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Proteolytic Processing ofBacillus thuringiensisCryIIIA Toxin and Specific Binding to Brush-Border Membrane Vesicles ofLeptinotarsa decemlineata(Colorado Potato Beetle)
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PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY ARTICLE NO. 0015
54, 115–122 (1996)
Proteolytic Processing of Bacillus thuringiensis CryIIIA Toxin and Specific Binding to Brush-Border Membrane Vesicles of Leptinotarsa decemlineata (Colorado Potato Beetle) A. C. MARTI´NEZ-RAMI´REZ1
AND
M. D. REAL
Departamento de Genética, Universitat de València, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain Received April 12, 1995; accepted December 4, 1995 The mode of action of Bacillus thuringiensis insecticidal proteins in lepidopteran insects is known to involve five steps: ingestion, solubilization, protease activation, binding to midgut membrane receptors, and disruption of the intestinal membrane. Two of these steps, protease activation and binding to midgut membrane receptors, have been analyzed in the major potato pest, the coleoptera Leptinotarsa decemlineata (Colorado potato beetle). Unlike recently proposed, after treatment of the coleopteran-specific B. thuringiensis toxin CryIIIA with gut content from the Colorado potato beetle, a 42-kDa processing polypeptide has been identified. The study of binding to midgut membrane receptors has demonstrated specific and saturable binding of chymotrypsinized CryIIIA to brush-border membrane vesicles from the Colorado potato beetle. The affinity constant and the concentration of binding sites values (Kd 4 37.5 ± 8.6 nM, Rt 4 17 ± 4 pmol/mg of protein) were in the range of the ones previously estimated for low affinity binding sites in lepidopteran insects. Taking into account that CryIIIA can be proteolytically processed by the Colorado potato beetle midgut proteases, along with the fact that, in our hands, binding can be demonstrated only if the toxin is chymotrypsin processed, these results suggest that the mode of action of the coleopteran-specific B. thuringiensis toxin CryIIIA is probably the same as that of lepidopteran-specific toxins. © 1996 Academic Press, Inc.
INTRODUCTION
Leptinotarsa decemlineata (Colorado potato beetle or CPB) is one of the most difficult potato pests to control because it has developed resistance to all synthetic insecticides (1). For this reason and in view of the negative environmental impact of the intensive use of certain pesticides, there is an increasing interest for alternatives to traditional insecticides. The insecticidal activity of the bacterium Bacillus thuringiensis is due to parasporal crystalline proteins called ICPs (insecticidal crystal proteins). According to their sequences and insecticidal spectrum, ICPs are grouped into five classes (2, 3). Class I, III, and IV proteins are active against species of the orders Lepidoptera, Coleoptera, and Diptera, respectively. Class II ICPs are specific for Lepidoptera and Diptera, and class V ICPs are larvicidal to some coleopteran and lepidopteran species. Most of the characterized ICPs show insecti1 To whom correspondence should be addressed. Fax:346-386-43-72.
cidal activity against lepidopteran and dipteran species, and since the isolation of the first coleopteran-specific ICP from B. thuringiensis sp. tenebrionis (4), only a few strains have also been reported to be toxic to coleopteran larvae (5–16). In the past few years, significant knowledge has been obtained on the mode of action of lepidopteran-specific B. thuringiensis ICPs. After ingestion, the ICP is solubilized and proteolytically activated by gut proteases. The processed ICP binds to receptors on the brushborder membrane of the insect midgut, and finally the permeability of the membrane is altered by pore formation. Recently, the structure of the coleopteranspecific ICP CryIIIA was revealed by crystallography (17), confirming the general model previously proposed for B. thuringiensis ICPs (2). The CryIIIA toxin consists of a core built from the five sequence segments conserved among all crystal proteins (2), a pore formation domain, and a binding region. The molecule with the specificity-determining regions situ-
115 0048-3575/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ated outside of the protein structure core perfectly accounts for their function. This structure represents the general fold of this family of insecticidal proteins, as it has recently been corroborated by the crystal structure of one lepidopteran-specific B. thuringiensis toxin, CryIA(a), that shows a similar fold to that of the CryIIIA protein (18). In a wide range of lepidopteran insects, a correlation between high-affinity binding and toxicity has been demonstrated (19–22). However, very little is known on ICP binding in Coleoptera. The CryIIIA ICP is highly toxic to a limited number of coleopteran insects, including CPB larvae (4, 5, 9, 10–12). Specific binding of this ICP in the caudal region of the CPB midgut has immunohistochemically been detected (23) and binding of [125I]-CryIIIA to CPB brushborder membrane vesicles (BBMV) has also been demonstrated (24). Although these findings suggested a similar mode of action of CryIIIA and CryI-type ICPs, a major difference has been reported. While the CryI ICPs are activated by proteolytic cleavage either in vitro (when treated with proteases) or in vivo (when exposed to insect digestive fluids), the intact CryIIIA has been proposed to be the toxic form (5, 24). Moreover, when ICP affinities for their receptors are compared, an additional difference with CryI-type ICPs can be noted. The estimated affinity of CryIIIA to BBMV from CPB is 80- to 4000-fold lower (Kd 4 410 nM) (24) than the ones of CryIA-type ICPs in lepidopteran insects (Kd 4 0.1–5 nM) (19–22, 25–27). Taking into account these two abovementioned differences with the mode of action of lepidopteran-specific ICPs, two steps of the known mode of action of B. thuringiensis ICPs (activation and binding) have been reexamined, by analyzing the in vivo CryIIIA processing and the binding of the in vitro digested CryIIIA. MATERIALS AND METHODS
Insects CPB larvae and adults were reared on potato leaves at 25 ± 1°C and a photoperiod of 16:8 (light:dark).
Bacillus thuringiensis ICPs CryIIIA and CryIIIC crystal proteins, prepared and purified as described in Lambert et al. (15), were kindly provided by Plant Genetic Systems (Ghent, Belgium). Chymotrypsin Digestion of CryIIIA CryIIIA ICP (67 kDa) was chymotrypsinized following the procedure of Carrol et al. (28). Solubilized CryIIIA (in 3.3 M NaBr, 200 mM Tris–HCl, pH 8.0) was digested with chymotrypsin (2:1, w/w, enzyme:substrate ratio) at 25°C for 8 hr. The digestion was stopped by addition of 0.1 mM PMSF. The mixture was dialyzed against 20 mM piperazine, pH 10.0, at 4°C and loaded on a Mono Q HR 5/5 column (Pharmacia). The proteins were eluted by a 0–0.6 M NaCl gradient in 20 mM piperazine, pH 10.0. Fractions containing the CryIIIA ICP were pooled and the amount of protein was determined according to Bradford (29). Iodination of Chymotrypsinized CryIIIA Chymotrypsinized CryIIIA was dialyzed against 20 mM Tris–HCl, 200 mM NaCl, pH 8.6, at 4°C. The digested CryIIIA was iodinated by the chloramine-T method (30). To 20 mg of processed CryIIIA, 1 mCi of Na125I and 20 mg of chloramine-T were added in phosphatebuffered saline. After 20 sec, the reaction was stopped by adding 53 mg of sodium metabisulfite. This mixture was loaded onto a Bio-Gel P-30 (Bio-Rad Laboratories) column to remove free iodine and possible degradation products. The specific radioactivity of iodinated chymotrypsinized CryIIIA was 11.6 mCi/mg of protein. Preparation of Brush-Border Membrane Vesicles (BBMV) BBMV were prepared from last instar CPB larvae according to the method of Biber et al. (31) as modified by Wolfersberger et al. (32). The final pellet was resuspended in ice-cold MET buffer (0.3 M mannitol, 5 mM EGTA, 17 mM Tris–HCl, pH 7.5) and immediately frozen and stored at −80°C until use.
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117 Binding Assays Immediately before use in the assays, the storage buffer of BBMV was replaced by microfuge centrifugation with PBS, pH 7.4, 0.1% BSA. BBMV and iodinated chymotrypsinized CryIIIA were incubated in PBS, pH 7.4, 0.1% BSA for 60 min at room temperature. The reaction volume was 100 ml and all samples were duplicated. After incubation, reactions were filtered through Whatman GF/F glass-fiber filters in a Millipore filtration manifold (1225 Unit). Filters were washed with 5 ml of ice-cold PBS, pH 7.4, 0.1% BSA and transferred to microtubes. The radioactivity retained in the filters was measured in a 1282 Compugamma CS counter (LKB). For competition experiments the reaction mixtures contained 10 mg of BBMV proteins and 0.62 nM labeled chymotrypsinized CryIIIA. The binding constants (Kd, affinity constant, and Rt, binding-site concentration) were estimated using the LIGAND program (33). Proteolytic Processing of Solubilized CryIIIA by CPB Gut Content The proteolytic stability of CryIIIA in CPB digestive content was analyzed by SDS–PAGE. The digestive content of CPB was obtained by forcing larvae to vomit (by gently pressing their heads). Solubilized CryIIIA (3 mg/ml in 50 mM NaHCO3, 5 mM dithiothreitol, pH 9.5 buffer) was added to 3% (v/v) CPB digestive fluid in PBS buffer (8 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl), pH 6.0, and incubated at room temperature. At intervals, aliquots were taken and processing was stopped by addition of Laemmli buffer and heating at 98°C for 5 min. Samples were then loaded on a 10% SDS– polyacrylamide gel. Incubated samples containing either CPB digestive fluid or CryIIIA were included as controls. SDS–PAGE gels were stained with Coomassie blue R. RESULTS
Binding of Chymotrypsinized CryIIIA Using 125I-labeled CryI ICPs, a correlation between toxicity and binding to BBMV has been demonstrated (19–22). Toxicity against
CPB of the purified CryIIIA used in this work was previously demonstrated by Lambert et al. (15) (LC50 3.5 mg protein/ml). However, in our hands, CryIIIA, either intact or trypsinized, could not bind to BBMV from CPB. Carroll (28) demonstrated that CryIIIA can be proteolytically processed by chymotrypsin to a 49-kDa resistant fragment. In order to investigate whether the chymotrypsinized CryIIIA binds to BBMV from CPB, the procedure of Carroll (28) was followed and binding of the resulting polypeptide to BBMV was analyzed. As expected, the action of chymotrypsin on CryIIIA resulted in a 49-kDa polypeptide purified by anion exchange chromatography, as described under Materials and Methods. Results are shown in Fig. 1. Chymotrypsinized CryIIIA eluted at 0.45 M NaCl from the Mono Q column. Fractions containing the chymotrypsin fragment were pooled and their purity was checked by SDS–PAGE (Fig. 2). Chymotrypsinized CryIIIA was iodinated as described under Materials and Methods. 125Ilabeled chymotrypsinized CryIIIA was incubated with increasing concentrations of BBMV from CPB. Binding in the presence of excess unlabeled ligand was substracted from the total binding for each data point (Fig. 3). Saturable binding was obtained, with maximum binding at a concentration of 300 mg of BBMV proteins/ ml. Only 7.6% of the 125I-labeled ICP was specifically bound to 200 mg BBMV proteins/ml. To obtain quantitative estimates of CryIIIA binding characteristics, homologous competition experiments (competition between a labeled ligand and its unlabeled analog) were performed (Fig. 4). From triplicate experiments, an estimated dissociation constant (Kd) of 37.5 ± 8.6 nM and a receptor concentration (Rt) of 17 ± 4 pmol/mg of protein were calculated. Heterologous competition experiments (competition between labeled chymotrypsinized CryIIIA and unlabeled CryIIIC ICP) showed no competition of CryIIIC for the CryIIIA binding site (Fig. 4). Proteolytic Processing of Solubilized CryIIIA by CPB Gut Content Since CryIIIA bound to BBMV from CPB
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FIG. 1. Chromatographic profile of the elution of chymotrypsin digestion of the CryIIIA toxin. Conditions: column, Mono Q HR 5/5; buffer A, 20 mM piperazine, pH 10.0, buffer B, 20 mM piperazine, pH 10.0, 0.6 M NaCl; gradient, 0–100% B in 20 min; flow rate, 1.0 ml/min; detection, absorbance at 280 nm, 0.05 AUFS.
only after chymotrypsin treatment, processing of the native CryIIIA by CPB gut content was analyzed. Solubilized CryIIIA was incubated with CPB gut content and the processing of this ICP was analyzed by SDS–PAGE (Fig. 5). The time required to completely process the CryIIIA toxin depended on the gut content concentration, and, under the conditions described under Materials and Methods, was approximately 60 min (lane 9). Just as the toxin was mixed with the gut content (lane 6), a 44-kDa band, absent in controls lacking either toxin or CPB intestinal juice, appeared. In the experiment shown in Fig. 5, from 15 min on, a gradual decrease of the 44kDa band, in addition to the accumulation of a 42-kDa band (lanes 7 and 8), absent in the corresponding controls (lanes 2 and 3), was observed. Bands at 60, 58, and 35 kDa were already present in the CryIIIA toxin used (data not shown). DISCUSSION
It has been proposed that CryIIIA is not pro-
teolytically activated when treated with papain, trypsin, or gut juice from Tenebrio molitor or CPB at a visible lower molecular weight form (5, 24). This feature would appear to distinguish CryIII class ICPs from other classes studied so far, which have been shown to be synthesized as protoxins that are proteolytically activated in the larval gut (34). However, Carroll and colleagues (28, 35) demonstrated that the 67-kDa CryIIIA protein is cleaved by proteases (trypsin,
FIG. 2. SDS/10% polyacrylamide gel (Coomassie blue stained) of Mono Q fractions containing the chymotrypsin fragment. Lane 1, molecular weight markers (97, 66, 45, 31, and 22 kDa); lane 2, CryIIIA toxin; chymotrypsinized CryIIIA.
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FIG. 3. Binding of 125I-chymotrypsinized CryIIIA as a function of the concentration of brush-border membrane vesicles of Leptinotarsa decemlineata. ■ , Total binding; u , nonspecific binding. Assay conditions are described under Materials and Methods.
chymotrypsin, papain, and Aspergillus acid proteinase) or insect gut content from T. molitor and Phaedon cochleariae to either a 55- or 49kDa product. Carroll also demonstrated that the toxicity of the 55- and 49-kDa digest products
against Phaedon cocheariae was comparable with the native CryIIIA and that the digests were also as effective as the native ICP in reducing the weight gain of T. molitor larvae (28). According to Carroll (28) we have found that
FIG. 4. Binding of 125I-chymotrypsinized CryIIIA to brush-border membrane vesicles of Leptinotarsa decemlineata at increasing concentrations of nonlabeled competitor. Binding is expressed as a percentage of the amount bound upon incubation with labeled toxin alone. Curve is the one predicted by the Ligand computer program. Each point is the mean or triplicate samples. v, Chymotrypsinized CryIIIA; u , CryIIIC.
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FIG. 5. Proteolytic digestion of CryIIIA toxin using Leptinotarsa decemlineata larval gut extracts. Lanes 1 to 4, 3% (v/v) digestive fluid in PBS buffer, pH 6.0, incubated at room temperature for 0, 15, 30, and 60 min. Lane 5, molecular weight markers (97, 66, 45, 31, and 22 kDa). Lanes 6 to 9, as in lanes 1 to 4 but plus 10 µg of CryIIIA. Arrow indicates the position of the CryIIIA toxin.
CryIIIA is processed to a 49-kDa polypeptide when exposed to chymotrypsin. After treatment of CryIIIA with gut content from CPB, we have identified a 42-kDa polypeptide, absent in the corresponding controls. This proteolytic fragment probably came from the 44-kDa product that immediately appeared when CryIIIA toxin and CPB gut content were mixed. This finding differs from that of Slaney et al. (24) who reported that treatment of CryIIIA with CPB intestinal juice did not yield a visible hydrolysis fragment. It is likely that some of their digestion conditions, such as the incubation buffer (2[N-morpholino]ethanesulfonic acid) or the addition of 0.5 M NaOH to arrest proteolysis, would alter the behavior of the digestive enzymes or the stability of the proteolysis products. Moreover, they did not provide details of the gut content:toxin ratios, thus it was not possible to reproduce their experiment. It seems clear that the ratios that Slaney et al. used were greater than ours because under their conditions the toxin was completely digested in approximately 10 min, whereas in our assay it was still visible after 1 hr of incubation at room temperature. Whether chymotrypsin and gut enzyme treat-
ment will be having the same effect on the toxin remains an unknown question that will require further studies to be elucidated. Nevertheless, the results showed that CryIIIA can be processed by either a specific protease (chymotrypsin) or gut enzymes from CPB as lepidopteranspecific toxins also are. The next step in the mode of action of CryI proteins, binding to the brush-border membrane of the insect gut, has also been studied with CryIIIA in CPB. Binding of chymotrypsinized CryIIIA to specific sites in BBMV prepared from CPB midgut has been demonstrated (24). In our hands neither intact nor trypsinized CryIIIA specifically bound to BBMV from CPB. However, ligand binding assays with chymotrypsinized CryIIIA showed a single binding site with Kd 4 37.5 nM, in BBMV from CPB. This result is in contrast to Slaney et al. (24) who reported binding of intact CryIIIA to BBMV from CPB. Nevertheless, it has to be pointed out that their dissociation constant for binding of intact CryIIIA to BBMV from CPB (Kd 4 410 nM) is about 10 times higher than the one we have estimated for chymotrypsinized CryIIIA. Moreover, to our knowledge, the dissociation constant of intact CryIIIA for its
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121 binding sites is the highest described to date for any B. thuringiensis toxin–insect combination. This means that its affinity is even lower than the lowest affinity described in lepidopteran insects, such as in Manduca sexta or Spodoptera littoralis with CryIE (M. sexta, CryIE Kd2 4 215 ± 155; S. littoralis, CryIE Kd2 4 352 ± 16) (22). On the other hand, a correlation between toxicity and binding to membranes from larval midgut has been previously suggested (19–22). Since CPB is about equally susceptible to CryIIIA (15, 24) than M. sexta or Heliothis virescens to active CryIA-type toxins, no significant differences in the affinity for their receptors would therefore be expected. Unlike what one might have assumed, the affinity of both intact and chymotrypsinized CryIIIA is significantly lower than that of lepidopteran toxins. Nevertheless, when comparing with lepidopteran insects, although the affinity constant of chymotrypsinized CryIIIA is not as great as one might hope for, it is 10-fold higher than the one obtained by Slaney for intact CryIIIA. This difference could indicate that other factors, such as an appropriate processing of the toxin prior to binding, will determine the toxicity levels. The present study on two steps of the mode of action of CryIIIA, activation and binding, has shown that this toxin can be proteolytically cleaved by CPB midgut proteases to a 42-kDa fragment and that the chymotrypsin-processed CryIIIA binds to midgut BBMV of this insect with higher affinity than the intact toxin. These results suggest that the mode of action of the coleopteran-specific B. thuringiensis toxin CryIIIA is similar to that of lepidopteranspecific toxins. ACKNOWLEDGMENTS We thank Plant Genetic Systems (Ghent, Belgium) and particularly J. Van Rie for his technical support on binding experiments. We are also very grateful to Dr. David Ellar and Dr. Joe Carroll for reading and helpful comments on the preparation of the manuscript. This work was funded by a grant from the EC under the ECLAIR program (Project AGRE-0003). A. C. Martínez-Ramírez was supported by a grant from the Consellería de Educación y Ciencia (Generalidad Valenciana).
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PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY ARTICLE NO. 0015
54, 115–122 (1996)
Proteolytic Processing of Bacillus thuringiensis CryIIIA Toxin and Specific Binding to Brush-Border Membrane Vesicles of Leptinotarsa decemlineata (Colorado Potato Beetle) A. C. MARTI´NEZ-RAMI´REZ1
AND
M. D. REAL
Departamento de Genética, Universitat de València, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain Received April 12, 1995; accepted December 4, 1995 The mode of action of Bacillus thuringiensis insecticidal proteins in lepidopteran insects is known to involve five steps: ingestion, solubilization, protease activation, binding to midgut membrane receptors, and disruption of the intestinal membrane. Two of these steps, protease activation and binding to midgut membrane receptors, have been analyzed in the major potato pest, the coleoptera Leptinotarsa decemlineata (Colorado potato beetle). Unlike recently proposed, after treatment of the coleopteran-specific B. thuringiensis toxin CryIIIA with gut content from the Colorado potato beetle, a 42-kDa processing polypeptide has been identified. The study of binding to midgut membrane receptors has demonstrated specific and saturable binding of chymotrypsinized CryIIIA to brush-border membrane vesicles from the Colorado potato beetle. The affinity constant and the concentration of binding sites values (Kd 4 37.5 ± 8.6 nM, Rt 4 17 ± 4 pmol/mg of protein) were in the range of the ones previously estimated for low affinity binding sites in lepidopteran insects. Taking into account that CryIIIA can be proteolytically processed by the Colorado potato beetle midgut proteases, along with the fact that, in our hands, binding can be demonstrated only if the toxin is chymotrypsin processed, these results suggest that the mode of action of the coleopteran-specific B. thuringiensis toxin CryIIIA is probably the same as that of lepidopteran-specific toxins. © 1996 Academic Press, Inc.
INTRODUCTION
Leptinotarsa decemlineata (Colorado potato beetle or CPB) is one of the most difficult potato pests to control because it has developed resistance to all synthetic insecticides (1). For this reason and in view of the negative environmental impact of the intensive use of certain pesticides, there is an increasing interest for alternatives to traditional insecticides. The insecticidal activity of the bacterium Bacillus thuringiensis is due to parasporal crystalline proteins called ICPs (insecticidal crystal proteins). According to their sequences and insecticidal spectrum, ICPs are grouped into five classes (2, 3). Class I, III, and IV proteins are active against species of the orders Lepidoptera, Coleoptera, and Diptera, respectively. Class II ICPs are specific for Lepidoptera and Diptera, and class V ICPs are larvicidal to some coleopteran and lepidopteran species. Most of the characterized ICPs show insecti1 To whom correspondence should be addressed. Fax:346-386-43-72.
cidal activity against lepidopteran and dipteran species, and since the isolation of the first coleopteran-specific ICP from B. thuringiensis sp. tenebrionis (4), only a few strains have also been reported to be toxic to coleopteran larvae (5–16). In the past few years, significant knowledge has been obtained on the mode of action of lepidopteran-specific B. thuringiensis ICPs. After ingestion, the ICP is solubilized and proteolytically activated by gut proteases. The processed ICP binds to receptors on the brushborder membrane of the insect midgut, and finally the permeability of the membrane is altered by pore formation. Recently, the structure of the coleopteranspecific ICP CryIIIA was revealed by crystallography (17), confirming the general model previously proposed for B. thuringiensis ICPs (2). The CryIIIA toxin consists of a core built from the five sequence segments conserved among all crystal proteins (2), a pore formation domain, and a binding region. The molecule with the specificity-determining regions situ-
115 0048-3575/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ated outside of the protein structure core perfectly accounts for their function. This structure represents the general fold of this family of insecticidal proteins, as it has recently been corroborated by the crystal structure of one lepidopteran-specific B. thuringiensis toxin, CryIA(a), that shows a similar fold to that of the CryIIIA protein (18). In a wide range of lepidopteran insects, a correlation between high-affinity binding and toxicity has been demonstrated (19–22). However, very little is known on ICP binding in Coleoptera. The CryIIIA ICP is highly toxic to a limited number of coleopteran insects, including CPB larvae (4, 5, 9, 10–12). Specific binding of this ICP in the caudal region of the CPB midgut has immunohistochemically been detected (23) and binding of [125I]-CryIIIA to CPB brushborder membrane vesicles (BBMV) has also been demonstrated (24). Although these findings suggested a similar mode of action of CryIIIA and CryI-type ICPs, a major difference has been reported. While the CryI ICPs are activated by proteolytic cleavage either in vitro (when treated with proteases) or in vivo (when exposed to insect digestive fluids), the intact CryIIIA has been proposed to be the toxic form (5, 24). Moreover, when ICP affinities for their receptors are compared, an additional difference with CryI-type ICPs can be noted. The estimated affinity of CryIIIA to BBMV from CPB is 80- to 4000-fold lower (Kd 4 410 nM) (24) than the ones of CryIA-type ICPs in lepidopteran insects (Kd 4 0.1–5 nM) (19–22, 25–27). Taking into account these two abovementioned differences with the mode of action of lepidopteran-specific ICPs, two steps of the known mode of action of B. thuringiensis ICPs (activation and binding) have been reexamined, by analyzing the in vivo CryIIIA processing and the binding of the in vitro digested CryIIIA. MATERIALS AND METHODS
Insects CPB larvae and adults were reared on potato leaves at 25 ± 1°C and a photoperiod of 16:8 (light:dark).
Bacillus thuringiensis ICPs CryIIIA and CryIIIC crystal proteins, prepared and purified as described in Lambert et al. (15), were kindly provided by Plant Genetic Systems (Ghent, Belgium). Chymotrypsin Digestion of CryIIIA CryIIIA ICP (67 kDa) was chymotrypsinized following the procedure of Carrol et al. (28). Solubilized CryIIIA (in 3.3 M NaBr, 200 mM Tris–HCl, pH 8.0) was digested with chymotrypsin (2:1, w/w, enzyme:substrate ratio) at 25°C for 8 hr. The digestion was stopped by addition of 0.1 mM PMSF. The mixture was dialyzed against 20 mM piperazine, pH 10.0, at 4°C and loaded on a Mono Q HR 5/5 column (Pharmacia). The proteins were eluted by a 0–0.6 M NaCl gradient in 20 mM piperazine, pH 10.0. Fractions containing the CryIIIA ICP were pooled and the amount of protein was determined according to Bradford (29). Iodination of Chymotrypsinized CryIIIA Chymotrypsinized CryIIIA was dialyzed against 20 mM Tris–HCl, 200 mM NaCl, pH 8.6, at 4°C. The digested CryIIIA was iodinated by the chloramine-T method (30). To 20 mg of processed CryIIIA, 1 mCi of Na125I and 20 mg of chloramine-T were added in phosphatebuffered saline. After 20 sec, the reaction was stopped by adding 53 mg of sodium metabisulfite. This mixture was loaded onto a Bio-Gel P-30 (Bio-Rad Laboratories) column to remove free iodine and possible degradation products. The specific radioactivity of iodinated chymotrypsinized CryIIIA was 11.6 mCi/mg of protein. Preparation of Brush-Border Membrane Vesicles (BBMV) BBMV were prepared from last instar CPB larvae according to the method of Biber et al. (31) as modified by Wolfersberger et al. (32). The final pellet was resuspended in ice-cold MET buffer (0.3 M mannitol, 5 mM EGTA, 17 mM Tris–HCl, pH 7.5) and immediately frozen and stored at −80°C until use.
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117 Binding Assays Immediately before use in the assays, the storage buffer of BBMV was replaced by microfuge centrifugation with PBS, pH 7.4, 0.1% BSA. BBMV and iodinated chymotrypsinized CryIIIA were incubated in PBS, pH 7.4, 0.1% BSA for 60 min at room temperature. The reaction volume was 100 ml and all samples were duplicated. After incubation, reactions were filtered through Whatman GF/F glass-fiber filters in a Millipore filtration manifold (1225 Unit). Filters were washed with 5 ml of ice-cold PBS, pH 7.4, 0.1% BSA and transferred to microtubes. The radioactivity retained in the filters was measured in a 1282 Compugamma CS counter (LKB). For competition experiments the reaction mixtures contained 10 mg of BBMV proteins and 0.62 nM labeled chymotrypsinized CryIIIA. The binding constants (Kd, affinity constant, and Rt, binding-site concentration) were estimated using the LIGAND program (33). Proteolytic Processing of Solubilized CryIIIA by CPB Gut Content The proteolytic stability of CryIIIA in CPB digestive content was analyzed by SDS–PAGE. The digestive content of CPB was obtained by forcing larvae to vomit (by gently pressing their heads). Solubilized CryIIIA (3 mg/ml in 50 mM NaHCO3, 5 mM dithiothreitol, pH 9.5 buffer) was added to 3% (v/v) CPB digestive fluid in PBS buffer (8 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl), pH 6.0, and incubated at room temperature. At intervals, aliquots were taken and processing was stopped by addition of Laemmli buffer and heating at 98°C for 5 min. Samples were then loaded on a 10% SDS– polyacrylamide gel. Incubated samples containing either CPB digestive fluid or CryIIIA were included as controls. SDS–PAGE gels were stained with Coomassie blue R. RESULTS
Binding of Chymotrypsinized CryIIIA Using 125I-labeled CryI ICPs, a correlation between toxicity and binding to BBMV has been demonstrated (19–22). Toxicity against
CPB of the purified CryIIIA used in this work was previously demonstrated by Lambert et al. (15) (LC50 3.5 mg protein/ml). However, in our hands, CryIIIA, either intact or trypsinized, could not bind to BBMV from CPB. Carroll (28) demonstrated that CryIIIA can be proteolytically processed by chymotrypsin to a 49-kDa resistant fragment. In order to investigate whether the chymotrypsinized CryIIIA binds to BBMV from CPB, the procedure of Carroll (28) was followed and binding of the resulting polypeptide to BBMV was analyzed. As expected, the action of chymotrypsin on CryIIIA resulted in a 49-kDa polypeptide purified by anion exchange chromatography, as described under Materials and Methods. Results are shown in Fig. 1. Chymotrypsinized CryIIIA eluted at 0.45 M NaCl from the Mono Q column. Fractions containing the chymotrypsin fragment were pooled and their purity was checked by SDS–PAGE (Fig. 2). Chymotrypsinized CryIIIA was iodinated as described under Materials and Methods. 125Ilabeled chymotrypsinized CryIIIA was incubated with increasing concentrations of BBMV from CPB. Binding in the presence of excess unlabeled ligand was substracted from the total binding for each data point (Fig. 3). Saturable binding was obtained, with maximum binding at a concentration of 300 mg of BBMV proteins/ ml. Only 7.6% of the 125I-labeled ICP was specifically bound to 200 mg BBMV proteins/ml. To obtain quantitative estimates of CryIIIA binding characteristics, homologous competition experiments (competition between a labeled ligand and its unlabeled analog) were performed (Fig. 4). From triplicate experiments, an estimated dissociation constant (Kd) of 37.5 ± 8.6 nM and a receptor concentration (Rt) of 17 ± 4 pmol/mg of protein were calculated. Heterologous competition experiments (competition between labeled chymotrypsinized CryIIIA and unlabeled CryIIIC ICP) showed no competition of CryIIIC for the CryIIIA binding site (Fig. 4). Proteolytic Processing of Solubilized CryIIIA by CPB Gut Content Since CryIIIA bound to BBMV from CPB
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FIG. 1. Chromatographic profile of the elution of chymotrypsin digestion of the CryIIIA toxin. Conditions: column, Mono Q HR 5/5; buffer A, 20 mM piperazine, pH 10.0, buffer B, 20 mM piperazine, pH 10.0, 0.6 M NaCl; gradient, 0–100% B in 20 min; flow rate, 1.0 ml/min; detection, absorbance at 280 nm, 0.05 AUFS.
only after chymotrypsin treatment, processing of the native CryIIIA by CPB gut content was analyzed. Solubilized CryIIIA was incubated with CPB gut content and the processing of this ICP was analyzed by SDS–PAGE (Fig. 5). The time required to completely process the CryIIIA toxin depended on the gut content concentration, and, under the conditions described under Materials and Methods, was approximately 60 min (lane 9). Just as the toxin was mixed with the gut content (lane 6), a 44-kDa band, absent in controls lacking either toxin or CPB intestinal juice, appeared. In the experiment shown in Fig. 5, from 15 min on, a gradual decrease of the 44kDa band, in addition to the accumulation of a 42-kDa band (lanes 7 and 8), absent in the corresponding controls (lanes 2 and 3), was observed. Bands at 60, 58, and 35 kDa were already present in the CryIIIA toxin used (data not shown). DISCUSSION
It has been proposed that CryIIIA is not pro-
teolytically activated when treated with papain, trypsin, or gut juice from Tenebrio molitor or CPB at a visible lower molecular weight form (5, 24). This feature would appear to distinguish CryIII class ICPs from other classes studied so far, which have been shown to be synthesized as protoxins that are proteolytically activated in the larval gut (34). However, Carroll and colleagues (28, 35) demonstrated that the 67-kDa CryIIIA protein is cleaved by proteases (trypsin,
FIG. 2. SDS/10% polyacrylamide gel (Coomassie blue stained) of Mono Q fractions containing the chymotrypsin fragment. Lane 1, molecular weight markers (97, 66, 45, 31, and 22 kDa); lane 2, CryIIIA toxin; chymotrypsinized CryIIIA.
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FIG. 3. Binding of 125I-chymotrypsinized CryIIIA as a function of the concentration of brush-border membrane vesicles of Leptinotarsa decemlineata. ■ , Total binding; u , nonspecific binding. Assay conditions are described under Materials and Methods.
chymotrypsin, papain, and Aspergillus acid proteinase) or insect gut content from T. molitor and Phaedon cochleariae to either a 55- or 49kDa product. Carroll also demonstrated that the toxicity of the 55- and 49-kDa digest products
against Phaedon cocheariae was comparable with the native CryIIIA and that the digests were also as effective as the native ICP in reducing the weight gain of T. molitor larvae (28). According to Carroll (28) we have found that
FIG. 4. Binding of 125I-chymotrypsinized CryIIIA to brush-border membrane vesicles of Leptinotarsa decemlineata at increasing concentrations of nonlabeled competitor. Binding is expressed as a percentage of the amount bound upon incubation with labeled toxin alone. Curve is the one predicted by the Ligand computer program. Each point is the mean or triplicate samples. v, Chymotrypsinized CryIIIA; u , CryIIIC.
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FIG. 5. Proteolytic digestion of CryIIIA toxin using Leptinotarsa decemlineata larval gut extracts. Lanes 1 to 4, 3% (v/v) digestive fluid in PBS buffer, pH 6.0, incubated at room temperature for 0, 15, 30, and 60 min. Lane 5, molecular weight markers (97, 66, 45, 31, and 22 kDa). Lanes 6 to 9, as in lanes 1 to 4 but plus 10 µg of CryIIIA. Arrow indicates the position of the CryIIIA toxin.
CryIIIA is processed to a 49-kDa polypeptide when exposed to chymotrypsin. After treatment of CryIIIA with gut content from CPB, we have identified a 42-kDa polypeptide, absent in the corresponding controls. This proteolytic fragment probably came from the 44-kDa product that immediately appeared when CryIIIA toxin and CPB gut content were mixed. This finding differs from that of Slaney et al. (24) who reported that treatment of CryIIIA with CPB intestinal juice did not yield a visible hydrolysis fragment. It is likely that some of their digestion conditions, such as the incubation buffer (2[N-morpholino]ethanesulfonic acid) or the addition of 0.5 M NaOH to arrest proteolysis, would alter the behavior of the digestive enzymes or the stability of the proteolysis products. Moreover, they did not provide details of the gut content:toxin ratios, thus it was not possible to reproduce their experiment. It seems clear that the ratios that Slaney et al. used were greater than ours because under their conditions the toxin was completely digested in approximately 10 min, whereas in our assay it was still visible after 1 hr of incubation at room temperature. Whether chymotrypsin and gut enzyme treat-
ment will be having the same effect on the toxin remains an unknown question that will require further studies to be elucidated. Nevertheless, the results showed that CryIIIA can be processed by either a specific protease (chymotrypsin) or gut enzymes from CPB as lepidopteranspecific toxins also are. The next step in the mode of action of CryI proteins, binding to the brush-border membrane of the insect gut, has also been studied with CryIIIA in CPB. Binding of chymotrypsinized CryIIIA to specific sites in BBMV prepared from CPB midgut has been demonstrated (24). In our hands neither intact nor trypsinized CryIIIA specifically bound to BBMV from CPB. However, ligand binding assays with chymotrypsinized CryIIIA showed a single binding site with Kd 4 37.5 nM, in BBMV from CPB. This result is in contrast to Slaney et al. (24) who reported binding of intact CryIIIA to BBMV from CPB. Nevertheless, it has to be pointed out that their dissociation constant for binding of intact CryIIIA to BBMV from CPB (Kd 4 410 nM) is about 10 times higher than the one we have estimated for chymotrypsinized CryIIIA. Moreover, to our knowledge, the dissociation constant of intact CryIIIA for its
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121 binding sites is the highest described to date for any B. thuringiensis toxin–insect combination. This means that its affinity is even lower than the lowest affinity described in lepidopteran insects, such as in Manduca sexta or Spodoptera littoralis with CryIE (M. sexta, CryIE Kd2 4 215 ± 155; S. littoralis, CryIE Kd2 4 352 ± 16) (22). On the other hand, a correlation between toxicity and binding to membranes from larval midgut has been previously suggested (19–22). Since CPB is about equally susceptible to CryIIIA (15, 24) than M. sexta or Heliothis virescens to active CryIA-type toxins, no significant differences in the affinity for their receptors would therefore be expected. Unlike what one might have assumed, the affinity of both intact and chymotrypsinized CryIIIA is significantly lower than that of lepidopteran toxins. Nevertheless, when comparing with lepidopteran insects, although the affinity constant of chymotrypsinized CryIIIA is not as great as one might hope for, it is 10-fold higher than the one obtained by Slaney for intact CryIIIA. This difference could indicate that other factors, such as an appropriate processing of the toxin prior to binding, will determine the toxicity levels. The present study on two steps of the mode of action of CryIIIA, activation and binding, has shown that this toxin can be proteolytically cleaved by CPB midgut proteases to a 42-kDa fragment and that the chymotrypsin-processed CryIIIA binds to midgut BBMV of this insect with higher affinity than the intact toxin. These results suggest that the mode of action of the coleopteran-specific B. thuringiensis toxin CryIIIA is similar to that of lepidopteranspecific toxins. ACKNOWLEDGMENTS We thank Plant Genetic Systems (Ghent, Belgium) and particularly J. Van Rie for his technical support on binding experiments. We are also very grateful to Dr. David Ellar and Dr. Joe Carroll for reading and helpful comments on the preparation of the manuscript. This work was funded by a grant from the EC under the ECLAIR program (Project AGRE-0003). A. C. Martínez-Ramírez was supported by a grant from the Consellería de Educación y Ciencia (Generalidad Valenciana).
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