©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The CryIA(c) Receptor Purified from Manduca sexta Displays Multiple Specificities (*)

(Received for publication, May 4, 1995)

Luke Masson (1)(§) Yang-jiang Lu (2) Alberto Mazza (1) Roland Brousseau (1) Michael J. Adang (2)

From the  (1)National Research Council of Canada, Biotechnology Research Institute, 6100 Royalmount Ave., Montreal H4P 2R2, Quebec, Canada and the (2)Department of Entomology, University of Georgia, Athens, Georgia 30602-2603

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 (10M s) and a slow reversible off rate (10 s). Although the second CryIA(c)-binding site retained a moderately fast association rate, it was characterized by a rate of dissociation from the aminopeptidase 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 (IC = 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.


INTRODUCTION

During sporulation the Gram-positive bacterium Bacillus thuringiensis produces a variety of intracellular insecticidal crystal proteins in the form of crystalline inclusions. These inclusions, when ingested by susceptible insects, are solubilized in the alkaline environment of the insect midgut where the protoxins undergo enzymatic conversion to the active toxin form by resident gut proteases(1) . After activation, CryI toxins have been shown to bind to specific high affinity receptors on the surface of the midgut epithelial cell layer(2, 3) . Independent studies have shown that B. thuringiensis toxins are able to form ion channels in susceptible cultured insect cells at low concentrations (4) or in artificial lipid bilayers in the complete absence of receptors at high toxin concentrations(5, 6) . Perturbation of the intracellular ionic homeostasis created by these ion channels is thought to ultimately result in cell death by lysis(7) .

Integral to our understanding of the toxin mode of action is the study of toxin interactions with their specific binding proteins. Most of the in vitro studies characterizing these proteins to date have utilized brush border membrane vesicles (BBMVs) (^1)purified from the gut epithelium of susceptible insects (8) or immunochemical staining of midgut sections(9) . Characterization of toxin-binding sites on BBMVs from a variety of larvae has revealed highly complex patterns of toxin binding. The existence of separate distinct classes of toxin-binding proteins as well as single binding sites capable of recognizing multiple toxins has been clearly demonstrated by numerous competition and ligand blotting studies(3, 10, 11, 12, 13, 14, 15, 16, 17) . However, the presence of multiple binding proteins on the surface of BBMVs, high levels of nonspecific binding, and inherent toxin integration into the membrane have made the interpretation of brush border binding results rather difficult and have led to some very complex interaction models(3, 17, 18) . In one notable case, a colony of Trichoplusia ni, having developed resistance to CryIA(b) by laboratory selection, failed to show resistance to CryIA(c) although in vitro binding studies demonstrated they share the same binding site(17) . These results suggest that binding site predictions based on BBMV studies do not necessarily correlate with in vivo toxicity. Clearly, studies on isolated toxin-binding proteins would be crucial in resolving the relationship between binding sites and binding proteins. Recently, three independent sources have described the purification of a 210-kilodalton (kDa) CryIA(b) (19, 20) and a 120-kDa CryIA(c) (21, 22) toxin-binding protein from Manduca sexta. The CryIA(c)-binding protein was functionally determined to be aminopeptidase N (21, 22) whereas the CryIA(b)-binding protein was reported to share sequence similarity with the cadherin family of glycoproteins(20) .

In this study, using surface plasmon resonance (SPR), we provide the first detailed kinetic analysis of the interaction between three B. thuringiensis CryIA toxin subclasses and a solubilized form of the 120-kDa CryIA(c)-binding protein purified from M. sexta. SPR is an optical detection technique which allows direct interaction analysis between a ligand immobilized on a modified dextran sensor chip and a specific analyte in a continuous flow system(23) . The reactants are monitored in real time without the use of labels thus permitting the determinations of kinetic rate constants, binding affinities, and binding site characterization(24, 25, 26) . SPR has been used previously to determine the kinetics of CryIA binding to BBMVs from both the spruce budworm (Choristoneura fumiferana) and the diamondback moth (Plutella xylostella)(27, 28) . In this report, we present a detailed kinetic analysis of CryIA toxin binding interactions with a specific membrane protein purified from the brush border of the lepidopteran larva M. sexta. Detailed evidence is presented showing that two toxins, CryIA(a) and CryIA(b), recognize a single binding site on the purified 115-kDa protein and that CryIA(c) binds to two binding sites. Furthermore, we show that the latter binding is selectively inhibited by the amino sugar N-acetylgalactosamine.


MATERIALS AND METHODS

Toxin Purification

The three recombinant CryIA protoxins from B. thuringiensis kurstaki (strain NRD-12) were expressed in Escherichia coli as cytoplasmic inclusions and purified as described elsewhere(29) . A second CryIA(c) protoxin was isolated from the single gene strain of B. thuringiensis kurstaki (HD-73). All protoxins were activated by solubilization in 40 mM carbonate buffer pH = 10.5 and treated with trypsin (0.1% (w/v) final concentration) for 3 h at ambient temperature. The 65-kDa trypsin-resistant proteins were purified by ion-exchange liquid chromatography using either Mono Q or Q-Sepharose anion exchangers (Pharmacia LKB AB) buffered with 40 mM carbonate buffer pH = 10.5. Bound toxins were eluted using a 50-500 mM NaCl gradient, dialyzed against four changes of distilled water, and the precipitated protein recovered and stored as a concentrated stock in water. Protein levels were quantitated by the dye-binding method of Bradford (30) using bovine serum albumin as a standard. To minimize the formation of multimers, a small aliquot of precipitated toxin was resuspended in HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA) before each experiment and kept on ice(31) . CryIA(c) toxin for radiolabeling was purified by fast protein liquid chromatography and directly iodinated by the chloramine-T method (32) as described in Garczynski et al.(14) . Specific activity of the labeled toxin was about 50 mCi/mg input toxin.

Preparation of Brush Border Membrane Vesicles

M. sexta eggs were obtained from the United States Department of Agriculture, Agricultural Research Services, Biosciences Research Laboratory (Fargo, ND) and larvae reared on artificial diet (Southland Products, Inc., Lake Village, AR). BBMVs were made from second day 5th instar larvae using the MgCl(2) precipitation method of Wolfersberger et al.(8) , except that 1 mM phenylmethylsulfonyl fluoride was included in the final suspension buffer. BBMVs were stored at -80 °C until needed.

Purification of M. sexta 115-kDa Protein

BBMVs (10 mg of protein) were solubilized at 4 °C for 30 min in 2 ml of Buffer A (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) containing 1% CHAPS (v/v). Undissolved material was removed by centrifugation at 100,000 g for 1 h at 4 °C. CHAPS-solubilized BBMVs were chromatographed in Buffer A plus 0.2% CHAPS on a Sephacryl 300 column (Pharmacia). Fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (33) and ligand blotting. Fractions containing the 115-kDa toxin-binding protein were dialyzed against Buffer B (20 mM Tris-HCl, pH 7.4, 0.2% CHAPS) at 4 °C overnight, loaded onto a Mono Q column pre-equilibrated with Buffer B, then the bound proteins eluted with 0-600 mM NaCl in Buffer B.

Ligand Blot Analyses

BBMVs or purified proteins separated by SDS-PAGE were electrophoretically transferred to nitrocellulose filters. Filters were blocked with 5% (w/v) dry milk in TBS-T (20 mM Tris-HCl, pH 8.5, 150 mM NaCl, 0.1% Tween-20) for 1 h, followed by three washes with TBS-T. Filters were bathed with I-CryIA(c) (10^8 counts/min/10 ml TBS-T) for 3 h, washed three times in TBS-T, blotted dry, and exposed to x-ray film at -80 °C.

Instrumentation and Reagents

The surface plasmon resonance detector (BIAcore) system and CM5 sensor chips were obtained from Pharmacia Biosensor. All chemical immobilizations of the M. sexta 115-kDa receptor were done using the standard BIAcore amine coupling protocol provided with the Pharmacia coupling kit. Bovine serum albumin (fraction 5, radioimmunoassay grade) was purchased from Sigma. The buffers used with the BIAcore machine contained the following: HBS-P20 (HBS containing 0.05% BIAcore Surfactant P20), regeneration buffer (50 mM CAPS, pH 11.0, and 150 mM NaCl), carboxymethylated dextran activation solutions (A = 0.1 MN-hydroxysuccinimide, B = 0.1 MN-ethyl-N`-(3-diethylaminopropyl)carbodiimide), coupling buffer (20 mM ammonium acetate, pH 4.5), and deactivation solution (1 M ethanolamine, pH 8.5). Approximately 700-1500 RUs of the 115-kDa binding protein (dissolved as a 0.1 mg/ml stock solution in 20 mM ammonium acetate, pH 5) was amine-coupled to the dextran. A reagent flow rate of 5 µl/min was used during all experiments except for dissociation rates which used 15 µl/min.

Binding Analyses

All sensorgram data transformations and analyses were performed with BIAevaluation software version 2.1. using non-linear least-squares curve fitting. This method of kinetic binding analysis permits the determination of both the dissociation rate constant (k) and the association rate constant (k) for each binding experiment(26, 34) . For all kinetic toxin binding experiments, k was determined first by plotting the log of the drop in response, ln(R(0)/R), against the time interval (t), where R(0) is the response at an arbitrarily chosen starting time soon after toxin flow is replaced by buffer, and Rand t are data points chosen every 0.5 s along the dissociation curve. The apparent k value was used to constrain values when determining the k constant. The maximum binding levels for each toxin was determined on surfaces containing approximately 700-1500 RUs (representing a surface of approximately 6.1-13.1 fmol/mm^2) of immobilized 115-kDa receptor. Since the molecular mass of a protein is proportional to the SPR response (35) , an immobilized surface, representing 1000 RU of the 115-kDa binding protein, could theoretically bind 565 RUs of a 65-kDa toxin analyte (A) in a simple one binding site model (A+B ⇔ AB) on ligand (B). To prevent any influence by toxin rebinding, the immobilized ligand was saturated with analyte and the start point arbitrarily chosen 5 s after the start of buffer flow where the ln(R(0)/R) against (t) curve initially becomes linear.

To determine k, eight different toxin concentrations ranging from 50 to 1500 nM were injected over immobilized APN. As with the kdetermination, all data were fitted to either a one-site (A+B ⇔ AB) or a two-site model (A+B1+B2 ⇔ AB1+AB2). Whenever possible, fitted lines from either model were compared to each other using an F-test comparison. All models were verified by residual plots which calculate the difference between the observed and the fitted curves for each data point. To determine the goodness of fit of the data to the model, both association and dissociation rate constants were chosen from sensorgrams producing a chi^2 value less than one. All models used in our kinetic evaluations were further evaluated by lag plots to determine the relationship of neighboring data points thus providing additional support for binding model selection (data not shown).

Competition Studies

Two types of competition studies were performed. For carbohydrate inhibition experiments, toxins were diluted in HBS-P20 buffer containing varying concentrations of either N-acetyl-D-galactosamine, N-acetyl-D-glucosamine (ICN Biomedicals, Aurora, OH), D-glucose (BDH, Inc., Toronto, Ontario), or D-mannose (Sigma) to a final concentration of 150 nM and injected over an immobilized receptor surface (approximately 1000 RUs). The highest response obtained for each sugar concentration injected over the receptor surface in the absence of toxin was subtracted from the highest RU value obtained in the presence of toxin and the results expressed as percent inhibition. For binding site competition experiments, saturating levels of toxin pairs were injected individually or co-injected over a surface of immobilized receptor.


RESULTS

Purification of 115-kDa CryIA(c)-binding APN

The 120-kDa APN that binds CryIA(c) toxin is cleaved to a 115 kDa form by treatment with PIPLC(36) . The same 120-115 kDa conversion occurs in solubilized preparations of M. sexta BBMVs due to an endogenous PIPLC. (^2)The 115-kDa APN form has the same N terminus, lacks the lipid moiety on the glycosylphosphatidyl inositol membrane anchor, and still binds CryIA(c) toxin. M. sexta BBMVs were solubilized in 1% CHAPS then fractionated by S-300 gel filtration in the presence of 0.2% CHAPS. Protein blots were probed with I-CryIA(c) to identify fractions containing toxin-binding 115-kDa APN. Toxin-binding 115-kDa protein was further purified by a Mono Q chromatography step. Fig. 1shows a stained gel and ligand blot of CHAPS-solubilized BBMVs and purified 115-kDa protein.


Figure 1: SDS-PAGE and ligand blot analyses of solubilized BBMVs and purified 115-kDa protein. Stained SDS-PAGE (A) and protein blot (B) probed with I-CryIA(c). Lane 1 represents the molecular mass markers (indicated in kilodaltons). Lane 2 contained solubilized M. sexta BBMV proteins and lane 3 purified 115-kDa protein.



Stoichiometry and Kinetic Analyses

Increasing concentrations of the different CryI toxin subclasses were injected over a low density surface of immobilized 115-kDa receptor to determine levels required for ligand saturation. As summarized in Fig. 2, CryIA(c) bound to almost a 2-fold higher level than either CryIA(a) or CryIA(b). Considering that the molecular masses of all three activated toxins are essentially the same (65 kDa) and that the SPR response corresponds linearly to the surface protein concentration (35) these results indicate that the immobilized 115-kDa protein possesses a single binding site for either CryIA(a) or CryIA(b) but that CryIA(c) binds to two sites on the same molecule. Injection of a similar concentration of CryIC resulted in little or no specific binding demonstrating the specificity of the immobilized ligand for the three CryIA toxins.


Figure 2: Stoichiometric analysis of CryIA binding. Saturating levels of CryIC, CryIA(a), or CryIA(b) (1500 nM) and of CryIA(c) (1000 nM) were injected at 5 µl/min over a surface of immobilized 115-kDa CryIA(c)-binding protein representing approximately 1000 RU. At the end of the injection, toxin flow was replaced by buffer alone and the sensorgram allowed to continue for an additional 100 s to demonstrate the rate of complex dissociation.



To confirm the number of toxin-binding sites and determine the kinetic rate constants, different toxin concentrations were injected over a surface of immobilized 115-kDa ligand. The binding data from each curve were fitted by non-linear least-squares fitting to either a one- or two-site model. The model fitting further suggested that CryIA(a) and CryIA(b) bound to a single site on the immobilized ligand whereas CryIA(c) best fit a two-site model (F-test comparison between the two models gave a probability = 1). The possibility that the second observed CryIA(c) dissociation rate was caused by the rebinding of toxin to immobilized aminopeptidase was eliminated since a plot of the log of the drop in response against time interval produced a linear rather than a curved response, a normal indicator of rebinding. (^3)Residual plots (i.e. a plot of the difference between the observed and the calculated response for each data point) of the dissociation segment of the response curves were created to verify the appropriateness of the binding model chosen. As shown in Fig. 3, A and B, the data point distribution is random around the x axis and the signal noise is no greater than background (±2 RU) thus indicating that the quality of fit was good for CryIA(a) and CryIA(b) to a single binding site model. The (2) values, testing for the goodness-of-fit, for all the sensorgrams from the different toxin concentrations were <1.0. Fig. 4shows a fitting of the CryIA(c) data to a one-site (A) and a two-site (B) model. The distribution of points for fitting of the data to a one-site model is clearly not random and is greater than background noise suggesting a poor fit. All (2) values for the one-site model were found to be >1.0. If the data are fitted to a model which accounts for two separate toxin-binding sites on the ligand, the point distribution becomes random indicating a good fit. Furthermore, all (2) values obtained from the different sensorgrams fall below 1.0 providing additional support for the two-site hypothesis. Residual plots performed on data from the association area of the sensorgram were similar to those shown above for the dissociation segments (data not shown).


Figure 3: Dissociation rate residual plots for CryIA(a) and CryIA(b). Residual plots, representing the randomness of data point distribution around a fitted curve, are shown for a typical binding data set taken 40 s after the start of complex dissociation for CryIA(a) (A) or CryIA(b) (B) when applied to a one-site model (A+B AB). In the Response plot, the actual dissociation data points are represented by a solid line and the fitted curve by a dashed line. In the Residual plot the response differences (residuals) in RU of the fitted line around the dissociation data are represented by solid dots. A zero difference reference line was added to help visulize the randomness of point distribution.




Figure 4: Dissociation rate residual plots for CryIA(c). A residual plot of a typical CryIA(c) binding data set applied to a one-site model (A+B AB) is shown in panel A, and applied to a two-site model (A+B1+B2 AB1+AB2) shown in panel B. As indicated in Fig. 3, the data set was taken 40 s after the start of complex dissociation. In the Response plot, the actual dissociation data points are represented by a solid line and the fitted curve by a dashed line. In the Residual plot the response differences (residuals) in RU of the fitted line around the dissociation data are represented by solid dots. A zero difference reference line was added to help visulize the randomness of point distribution.



A summary of the apparent rate constants is shown in Table 1. Each toxin studied displayed a moderately fast association rate showing at most a 5-fold variation. The most interesting differences were found in the various dissociation rates. In general, all the E. coli produced NRD-12 toxins showed similar k values; however, the second CryIA(c)-binding site demonstrated a much faster rate (geq an order of magnitude) of toxin-receptor complex dissociation than that calculated for the other CryIA toxins. Furthermore, the B. thuringiensis produced CryIA(c) toxin, although showing k rates indistinguishable from the E. coli produced CryIA(c), demonstrated a 2-fold faster dissociation rate for both sites than the E. coli produced toxin. Despite the observed variations in kinetic rates, the three CryIA toxins from NRD-12 essentially share the same affinity for the immobilized aminopeptidase with CryIA(c) also binding to a second site at a lower affinity.



Competition Analyses

In order to verify whether the multiple binding sites were unique or shared among the three CryIA toxins, saturating levels of toxins were paired together and co-injected over a surface of immobilized ligand. If the toxins bind to separate sites, an additive effect should be observed when comparing the maximal binding response (R(max)) to single toxin injections. As shown in Fig. 5A, pairing of the CryIA(a) and CryIA(b) toxins produced a maximal binding level (R(max)) similar to either of the individual injections. This result agrees with the finding of van Rie et al.(3) using BBMVs from M. sexta that these two toxins share the same site. When the CryIA(b) toxin is paired with the CryIA(c) toxin, an additive effect is again absent. The maximal binding level obtained with the co-injected toxins was similar to CryIA(c) alone suggesting that one of the two CryIA(c)-binding sites is shared with CryIA(b) and by extension, CryIA(a). Experiments using consecutive rather than simultaneous toxin injections were attempted, but the ability of toxins to bind to each other at high concentrations (28) made the data difficult to interpret.


Figure 5: Co-injection of CryIA toxin pairs. Saturating levels of CryIA(a) or CryIA(b) were injected either alone or together over 1000 RU of immobilized aminopeptidase (A). Saturating levels of CryIA(b) or CryIA(c) were injected either alone or together over a similar surface (B).



Carbohydrate Inhibition of Toxin Binding

Various reports have suggested a role for the amino sugar N-acetylgalactosamine (14, 37, 38) as well as mannose (39) in the molecular interaction of toxin with receptor. Varying concentrations of mannose, N-acetylglucosamine, N-acetylgalactosamine, and dextrose were co-injected with a standard concentration (150 nM) of each CryIA toxin. With the exception of N-acetylgalactosamine, no sugar examined had an inhibitory effect on any of the three toxins examined.^3 However, co-injection of N-acetylgalactosamine resulted in binding inhibition with CryIA(c) but not CryIA(a) or CryIA(b) toxins. As shown in Fig. 6, this amino sugar prevented CryIA(c) binding at concentrations as low as 1 mM having an IC = 5 mM. The lack of effect by N-acetylglucosamine suggests that the binding inhibition observed by N-acetylgalactosamine is specific since N-acetylglucosamine is identical in size and charge. Binding inhibition higher than 90% was never obtained even at 75 mM of N-acetylgalactosamine suggesting that factors other than the amino sugar (e.g. protein sequence of the ligand or a different sugar not tested here) are involved in CryIA(c) toxin recognition of the receptor.


Figure 6: Inhibition of CryIA(c) binding by N-acetylgalactosamine. A 150 nM stock solution of CryIA(c) was preincubated with various concentrations of N-acetylgalactosamine for 30 min followed by injection over a low density surface (1000 RU) of APN. The maximum binding level was determined and corrected for refractive index changes caused by the amino sugar, and the level of inhibition when compared to the response in the absence of sugar preincubation was plotted as a function of amino sugar concentration.




DISCUSSION

An essential step in the mode of B. thuringiensis toxin action is the recognition and binding to high affinity sites on the intestinal brush border surface of susceptible insects. Much of our current knowledge of toxin-receptor interactions has been based on studies using vesicles purified from this tissue; however, numerous factors such as multiple Cry-binding proteins, radiolabeling of toxins, and the inherent ability of B. thuringiensis Cry toxins to integrate into lipid bilayers serve to complicate interpretations of binding data from these vesicles. The recent purification and functional identification of a CryIA(c) toxin-binding protein (21) provided a unique opportunity to assess how insecticidal toxins of B. thuringiensis specifically interact, at the molecular level, with a single binding protein in the absence of these complicating factors. This 120-kDa aminopeptidase is normally anchored in brush border membranes of M. sexta by a glycosylphosphatidylinositol anchor(36) . When solubilized by detergents, this form of the protein is tightly complexed with four other proteins. Therefore, in order to purify the CryIA(c)-binding protein to homogeneity, removal of the anchor by a phospholipase C was required. To establish that the removal of this glycosylphosphatidylinositol linkage did not affect the binding characteristics of this protein, CryIA toxin interactions with the purified complex were examined. Preliminary evidence showed that all three CryIA toxins can also bind to the immobilized complex^3 suggesting that the solubilized form of the aminopeptidase does not show any gross alterations in its binding properties.

In this study we used an optical biosensor approach which eliminated the additional complication of using labeled toxin while permitting the measurement of toxin binding in real time. Using four different toxins known to display toxicity toward M. sexta(40) , we show that three CryIA toxins but not CryIC specifically recognized and bound to APN. Two separate lines of evidence involving stoichiometric binding data and non-linear fitting of binding data clearly demonstrated the presence of a single binding site on the aminopeptidase for either CryIA(a) or CryIA(b) toxins whereas binding by a third toxin, CryIA(c), best fitted a two-site model. The fact that multiple toxins bind to a single molecule is not necessarily surprising as Cry toxins tend to be very similar in overall structure despite a large disparity in amino acid composition as recently demonstrated by Grochulski et al.(48) with CryIA(a) (41) and CryIIIA(42) . In contrast to our results, CryIA(b) binding to the 120 kDa protein (glycosylphosphatidylinositol-linked APN) was not observed by Vadlamudi et al.(19) in ligand blots of BBMVs prepared from M. sexta. Instead, binding of CryIA(b) to a 210-kDa protein band was observed. Moreover, two reports (20, 43) suggested that the 210-kDa protein is the binding protein, noted by van Rie et al.(3) , which binds all three CryIA toxins. Unfortunately, with the exception of CryIA(b), this speculation was based solely on ligand blots and not quantitative kinetic or affinity binding data. In light of our results, this discrepancy presumably reflects either the limitations of using denatured membrane proteins with the ligand blotting technique or an even more complex toxin binding pattern than that originally proposed by van Rie et al.(3) . CryIA(c) bound to one site on the APN molecule with an affinity essentially identical to the calculated K values determined for both CryIA(a) and CryIA(b). However, CryIA(c) bound to a second site on the same molecule at a 3-4-fold lower affinity. The kinetics of this lower affinity site were interesting from the viewpoint that the lower affinity was due primarily to a faster dissociation rate especially since the rate of complex association of the lower affinity site was actually higher (more than 3-fold faster) than measured for the high affinity site. This faster dissociation rate was even more pronounced for the CryIA(c) protein produced by the HD-73 strain of B. thuringiensis rather than by E. coli indicating that subtle differences in the primary structure of Cry toxins from the same subgroup may exert a direct effect on binding rates. The NRD-12 CryIA(c) protein used in our studies has four amino acid differences with the HD-73 CryIA(c) sequence as described by Adang et al.(44) . Three of these differences are localized to Domain II, the specificity domain, with two relatively conservative amino acid substitutions (L366F and F439S) and one drastic change, which is the deletion of a negatively charged residue (Asp) at position 442(45) . Additional experiments using E. coli-produced HD-73 toxin should be performed to eliminate the possibility that the differences are due to expression in different hosts rather than sequence differences. However, since only the dissociation rate was affected, it is reasonable to assume that sequence differences rather than some undetermined host factor like toxin glycosylation (46) is the primary reason for the altered dissociation rates.

The affinity constants for the three CryIA toxins determined by SPR varied substantially both in overall affinity and in relation to each other when compared to values derived from M. sexta BBMVs using either displacement or homologous competition experiments(3) . Vesicle binding experiments showed that CryIA(a) and CryIA(c) share similar K values in the 0.2-0.4 nM range with CryIA(b) showing a 3-5-fold lower affinity. Our results show that these three toxins share similar affinities to the solubilized aminopeptidase and that the K values are approximately two orders of magnitude higher than those determined by van Rie et al. (3). We cannot say, at this stage, exactly which factor(s) between the two methods are responsible for the observed differences, but the presence of other biomolecules, particularly lipids, may be partly responsible. The recent affinity determination of CryIA(c) for BBMVs from P. xylostella using SPR(28) , which produced a Konly 2-fold higher than the value determined by equilibrium binding using labeled CryIA(c) and P. xylostella BBMVs(47) , is consistent with this hypothesis and shows that the different techniques can produce similar results.

Competition studies are a limitation of the SPR technique unless one molecule is small and exerts little or no shift in refractive index. In our SPR experiments the optical biosensor could not discriminate between different toxin classes; therefore, we relied on an alternative approach using total binding. In all binary combinations studied, an additive effect was never observed suggesting that the toxins shared a common site. However, considering the size of the toxin molecules in comparison to the toxin-binding protein, one cannot rule out the blocking of separate unique sites on the molecule due to steric hindrance. Because two sites were observed during CryIA(c) binding, it is probable that these two sites are further away from each other. On the other hand, since an additive effect was not observed using CryIA(c) in combination with either of the other CryIA toxins, it is possible that one site is shared whereas the second CryIA(c) site is unique to CryIA(c). An alternative explanation as to why two sites are observed during CryIA(c) binding is that two ligand populations are present (one with and one without a sugar moiety). Therefore, there would only be one binding site normally, but it may or may not be occupied by a sugar molecule. The fact that CryIA(c) toxin binds twice as much as the other two suggests that this is not the case and that there is only one ligand population. Furthermore, both CryIA(c) sites were inhibited by the amino sugar. To eliminate the idea that the 150 nM toxin concentration preferentially showed the high affinity site only, therefore concealing the possibility that the low affinity site was not inhibited, the inhibition experiments were repeated at saturating levels of CryIA(c) toxin. Since no overt differences to the lower toxin concentration were observed (data not shown), it is reasonable to assume that the ligand population was homogeneous in possessing two N-acetylgalactosamine-sensitive sites.

The existence of multiple N-acetylgalactosamine-sensitive CryIA(c)-binding sites on the same molecule may provide an explanation for the observed resistance in the T. ni colony described by Estada and Ferrè(17) . These authors showed that although CryIA(b) and CryIA(c) compete for the same binding site, resistance to CryIA(b) did not automatically translate into resistance to CryIA(c). If CryIA(c) binding to T. ni is also N-acetlygalactosamine sensitive as is the case with M. sexta and other insects(14, 37, 38) , alterations in a CryIA(b)-binding site may not necessarily affect CryIA(c) binding. Alternatively, if the shared site was indeed altered for both toxins, the existence of a second N-acetylgalactosamine CryIA(c)-binding site on the same molecule could account for continued CryIA(c) sensitivity.

So far, with only one exception, carbohydrate inhibition of toxin binding has only been observed with CryIA(c). In accordance with this observation, our results clearly show that in the case of M. sexta, N-acetylgalactosamine is a component of the 115-kDa binding protein. The recognition of this amino sugar occurs only with CryIA(c) thus illustrating the broad heterogeneity of toxin receptors. The combination of having multiple sites on a single receptor molecule and separate receptors for the same toxin may well account for the difficulty in developing insect resistance to B. thuringiensis.


FOOTNOTES

*
This research was supported in part by United States Department of Agriculture/Cooperative State Research Service Competitive Grant 9502312 (to M. J. A.). 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.: 514-496-6150; Fax: 514-496-6213; Masson{at}biotech.lan.nrc.ca.

(^1)
The abbreviations used are: BBMVs, brush border membrane vesicles; SPR, surface plasmon resonance; HBS, HEPES-buffered saline; RU, resonance units; APN, aminopeptidase N; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate; PIPLC, phosphatidylinositol-specific phospholipase C; PAGE, polyacrylamide gel electrophoresis.

(^2)
Lu, Y. J., and Adang, M.(1995) Biochem. Mol. Biol., in press.

(^3)
L. Masson, unpublished observations.


ACKNOWLEDGEMENTS

We thank C. Whalen from Pharmacia for help on model selection and kinetic evaluation, A. M. Mes-Masson and M. O'Connor-McCourt for insightful discussions and critical reading of the manuscript, and finally M. Beauchemin for toxin purification.


REFERENCES

  1. Knowles, B. H. (1994) Adv. Insect Physiol. 24,275-308
  2. Hofmann, C., Luthy, P., Hutter, R., and Pliska, V. (1988) Eur. J. Biochem. 173,85-91 [Abstract]
  3. van Rie, J., Jansens, S., Höfte, H., Degheele, D., and van Mellaert, H. (1989) Eur. J. Biochem. 186,239-247 [Abstract]
  4. Schwartz, J.-L., Garneau, L., Masson, L., and Brousseau, R. (1991) Biochim. Biophys. Acta 1065,250-260 [Medline] [Order article via Infotrieve]
  5. Slatin, S. L., Abrams, C. K., and English, L. (1990) Biochem. Biophys. Res. Commun. 169,765-772 [CrossRef][Medline] [Order article via Infotrieve]
  6. Schwartz, J.-L., Garneau, L., Savaria, D., Masson, L., Brousseau, R., and Rousseau, E. (1993) J. Membr. Biol. 132,53-62 [Medline] [Order article via Infotrieve]
  7. Knowles, B. H., and Ellar, D. J. (1987) Biochim. Biophys. Acta 924,509-518
  8. Wolfersberger, M., Luthy, P., Mauer, A., Parenti, P., Sacchi, V., Giordana, B., and Hanozet, G. (1987) Comp. Biochem. Physiol. 86,301-308 [CrossRef]
  9. Bravo, A., Hendrickx, K., Jansens, S., and Peferoen, M. (1992) J. Invert. Pathol. 60,247-253
  10. Hofmann, C., Vanderbruggen, H., Höfte, H., van Rie, J., Jansens, S., and van Mellaert, H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,7844-7848 [Abstract]
  11. van Rie, J., Jansens, S., Höfte, H., Degheele, D., and van Mellaert, H. (1990) Appl. Env. Microbiol. 56,1378-1385 [Medline] [Order article via Infotrieve]
  12. van Rie J., McGaughey, W. H., Johnson, D. E., Barnett, B. D., and van Mellaert, H. (1990) Science 247,72-74 [Medline] [Order article via Infotrieve]
  13. Wolfersberger, M. G. (1990) Experientia 46,475-477 [Medline] [Order article via Infotrieve]
  14. Garczynski, S. F., Crim, J. W., and Adang, M. J. (1991) Appl. Environ. Microbiol. 57,2816-2820 [Medline] [Order article via Infotrieve]
  15. Ferré, J., Real, M. D., van Rie, J., Jansens, S., and Peferoen, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,5119-5123 [Abstract]
  16. Oddou, P., Hartmann, H., Radecke, F., and Geiser, M. (1993) Eur. J. Biochem. 212,145-150 [Abstract]
  17. Estada, U., and Ferre, J. (1994) Appl. Env. Microbiol. 60,3840-3846 [Abstract]
  18. MacIntosh, S. C., Stone, T. B., Jokerst, R. S., and Fuchs, R. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,8930-8933 [Abstract]
  19. Vadlamudi, R. K., Ji, T. H., and Bulla, L. A., Jr. (1993) J. Biol. Chem. 268,12334-12340 [Abstract/Free Full Text]
  20. Vadlamudi, R. K., Weber, E., Ji, I., Ji, T. H., and Bulla, L. A., Jr. (1995) J. Biol. Chem. 270,5490-5494 [Abstract/Free Full Text]
  21. Sangadala, S., Walters, F. W., English, L. H., and Adang, M. J. (1994) J. Biol. Chem. 269,10088-10092 [Abstract/Free Full Text]
  22. Knight, P. J. K., Crickmore, N., and Ellar, D. J. (1994) Mol. Microbiol. 11,429-436 [Medline] [Order article via Infotrieve]
  23. J ö nsson, U., F ä gerstam, L., Ivarsson, B., Johnsson, B., Karlsson, R., Lundh, K., L ö f, S., Persson, B., Roos, H., R ö nnberg, I., Sj ö lander, S., Stenberg, E., St, R., Urbaniczky, C., Ö stlin, H., and Malmqvist, M. (1991) BioTechniques 11, 620-627 [Medline] [Order article via Infotrieve]
  24. Karlsson, R., Michalesson, A., and Mattsson, L. (1991) J. Immunol. Methods 145,229-240 [CrossRef][Medline] [Order article via Infotrieve]
  25. Fägerstam, L. G., Frostell-Karlsson, Å., Karlsson, R., Persson, B., and Rönnberg, I. (1992) J. Chromatogr. 597,397-410 [CrossRef][Medline] [Order article via Infotrieve]
  26. O'Shannessy, D. J., Brigham-Burke, M., Soneson, K. K., Hensley, P., and Brooks, I. (1993) Anal. Biochem. 212,457-468 [CrossRef][Medline] [Order article via Infotrieve]
  27. Masson, L., Mazza, A., and Brousseau, R. (1994) Anal. Biochem. 218,405-412 [CrossRef][Medline] [Order article via Infotrieve]
  28. Masson, L., Mazza, A., Brousseau, R., and Tabashnik, B. (1995) J. Biol. Chem. 270,11887-11896 [Abstract/Free Full Text]
  29. Masson, L., Préfontaine, G., Péloquin, L., Lau, P. C. K., and Brousseau, R. (1990) Biochem. J. 269,507-512 [Medline] [Order article via Infotrieve]
  30. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 [CrossRef][Medline] [Order article via Infotrieve]
  31. Feng, Q., and Becktel, W. J. (1994) Biochemistry 33,8521-8526 [Medline] [Order article via Infotrieve]
  32. Hunter, W., and Greenwood, F. (1962) Nature 194,495-496
  33. Laemmli, U. K. (1970) Nature 227,680-685 [Medline] [Order article via Infotrieve]
  34. O'Shannessy, D. J. (1994) Curr. Opin. Biotechnol. 5,65-71 [Medline] [Order article via Infotrieve]
  35. Stenberg, E., Persson, B., Roos, H., and Urbaniczky, C. (1991) J. Coll. Int. Sci. 143,513-526
  36. Garczynski, S. F., and Adang, M. J. (1995) Insect Biochem. Mol. Biol., 25, 409-415 [CrossRef]
  37. Knowles, B. H., Knight, P. J., and Ellar, D. J. (1991) Proc. R. Soc. Lond. B. 245,31-35 [Medline] [Order article via Infotrieve]
  38. Knowles, B. H., and Ellar, D. J. (1986) J. Cell Sci. 83,89-101 [Abstract]
  39. Indrasith, L. S., and Hori, H. (1992) Comp. Biochem. Physiol. 102B,605-610
  40. van Frankenhuyzen, K., Gringorten, J. L., Gauthier, D., Milne, R. E., Masson, L., and Peferoen, M. (1993) J. Invert. Pathol. 62,295-301 [CrossRef]
  41. Borisova, S., Grochulski, P., van Faassen, H., Pusztai-Carey, M., Masson, L., and Cygler, M. (1994) J. Mol. Biol. 243,530-532 [CrossRef][Medline] [Order article via Infotrieve]
  42. Li, J., Carrol, J., and Ellar, D. J. (1991) Nature 353,815-821 [CrossRef][Medline] [Order article via Infotrieve]
  43. Martinez-Ramirez, A. C., Gonzalez-Nebauer, S., Escriche, B., and Real, M. D. (1994) Biochem. Biophys. Res. Commun. 201,782-787 [CrossRef][Medline] [Order article via Infotrieve]
  44. Adang, M. J., Staver, M. J., Rocheleau, T. A., Leighton, J., Barker, R. F., and Thompson, D. V. (1985) Gene (Amst.) 36,289-300 [CrossRef][Medline] [Order article via Infotrieve]
  45. Masson, L., Mazza, A., Gringorten, L., Baines, D., Aneliunas, V., and Brousseau, R. (1994) Mol. Microbiol. 14,861-860 [Medline] [Order article via Infotrieve]
  46. Pfannenstiel, M. A., Muthukumar, G., Couche, G. A., and Nickerson, K. (1987) J. Bacteriol. 169,796-801 [Medline] [Order article via Infotrieve]
  47. Tabashnik, B. E., Finson, N., Groeters, F. R., Moar, W. J., Johnson, M. W., Luo, K., and Adang, M. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,4120-4124 [Abstract]
  48. Grochulski, P., Borisova, S., Pusztai-Carey, M., Masson, L., and Cygler, M. (1994) Three-dimensional Crystal Structure of Lepidopteran-specific -Endotoxin CryIA(a) , VIth International Colloquium on Invertebrate Pathology and Microbial Control, Aug. 26-Sept. 2, 1994, Montpellier, France

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