Molecular Microbiology (1999) 31(2), 463–471

Domain III of the delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N

Ruud A. de Maagd,1* Petra L. Bakker,1 Luke Masson,2 suggest that domain III of delta-endotoxins play a role Michael J. Adang,3 Sreedhara Sangadala,3 Willem in insect specificity through their involvement in speci- Stiekema1 and Dirk Bosch1 fic binding to insect gut epithelial receptors. 1Department of Molecular Biology, DLO-Centre for Breeding and Reproduction Research (CPRO-DLO), Introduction PO Box 16, 6700 AA Wageningen, the Netherlands. 2Biotechnology Research Institute, National Research During sporulation Bacillus thuringiensis (Bt) produces Council of Canada, 6100 Royalmount Avenue, Montreal, crystalline inclusions that consist of one or several insecti- Quebec H4P 2R2, Canada. cidal delta-endotoxins (Ho¨fte and Whiteley, 1989). After 3Department of Entomology, University of Georgia, ingestion by an insect the crystal is solubilized in the alka- Athens, GA 30602-2603, USA. line midgut, and the proteins are released in the form of protoxins. These are further processed by gut proteases to yield the active that bind to specific receptors Summary on the surface of the epithelial cells of the midgut brush Three types of binding assays were used to study the border. Subsequently, through a still not well-understood binding of Bacillus thuringiensis delta-endotoxin mechanism, the bound toxins insert into the epithelial Cry1Ac to brush border membrane vesicle (BBMV) cell membranes to form pores that cause colloid-osmotic membranes and a purified putative receptor of the lysis of the cells and finally death of the insect (Knowles target insect Manduca sexta. Using hybrid proteins and Ellar, 1987 Knowles and Dow, 1993). consisting of Cry1Ac and the related Cry1C protein, The host range of the Bt delta-endotoxins, which form a it was shown that domain III of Cry1Ac is involved in large family with distinct insect specificities, is thought to specificity of binding as observed by all three tech- be determined to a large extent by the specific interaction niques. In ligand blotting experiments using SDS– of the toxins with their receptors on the epithelial cell mem- PAGE-separated BBMV proteins as well as the purified branes (van Rie et al., 1990). Emphasizing the importance putative receptor aminopeptidase N (APN), the pre- of this interaction, resistance to Bt toxins that occurs as a sence of domain III of Cry1Ac in a hybrid with Cry1C result of prolonged exposure of insect populations in the was necessary and sufficient for specific binding to laboratory or in the field has been shown to be caused by APN. Using the surface plasmon resonance (SPR) changes in these receptors in several cases (Ferre´ et al., technique with immobilized APN, it was shown that 1995). In the last few years several -binding proteins the presence of domain III of Cry1Ac in a hybrid is suf- have been purified to homogeneity, and their encoding ficient for binding to one of the two previously identi- have been cloned, thus allowing the detailed study fied Cry1Ac binding sites, whereas the second site of the toxin/receptor interaction at the molecular level. requires the full Cry1Ac toxin for binding. In addition, One Cry1Ac receptor of the tobacco hornworm, Manduca the role of domain III in the very specific inhibition of sexta, has been postulated to be an aminopeptidase N Cry1Ac binding by the amino sugar N-acetylgalactos- (APN) (Knight et al., 1994; Sangadala et al., 1994), bound amine (GalNAc) was determined. Both in ligand blot- to the epithelial cell membrane through a glycosyl-phospha- ting and in surface plasmon resonance experiments, tidylinositol anchor (Garczynski and Adang, 1995). Binding as well as in binding assays using intact BBMVs, it experiments using surface plasmon resonance subse- was shown that the presence of domain III of Cry1Ac quently showed the presence of two binding sites for in a toxin molecule is sufficient for the inhibition of Cry1Ac on the APN, one of which is shared with the related binding by GalNAc. These and other results strongly Cry1Aa and Cry1Ab toxins (Masson et al., 1995). Binding of Cry1Ac to both intact membrane vesicles as well as to isolated APN is inhibited by the sugar N-acetylgalactos- Received 21 July, 1998; revised 26 September, 1998; accepted 15 October, 1998. *For correspondence. E-mail R.A.deMaagd@CPRO. amine (GalNAc), whereas binding of Cry1Aa and Cry1Ab DLO.NL; Tel. (317) 477128; Fax (317) 418094. is not affected (Garczynski et al., 1991; Knowles et al.,

ᮊ 1999 Blackwell Science Ltd 464 R. A. de Maagd et al. 1991; Masson et al., 1995). As the APN is a glycoprotein that binds the galactosamine-specific lectin SBA (soybean agglutinin), it is thought that GalNAc or a structurally related sugar is part of the toxin binding site that is unique to Cry1Ac (Knight et al., 1994). On the toxin side, elucidation of the three-dimensional structures of Cry3Aa (Li et al., 1991) and Cry1Aa (Gro- chulski et al., 1995) have revealed a three-domain struc- ture. The N-terminal domain I is thought to be responsible Fig. 1. Ligand blot of purified Manduca sexta aminopeptidase N incubated with toxins: Cry1Ac (lane 1), Cry1Ab (lane 2), Cry1C for penetration of the epithelial cell membrane and pore (lane 3), hybrid H130 (1Ac/1Ac/1C, lane 4) and hybrid H201 formation. Results from mutagenesis studies strongly sug- (1C/1C/1Ac, lane 5). The position of the 115 kDa APN band is gest that the highly variable domain II is involved in specific indicated with an arrow. receptor recognition, thereby being one of the determinants of insect specificity of the toxin (reviewed in Dean et al., 1996). Experiments with hybrid toxins containing different in Fig. 1. Only Cry1Ac and hybrid H201, containing domain combinations of domains (‘domain swapping’) have shown III of Cry1Ac, showed detectable binding to a 115 kDa band that domain III can also be a major determinant of specifi- in the APN preparation (lanes 1 and 5 respectively). In city (Ge et al., 1991; Bosch et al., 1994; Masson et al., addition, there was some binding of Cry1Ac and H201 to 1994). Domain III exchanges can result in new hybrid toxins a slightly faster migrating band, possibly a degradation with altered specificity and increased toxicity to a level not product of APN as a result of autolytic activity of the enzyme found in the original parental toxins (Bosch et al., 1994; de when stored in the absence of protease inhibitors (S. San- Maagd et al., 1996a). Using these hybrids as well as muta- gadala, unpublished observation). Cry1C and the hybrid tions in domain III, it was shown that, in addition to domain H130, containing domain I and II of Cry1Ac, did not bind II, domain III also plays an important role in specific bind- (lanes 3 and 4 respectively). Therefore, the observed bind- ing to putative receptors as visualized on ligand blots of ing of Cry1Ac to APN on ligand blots was mediated through BBMV (brush border membrane vesicle) proteins of target domain III. We did not observe binding of Cry1Ab to the insects (Aronson et al., 1995; Lee et al., 1995; de Maagd APN (lane 2), although the presence of a common Cry1A et al., 1996a,b) as well as in binding of native APN to toxins binding site on the APN had been detected before in sur- on slot blots (Lee et al., 1995). face plasmon resonance (SPR) experiments (Masson et The purpose of the study described here was to use a al., 1995). This suggests that under the conditions used combination of binding assay techniques to gain insight here for ligand blotting, binding to this common Cry1A site about the role of domain III in binding to SDS–PAGE-sepa- is either too weak to detect, or the binding site is destroyed rated BBMV proteins, purified APN and intact BBMVs of by the denaturation preceding the detection. M. sexta. Using this approach, we were able to show the direct involvement of domain III of Cry1Ac in insect epithe- Binding of domain-swap hybrids to the lial gut protein binding and in inhibition of putative receptor aminopeptidase N detected by surface plasmon binding by GalNAc. resonance To study the role of domain III of Cry1Ac in binding to the Results non-denatured, purified APN we used the SPR technique. This technique measures the increase in mass of protein Cry1 toxin and hybrid toxin binding on ligand blots (toxin) binding to an immobilized receptor, in this case with purified M. sexta aminopeptidase N APN. It was shown previously that APN can bind twice In previous experiments we have shown that Cry1Ac as much Cry1Ac as it can bind Cry1Ab or Cry1Aa, and binds to a 120 kDa protein as well as to a 210 kDa protein that this is probably due to the presence of one common on ligand blots of SDS–PAGE separated M. sexta BBMV binding site for all Cry1As and of one unique site for proteins. Binding to the 120 kDa protein (assumed to be Cry1Ac (Masson et al., 1995). Cry1C did not bind to APN the putative Cry1Ac receptor APN) was shown to be depen- in these experiments. In this study, we tested binding of dent on domain III of Cry1Ac (de Maagd et al., 1996b). To Cry1Ac and its hybrids H201 (1C/1C/1Ac) and H130 confirm that domain III is indeed involved in binding to the (1Ac/1Ac/1C) as well as that of Cry1Ab/Cry1C hybrids APN, we incubated a ligand blot of purified APN with H04 (1Ab/1Ab/1C) and H205 (1C/1C/1Ab) to the purified Cry1Ac, Cry1C and their hybrids H201 (domains I and II APN. The results are shown in Fig. 2. Cry1Ac bound to the of Cry1C, domain III of Cry1Ac) and H130 (domain I and APN under these conditions, whereas Cry1C did not bind. II of Cry1Ac, domain III of Cry1C). The results are shown The SPR response, in resonance units (RU), is linearly

ᮊ 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 463–471 Role of B. thuringiensis toxin Cry1Ac domain III in binding 465 Cry1Ac domain III involvement in inhibition of binding to putative receptors on ligand blots by GalNAc

GalNAc has been shown to inhibit binding of Cry1Ac both to intact BBMVs of M. sexta (Knowles et al., 1991) as well as to the purified APN on ligand blots (Knight et al., 1994) and in SPR experiments (Masson et al., 1995). As shown in Fig. 3, GalNAc in increasing concentrations (0–80 mM) progressively inhibited binding of Cry1Ac to both the puta- tive APN band at 120 kDa, as well as to the 210 kDa pro- tein band on a ligand blot of M. sexta BBMV proteins (lanes 1–4). Equal concentrations of the structurally simi- lar sugar GlcNAc had no inhibitory effect on binding to both BBMV proteins (Fig. 3, lanes 5–7). Binding of Cry1Ab to Fig. 2. Analysis of toxin binding to purified, immobilized APN the 210 kDa protein was not affected by GalNAc, whereas measured by surface plasmon resonance. All toxins were diluted binding to the Cry1Ab domain III-specific 250 kDa protein in HBS to a final concentration of 1500 nM. The toxins were then was slightly inhibited by 80 mM GalNAc (Fig. 3, lanes 8 injected over a 900 RU surface of immobilized aminopeptidase N at a rate of 5 ␮l min¹1 for 300 s after which toxin flow was and 9). These results show that binding of Cry1Ac to both replaced by buffer alone to monitor dissociation of the APN/toxin the domain III-specific 120 kDa band as well as to the complex. domain II-specific 210 kDa protein are specifically inhibited by the sugar GalNAc. Whereas the first part confirms earlier observations with purified APN, it is surprising that also proportional to the surface protein concentration, there- domain I/II-specific binding of Cry1Ac to the 210 kDa pro- fore allowing the examination of the stoichiometry of the tein is inhibited, although binding of Cry1Ab, which has a binding reaction. The amount of APN bound to the sensor very similar domain IþII (only six amino acid residue differ- chip corresponded to 900 RU, and the maximum amount ences), was not affected. of Cry1Ac bound also approached 900 RU. With the mole- We tested further the role of domain III in the inhibition cular weight of the activated toxin being approximately half of binding by GalNAc described above by looking at the of that of the receptor, it follows that the molar ratio of effect of this sugar on binding of the Cry1C/Cry1Ac Cry1Ac/receptor is Ϸ2:1, as was found previously (Mas- hybrids H201 and H130 to ligand blots. Using these son et al., 1995). hybrids, we have shown previously that domains I and/or H201, which contains domain III of Cry1Ac, and H04 II of Cry1Ac are involved in the specificity of binding to showed binding activity, although the latter to a much lesser the 210 kDa BBMV protein of M. sexta, whereas the pre- extent. The other two hybrids, H205 and H130, did not bind sence of domain III of Cry1Ac in a toxin is necessary to the immobilized APN (data not shown). For H201, bind- and sufficient for the specificity of binding to the 120 kDa ing levels reached Ϸ50% of that of Cry1Ac, suggesting protein (de Maagd et al., 1996b). The 210 kDa protein that domain III of Cry1Ac is sufficient for binding to just one of the two Cry1Ac binding sites observed previously (Masson et al., 1995). The apparent binding affinity con- stant of H201 was calculated to be 5.07 × 10¹7 M, which is approximately eight times higher (i.e. binding is weaker) than that reportedly previously for Cry1Ac (Masson et al., 1995). Most of this difference was caused by a sub- stantially lower association rate of H201 [(7.54 Ϯ 0.32) × 103 M¹1 s¹1 for H201 versus (9.0 Ϯ 1.1) × 104 M¹1 s¹1 for Cry1Ac reported previously], whereas their dissociation rates were not significantly different [(3.82 Ϯ 0.69) × 10¹3 s¹1 and (3.66 Ϯ 0.13) × 10¹3 s¹1 respectively]. H04 (1Ab/ 1Ab/1C) showed binding with a very low affinity compared with Cry1Ac and H201 and did not reach saturation during Fig. 3. Ligand blot of SDS–PAGE separated Manduca sexta the toxin injection period. After the end of toxin injection, BBMV proteins incubated with Cry1Ac (lanes 1–7) and Cry1Ab the binding signal did not rapidly decrease, suggesting (lanes 8 and 9) either in the absence of aminosugars (lanes 1 and that rebinding of dissociated toxin readily takes place 8) or in the presence of 10, 20, and 80 mM GalNAc (lanes 2, 3, 4 and 9) or 10, 20, and 80 nM GlcNAc (lanes 5, 6, and 7). because of the availability of free receptor (Masson et Positions of the 120, 210 and 250 kDa bands are indicated with al., 1995). arrows.

ᮊ 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 463–471 466 R. A. de Maagd et al. Effects of GalNAc on Cry1Ac domain III-dependent binding to non-denatured APN in SPR experiments

In SPR experiments using the purified APN of M. sexta, Cry1Ac binding to both detected sites was inhibited by GalNAc, whereas binding of Cry1Aa and Cry1Ab to a single common site was not inhibited (Masson et al., 1995). In SPR experiments with the hybrid H201, which showed Cry1Ac domain III-mediated binding to the purified APN (see above), binding was increasingly inhibited up to Ϸ80% by GalNAc concentrations from 0 to 75 mM (Fig. 6). This inhibition level is similar to that previously observed for Cry1Ac (Masson et al., 1995). This shows that as in the experiments with ligand blots, also with the non-denatured purified APN, the presence of domain III of Cry1Ac in a toxin is sufficient for both binding to one binding site as Fig. 4. Ligand blot of Manduca sexta BBMV incubated with hybrid well as for inhibition of this binding by GalNAc. toxins H201 (1C/1C/1Ac, lanes 1–3) and H130 (1Ac/1Ac/1C, lanes 4–6) incubated without (lanes 1 and 4) or in the presence of 40 mM (lanes 2 and 5) or 80 mM (lanes 3 and 6) GalNAc. Positions Binding of Cry1Ac and hybrid toxins to intact M. sexta of the 120 and 210 kDa toxin binding proteins are indicated with BBMVs arrows. Both ligand blot experiments and SPR experiments with purified APN have potential drawbacks when trying to elu- is a common binding protein for Cry1Aa, Cry1Ab and cidate the process of specific binding to target membranes Cry1Ac; it has been observed by several groups (Vad- and the role of the different toxin domains therein. Denatur- lamudi et al., 1993; Martinez-Ramirez et al., 1994; de ation of BBMV membranes during ligand blotting may Maagd et al., 1996b; Keeton et al., 1998) and has been destroy important binding sites, or expose sites that are purified, cloned and shown to be a cadherin-like glyco- not taking part in binding of intact membranes. Even protein (Vadlamudi et al., 1995). In Fig. 4 (lanes 1–3), it experiments with purified non-denatured APN can over- is shown that increasing concentrations of GalNAc inhibi- look the potentially important role of interactions between ted binding of H201 to the 120 kDa (APN–) band at least different membrane proteins and lipids for toxin binding. equally well and possibly more efficient than binding of Therefore, we studied binding of parental and hybrid the parental toxin Cry1Ac. On the other hand, in contrast to the observed inhibition for the parental Cry1Ac, the binding of hybrid H130 to the 210 kDa protein was not visibly inhibited by the highest GalNAc concentration used (Fig. 4, lanes 4–6). Also on a ligand blot of a purified APN preparation (Fig. 5) both the binding of Cry1Ac (lanes 1 and 2) as well as that of hybrid H201 (lanes 3 and 4) was inhibited by GalNAc, confirming the results with separated BBMV proteins shown above.

Fig. 6. Inhibition of hybrid H201 (1C/1C/1Ac) binding to purified APN. The hybrid toxin dissolved in HBS was mixed with various concentrations of GalNAc to a final toxin concentration of 1500 nM and variable sugar concentrations. The different toxin solutions Fig. 5. Ligand blot of purified Manduca sexta APN incubated with were then injected over a 600 RU surface of APN. Corrections for Cry1Ac (lanes 1 and 3) and hybrid H201 (1C/1C/1Ac, lanes 3 and refractive index changes were made as described earlier in 4) without (lanes 1 and 3) and in the presence of 80 mM GalNAc materials and methods and the results expressed as a percentage (lanes 2 and 4). inhibition of total binding in the absence of sugar.

ᮊ 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 463–471 Role of B. thuringiensis toxin Cry1Ac domain III in binding 467 Aminopeptidase N was first identified as a putative recep- tor for Cry1Ac in M. sexta (Knight et al., 1994; Sangadala et al., 1994), after which is was shown by SPR experi- ments that the related Cry1Aa, Cry1Ab and Cry1Ac could all recognize and interact with APN (Masson et al., 1995). Subsequently, a similar role as (putative) cry1A receptors was suggested for APN of Heliothis virescens (Luo et al., 1997a), Plutella xylostella (Denolf et al., 1997; Luo et al., 1997b), Trichoplusia ni (Lorence et al., 1997), Lymantria Fig. 7. Assay of biotin-labelled toxin binding to intact Manduca dispar (Lee et al., 1996) and Bombyx mori (Yaoi et al., sexta BBMVs for Cry1Ac, Cry1C, hybrid H201 (1C/1C/1Ac) and 1997). The case for APN being a functional receptor (i.e. hybrid H130 (1Ac/1Ac/1C). Binding levels were determined in the being involved in binding that leads to toxicity for the target absence of competitor (lanes 1), with 100 mM GalNAc (lanes 2), with 100 mM GlcNAc (lanes 3) or with a 50-fold excess of insect) was made stronger by showing that pore formation unlabelled homologous competitor (lanes 4). in lipid membranes by Cry1 toxins is strongly facilitated by incorporation of APN of M. sexta (Schwartz et al., 1997), T. ni (Lorence et al., 1997) or H. virescens (Luo et al., toxins to intact, non-denatured M. sexta BBMVs in a bind- 1997a). ing assay. Binding of biotin-labelled toxins in the presence Our experiments using toxin hybrids with ligand blots of or absence of various competitors was detected with separated BBMV proteins and of purified APN of M. sexta streptavidin/peroxidase after blotting of the SDS–PAGE- show that the presence of domain III of Cry1Ac in the toxin separated BBMV proteins (including the bound, labelled is sufficient for binding to the 120 kDa membrane anchored toxin) to a nitrocellulose membrane. Results are shown APN as well as its purified, GPI anchorless 115 kDa form. in Fig. 7, where the bands shown represent the total It must be stressed that this finding does not preclude a amount of bound, labelled toxin. All four toxins tested, role for domains I and II in binding. It seems likely that Cry1Ac, Cry1C and hybrids H201 and H130, showed other residues, especially in domain II, take part in (recep- specific binding, as can be deduced from comparison of tor) binding as well, yet in the experiments as described the levels of binding in the absence (lanes 1) and in the here specificity of much of the observed binding is deter- presence of a 50-fold excess of homologous unlabelled com- mined by domain III of Cry1Ac. In those cases, domains petitor toxin (lanes 4). However, only binding of Cry1Ac I and/or II of Cry1Ac may play a role that can also be per- and H201 could be inhibited by the presence of 100 mM formed by the corresponding domains of Cry1C. GalNAc (lanes 2). The lack of inhibition of binding in the In contrast to the binding of Cry1Aa and Cry1Ab to APN presence of the similar sugar GlcNAc again confirmed observed by SPR (Masson et al., 1995), binding on ligand the specificity of the inhibition (lanes 3). These results blots was not detected with either BBMV preparations (de show that also on intact membranes, although all tested Maagd et al., 1996b) or purified APN (this study). This sug- toxins and hybrids do bind, the presence of domain III of gests that the (common) binding site for Cry1A toxins Cry1Ac is required for inhibition of binding by GalNAc. postulated for M. sexta APN (Masson et al., 1995) may not survive the denaturation procedure that is part of the ligand blot protocol. In our SPR experiments we found Discussion that the presence of domain III of Cry1Ac is also sufficient In this study, we have compiled data from three different for binding to one of the two Cry1Ac binding sites detected types of binding assays to determine the role of domain in this assay. However, the complementary hybrid H130 III of Cry1Ac in binding to midgut epithelial membranes (1Ac/1Ac/1C) did not interact with immobilized APN, sug- of its target insect M. sexta. The results of all three assays gesting that for the binding to the second Cry1Ac site using natural and hybrid toxins in which domains were domain I and II of Cry1Ac are not sufficient but that a exchanged show that domain III of Cry1Ac plays an essen- proper combination of domains (Iþ) II and III is required. tial role in recognition of the putative receptor APN as well Curiously, hybrid H04 (1Ab/1Ab/1C), which differs only as in binding to intact membranes. Moreover, this role of in six amino acids from H130, did show some residual domain III of Cry1Ac is tightly linked to a unique feature of binding. Nonetheless, all hybrid toxins used in this study Cry1Ac binding, i.e. inhibition by the amino sugar GalNAc. are active in M. sexta (data not shown). These results The combination of our results with others showing that strongly suggest that domain III is participating in actual GalNAc can decrease pore formation (Lorence et al., 1997) binding contacts to both binding sites, although alternative or swelling in BBMVs (Carroll et al., 1997) provides strong explanations remain possible. support for the involvement of domain III in in vivo binding GalNAc or a similar sugar seems to be part of a Cry1Ac and consequently toxicity. binding site on APN of both M. sexta and L. dispar,because

ᮊ 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 463–471 468 R. A. de Maagd et al. they both bind the lectin soybean agglutinin (Knight et al., far as the interaction with Cry1Ac is concerned (2 Cry1Ac 1994; Lee et al., 1996). Moreover, for these and other binding sites per APN molecule). Using non-denaturing Cry1Ac-binding APN, GalNAc specifically inhibits Cry1Ac detergents, APN of M. sexta can be isolated as a complex binding (Knight et al., 1994; Lee et al., 1996; Luo et al., containing various other GPI-anchored proteins and pos- 1997a,b; Valaitis et al., 1997). GalNAc also inhibits binding sibly lipids (Lu and Adang, 1996). The toxin-binding prop- of Cry1Ac to intact membranes of M. sexta and, to a lesser erties of this complex are different from those of the purified extent, of T. ni (Haider and Ellar, 1987) as well as causing APN (Schwartz et al., 1997). Furthermore, it has been a reduction in pore formation in membrane preparations shown that these proteins are clustered with specific glyco- from these insects, indicating that this type of binding lipids when purified from brush border membranes, some is essential for toxicity to insects (Carroll et al., 1997; of which can bind Cry1 toxins on thin-layer chromato- Lorence et al., 1997). Our experiments have shown that graphy overlays (Adang et al., 1997). Although this com- the presence of domain III of Cry1Ac in a toxin is neces- plex does not necessarily represent the in vivo situation, sary and sufficient for this inhibitory effect of GalNAc on this result does raise the possibility that receptors for differ- binding. This suggests that domain III of Cry1Ac is speci- ent domains (II or III respectively) act as a single complex fically interacting with this or a similar sugar on the recep- binding site in the intact membrane. Further experimenta- tor complex, and that this binding contributes to toxicity. tion using various techniques for studying binding and In SPR experiments GalNAc has been shown to inhibit using toxin mutants specifically affected in domain II or binding of Cry1Ac to both sites on the M. sexta APN (Mas- domain III-dependent binding may in the future clarify son et al., 1995), which again suggests the involvement of this complex picture. domain III in binding to both these sites. As GalNAc almost completely inhibited binding of Cry1Ac to intact M. sexta BBMVs, it is likely that domain III plays a specific binding Experimental procedures role in vivo and, consequently, in toxicity. Recently it was Toxin isolation and purification shown that GalNAc-inhibited Cry1Ac pore formation activ- ity only occurs in the posterior midgut of M. sexta,whereas All wild-type toxins and hybrid toxins were produced by expres- both in posterior and in anterior midgut BBMVs there is a sion in Escherichia coli strain XL-1 Blue, solubilization, trypsin second, GalNAc-independent pore formation mechanism activation of the protoxin, and FPLC purification of the mature toxin as described elsewhere (Bosch et al., 1994), except that (Carroll et al., 1997). It is not known whether the second 0.5 mM PMSF (phenylmethylsulphonyl fluoride) was added mechanism involves saturable, specific binding to the after incubation of protoxins with trypsin to prevent further membrane. From our results we conclude that domain III degradation. Hybrid toxins H201 (domain composition: 1C/ of Cry1Ac is involved in at least the former of these two 1C/1Ac), H130 (1Ac/1Ac/1C), H04 (1Ab/1Ab/1C) and H205 mechanisms. (1C/1C/1Ab) have been described previously (de Maagd et The combined results of the different binding techniques al., 1996a, b). used here and those found in the literature do not as yet allow a fully comprehensive model of Cry1Ac binding to Isolation of brush border membrane vesicles M. sexta membranes. Clearly defined roles of the different putative receptors (APN and cadherin-like glycoprotein, Brush border membrane vesicles (BBMVs) were isolated and possibly others) and how they interact with the differ- essentially as described by Wolfersberger et al. (1987) from dissected midguts of 7-day-old M. sexta larvae, except for ent toxin domains will require further experimentation. the addition of 1 mM PMSF and 100 ␮M chymostatin in the Binding studies with intact M. sexta membranes revealed MET (300 mM mannitol, 5 mM EGTA, 17 mM Tris-HCL a single high-affinity binding site for Cry1Ac, shared with pH 7.5) buffer during the initial homogenization step. Isolated Cry1Aa and Cry1Ab (van Rie et al., 1989). However, ligand BBMVs were resuspended in 0.5× MET buffer and stored at blotting shows that these toxins can recognize and bind to ¹80ЊC. different BBMV proteins through domain II and III (de Maagd et al., 1996b). In addition, SPR experiments Ligand blotting show that a single putative receptor molecule, APN, may have two distinct binding sites (Masson et al., 1995). Proteins from isolated BBMVs were separated by SDS–PAGE. Although the purified APN shows a single band on a gel, For ligand blotting, 50 ␮g of BBMV protein or 12.5 ␮g of puri- the apparent ‘fuzziness’ of the APN band on ligand blots fied APN from M. sexta was mixed with SDS–PAGE sample suggests a heterogeneity that could be caused by the buffer, heated for 5 min at 100ЊC and loaded onto a 7.5% acryl- amide gel in a continuous, 7.4-cm-wide sample slot. Prestained association with lipids or by differences in the extent of gly- molecular-mass marker proteins were run alongside in a sepa- cosylation. However, the apparent 2:1 stoichiometry of the rate single slot. After electrophoresis, separated proteins were Cry1Ac/APN interaction in these experiments suggests electrophoretically transferred to nitrocellulose (0.22 ␮m pore that the APN preparation is likely to be homogeneous as size, Schleicher and Schull). Strips (3 mm wide) were cut from

ᮊ 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 463–471 Role of B. thuringiensis toxin Cry1Ac domain III in binding 469 the blots for incubation with toxins (2 ␮g of toxin per strip), and binding analyses were performed exactly as described followed by incubation with a Cry1 antiserum and detection elsewhere (Masson et al., 1995). All sensorgram data trans- with ECL reagent as described previously (de Maagd et al., formations and analyses were performed with BIAEVALUATION 1996a). software version 2.1. using non-linear least-squares curve fit- ting. Carbohydrate inhibition studies were performed by inject- ing a 1.5 ␮M solution of toxin diluted in HBS-P20 (10 mM Binding experiments on intact BBMVs HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.05% BIAcore Surfactant P20) buffer possessing various concentrations of Toxins were labelled with biotin using biotin-N-hydroxysucci- GalNAc. The highest response (in resonance units) obtained nimide ester (BNHS, Boehringer) as described previously for each toxin/sugar combination at the end of an injection (Bosch et al., 1994). For binding experiments, BBMVs (5 ␮g over the receptor surface after subtraction of a sugar blank of protein) were mixed with 2 ng of biotin-labelled toxin and, if (i.e. no toxin) was expressed as percentage inhibition using appropriate, 100 ng of unlabelled competitor toxin or 100 mM toxin binding in the absence of sugar as the 100% binding N-acetylaminosugar (-galactosamine or -glucosamine) in point. 100 ␮l of PBS/Tween (phosphate-buffered saline, pH 7.4, con- taining 0.1% Tween-20). After incubation for 1 h at room tem- perature, vesicles and unbound toxin were separated by References centrifugation, and the pellet was washed once briefly with PBS/Tween. Subsequently, BBMV proteins (including bound, Adang, M., Luo, K., Sangadala, S., and Garczynski, S. (1997) labelled toxin) were separated by SDS–PAGE and electro- Glycolipids in insect brush border membranes: exploring blotted onto a nitrocellulose membrane. Bound, labelled toxin their roles in membrane structure, protein anchorage and on the blot was detected with streptavidin/peroxidase as Bacillus thuringiensis toxin action. In Abstracts of the described previously (Bosch et al., 1994). 30th Annual Meeting of the Society for Pathol- ogy, Banff, 24–29 August 1997. Aronson, A.I., Wu, D., and Zhang, C. (1995) Mutagenesis of Purification of M. sexta 115 kDa aminopeptidase N specificity and toxicity regions of a Bacillus thuringiensis protoxin . J Biol Chem 177: 4059–4065. Aminopeptidase N (120 kDa) was purified from M. sexta brush Bosch, D., Schipper, B., van der Klei, H., de Maagd, R., border membrane vesicles as described previously with a few and Stiekema, W. (1994) Recombinant Bacillus thurin- modifications (Lu and Adang, 1996). Briefly, frozen BBMVs giensis crystal proteins with new properties: possibilities were thawed on ice and resuspended at 5 mg protein ml¹1 in for resistance management. Bio/Technology 12: 915–918. a buffer containing 20 mM Tris-HCl, pH 8.5, 100 mM NaCl, Carroll, J., Wolfersberger, M.G., and Ellar, D.J. (1997) The 5 mM EDTA, 1 mM PMSF and 1% CHAPS (3-((3-cholamido- Bacillus thuringiensis Cry1Ac toxin-induced permeability propyl)dimethylammonio)-1-propanesulphonate) and stirred change in Manduca sexta midgut brush border membrane at 4ЊC overnight for solubilization. Under these conditions, vesicles proceeds by more than one mechanism. J Cell Sci the 115 kDa soluble form of aminopeptidase is released by 110: 3099–3104. endogenous phospholipase C from its lipid anchor. Insoluble Dean, D.H., Rajamohan, F., Lee, M.K., Wu, S.J., Chen, X.J., material was removed by centrifugation at 100 000 × g for Alcantara, E., and Hussain, S.R. (1996) Probing the 1 h at 4ЊC. The supernatant was diluted fivefold with 20 mM mechanism of action of Bacillus thuringiensis insecticidal Tris-HCl, pH 8.5; 0.05% CHAPS and syringe filtered through proteins by site directed mutagenesis: a minireview. Gene a 0.2 ␮m membrane for anion exchange chromatography 179: 111–117. using two consecutively connected 5 ml EconoQ anion Denolf, P., Hendrickx, K., Vandamme, J., Jansens, S., exchange columns (Bio-Rad) linked to a FPLC system (Phar- Peferoen, M., Degheele, D., and van Rie, J. (1997) Cloning macia). Buffer A contained 20 mM Tris-HCl, pH 8.5; 0.05% and characterization of Manduca sexta and Plutella xylo- CHAPS, and buffer B contained 20 mM Tris-HCl, pH 8.5; stella midgut Aminopeptidase N enzymes related to Bacil- 0.05% CHAPS; 1.0 M NaCl. Solubilized BBMV proteins lus thuringiensis toxin binding proteins. Eur J Biochem (50 mg) were loaded onto the column and the bound proteins 248: 748–761. were eluted with a two-step salt gradient. The chromato- Ferre´, J., Escriche, B., Bel, Y., and van Rie, J. (1995) Bio- graphic run started at 1% min¹1 gradient of buffer B at a flow chemistry and genetics of insect resistance to Bacillus rate of 2 ml min¹1 for 40 min followed by a 3% min¹1 gradient thuringiensis insecticidal crystal proteins. FEMS Microbiol of buffer B for 20 min. Fractions (1 ml) were collected and ana- Lett 132: 1–7. lysed by SDS–PAGE and toxin binding. The soluble form of Garczynski, S.F., Crim, J.W., and Adang, M.J. (1991) Identi- aminopeptidase (115 kDa) was eluted in fractions 5–8. These fication of putative insect brush border membrane-binding fractions were combined, concentrated and stored at ¹70ЊC molecules specific to Bacillus thuringiensis delta-endotoxin until further use. by protein blot analysis. Appl Environ Microbiol 57: 2816– 2820. Surface plasmon resonance experiments Garczynski, S.F., and Adang, M.J. (1995) Bacillus thurin- giensis CryIA(C) delta-endotoxin binding Aminopeptidase The binding of hybrid toxins to the purified soluble 115 kDa N in the Manduca sexta midgut has glycosyl-phospha- APN from M. sexta was studied by SPR using the BIAcore tidylinositol anchor. Insect Biochem Mol Biol 25: 409– system and CM5 sensor chips, both from Pharmacia Biosen- 415. sor. The aminopeptidase was immobilized by amine coupling, Ge, A.Z., Rivers, D., Milne, R., and Dean, D.H. (1991)

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