Location of the Bombyx mori 175 kDa cadherin-like protein-binding site on Bacillus thuringiensis Cry1Aa toxin Shogo Atsumi 1,2 , Yukino Inoue 1 , Takahisa Ishizaka 1 , Eri Mizuno 1 , Yasutaka Yoshizawa 1 , Madoka Kitami 1 and Ryoichi Sato 1 1 Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan 2 Division of Insect Sciences, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Ibaraki, Japan Bacillus thuringiensis, a Gram-positive bacterium, pro- duces various insecticidal proteins called Cry toxins. These bacteria are used as microbial insecticides and for the genetic development of insect-resistant plants, because they are specific to their target insects. Cry toxins are expressed in inclusion bodies as protoxins (70–140 kDa) during sporulation. When a protoxin is ingested by the target insect, it is solubilized in the insect midgut and digested by proteolytic enzymes [1]. After enzymatic activation, the toxic protease- resistant fragment, which is the 60–70 kDa activated toxin, binds to specific receptors located in the columnar cells of the midgut apical brush border membrane [2]. Keywords Bacillus thuringiensis; BmAPN1; Bombyx mori; BtR175; Cry1Aa Correspondence R. Sato, Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184 8588, Japan Fax: +81 42 388 7277 Tel: +81 42 388 7277 E-mail: ryoichi@cc.tuat.ac.jp (Received 9 June 2008, revised 25 July 2008, accepted 8 August 2008) doi:10.1111/j.1742-4658.2008.06634.x To identify and gain a better understanding of the cadherin-like receptor- binding site on Bacillus thuringiensis Cry toxins, it is advantageous to use Cry1Aa toxin, because its 3D structure is known. Therefore, Cry1Aa toxin was used to examine the locations of cadherin-like protein-binding sites. Initial experiments examining the binding compatibility for Cry1Aa toxin of partial fragments of recombinant proteins of a 175 kDa cadherin-like protein from Bombyx mori (BtR175) and another putative receptor for Cry1Aa toxin, aminopeptidase N1, from Bo. mori (BmAPN1), suggested that their binding sites are close to each other. Of the seven mAbs against Cry1Aa toxin, two mAbs were selected that block the binding site for BtR175 on Cry1Aa toxin: 2A11 and 2F9. Immunoblotting and alignment analyses of four Cry toxins revealed amino acids that included the epitope of mAb 2A11, and suggested that the area on Cry1Aa toxin blocked by the binding of mAb 2A11 is located in the region consisting of loops 2 and 3. Two Cry1Aa toxin mutants were constructed by substituting a Cys on the area blocked by the binding of mAb 2A11, and the small blocking mol- ecule, N-(9-acridinyl)maleimide, was introduced at each Cys substitution to determine the BtR175-binding site. Substitution of Tyr445 for Cys had a crippling effect on binding of Cry1Aa toxin to BtR175, suggesting that Tyr445 may be in or close to the BtR175-binding site. Monoclonal anti- bodies that blocked the binding site for BtR175 on Cry1Aa toxin inhibited the toxicity of Cry1Aa toxin against Bo. mori, indicating that binding of Cry1Aa toxin to BtR175 is essential for the action of Cry1Aa toxin on the insect. Abbreviations ACN, aminopeptidase N; BmAPN1, Bombyx mori aminopeptidase N1; BtR175, Bombyx mori 175 kDa cadherin-like protein; GST 27 kDa BtR175, a recombinant fusion protein of glutathione S-transferase and the 27 kDa fragment of Bombyx mori 175 kDa cadherin-like protein; GST 7 kDa BmAPN1, a recombinant fusion protein of glutathione S-transferase and the 7 kDa fragment of Bombyx mori aminopeptidase N; GST, glutathione S-transferase; HRP, horseradish peroxidase; NAM, N-(9-acridinyl)maleimide. FEBS Journal 275 (2008) 4913–4926 ª 2008 The Authors Journal compilation ª 2008 FEBS 4913 The 3D structures of Cry1Aa toxin have been deter- mined by X-ray diffraction crystallography [3]. This protein is composed of three domains. Domain I is composed of a seven-a-helix bundle and is involved in membrane insertion [3,4]. Domain II consists of three antiparallel b-sheets and plays a role in binding to receptor molecules [3,5,6]. Domain III is a b-sandwich and has several functions, including the recognition of N-acetylgalactosamine on receptor molecules [3,7–9]. In early studies, various aminopeptidase N (APN) isoforms from several insect species were identified as candidate receptors for B. thuringiensis Cry toxins [10– 14]. Expression of an APN in Drosophila melanogaster made the larvae sensitive to Cry1Ac toxin [15]. RNA interference experiments have indicated that silencing of midgut APN in Spodoptera litura and Heli- coverpa armigera reduces sensitivity to Cry1C and Cry1Ac toxins [16,17]. The 170 kDa APN from Helio- this virescens plays a role in pore formation in membrane vesicles [18]. The 120 kDa APN from Manduca sexta mediates channel formation in planar lipid bilayers [19]. These results suggest that APN functions as a Cry1 receptor and is involved in the lytic activity of Cry1 toxins. In addition, cadherin-like proteins are selected as candidate receptors [20–23]. Expression of the Bombyx mori 175 kDa cadherin-like protein (BtR175) on the surface of Sf9 insect cells made these cells sensitive to Cry1Aa toxin [12]. Our previous work indicated inhibition of the binding of Cry1Aa and Cry1Ac toxins to BtR175 after pretreat- ment with antibody against BtR175, as this suppressed the lytic activity of the toxins on collagenase-dissoci- ated Bo. mori midgut epithelial cells [24]. On genetic mapping analysis, the disruption of a cadherin-like protein gene (BtR-4) by retrotransposon-mediated insertion was shown to be linked to high levels of resis- tance to Cry1Ac toxin in H. virescens [25]. RNA inter- ference experiments have indicated that silencing of midgut BT-R 1 in M. sexta reduces sensitivity to Cry1Ab toxin [26]. These findings suggest that the cadherin-like protein plays an important role in Cry toxin susceptibility and that the cadherin-like protein is the functional Cry toxin receptor in the insect midgut. One hypothesis regarding the mode of action of Cry toxin for M. sexta is that binding of monomeric Cry1Ab toxin to BT-R 1 promotes additional proteo- lytic cleavage in the N-terminal end of the toxin, facili- tating the formation of a prepore oligomeric structure that is competent for membrane insertion, and that oligomer formation is important for toxicity [27,28]. The prepore oligomer has a higher affinity for APN [29,30]. The oligomeric Cry1A toxin structure then binds to the APN receptor, leading to its insertion into membrane lipid rafts, implying a sequential binding mechanism of Cry1A toxins with BT-R 1 and APN receptor molecules [30,31]. However, a different mech- anism of action of Cry toxins was recently proposed, based on a study of the effect of Cry1Ab toxin on cul- tured Trichoplusia ni H5 insect cells expressing M. sex- ta BT-R 1 [32,33], and was called the signaling model. However, modified Cry1A toxins lacking helix 1 formed a prepore oligomeric structure and killed insects without needing intact BT-R 1 [26]. Although no consensus regarding the mode of action of Cry toxins has yet been reached, this result favors the membrane insertion model. The Cry toxin-binding site on cadherin-like protein receptors and the cadherin-like protein receptor-bind- ing site on Cry toxins are being mapped. Three Cry1A toxin-binding sites have been mapped in BT-R 1 . The first site, NITIHITDTNN(865–875), was mapped using phage display and is involved in binding loop 2 (b6–b7 loop) of Cry1Aa and Cry1Ab toxins [5,34]. A second region, 1291–1360, is important for toxin bind- ing [35], and was subsequently narrowed down to a 12 amino acid region, IPLPASILTVTV(1331–1342), which binds loop a8(a8a–a8b loop) [6]. A third region, 1363–1464, is involved in toxin binding and cytotoxicity [36]. In the case of the H. virescens cadh- erin, this binding region was narrowed down to a 19 amino acid region, 1422–1440, which binds domain II loop 3 (b10–b11 loop) of Cry1Ab and Cry1Ac toxins [23]. In Bo. mori, a 219 amino acid region (1245–1464) of BtR175 is responsible for Cry1Aa toxin binding [37]. However, the binding site on Cry1Aa toxin for BtR175 is not yet known. It is possible to consider the structure of the cadherin-binding site three-dimension- ally, because the 3D structure of Cry1Aa toxin is known. Three-dimensional visualization is very useful for understanding the mechanism of selective toxin– receptor interactions. We analyzed the binding site on Cry1Aa toxin for one of the Cry1Aa toxin receptors in Bo. mori,a 115 kDa APN type 1 (BmAPN1), using mAbs that block binding between the binding site and the recep- tor [38]. In this study, to analyze the binding site on Cry1Aa toxin for the other Cry1Aa toxin receptor, BtR175, using mAbs that block binding between the binding site and the receptor, mAbs that block the binding site for BtR175 on Cry1Aa toxin were selected. These antibodies also reduce the toxicity of Cry1Aa toxin against Bo. mori. Monoclonal antibodies that block the binding site for BmAPN1 on Cry1Aa toxin have no effect on the toxicity of Cry1Aa toxin against Bo. mori. We found that the binding of BtR175 binding site on the Cry1Aa toxin S. Atsumi et al. 4914 FEBS Journal 275 (2008) 4913–4926 ª 2008 The Authors Journal compilation ª 2008 FEBS BmAPN1 to Cry1Aa toxin blocked the binding of BtR175. We discuss the location of the BtR175-bind- ing site on Cry1Aa toxin on the basis of location infor- mation obtained with the epitopes of mAbs that block the binding sites for BtR175 and BmAPN1. Results Competitive binding of glutathione S-transferase (GST) 27 kDa BtR175 and GST 7 kDa BmAPN1 to Cry1Aa toxin A recombinant fusion protein of GST and 7 kDa frag- ment of BmAPN1 containing the Cry1Aa toxin-bind- ing region (GST 7 kDa BmAPN1) [39] and a recombinant fusion protein of GST and 27 kDa frag- ment of BtR175 containing the Cry1Aa toxin-binding region (GST 27 kDa BtR175) [37] were tested for their ability to block binding of Cry1Aa toxin to GST 27 kDa BtR175. GST 7 kDa BmAPN1 and GST 27 kDa BtR175 blocked the binding of Cry1Aa toxin to GST 27 kDa BtR175 in a dose-dependent manner (Fig. 1). These results suggest that the surface of Cry1Aa toxin blocked by GST 7 kDa BmAPN1 is located close to the GST 27 kDa BtR175-binding site. Determination of binding compatibility for GST 27 kDa BtR175, GST 7 kDa BmAPN1, and mAb 1B10, using an IAsys optical sensor To investigate the relative locations of the binding sites for GST 27 kDa BtR175 and GST 7 kDa BmAPN1 on the surface of Cry1Aa toxin, tests were performed using an IAsys resonant mirror optical biosensor. GST 27 kDa BtR175, GST 7 kDa BmAPN1 or mAb 1B10 was bound to Cry1Aa toxin immobilized on a cuvette surface, as demonstrated by the equal heights of the sensorgrams (Fig. 2A,B,F). To test for binding compe- tition with the same protein, a solution of GST 7 kDa BmAPN1 was added to the cuvette after GST 7 kDa BmAPN1 had been bound to the immobilized Cry1Aa toxin, and the binding of the added protein to the sur- face was determined (Fig. 2C). When additional GST 7 kDa BmAPN1 was added, almost no further binding to the immobilized Cry1Aa toxin occurred, suggesting that the binding sites for GST 7 kDa BmAPN1 on the surface had been largely blocked by the initial GST 7 kDa BmAPN1 solution. This type of binding curve, which demonstrates that there was no additional pro- tein binding, is observed if the second protein (in this case, the same protein) competes with the first protein. To test for binding competition between BmAPN1 and BtR175, GST 27 kDa BtR175 was added to the cuv- ette after GST 7 kDa BmAPN1 had been bound to the immobilized Cry1Aa toxin, and the binding of the added protein to the surface was determined (Fig. 2D). When additional GST 27 kDa BtR175 was added, almost no further binding to the immobilized Cry1Aa toxin occurred, suggesting that the binding sites for GST 27 kDa BtR175 had been largely blocked by the initial solution of GST 7 kDa BmAPN1. To test for binding competition in the reverse order, a solution of GST 7 kDa BmAPN1 was added to the cuvette after GST 27 kDa BtR175 had been bound to the immobi- lized Cry1Aa toxin, and the binding of the added pro- tein to the surface was determined (Fig. 2E). When GST 7 kDa BmAPN1 was added, almost no further binding to the immobilized Cry1Aa toxin occurred, suggesting that the binding sites for GST 7 kDa BmAPN1 had been largely blocked by the initial solu- tion of GST 27 kDa BtR175. As a control, the com- patibility of binding of BtR175 and mAb 1B10 on the surface of Cry1Aa toxin at the same time was tested. Monoclonal antibody 1B10 was raised against Cry1Aa toxin in a BALB ⁄ c mouse, and can block the binding of Cry1Aa toxin to GST 7 kDa BmAPN1 [38]. To test Fig. 1. Dose-dependent inhibition of Cry1Aa toxin binding to BtR175 by GST 27 kDa BtR175 and GST 7 kDa BmAPN1. Biotiny- lated Cry1Aa toxin was preincubated with various concentrations of GST 27 kDa BtR175 or GST 7 kDa BmAPN1 for 90 min and then added to wells coated with GST 27 kDa BtR175. It was then incubated for 90 min with HRP-conjugated streptavidin. Bound streptavidin was detected after incubation with 2,2¢-azinobis(3-ethyl- benzo-6-thiazolinesulfonic acid) solution for 15 min. Absorbance at 415 nm was measured using a microtiter plate reader. The assays were repeated three times, and mean values are shown. S. Atsumi et al. BtR175 binding site on the Cry1Aa toxin FEBS Journal 275 (2008) 4913–4926 ª 2008 The Authors Journal compilation ª 2008 FEBS 4915 for binding competition between BtR175 and mAb 1B10, a solution of mAb 1B10 was added to the cuv- ette after GST 27 kDa BtR175 had been bound to the immobilized Cry1Aa toxin, and the binding of the added protein to the surface was determined (Fig. 2G). When additional mAb 1B10 was added, antibody binding was the same as that seen with mAb 1B10 alone (Fig. 2F), suggesting that the binding sites for mAb 1B10 had not been blocked by the initial solution of GST 27 kDa BtR175. This type of binding curve indicates that mAb 1B10 does not compete for binding sites with GST 27 kDa BtR175. Monoclonal antibodies that block the binding of Cry1Aa toxin to GST 27 kDa BtR175 Seven mAbs (1B10, 1E10, 1G10, 2A11, 2C2, 2F9, and 3C7) were raised against Cry1Aa toxin in a BALB ⁄ c mouse [38]. These seven mAbs against Cry1Aa toxin were tested for their ability to block the binding of Cry1Aa toxin to BtR175. The ability of each mAb to block the binding of Cry1Aa toxin to GST 27 kDa BtR175 was investigated by preincubation of each mAb with biotinylated Cry1Aa toxin before reaction with GST 27 kDa BtR175. Of the seven mAbs, only mAbs 2A11 and 2F9 blocked the binding of Cry1Aa toxin to GST 27 kDa BtR175. The other mAbs (1B10, 1E10, 1G10, 2C2, and 3C7) had little or no effect on the binding of Cry1Aa toxin to GST 27 kDa BtR175 (Fig. 3). The concentration dependence of the blocking effects of the two blocking mAbs was determined in a similar experiment. Monoclonal antibodies 2A11 and 2F9 blocked the binding of Cry1Aa toxin to GST 27 kDa BtR175 in a dose-dependent manner, but mAbs 1B10 and 2C2 did not block the binding of Cry1Aa toxin to GST 27 kDa BtR175 even at the highest concentration used (data not shown). These results suggest that the surface of Cry1Aa toxin A B C D E F G Fig. 2. Determination of binding compatibility for BtR175, BmAPN1 and mAb 1B10, using the IAsys optical sensor. Cry1Aa toxin was cova- lently immobilized on the carboxylated surface of a cuvette. To measure the binding of receptor molecules to Cry1Aa molecules, BmAPN1 (A) or BtR175 (B) was added to the Cry1Aa-immobilized surface and the association was observed for 5 min. Subsequently, each receptor molecule solution was replaced with protein-free binding buffer (NaCl ⁄ P i ), and the dissociation of binding was observed for 5 min. At the end of this cycle, the NaCl ⁄ P i in the cuvette was replaced with 6 M guanidine hydrochloride to regenerate the sensor surface. Next, we investigated the relative locations of the binding sites for BtR175 and BmAPN1 on the surface of Cry1Aa toxin. First, BmAPN1 was added to block its binding site on the immobilized Cry1Aa toxin. Then, BmAPN1 (C) or BtR175 (D) was added as a second molecule, and the addi- tive association was observed for 5 min. BtR175 was added to block its binding site on the immobilized Cry1Aa toxin. Then, BmAPN1 (E) was added, and the additive association was observed for 5 min. As a control, the simultaneous binding of BtR175 and mAb 1B10 on the surface of Cry1Aa toxin was examined (F, G). Fig. 3. Inhibition of Cry1Aa toxin binding to GST 27 kDa BtR175 by mAbs. Biotinylated Cry1Aa toxin was preincubated with or without each mAb at 500 n M for 90 min and then added to wells coated with GST 27 kDa BtR175. After washing, it was incubated for 90 min with HRP-conjugated streptavidin. Bound streptavidin was detected by incubation with 2,2¢-azinobis(3-ethylbenzo-6-thiazoline- sulfonic acid) solution for 15 min. Absorbance at 415 nm was mea- sured using a microtiter plate reader. The assays were repeated three times, and mean values are shown. BtR175 binding site on the Cry1Aa toxin S. Atsumi et al. 4916 FEBS Journal 275 (2008) 4913–4926 ª 2008 The Authors Journal compilation ª 2008 FEBS blocked by mAbs 2A11 and 2F9 overlaps with or is close to the GST 27 kDa BtR175-binding site. Binding of mAbs 2A11 and 2F9 to Cry toxins We reported previously that the epitopes of mAbs 2A11 and 2F9 on Cry1Aa toxin are located in domain II [38]. We used two other approaches for epitope mapping of mAbs 2A11 and 2F9, immunoblotting using deletion mutants of Cry1Aa toxin, and a binding assay using synthetic peptides, but it was impossible to determine details at the amino acid level using these methods [38]. As the epitopes for mAbs 2A11 and 2F9 may consist of several discontinuous segments of polypeptide chains, they may be conformational epitopes. We looked for Cry toxins other than Cry1Aa toxin, and used immuno- blotting analysis to determine the epitope of each mAb on Cry1Aa toxin. We tested the binding of mAbs 2A11 and 2F9 to membrane blots of four Cry toxins (Cry1Aa1, Cry1Ab8, Cry1Ac1, and Cry9Da2). Three molecules, Cry1Aa1, Cry1Ab8 and Cry1Ac1 toxins, are phylogenetically closely related, but Cry9Da2 toxin is not closely related to the others. Aliquots of approxi- mately 1 lg of each toxin were subjected to SDS ⁄ PAGE, transferred onto nitrocellulose mem- branes, and probed with mAbs 2A11 (Fig. 4Bb) and 2F9 (Fig. 4Bc). The proteins on the gels were visualized by staining with Coomassie brilliant blue (Fig. 4Ba). With Cry9Da2 toxin, a 55 kDa band (fragment) was equivalent to 1 lg. Monoclonal antibody 2F9 recog- nized only Cry1Aa toxin but not Cry1Ab, Cry1Ac or Cry9Da2 toxins (Fig. 4Bc). Monoclonal antibody 2A11 recognized Cry1Aa1 toxin and the 55 kDa protein of the activated Cry9Da2 toxin, but not Cry1Ab8 or Cry1Ac1 toxins (Fig. 4Bb). Comparison of the domain II regions of Cry1Aa1, Cry1Ab8, Cry1Ac1 and Cry9Da2 toxins The sequences of Cry1Aa1 [40], Cry1Ab8 [41], Cry1Ac1 [42] and Cry9Da2 [43] toxins were compared (Fig. 4A). Amino acids that were conserved in three or A B a b c Fig. 4. Comparison of the domain II regions in Cry1Aa1, Cry1Ab8, Cry1Ac1 and Cry9Da2 toxins (A) and immunoblotting analysis of the reac- tivity of mAbs 2A11 and 2F9 with these toxins (B). (A) The sequences of Cry1Aa1 [40], Cry1Ab8 [41], Cry1Ac1 [42] and Cry9Da2 [43] toxins were aligned using GENETYX-WIN v. 5.0.0 software. Amino acids conserved in three or four toxins are boxed. Amino acids conserved in Cry1Aa1 and Cry9Da2 toxins, but not Cry1Ab8 and Cry1Ac1 toxins, are highlighted (black). The numbers beneath the alignment indicate amino acid numbers in Cry1Aa1 toxin. (B) Samples of approximately 1 lg of each toxin [i.e. Cry1Aa1 (lane 1), Cry1Ab8 (lane 2), Cry1Ac1 (lane 3), and Cry9Da2 (lane 4)] were subjected to SDS ⁄ PAGE, transferred onto nitrocellulose membranes, and probed with mAb 2A11 (b) or mAb 2F9 (c). The proteins on the gels were visualized by Coomassie brilliant blue staining (a). Arrowheads indicate the 55 kDa activated Cry9Da2 toxin (lane 4). S. Atsumi et al. BtR175 binding site on the Cry1Aa toxin FEBS Journal 275 (2008) 4913–4926 ª 2008 The Authors Journal compilation ª 2008 FEBS 4917 four toxins were noted. Amino acids conserved in Cry1Aa1 and Cry9Da2 toxins but not Cry1Ab8 and Cry1Ac1 toxins were highlighted. Thirteen amino acids were conserved in Cry1Aa1 and Cry9Da2 toxins, but not Cry1Ab8 and Cry1Ac1 toxins: Ile357, Gly372, Ser373, Phe381, Ser389, Glu404, Arg405, Thr435, Glu439, Ala440, Gly442, Thr446, and Thr451. Of these 13 amino acids, Glu439, Ala440, Gly442 and Thr446 were located on loop 3 of domain II in the Cry1Aa toxin 3D structure reported by Grochulski et al. [3] (Fig. 5). Expression and stability of Cry1Aa toxin Cys-substitution mutants In Cry1Aa, Cry1Ab, and Cry1Ac toxins, loop 3 of domain II has been reported to be involved in binding to cadherin-like proteins or brush border membrane vesicles [23,44]. To determine whether amino acids on loop 3 are involved in binding to cadherin-like pro- teins, we introduced five Cys substitutions – Y315C, V444C, N340C, Y445C, and R448C – in and near loop 3, and reacted these sites with a small blocking molecule, N-(9-acridinyl)maleimide (NAM). The sin- gle-Cys-substitution mutants of Cry1Aa toxin (amino acids 11–615) were constructed as GST fusion proteins, which were purified from Escherichia coli, solubilized, activated by trypsin, and analyzed by SDS ⁄ PAGE. Almond & Dean [45] reported that poor expression can be correlated with an unstable or mis- folded protein, but this finding was not applicable to our results, because we found that all mutant proteins (26 kDa GST plus 60 kDa mutant toxins, resulting in 86 kDa proteins) were expressed as strongly as the wild-type Cry1Aa toxin (data not shown). After tryptic digestion, the three single-Cys-substitution mutations of Cry1Aa toxin (i.e. Y315C, V444C, and R448) did not yield a 60 kDa trypsin-resistant core fragment (data not shown). These amino acid substitutions seem to be involved in a conformational change of Cry1Aa toxin, resulting in the loss of trypsin resistance. The two single-Cys-substitution mutations of Cry1Aa toxin (i.e. N340C and Y445C) yielded a 60 kDa trypsin- resistant core fragment (data not shown), suggesting that the substitutions did not cause any structural alterations to the protein. These two mutant proteins expressed in E. coli had four Cys residues (amino acids 84, 137, 168, and 177) in the GST region and only two Cys residues (amino acids 15 and 340 or 445) in the Cry1Aa toxin region. However, the GST region and the N-terminal part of the Cry1Aa toxin region containing five of the six Cys residues were removed by tryptic digestion. Thus, each of the two single-Cys- substitution mutants of Cry1Aa toxin had only one Cys residue (amino acid 340 or 445) after digestion with trypsin. We introduced NAM at a region on or near loop 3 of Cry1Aa toxin . NAM contains a malei- mide group, which can react specifically with the thiol group of Cys in protein molecules to covalently bind it to the protein. When NAM was reacted with the single-Cys-substitution mutants, it bound specifically to the Cys residues of the N340C and Y445C mutants of Cry1Aa toxin. NAM molecules introduced on the surface of Cry1Aa toxin at either of these two sites may block cadherin-like protein binding at the site, due to steric hindrance. Binding of Cry1Aa toxin constructs to GST 27 kDa BtR175 To investigate the location of BtR175-binding sites on Cry1Aa toxin, the binding of five Cry1Aa toxin con- structs (wild-type Cry1Aa, N340C, Y445C, N340C– NAM, and Y445C–NAM) to immobilized GST 27 kDa BtR175 molecules was measured using an Fig. 5. Epitope of mAb 2A11 on a 3D model of Cry1Aa toxin. The amino acids in domains I, II and III are colored pink, light green, and light blue, respectively. The amino acids conserved in Cry1Aa and Cry9Da toxins, but not Cry1Ab and Cry1Ac toxins, are shown in red. Tyr445 and Asn340, which were substituted for Cys in Fig. 6, are shown in blue. BtR175 binding site on the Cry1Aa toxin S. Atsumi et al. 4918 FEBS Journal 275 (2008) 4913–4926 ª 2008 The Authors Journal compilation ª 2008 FEBS IAsys resonant mirror optical biosensor. The profiles of binding of the toxin constructs with the immobilized GST 27 kDa BtR175 were monitored (Fig. 6), and kinetic constants were evaluated from profiles for the binding of each toxin construct to the immobilized GST 27 kDa BtR175 molecules (Table 1). There were only subtle differences in the B max , k diss and K D of wild-type Cry1Aa toxin and the N340C mutant (Table 1). There was almost no difference in the B max , k ass , k diss and K D of the N340C and N340C–NAM mutants (Table 1). These results indicate that substitu- tion of Asn340 for Cys and the introduction of NAM on the N340C mutant have rather subtle effects on binding. In contrast, the K D of the Y445C mutant was significantly lower than that of wild-type Cry1Aa toxin (Table 1, Fig. 6). This result indicates that the substi- tution of Tyr445 for Cys has a crippling effect on binding. Effects of mAbs that inhibit binding of Cry1Aa toxin to receptors on toxicity Monoclonal antibodies 1B10 and 2C2 inhibit the binding of Cry1Aa toxin to GST 7 kDa BmAPN1 [38]. Monoclonal antibodies 2A11 and 2F9 inhibited the binding of Cry1Aa toxin to GST 27 kDa BtR175 (Fig. 3). To investigate the effects of mAb binding on Cry1Aa toxicity, Cry1Aa toxin was preincubated with or without each antibody. Twenty second-instar larvae were fed an artificial diet containing Cry1Aa toxin with or without previous binding to antibodies. The LC 50 value of Cry1Aa toxin for Bo. mori was 1.43 lgÆg )1 artificial diet. The mortality rate was 63.3% when 20 second-instar larvae were fed an arti- ficial diet containing Cry1Aa toxin (2.0 lgÆg )1 artifi- cial diet) without any antibodies. There was no mortality if the toxin was preincubated with mAb 2A11 (Table 2). This indicated that the toxicity of Cry1Aa toxin against Bo. mori was blocked com- pletely when the toxin was preincubated with mAb 2A11. The mortality rate was 13.3% when the toxin was preincubated with mAb 2F9. This indicated that the toxicity of Cry1Aa toxin against Bo. mori was reduced significantly when the toxin was preincubated with mAb 2F9. However, the mortality rates were 73.3% and 66.7% when the toxin was preincubated with mAbs 1B10 and 2C2, respectively (Table 2). These results indicate that the toxicity of Cry1Aa toxin against Bo. mori was not reduced when the toxin was preincubated with mAbs 1B10 or 2C2, which can block the binding of Cry1Aa toxin to GST 7 kDa BmAPN1 [38]. Fig. 6. Sensorgrams for the binding of wild-type and mutant Cry1Aa toxins to GST 27 kDa BtR175 immobilized on the biosensor surface. The GST 27 kDa BtR175-immobilized cuvette was equili- brated in NaCl ⁄ P i for 2 min. Then, the wild-type toxin or one of the four mutant Cry1Aa toxins (1 l M) was added to the cuvette and allowed to bind for 5 min. The toxin was then removed from the reaction chamber, and NaCl ⁄ P i was added. Dissociation occurred for 5 min after addition of NaCl ⁄ P i . Table 1. Equilibrium and kinetic binding parameters for the binding of wild-type and mutant Cry1Aa toxins to BtR175 immobilized on the biosensor surface. B max , maximum binding amount; k ass , association rate constant; k dis , dissociation rate constant; K D , dissociation equilibrium constant; NAM, single Cys-substitution mutant-bound NAM. Toxins B max (pgÆmm )2 Æ5 min )1 ) k ass (· 10 3 M )1 Æs )1 ) k dis (· 10 3 Æs )1 ) K D (nM) Cry1Aa 21.10 87.5 8.20 93.7 N340C 20.05 15.9 2.92 184 N340C–NAM 15.19 7.42 2.57 346 Y445C 3.320 1.19 2.70 2260 Y445C–NAM 1.880 0.546 1.39 2550 Table 2. Effects of mAbs 2F9 and 2A11 on the toxicity of Cry1Aa toxin against second-instar larvae. Cry1Aa toxins were preincubated for 1 h with or without each antibody. Twenty second-instar larvae were fed 1.0 g of artificial diet mixed with 2.00 lg of Cry1Aa toxin with or without previous binding to antibodies. Mortality was recorded after 3 days. The LC 50 value of Cry1Aa toxin against Bo. mori was 1.43 lgÆg )1 artificial diet. The assays were repeated three times, and mean values are shown. Treatment Mortality (%) – 0.0 ± 0.0 Cry1Aa 63.3 ± 2.4 Cry1Aa + 2C2 66.7 ± 2.4 Cry1Aa + 1B10 73.3 ± 4.7 Cry1Aa + 2F9 13.3 ± 4.7 Cry1Aa + 2A11 0.0 ± 0.0 S. Atsumi et al. BtR175 binding site on the Cry1Aa toxin FEBS Journal 275 (2008) 4913–4926 ª 2008 The Authors Journal compilation ª 2008 FEBS 4919 The effect of substituting Tyr445 for Cys on the toxicity of Cry1Aa toxin Twenty third-instar larvae of Bo. mori were fed solu- tions of three serially diluted Cry1Aa toxin constructs (wild-type Cry1Aa toxin, Y445C, and Y445C–NAM), and the LD 50 was determined by a probit analysis for each toxin. As a result, the LD 50 ± 95% confidence limits of wild-type Cry1Aa toxin, mutant Y445C and mutant Y445C–NAM were 1.02 (+0.08 ⁄ )0.08), 166.85 (+20.49 ⁄ )13.58), and 154.55 (+11.22 ⁄ )9.38) ng, respectively. The LD 50 values of the Y445C and Y445C–NAM mutants were 164 and 152 times higher than that of wild-type Cry1Aa toxin, respectively. Discussion Knowledge of the receptor-binding site on a Cry toxin would allow improvements in the specificity and toxic- ity of these toxins. In the case of Cry1Ab toxin, several binding sites for M. sexta cadherin BT-R 1 have been identified. However, the binding site on Cry1Aa toxin for the Bo. mori cadherin-like protein has not been reported. As the 3D structure of Cry1Aa toxin is known, it is possible to understand the BtR175-binding site on Cry1Aa toxin three-dimensionally. In this study, we attempted to determine the binding site on Cry1Aa toxin for the candidate Cry1Aa toxin receptor in Bo. mori, a cadherin-like protein, BtR175 [37] (Gen- Bank accession number AB026260.1). Locational relationship between the BmAPN1- binding site and the BtR175-binding site To locate the BtR175-binding site, the relationship between the BmAPN1-binding and BtR175-binding sites on Cry1Aa toxin was investigated using two methods (Figs 1 and 2). The results suggest that the BtR175-binding site is close to the BmAPN1-binding site, and that the binding sites are not located on opposite surfaces of the Cry1Aa toxin molecule. Monoclonal antibodies 2A11 and 2F9 competed with BtR175 for binding to Cry1Aa toxin (Fig. 3), but did not compete with BmAPN1 [38]. Monoclonal anti- bodies 1B10 and 2C2 competed with BmAPN1 for binding to Cry1Aa toxin [38], but did not compete with BtR175 (data not shown). These results suggest that the BtR175-binding and BmAPN1-binding sites are not the same. In conclusion, the BtR175-binding site is on the same face of Cry1Aa toxin as the BmAPN1-binding site, as the areas occupied by GST 27 kDa BtR175 and GST 7 kDa BmAPN1 overlap or are close to each other. In our previous report, the BmAPN1-binding site on Cry1Aa toxin was hypothe- sized to consist of conserved amino acids (Gln292, Pro294, His295, Leu296, His433, Leu481, Val444, Val487, Arg500, and Gly505) between Cry1Aa, Cry1Ac and Cry9Da toxins, and nonconserved amino acids surrounding the conserved amino acids [38]. In fact, this region is on the same face as loop 3 of domain 2 and is close enough to it to be hidden by the protein that binds to loop 3. The location of the mAb 2A11 epitope that blocks binding of Cry1Aa toxin to GST 27 kDa BtR175 The results of competitive binding assays indicated that the epitope of mAb 2A11 and the BtR175-bind- ing site overlap or are close to each other on Cry1Aa toxin (Fig. 3). To analyze the area in which the BtR175-binding site may be located, the epitope of mAb 2A11 was investigated. Immunoblotting anal- ysis using four Cry toxins showed that mAb 2A11 recognized Cry1Aa1 and Cry9Da2 toxins, but not Cry1Ab8 or Cry1Ac1 toxin (Fig. 4Bb). These obser- vations suggest that the epitope of mAb 2A11 is localized in the region consisting of the conserved amino acids in Cry1Aa1 and Cry9Da2 toxins but not in Cry1Ab8 or Cry1Ac1 toxins. The epitope of mAb 2A11 on Cry1Aa toxin is in domain II [38]. Align- ment of Cry toxin domain II showed that 13 amino acids were conserved in Cry1Aa1 and Cry9Da2 tox- ins, but not in Cry1Ab8 or Cry1Ac1 toxin (Fig. 4A). Seven of the 13 amino acids (Gly372, Ser373, Glu439, Ala440, Gly442, Thr435, and Thr446) are located on the same face as the BmAPN1-binding site on the Cry1Aa toxin 3D structure (Fig. 5) [38]. It seems unlikely that Thr435, which is adjacent to the BmAPN1-binding site, is contained in the epitope of mAb 2A11, because this mAb did not block the bind- ing of Cry1Aa toxin to BmAPN1. The interaction between an antibody and antigen occurs over a large area, with approximate dimensions of 20 · 30 A ˚ [46], the epitope contains 15–20 amino acids [47], and the epitope is made up of four discontinuous segments of polypeptide chain on average [48]. Thus, the epitope of mAb 2A11 might be located in the region consist- ing of six amino acids (Gly372, Ser373, Glu439, Ala440, Gly442, and Thr446) (Fig. 5), because this region fulfills the above conditions. Region in which the amino acid substitution influences the binding of Cry1Aa toxin to BtR175 Substitution for Cys and the steric hindrance by NAM at Asn340 had subtle effects on the binding of Cry1Aa BtR175 binding site on the Cry1Aa toxin S. Atsumi et al. 4920 FEBS Journal 275 (2008) 4913–4926 ª 2008 The Authors Journal compilation ª 2008 FEBS toxin to BtR175 (Fig. 6, Table 1), suggesting that Asn340 may be outside of the BtR175-binding site. Meanwhile, substitution of Tyr445 for Cys had a crip- pling effect on binding of Cry1Aa toxin to BtR175, indicating that Tyr445 is located in the BtR175-bind- ing site. Otherwise, the mutation might have altered the conformation of loop 3 enough to reduce the abil- ity to bind to BtR175. Asn340 and Tyr445 are close to each other in the Cry1Aa toxin 3D structure. How- ever, they are on different strands and different faces on the Cry1Aa toxin 3D structure (Fig. 5). The BmAPN1-binding [38] and BtR175-binding sites seemed to be close to each other in the Cry1Aa toxin 3D structure (Figs 1 and 2). Glu439, Ala440, Gly442, and Thr446, which comprise the epitope of mAb 2A11, are located on loop 3 of Cry1Aa toxin (Fig. 5). On the basis of the above considerations, it is possible that the BtR175-binding site is located near loop 3 in domain II (Fig. 5). An in vitro binding assay and an in vivo bioassay using several Ala substitution mutants of Cry1Ab toxin showed that Ala substitution of amino acids in loop 3 affects initial receptor binding and toxicity in M. sexta and H. virescens [44]. Competition binding assays using loop region peptides have shown that H. virescens cadherin binds loop 3 of Cry1Ab and Cry1Ac toxins [23]. These results also support the conclusion that the loop 3 region of Cry1Aa toxin is involved in binding to BtR175. Recently, it was shown that sub- stitutions of loops, including loop 3 of Cry1Aa toxin for the third loop of the complementarity-determining region of the immunoglobulin heavy chain, do not affect the binding property of the toxin for H. vires- cens cadherin-like protein [49]. This result contradicts our conclusion mentioned here and other reported hypotheses [23,36]. Impact of Cry1Aa–BtR175 binding on toxicity In this study, mAbs 2A11 and 2F9 inhibited the binding of Cry1Aa toxin to BtR175 (Fig. 3). To investigate the effects of these mAbs on Cry1Aa toxicity, second-instar larvae were fed Cry1Aa toxin, either alone or together with a 1000-fold molar excess of each mAb. The mortal- ity rate decreased markedly with mAbs 2A11 and 2F9 (Table 2). Inhibition of the binding of Cry1Aa and Cry1Ac toxins to BtR175 by pretreatment with antibody against BtR175 suppresses the lytic activity of toxins on collagenase-dissociated Bo. mori midgut epithelial cells [24]. Substitution of Tyr445 for Cys had a crippling effect on binding to BtR175 (Table 1). This substitution caused a 164-fold decrease in toxicity. These results showed that BtR175 is involved in Cry1Aa toxicity. Experimental procedures Preparation of GST 7 kDa BmAPN1 and GST 27 kDa BtR175 A partial fragment of BmAPN1 (7 kDa BmAPN1) contain- ing the Cry1Aa toxin-binding region (Ile135 to Pro198) was prepared as a GST fusion protein as described by Yaoi et al. [39]. A partial fragment of BtR175 (27 kDa BtR175) contain- ing the Cry1Aa toxin-binding region (Glu1108 to Val1464, including cadherin repeat-8 and repeat-9, and the N-terminal half of the membrane proximal region) was prepared as a GST fusion protein, as described by Hara et al. [24]. Preparation of Cry1Aa, Cry1Ab, Cry1Ac and Cry9Da2 toxins Cry1Aa1 and Cry1Ab8 toxins were prepared from recombi- nant E. coli strains as described by Atsumi et al. [38]. Cry1Ac and Cry9Da2 toxins were prepared from B. thurin- giensis strains as described by Shinkawa et al. [50]. Biotinylation of Cry1Aa toxin Cry1Aa toxin was biotinylated with EZ-LinkSulfo-NHS- LC-LC-biotin (Pierce, Chester, UK) as described by Atsumi et al. [38]. Competitive binding assay using ELISA plate Ninety-six-well flat-bottomed ELISA plates (Corning Inc., Corning, NY, USA) were coated with 1 lm GST 27 kDa BtR175 solution in NaC ⁄ P i (100 lL per well) at 37 °C for 2 h. The wells were blocked by incubating the plates with NaCl ⁄ P i containing 2% BSA (150 lL per well) at 37 °C for 2 h and using an avidin ⁄ biotin blocking kit in accordance with the manufacturer’s instructions (Vector Laboratories, Burlingame, CA, USA). The wells were washed three times with NaCl ⁄ P i . Biotinylated Cry1Aa toxin (20 nm) was preincubated with various concentrations (0, 2, 5, 10, 20, 50, 100, 200 or 500 nm) of GST 7 kDa BmAPN1 or GST 27 kDa BtR175 solution or preincubated with various con- centrations (0, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000 or 5000 nm) of each mAb at 37 °C for 90 min and then added to the wells (100 lL per well). Biotinylated Cry1Aa toxin bound to GST 27 kDa BtR175 was detected with horseradish peroxidase (HRP)-conjugated streptavidin as described by Atsumi et al. [38]. The results are expressed as B ⁄ B max , where B is the enzymatic activity bound to the solid phase measured at various concentrations of GST 7 kDa BmAPN, GST 27 kDa BtR175 or mAbs, and B max is the enzymatic activity in the absence of these proteins. The assays were repeated three times, and mean values were plotted. S. Atsumi et al. BtR175 binding site on the Cry1Aa toxin FEBS Journal 275 (2008) 4913–4926 ª 2008 The Authors Journal compilation ª 2008 FEBS 4921 Immobilization of Cry1Aa toxin on the sensing surface of a biosensor Single-well cuvettes with carboxylate surfaces were used with the N-hydroxysuccinimide and 1-ethyl-3-(3-dimethyl- aminopropyl)-carbodiimide coupling system (Affinity Sensors, Cambridge, UK). The well of each cuvette was coated with activated Cry1Aa toxin as described by Atsumi et al. [38]. Determination of binding compatibility for BtR175, BmAPN1 and mAb 1B10 using the IAsys optical sensor After covalent immobilization of Cry1Aa toxin on the car- boxylate sensor surface, the compatible binding of GST 7 kDa BmAPN1, GST 27 kDa BtR175 and mAb 1B10 against Cry1Aa toxin to the immobilized Cry1Aa toxin was determined using an IAsys resonant mirror optical bio- sensor (Affinity Sensors). The concentrations of the GST 7 kDa BmAPN1, GST 27 kDa BtR175 and mAb 1B10 solutions were 55.5, 27.6 and 10 nm, respectively. To mea- sure the binding of individual receptors to Cry1Aa toxin molecules, GST 7 kDa BmAPN1 or GST 27 kDa BtR175 was added to the Cry1Aa toxin-immobilized surface at the concentrations given above, and the association was observed for 5 min (binding response). Subsequently, each receptor molecule solution was replaced with protein-free binding buffer (NaCl ⁄ P i ), and the dissociation of binding was observed for 5 min (dissociation response). At the end of this cycle, the NaCl ⁄ P i in the cuvette was replaced with 6 m guanidine hydrochloride solution to regenerate the sen- sor surface. Next, to investigate the relative locations of the binding sites for GST 27 kDa BtR175 and GST 7 kDa BmAPN1 on the surface of Cry1Aa toxin, the following compounds were injected. First, GST 7 kDa BmAPN1 was added to block the binding site on the immobilized Cry1Aa toxin. Then, GST 7 kDa BmAPN1 or GST 27 kDa BtR175 was added as a second molecule, and the additive associa- tion was observed for 5 min. GST 27 kDa BtR175 was added to block the binding site on the immobilized Cry1Aa toxin. Then, GST 7 kDa BmAPN1 was added, and the additive association was observed for 5 min. As a control, the compatibility of binding of GST 27 kDa BtR175 and mAb 1B10 on the surface of Cry1Aa toxin at the same time was tested. The plots during regeneration of the sensor sur- face were omitted from the sensorgram because the values exceeded 20 000 arcsec during surface regeneration. Preparation of hybridomas and monoclonal antibodies against Cry1Aa toxin A BALB ⁄ c mouse was immunized with 20 lg of activated Cry1Aa toxin, and hybridomas were produced. Ascites were obtained from the hybridomas. Seven mAbs were raised against Cry1Aa toxin: mAbs 1B10, 1E10, 1G10, 2A11, 2C2, 2F9 and 3C7 were purified from ascites [38]. Aliquots of purified antibodies were conjugated with HRP using the methods of Wilson & Nakane [51]. Immunoblotting Prepared proteins (1 lg per lane) were subjected to SDS ⁄ PAGE (10% polyacrylamide gel) according to the method of Laemmli [52]. After SDS ⁄ PAGE, the proteins were transferred onto Immobilon nitrocellulose membranes (Millipore, Bedford, MA, USA), blocked with 2% BSA in 10 mm Tris ⁄ HCl (pH 8.3) with 150 mm NaCl and 0.05% (v ⁄ v) Tween-20 (TNT) for 12 h at 4 °C, and then incubated for 2 h at room temperature with HRP-conjugated mAb diluted 1 : 10 000. The membranes were washed three times with TNT. The bands on the blots were visualized using an ECL detection system (GE Healthcare UK Ltd., Little Chalfont, UK). Site-directed mutagenesis Five pairs of complementary mutagenic oligonucleotide primers (Table 3) were designed to introduce Cys substitu- tions. Site-directed mutations were introduced into the Cry1Aa toxin gene using pN615, a plasmid containing the B. thuringiensis activated Cry1Aa toxin gene as a template as described by Atsumi et al. [38]. The mutated DNA was used to transform E. coli BL21(DE3) cells. Mutations were confirmed by DNA sequencing. Introduction of small blocking molecules into single-Cys-substitution Cry1Aa toxin mutants Two Cys-substitution mutants of Cry1Aa toxin were reacted with a small blocking molecule, NAM (Dojindo Table 3. Mutagenic primers used for site-directed mutagenesis of Cry1Aa toxin genes. The mutated cysteine codons are underlined and the nucleotide point mutations are shown in upper case. Mutants Primers Oligonucleotide sequence (5¢-to-3¢) Y315C Y315C atagaggctttaat tGttggtcagggcatc Y315C gatgccctgacca aCaattaaagcctctat N340C N340C tccctttatttggg TGtgcggggaatgcag N340C ctgcattccccgc aCAcccaaataaaggga V444C V444C aagcagctggagca TGttacaccttgagag V444C ctctcaaggtgta aCAtgctccagctgctt Y445C Y445C agctggagcagtt tGTaccttgagagctcc Y445C ggagctctcaaggt ACaaactgctccagct R448C R448C cagtttacaccttg TgTgctccaacgtttt R448C aaaacgttggagc AcAcaaggtgtaaactg BtR175 binding site on the Cry1Aa toxin S. Atsumi et al. 4922 FEBS Journal 275 (2008) 4913–4926 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... Binding of Cry1Aa toxin constructs to GST 27 kDa BtR175 After covalent immobilization of GST 27 kDa BtR175 on the carboxylate sensor surface, the binding of five Cry1Aa toxin constructs (i.e Cry1Aa, N340C, Y4 45C, N340C– NAM and Y4 45C–NAM) to GST 27 kDa BtR175 was determined using an IAsys resonant mirror optical biosensor To measure the binding of each toxin construct with GST 27 kDa BtR175, each toxin construct... (2005) Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin Appl Environ Microbiol 71, 3966–3977 39 Yaoi K, Nakanishi K, Kadotani T, Imamura M, Koizumi N, Iwahana H & Sato R (1999) Bacillus thuringiensis Cry1Aa toxin- binding region of Bombyx mori aminopeptidase N FEBS Lett 463, 221–224 40 Schnepf HE, Wong HC & Whiteley HR (1985) The amino acid sequence of. .. et al BtR175 binding site on the Cry1Aa toxin Laboratories, Tokyo, Japan) and dialyzed as described by Atsumi et al [38] ciation constant was determined on the basis of the equation KD = kdiss ⁄ kass Immobilization of BtR175 on the sensing surface of a biosensor Insects Single-well cuvettes with carboxylate surfaces were used with the N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide... cadherin repeat 12 mediates Bacillus thuringiensis Cry1Ab binding and cytotoxicity J Biol Chem 279, 28051–28056 Nagamatsu Y, Koike T, Sasaki K, Yoshimoto A & Furukawa Y (1999) The cadherin-like protein is essential to specificity determination and cytotoxic action of the Bacillus thuringiensis insecticidal CryIAa toxin FEBS Lett 460, 385–390 BtR175 binding site on the Cry1Aa toxin 38 Atsumi S, Mizuno... regenerate the sensor surface Kinetic constants were evaluated from profiles for the binding of each Cry1Aa toxin construct to GST 27 kDa BtR175 using the kinetic analysis program fast fit (Affinity Sensors) The apparent association rate constant, kon (s)1), was calculated from a single exponential fit of the binding response curve, and the apparent dissociation rate constant, koff (s)1), was calculated from the. .. Identification, isolation, and cloning of a Bacillus thuringiensis Cry1Ac toxin- binding protein from the midgut of the lepidopteran insect Heliothis virescens J Biol Chem 270, 27277– 27282 12 Nakanishi K, Yaoi K, Shimada N, Kadotani T & Sato R (1999) Bacillus thuringiensis insecticidal Cry1Aa toxin binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary success... from the dissociation response curve The dissociation constant, kdiss (m)1Æs)1), was determined using the expression koff = kdiss, and the association rate constant, kass (m)1Æs)1), was determined on the basis of the equation kass = (kon ⁄ koff) ⁄ [Ligate] [53] The equilibrium disso- Kinshu · Showa, a hybrid strain of Bo mori, was purchased from Ueda-Sanshu Co (Ueda, Japan) and reared on an artificial... construct was added to the GST 27 kDa BtR175 immobilized surface at a concentration of 1 lm, and the association was observed for 5 min (binding response) Subsequently, each toxin construct solution was replaced with protein-free binding buffer (NaCl ⁄ Pi), and the dissociation of binding was observed for 5 min (dissociation response) At the end of this cycle, the NaCl ⁄ Pi in the cuvette was replaced... Single amino acid mutations in the cadherin receptor from heliothis virescens affect its toxin binding ability to Cry1A toxins J Biol Chem 280, 8416–8425 24 Hara H, Atsumi S, Yaoi K, Nakanishi K, Higurashi S, Miura N, Tabunoki H & Sato R (2003) A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae FEBS Lett... Bulla LA Jr (2006) A mechanism of cell death involving an adenylyl cyclase ⁄ PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis Proc Natl Acad Sci USA 103, 9897–9902 ´ ´ Gomez I, Oltean DI, Gill SS, Bravo A & Soberon M (2001) Mapping the epitope in cadherin-like receptors involved in Bacillus thuringiensis Cry1A toxin interaction using phage display J Biol Chem 276, 28906– 28912 . blocked the binding of BtR175. We discuss the location of the BtR175-bind- ing site on Cry1Aa toxin on the basis of location infor- mation obtained with the. of Cry1Aa toxin constructs to GST 27 kDa BtR175 To investigate the location of BtR175-binding sites on Cry1Aa toxin, the binding of five Cry1Aa toxin con- structs