected incubation times. A, wild type IAb; B, A92E; C, Y153D; D, Y153R (Y153A was similar to Y153R in this study).






DISCUSSION

Recently, there has been considerable interest in understanding the toxic mechanism of -endotoxins(28, 29) . The mode of action of B. thuringiensis toxins has been described as a multicomponent process, involving solubilization, protoxin activation, receptor binding, membrane integration, and pore formation. The resolution of the CryIIIA tertiary structure, consisting of three domains, provides us with more insights into the potential mode of action of -endotoxin. Membrane integration and channel formation have been linked to the function of domain I.

In this study, we have constructed several mutants in domain I of CryIAb to determine the region responsible for membrane insertion. Ala-92, Phe-148, and Tyr-153, carefully selected based on amino acid alignment with CryIIIA, are located on the side that could directly interact with the membrane. Substitution of phenylalanine at position 148 with aspartic acid or arginine results in no detectable expression of protoxin. We assume that phenylalanine, located in helix 4, is a buried residue, crucial in maintaining domain I structural integrity. Thus, introducing a charged aspartic acid or arginine at this position could disrupt protein structure. Mutation A92E, which is located at the beginning of helix 3 based on the alignment, almost totally abolishes toxicity. Substitution of tyrosine at position 153 in the loop region between helixes 3 and 4 with alanine, aspartic acid, or arginine causes different effects. Mutation Y153D greatly reduces toxicity, whereas mutations Y153A and Y153R slightly affect biological activity.

To understand why these mutations affect toxicity, we have performed a series of experiments to examine factors known to be involved in toxicity. Mutants A92E, Y153A, Y153D, and Y153R express protoxins at a level similar to that of wild type IAb, and their protoxins yield trypsin-resistant cores. All these mutant toxins are as stable as wild type IAb toxin in the presence of M. sexta gut juice and exhibit essentially identical CD spectrum curves (data not shown). Therefore, structural alteration is not the likely reason for the lowered toxicity of these mutants.

The binding capacity of the mutant proteins has been tested by competition binding analysis and immunoblotting assay. Only minor changes in K(d) or B(max) values are detected for the toxins that have the greatest loss in toxicity, A92E and Y153D (Table 1), and the changes that are observed for increases in B(max) cannot account for the loss of toxicity. For Y153A and Y153R, the changes in the dissociation constant (K(d)) calculated by heterologous competition binding may account for the slight loss in toxicity, but the K(d) calculated from homologous competition binding indicates no difference in binding. The homologous competition assays demonstrate similar high affinity binding to M. sexta BBMV by mutant and wild type toxins (Fig. 2B). The heterologous competition assays reveal that mutant and wild type toxins recognize the identical binding site on the BBMV and compete with each other at similar levels for binding to the M. sexta BBMV (Fig. 2, A and B). Therefore, competition binding results indicate that these mutations do not affect initial receptor binding of CryIAb toxin. This conclusion is supported by our immunoblotting experimental data, which show that wild type IAb and mutant toxins bind to the same M. sexta BBMV-binding proteins (Fig. 3). Binding detected by blotting experiments has been demonstrated to be specific (30) and most likely represents only the reversible binding since membrane insertion cannot take place. Wu and Aronson (22) present data showing that mutations within conserved block 1 (helix 5) result in a loss of toxicity. Surprisingly, an A92D mutant of CryIAc, very similar to the A92E mutant in this work, lost both toxicity and receptor binding capacity(22) . This differs from our results. At this point, it is not clear what causes the difference in results, but we have repeated our observations using the CryIAc A92D mutant sent to us by D. Wu. Under the binding conditions that they published, we observe no difference in heterologous competition between A92D and CryIAc toxins (data not shown).

The voltage clamp method was used to measure channel function of mutant toxins. In contrast to the binding data, dramatic differences in the inhibition of I are observed ( Fig. 4and Table 1). The differences in the inhibition of I to a large extent are correlated with the toxicity of the mutant toxins. This relationship suggests that steps following initial receptor binding, such as membrane insertion and pore formation, have been altered in these mutant toxins.

The binding of toxins to BBMV may include two major steps: an initial reversible binding and an irreversible binding. The process could be simply expressed as follows: toxin + receptor reversible binding irreversible binding (membrane insertion). Van Rie et al.(5) have reported that toxin binding essentially becomes irreversible. Wolfersberger et al.(31) could not detect toxins on the outer surface or in the inner space of BBMV after a 10-min incubation of toxins with BBMV. However, they have found the intact toxin molecules by denaturing BBMV with detergent. They proposed that soon after binding to cell membrane components, the toxin molecules become integrated into the cell membrane. It is reasonable to consider that irreversible binding corresponds to the membrane integration process. If more toxins are inserted into the membrane, more pores could be formed, and higher toxicity would be observed. The method commonly used to study toxin-BBMV interaction is a competition binding system based on the Scatchard plot, which is best known to fit the reversible ligand binding process, such as antibody-antigen interaction(27) . Application of the Scatchard plot and competition binding analysis to the toxin-BBMV interaction may oversimplify this process. There is no doubt that this type of ligand binding assay can demonstrate the specific or nonspecific binding of a toxin to BBMV, but the binding data cannot be clearly interpreted in terms of both reversible binding (initial binding) and irreversible binding (membrane insertion). English and Slatin (32) proposed that the binding statistics not only represented the initial binding reaction, but also included the contributions from intercalation, oligomerization, and even stable ion channel formation. MacIntosh et al.(33) and Ferréet al.(34) seemed to assume that the competition binding data reflected only the initial association between toxins and BBMV. Our results support the conclusion of these two groups.

The limitations of the competition binding method have led us to design dissociation and association binding assays to study irreversible binding. In the dissociation binding studies, wild type IAb and mutants Y153A and Y153R, both with slightly lower toxicity than the wild type, display much more stable irreversible interaction with BBMV than do the partially toxic Y153D and the nontoxic A92E mutants (Fig. 5). This indicates that toxicity differences among wild type and mutant toxins are correlated with their irreversible binding. The association of toxin with BBMV was further analyzed as a function of incubation time. The association assays clearly demonstrate that the binding of toxin to BBMV includes reversible and irreversible steps. As we can see from Fig. 6, at the beginning of the incubation, the most specific binding is reversible binding, but as the incubation progresses, irreversible binding gradually increases. Wild type toxin irreversibly binds to BBMV in a much faster and more efficient way than do the nontoxic A92E and the partially toxic Y153D mutants. If irreversible binding represents membrane insertion, mutants A92E and Y153D have a very low efficiency of insertion. Therefore, the loss of or large decrease in toxicity observed in these two mutants could be caused by a severely disturbed insertion process. In contrast to A92E and Y153D, mutations Y153A and Y153R result in a slight change in irreversible binding, which correlates with a minor change in toxicity. Ihara et al.(35) have demonstrated that CryIAa is 17-fold more toxic to B. mori than is CryIAb and have observed that the two toxins exhibit similar binding affinity and binding site concentrations with B. mori BBMV, but that CryIAa toxin shows much higher irreversible binding than does CryIAb. They have proposed that the difference in irreversible binding between CryIAa and CryIAb leads to the difference in toxicity. Our results are in agreement with theirs. It is important to point out that insertion is a necessary step for channel formation, and if a B. thuringiensis toxin cannot insert into the cell membrane, the toxin is unlikely to be toxic. Theoretically, insertion may not absolutely result in channel formation. In other words, some insertion could be ineffective because the molecules may not form pores or may form defective pores in the membrane. However, more research needs to be done to understand fully the -endotoxin membrane insertion process.

Another suggestion from this work is that the membrane region where domain I first contacts may be rich in negative charges. Introduction of a negative charge (A92E or Y153D) into the domain I region, which is proposed to start initial insertion after initial receptor binding, disrupts the insertion process. Mutation A92E or Y153D may disrupt the charge balance on the initial membrane contact region of domain I, making this region more negatively charged. If the membrane region is negatively charged, these two regions will repulse instead of attract each other. Results with Y153A and Y153R mutants support this assumption. This conclusion is also in agreement with the observation of Wu and Aronson (22) that mutations at Arg-93 of CryIAc, except R93K, lower toxicity. The different effect of A92E and Y153R could be explained if the region around Ala-92 is more important in membrane insertion than the one around Tyr-153.

In studies of resistance of H. virescens to CryIA toxins, two different groups have found no clear correlation between initial toxin binding and resistance levels(33, 34) . These results indicate that events other than binding could contribute to resistance, affecting the biological activity. Our data have shown that modification of the membrane integration capacity could dramatically change its toxicity. These post-binding steps might be other potential mechanisms of resistance of insects to -endotoxins besides protease activation (36) and receptor binding(8) .

In summary, our results support the model that domain I plays a crucial role in insertion and pore formation rather than receptor binding. The work reported here demonstrates that reduction in irreversible binding (insertion) correlates with reduction in toxicity, broadens our understanding of toxin-BBMV interaction, and favors a multistep toxic mechanism for -endotoxins.


FOOTNOTES

*
This work was supported by Grant R01 AI29092-04A1 from the National Institutes of Health (to D. H. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 614-292-8829; Fax: 614-292-3206 or 6773; dean.10{at}osu.edu.

(^1)
The abbreviations used are: BBMV, brush border membrane vesicle(s); I, short circuit current.

(^2)
X. J. Chen and D. H. Dean, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. T. Yamamoto for providing the oligonucleotides and Dr. D. Zeigler for critical review of the manuscript. We are grateful to Drs. F. Rajamohan and S.-J. Wu for assistance in the immunoblotting assay.


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