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Identification of a Bacillus thuringiensis Cry8Da toxin-binding glucosidase from the adult Japanese beetle, Popillia japonica
Journal of Invertebrate Pathology 113 (2013) 123–128
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Identification of a Bacillus thuringiensis Cry8Da toxin-binding glucosidase from the adult Japanese beetle, Popillia japonica Takuya Yamaguchi, Hisanori Bando, Shin-ichiro Asano ⇑ Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, N9 W9, Sapporo 060-8589, Japan
a r t i c l e
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Article history: Received 19 November 2012 Accepted 18 March 2013 Available online 28 March 2013 Keywords: Bacillus thuringiensis Receptor Japanese beetle Glucosidase Cry8
a b s t r a c t Cry8Da from Bacillus thuringiensis galleriae SDS-502 has insecticidal activity against both the larvae and adult Japanese beetle (Popillia japonica Newman). The receptor determines the specificity of the insecticidal activity of Cry proteins and hence, in order to reveal the mode of action of Cry toxin, receptor identification is a necessary step. However, a receptor for Cry8-type toxin has not been identified in the Scarabaeidae family of insects. Therefore, we aimed to identify the receptor of Cry8Da toxin in adult P. japonica BBMV. A ligand blot showed the Cry8Da toxin only bound to a 150 kDa protein in the BBMV of adult P. japonica. In order to identify the Cry8Da toxin binding protein, it was purified by column chromatography and three internal amino acid sequences were determined. Two of the three internal amino acid sequences shared homology with Coleopteran b-glucosidases. In addition, the fraction containing the Cry8Da toxin binding protein had b-glucosidase activity but no aminopeptidase N and alkaline phosphatase activity, both of which are commonly reported as receptors for Cry toxins in Lepidopteran and Dipteran insects. The b-glucosidase homologous genes could be amplified by PCR using degenerate oligonucleotide primers designed from a conserved sequence of Coleopteran b-glucosidases and an internal amino acid sequence of the Cry8Da toxin binding protein. Taken together, the b-glucosidase in adult P. japonica BBMV is the receptor for B. thuringiensis Cry8Da toxin. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Bacillus thuringiensis (Bt) is a rod shaped, Gram-positive, sporeforming bacterium. Bt produces parasporal crystal (Cry) proteins during sporulation. Since the crystal proteins often are insecticidal to specific species within the Lepidoptera, Diptera and Coleoptera orders, especially the larvae of these insects, Bt is widely used in pest control agents. Control of Coleopteran pests such as larvae of the Scarabaeidae family, which damage the roots of turf grass and other horticultural and agricultural plants, is difficult because they live in soil making it difficult for the sprayable Bt formulation to reach them. The Cry8-type proteins are insecticidal to the larvae of scarab beetles (Sato et al., 1994; Yu et al., 2006; Shu et al., 2009a, 2009b), but not insecticidal to adults. Therefore, it is necessary to identify a Bt Cry protein that effectively controls both the larvae and adults of the target beetles. We previously reported that Cry8Da and Cry8Db are toxic against not only larvae but also adults of Japanese beetle (Popillia japonica Newman) (Asano et al., 2003; Yamaguchi et al., 2008). It has also been reported that among the Coleopteran beetle order, few adult beetles were sensitive to Cry proteins like P. japonica. For instance, adult Batocera horsfieldi Hope is sensitive to Cry1Ac (Qi et al., 2011), Agrilus planipennis is sensitive ⇑ Corresponding author. Fax: +81 11 706 2423. E-mail address: [email protected] (S.-i. Asano). 0022-2011/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2013.03.006
to Cry8Da (Bauer and Londoño, 2010), and adult Agelastica coerulea Baly is sensitive to Cry8Da and Cry8Db (Yamaguchi et al., 2008). Generally Cry proteins, which are toxic to Lepidopteran larvae, are first solubilized in the larval midgut and are processed into active toxins by gut proteases such as trypsin and chymotrypsin. Activated Cry toxins bind specific receptors on the midgut epithelial cell brush border membrane vesicles (BBMVs). Following certain conformational changes and oligomerization, the toxins insert into the membrane to form cation-permeable pores, which cause swelling and death of epithelial cells by colloid-osmotic lysis (Schnepf et al., 1998; Soberón et al., 2009). Our previous study showed the 130 kDa Cry8Da protoxin is processed to 64 kDa, 54 kDa and 8 kDa fragments by the gut juice of larvae and adult P. japonica. The fragments of 54 kDa and 8 kDa are generated by intramolecular cleavage at the loop between the a3 and a4-helix of Domain I. Only the 54 kDa fragment binds to larvae and adult P. japonica BBMV, however it binds to different proteins in each (Yamaguchi et al., 2010). This indicates that the mode of action of Cry8Da against larvae and adult P. japonica is similar except the Cry8Da toxin utilizes different receptors. The binding of a toxin to a receptor is a critical step leading to cell death and is a key factor of toxin specificity (Bravo et al., 2011). In Lepidopteran insects and Dipteran insects, the cadherin like proteins, glycophosphatidiylinositol (GPI) anchored aminopeptidase N (APN) and GPI anchored alkaline phosphatase (ALP), have
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been reported as receptors of Cry toxins (Pigott and Ellar, 2007; Likitvivatanavong et al., 2011). In Coleopteran insects, cadherin like proteins have been identified as the receptor of Cry3A toxin in common with Lepidopteran and Dipteran insects (Fabrick et al., 2009; Park et al., 2009). In addition, Cry3A toxin interacts with a disintegrin and metalloprotease from Leptinotarsa decemlineata (Say) and a-amylase and vacuolar ATPase from Tenebrio molitor L. (Ochoa-Campuzano et al., 2007; Bulushova et al., 2011). In Scarabaeidae beetles, a Cry toxin binding protein has not been identified. Identification of a binding protein should prove useful in gaining a better understanding of how Cry8Da kills not only larval P. japonica but also adult. In this paper, the 150 kDa Cry8Da binding protein was purified from adult P. japonica BBMV and was identified as a b-glucosidase. The b-glucosidase genes were also partially cloned from the midgut of adult P. japonica. 2. Materials and methods 2.1. Insects and strains Adult beetles were collected around our University in Sapporo, Japan. Beetles were reared with Virginia creeper (Parthenocissus quinquefolia (L.) Planch.). BT51-8Da carrying the Cry8Da expression vector (Yamaguchi et al., 2010) was used for Cry8Da crystal protein expression. 2.2. Preparation and biotinylation of Cry8Da toxin The Cry8Da crystal protein and gut juice of adult P. japonica were prepared as described previously (Yamaguchi et al., 2008). The concentration of the Cry8Da protein was adjusted to 1 mg/ ml with 50 mM sodium carbonate buffer, pH 10.5 and activated with the gut juice of adult P. japonica (1:5, w/w) overnight at 25 °C. Activated Cry8Da toxin was purified by anion exchange chromatography followed by gel filtration chromatography as described previously (Yamaguchi et al., 2010). The purity of the Cry8Da toxin was determined by Tricine–SDS–PAGE. Purified Cry8Da toxin was biotinylated with ECL Protein Biotinylation Module (GE Healthcare, Chalfont St. Giles, UK) according to the manufacturer’s protocol. 2.3. Preparation of P. japonica BBMV BBMV were prepared from the dissected midguts of adult P. japonica by a differential magnesium precipitation method (Wolfersberger et al., 1987). Protein concentration was determined with a Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin (BSA) as the standard. 2.4. Ligand blot Proteins of adult P. japonica BBMV (20 lg) were separated by 8% SDS–PAGE and transferred to a PVDF membrane. The membranes were incubated in PBS containing 3% ECL Blocking Agent (GE Healthcare) and 0.1% Tween 20, pH 7.4 for 90 min at 25 °C. After washing three times with PBST for 10 min each, the membranes were incubated with PBST containing 3% ECL Blocking Agent and biotinylated Cry8Da toxin (1 lg/ml, final concentration) for 16 h at 4 °C. After washing three times with PBST for 10 min each, the membranes were incubated with a 1:1000 dilution of streptavidin-conjugated horseradish peroxidase (GE Healthcare) for 60 min at 25 °C. After another set of three washes, membranes were developed using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).
2.5. Solubilization of P. japonica BBMV A total of 25 mg of BBMV prepared from adult P. japonica was solubilized by gentle rotation at 4 °C for 1 h in 1% 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS) in 10 mM sodium phosphate buffer (SPB), pH 7.4. Insoluble materials were precipitated by ultracentrifugation (100,000g, 60 min, 4 °C) and CHAPS solubilized proteins were recovered. CHAPS soluble and insoluble fractions were subjected to ligand blot to evaluate the solubilization efficiency of the 150 kDa Cry8Da binding protein. 2.6. Purification of the Cry8Da binding protein on P. japonica BBMV Solubilized BBMV was fractionated by HiTrap Q HP (GE Healthcare) in 0.6% CHAPS containing 10 mM SPB, pH 7.4 using a NaCl step gradient elution (0.2, 0.4, 0.6, 1.0 M). The 150 kDa Cry8Da binding protein eluted fractions were identified by ligand blot and pooled followed by concentration using an Amicon Ultra concentrator (MWCO: 10 kDa). Concentrated fractions were loaded onto a Superdex 200 HR column (GE Healthcare) in 10 mM SPB containing 0.6% CHAPS and 0.1 M NaCl. The fractions containing the Cry8Da binding protein were identified by ligand blot, pooled and further purified on a UNO-Q column (Bio-Rad) in 10 mM SPB containing 0.6% CHAPS using a NaCl linear gradient (0.2–0.7 M). The Cry8Da binding protein containing fractions were identified by ligand blot and pooled. 2.7. N-terminal amino acid sequencing N-terminal amino acid sequencing of the 150 kDa Cry8Da binding protein was performed at the Center for Instrumental Analysis, Hokkaido University using Procise 491 cLC (Applied Biosystems, Foster City, CA, USA). Cry8Da binding protein was precipitated with 10% (final concentration) trichloroacetic acid. Precipitated protein was washed twice with ice cold acetone and solubilized with 5 M urea, 1.5 M thiourea, 2% CHAPS in 125 mM Tris–HCl, pH 6.5. The solubilized proteins were separated by 8% SDS–PAGE followed by transfer to a PVDF membrane. The membrane was stained by CBB-R250, the 150 kDa band was cut out from membrane and sequenced. 2.8. Peptide mapping V8 protease digestion of the Cry8Da binding protein was done by the method of Cleveland et al. (1977). Generated peptides were separated by 14% SDS–PAGE, transferred to a PVDF membrane and sequenced as described above. 2.9. Detection of b-glucosidase activity b-glucosidase activity was detected using the substrate pnitrophenyl-b-D-galactopyranoside (pNPG) purchased from Wako Chemicals. Adult P. japonica BBMV (2 lg) and the 150 kDa Cry8Da binding protein containing fraction (5 ll) eluted from the UNO-Q column were mixed with 100 ll of PBS containing pNPG for 10 min at room temperature. Color change resulting from the release of p-nitrophenol was quantified. 2.10. Amplification of b-glucosidase genes by DOP–PCR Midguts from adult P. japonica were obtained by dissection and maintained in RNAlater (Ambion) at 30 °C until total RNA preparation was carried out. Total RNA was prepared with Trizol (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using the PrimeScript RTreagent kit (Takara, Japan) according to manufacturer’s protocol. Two degenerated oligonucleotide primers (DOPs)
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BLAST X (http://blast.ncbi.nlm.nih.gov/) and Clustal X (Larkin et al., 2007).
Table 1 Degenerate oligonucleotide primes for PCR. Primers
Sequences (50 –30 )
Degeneracy
APj-bGlu-450FW APj-15060-RV
aWYcaYtgggaYYtRccNca ggagtNNNRtcRgaRaaNcc
x256 x2048
3. Results 3.1. Detection and partial purification of Cry8Da binding protein
200 116 97 66
200 116 97 66
45
45
To detect the Cry8Da binding protein, a ligand blot was performed. The results showed the Cry8Da toxin bound to a 150 kDa protein in adult P. japonica BBMV (Fig. 1). Adult P. japonica BBMV was solubilized with 1% CHAPS. Ligand blot and densitometry analysis showed more than 90% of the Cry8Da binding protein was contained in the CHAPS solubilized fraction (data not shown). CHAPS solubilized P. japonica BBMV was fractionated by anion exchange chromatography (Hitrap Q HP). Each column fraction was subjected to ligand blot for identification of Cry8Da toxin binding protein. Cry8Da toxin binding protein was eluted with 0.6 M NaCl (data not shown) and pooled. Pooled fractions were concentrated with an Amicon Ultra and further fractionated by gel filtration chromatography. The fractions containing the150 kDa Cry8Da toxin binding protein were pooled and were further fractionated by anion exchange chromatography using an UNO-Q column. The presence of the 150 kDa Cry8Da binding protein was confirmed by SDS–PAGE and ligand blot (Fig. 2). The 150 kDa Cry8Da binding protein eluted at around 0.5 M NaCl.
31
31
3.2. Peptide mapping and N-terminal amino acid sequencing of Cry8Da binding protein
21
21
14
14
were designed: APj-bGlu-450FW and APj-15060-RV (Table 1) from (F/Y)HWDLPQ, a conserved sequence of Coleopteran b-glucosidase and GFSDXTG, an internal sequence of the 150 kDa Cry8Da binding protein. Thirty cycles of stepwise amplification (94 °C for 1 min, 40 °C for 1 min, and 72 °C for 3 min) were carried out. After gel purification, the 900 bp of the PCR product was cloned into pCRII-TOPO (Invitrogen) and sequenced. Homology search and alignment of amino acid sequences were carried out using NCBI
(kDa)
(kDa)
Fig. 1. Ligand blot of biotinylated Cry8Da toxin to adult P. japonica BBMV. Twenty micro gram of adult P. japonica BBMV was subjected to 9% SDS–PAGE. And then, proteins were visualized by CBB G-250 stain (left panel) or transferred to PVDF membrane and probed with biotinylated Cry8Da toxin (right panel).
A
Partially purified 150 kDa Cry8Da binding protein was subjected to N-terminal amino acid sequencing but Edman degradation was unsuccessful. Instead, a peptide map of Cry8Da binding protein was prepared to allow identification of internal amino acid sequences. The 150 kDa Cry8Da binding protein was digested with V8 protease according to the method of Cleveland et al. (1977). Three generated peptides were subjected to N-terminal amino acid sequencing. The 60 kDa peptide named BG150-60 had the sequence XGFSDXTGXL, the 40 kDa peptide, BG150-40, had the sequence XAXNEDXKGE, and the 10 kDa peptide, BG150-10, had
B
0.15
(kDa) 250 150
absorbance
0.1
100 75 0.05
1
50 NaCl (M)
0
35
(kDa) 250 150 100 75
50
35
0
0
5
10
15
20
25
30
run volume (ml) Fig. 2. UNO-Q anion exchange chromatography. Pre-purified the 150 kDa Cry8Da binding protein from solubilized adult P. japonica BBMV using Hitrap Q HP and Superdex 200 HR were finally purified using UNO-Q. (A) Chromatogram of anion exchange chromatography. The absorbance at 280 nm (black line) is indicated at the left, the NaCl concentration (gray line) is indicated at the right and the run volume is indicated at the bottom. The 150 kDa Cry8Da binding protein was eluted at around 0.5 M NaCl and (B) detection of the 150 kDa Cry8Da binding protein. The Cry8Da binding protein eluted fraction was subjected to SDS–PAGE and visualized by CBB G-250 stain (left panel) or ligand blot (right panel).
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the sequence XRALVDGEAA. Homologous sequences were identified in the NCBI Coleopteran protein database using the BLAST P program. The sequence BG150-60 shared amino acid sequence homology with Caryedon serratus cytochrome b (AE072230), T. castaneum phospholipid transporting ATPase (XP_974455, XP_970033) and T. molitor b-glucosidase (AAG26008). The sequence 150-40 shared homology with T. castaneum and T. molitor b-glucosidase (XP_975665, XP_972082 AAG26008). The sequence of BG150-10 was homologous with various proteins however no homology was detected with a b-glucosidase. 3.3. Detection of b-glucosidase activity Two internal sequences of the 150 kDa Cry8Da binding protein shared homology with known b-glucosidase sequences. We attempted to detect b-glucosidase activity in adult P. japonica BBMV and the 150 kDa Cry8Da binding protein containing fraction from the UNO-Q column using the colorimetric substrate, pNPG. If a bglucosidase hydrolyzes pNPG, the reaction solution will turn yellow because of the release of p-nitrophenol. As shown in Fig. 4, BBMV and the 150 kDa Cry8Da binding protein containing fraction had b-glucosidase activity. 3.4. Amplification and sequencing of b-glucosidase genes To amplify the b-glucosidase, the cDNA was synthesized from the total RNA prepared from the midguts of adult P. japonica. Degenerated oligonucleotide primers (DOPs) were designed from an internal sequence of the 150 kDa Cry8Da binding protein and a conserved region of Coleopteran b-glucosidases. Around 900 bp of DNA was amplified, cloned into a vector and the sequence determined. We obtained four sequences homologous to b-glucosidase and named them APj-bGlu-1, APj-bGlu-2, APj-bGlu-3 and APj-bGlu4. The deduced amino acid sequences of these genes are shown in Fig. 5. APj-bGlu-1 had an amino acid sequence, RALVDGEAA, corresponding to 150-10, which is not similar to any known bglucosidase.
databases of Scarabaeidae beetle are still insufficient for this purpose. Proteins from adult P. japonica BBMV were solubilized in 1% CHAPS. The ligand blot showed that CHAPS treatment solubilized more than 90% of the total amount of 150 kDa Cry8Da binding protein (data not shown). CHAPS is a good detergent to solubilize Cry8Da binding protein in BBMV since after the purification, the Cry8Da binding protein was still remaining the b-glucosidase activity (Fig. 4). Cry8Da binding protein was purified to a sufficient level to attempt N-terminal amino acid sequencing as shown in Fig. 2. However, the N-terminal amino acid sequence of the 150 kDa Cry8Da binding protein could not be determined by automated Edman degradation using a protein sequencer likely because of post-translational modifications at the N-terminal amino acid such as acylation and/or polyglutamylation. Therefore, the 150 kDa Cry8Da binding protein was digested with V8 protease and three internal amino acid sequences were determined, BG150-60, BG150-40 and BG150-10 (Fig. 3). Homology searches revealed BG150-60 and BG150-40 shared amino acid sequence homology with Coleopteran b-glucosidases. APj-bGlu-1, one of the b-glucosidase genes amplified by PCR using DOP designed from BG150-60 and conserved region of Coleopteran glucosidase contained BG150-10, which had no homology with known glucosidases (Fig. 5). Taken together, these results strongly suggested receptor of Cry8Da is a b-glucosidase encoded by the APj-bGlu-1. The precise role of the receptor has been described for Manduca sexta and Cry1Ab toxin (Gómez et al., 2007). Activated Cry1Ab toxin binds first to the receptor, a cadherin like protein. This binding
(kDa) 97
45
150-40 XAXNEDXKGE
31
4. Discussion We previously reported Cry8Da showed insecticidal activity against not only the larvae of P. japonica but also the adult (Yamaguchi et al., 2008). Cry proteins are solubilized in the larval midgut and are processed into active toxins by gut juice proteases such as trypsin and chymotrypsin. Activated Cry toxins bind specific receptors on the midgut epithelial cell brush border membrane vesicles (BBMVs). Following certain conformational changes and oligomerization, the toxins insert into the membrane to form cation-permeable pores, which cause swelling and death of epithelial cells by colloid-osmotic lysis (Schnepf et al., 1998). The insect specificity of Cry toxins is largely determined by the specific binding of the toxins to receptors on the midgut epithelial cell BBMV (Bravo et al., 2011). In lepidopteran and dipteran insects, the cadherin like proteins, GPI anchored APN and ALP, have been reported as receptors of Cry toxins (Pigott and Ellar, 2007; Likitvivatanavong et al., 2011). Binding of these receptor proteins and Cry toxins can be demonstrated by ligand blot. In our previous study, the binding of Cry8Da toxin and the 150 kDa protein from adult P. japonica BBMV was demonstrated by ligand blot (Yamaguchi et al., 2010). To identify the 150 kDa Cry8Da binding protein from adult P. japonica BBMV, we partially purified the 150 kDa Cry8Da binding protein from BBMV and determined its internal amino acid sequence. Although peptide mass spectrometry finger printing is a powerful tool to identify proteins, it requires a comprehensive DNA and protein database. Unfortunately, the DNA and protein
150-60 XGFSDXTGXL
66
21 14
150-10 XRALVDGEAA
6.5
Fig. 3. Peptide mapping and N-terminal amino acid sequencing. The 150 kDa Cry8Da binding protein from adult P. japonica BBMV was digested with V8 proteinase according to Cleveland method. The peptides were subjected to Nterminal amino acid sequencing. Arrows indicate peptides, subjected to N-terminal amino acid sequencing and N-terminal amino acid sequences are shown in right side of gel picture.
1
2
3
Fig. 4. Detection of b-glucosidase activity. To detect b-glucosidase activity, PBS (1), BBMV (2) and the 150 kDa Cry8Da binding protein eluted fraction (3) were incubated in pNPG containing PBS. b-glucosidase hydrolyzes pNPG and then aliquot will turn to yellow because of released p-nitoroanilline.
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Fig. 5. Amino acid sequence alignment of DOP–PCR amplified b-glucosidases. Partial b-glucosidase genes were amplified using degenerated oligonucleotide primers (DOPs) designed from a conserved amino acid sequence of Coleopteran b-glucosidases and internal amino sequence (150-60) of the 150 kDa Cry8Da binding protein. Amino sequences responding to DOP were indicated by doted box. A sequence corresponding to a internal amino acid sequence, 150-10 were indicated by black box.
promotes the proteolytic cleavage of the a1-helix of Domain I, inducing oligomerization. Oligomerized Cry1Ab toxin binds to a second receptor, such as GPI anchored APN or ALP, and then inserts into the membrane to form pores that causes cell lysis. Although little is known specifically about toxin binding in Coleopteran insects, Cry3A seems to follow a similar mode of action to Cry8. In Coleopteran insects, Cry3A recognizes a cadherin like protein on BBMV (Fabrick et al., 2009; Park et al., 2009). The binding of Cry3A toxin and the cadherin like protein induces oligomerization of Cry3A (Fabrick et al., 2009). Cry3A is capable of creating pores on the BBMV of Cry3A sensitive insects such as L. decemlineata (Rausell et al., 2004). In contrast, chymotrypsin activated Cry8Ea, which is toxic to a Coleopteran chafer, only has pore-forming activity on artificial liposomes (Guo et al., 2009). b-glucosidase is a glycoside hydrolase, which hydrolyzes the bglycoside bond of a sugar. b-glucosidase is involved in the degradation of cellulose, which is a polysaccharide consisting of a linear chain of b-1, 4-linked D-glucose units and is a structural component of the cell walls of plant cells. b-glucosidases include APjbGlu-1 will be involved in cellulose digestion of adult P. japonica, since these glucosidases were found in the digestive system of adult P. japonica, voracious plant leaf eaters. Binding of glycohydrolase and Cry toxins have been previously reported. Cry4Ba and Cry11Aa bind to GPI anchored a-amylase of Anopheles albimanaus (Fernandez-Luna et al., 2010). In addition, binding of beetle specific Cry3A toxin and a-amylase of T. molitor is reported (Bulushova et al., 2011). Although precise role of these Cry toxins binding glycoside hydrolases is not described, APj-bGlu-1 is probably involved in oligomerization and pore formation of Cry8Da toxin. In this study receptor of Cry8Da toxin was identified as a b-glucosidase in adult P. japonica BBMV. Further research including complete gene cloning and studies into the interactions of Cry8Da toxin and b-glucosidase is needed to determine the role of this Cry8Da receptor in the mechanism of Cry8Da insecticidal activity. Acknowledgment This work was supported in part by the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (T.Y.).
References Asano, S.-I., Yamashita, C., Iizuka, T., Takeuchi, K., Yamanaka, S., Cerf, D., Yamamoto, T., 2003. A strain of Bacillus thuringiensis subsp. galleriae containing a novel cry8 gene highly toxic to Anomala cuprea (Coleoptera: Scarabaeidae). Biol. Control 28, 191–196. Bauer, L., Londoño, D., 2010. Effects of Bacillus thuringiensis SDS-502 on adult emerald ash borer. In: Proceedings, 21st U.S. Department of Agriculture Interagency Research Forum on Invasive Species 2010, 74–75. Bulushova, N., Zhuzhikov, D., Lyutikova, L., Kirillova, N., Zalunin, I., Chestukhina, G., 2011. Toxin-binding proteins isolated from yellow mealworm Tenebrio molitor and wax moth Galleria mellonella. Biochemistry Moscow 76, 202–208. Bravo, A., Likitvivatanavong, S., Gill, S.S., Soberón, M., 2011. Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem. Mol. Biol. 41, 423–431. Cleveland, D.W., Fischer, S.G., Kirschner, M.W., Laemmli, U.K., 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102–1106. Fabrick, J.A., Oppert, C., Lorenzen, M.D., Morris, K., Oppert, B., Jurat-Fuentes, J.L., 2009. A novel Tenebrio molitor cadherin is a functional receptor for Bacillus thuringiensis Cry3Aa toxin. J. Biol. Chem. 284, 18401–18410. Fernandez-Luna, M.T., Lanz-Mendoza, H., Gill, S.S., Bravo, A., Soberón, M., MirandaRios, J., 2010. An a-amylase is a novel receptor for Bacillus thuringiensis ssp. israelensis Cry4Ba and Cry11Aa toxins in the malaria vector mosquito Anopheles albimanus (Diptera: Culicidae). Environ. Microbiol. 12, 746–757. Gómez, I., Pardo-López, L., Muñoz-Garay, C., Fernandez, L.E., Pérez, C., Sánchez, J., Soberón, M., Bravo, A., 2007. Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis. Peptides 28, 169–173. Guo, S., Ye, S., Liu, Y., Wei, L., Xue, J., Wu, H., Song, F., Zhang, J., Wu, X., Huang, D., Rao, Z., 2009. Crystal structure of Bacillus thuringiensis Cry8Ea1: an insecticidal toxin toxic to underground pests, the larvae of Holotrichia parallela. J. Struct. Biol. 168, 259–266. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G., 2007. Clustal W and clustal X version 2.0. Bioinformatics 23, 2947–2948. Likitvivatanavong, S., Chen, J., Evans, A.M., Bravo, A., Soberón, M., Gill, S.S., 2011. Multiple receptors as targets of Cry toxins in mosquitoes. J. Agric. Food Chem. 59, 2829–2838. Ochoa-Campuzano, C., Real, M.D., Martínez-Ramírez, A.C., Bravo, A., Rausell, C., 2007. An ADAM metalloprotease is a Cry3Aa Bacillus thuringiensis toxin receptor. Biochem. Biophys. Res. Commun. 362, 437–442. Park, Y., Abdullah, M.A.F., Taylor, M.D., Rahman, K., Adang, M.J., 2009. Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb toxicities to Coleopteran larvae by a toxin-binding fragment of an insect cadherin. Appl. Environ. Microbiol. 75, 3086–3092. Pigott, C.R., Ellar, D.J., 2007. Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol. Mol. Biol. Rev. 71, 255–281. Qi, G., Lu, J., Zhang, P., Li, J., Zhu, F., Chen, J., Liu, Y., Yu, Z., Zhao, X., 2011. The cry1Ac gene of Bacillus thuringiensis ZQ-89 encodes a toxin against long-horned beetle adult. J. Appl. Microbiol. 110, 1224–1234.
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Rausell, C., García-Robles, I., Sánchez, J., Muñoz-Garay, C., Martínez-Ramírez, A.C., Real, M.D., Bravo, A., 2004. Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata (Say). Biochim. Biophys. Acta 1660, 99–105. Sato, R., Takeuchi, K., Ogiwara, K., Minami, M., Kaji, Y., Suzuki, N., Hori, H., Asano, S., Ohba, M., Iwahana, H., 1994. Cloning, heterologous expression, and localization of a novel crystal protein gene from Bacillus thuringiensis serovar japonensis strain Buibui toxic to scarabaeid insects. Curr. Microbiol. 28, 15–19. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J.A., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775–806. Shu, C., Yan, G., Wang, R., Zhang, J., Feng, S., Huang, D., Song, F., 2009a. Characterization of a novel cry8 gene specific to Melolonthidae pests: Holotrichia oblita and Holotrichia parallela. Appl. Microbiol. Biotechnol. 84, 701–707. Shu, C., Yu, H., Wang, R., Fen, S., Su, X., Huang, D., Zhang, J., Song, F., 2009b. Characterization of two novel cry8 genes from Bacillus thuringiensis strain BT185. Curr. Microbiol. 58, 389–392.
Soberón, M., Gill, S.S., Bravo, A., 2009. Signaling versus punching hole: how do Bacillus thuringiensis toxins kill insect midgut cells? Cell Mol. Life Sci. 66, 1337– 1349. Wolfersberger, M., Luethy, P., Maurer, A., Parenti, P., Sacchi, F., Giordana, B., Hanozet, G., 1987. Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp. Biochem. Phys. A 86, 301–308. Yamaguchi, T., Sahara, K., Bando, H., Asano, S.-I., 2008. Discovery of a novel Bacillus thuringiensis Cry8D protein and the unique toxicity of the Cry8D-class proteins against scarab beetles. J. Invertebr. Pathol. 99, 257–262. Yamaguchi, T., Sahara, K., Bando, H., Asano, S.-I., 2010. Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle. J. Invertebr. Pathol. 105, 243–247. Yu, H., Zhang, J., Huang, D., Gao, J., Song, F., 2006. Characterization of Bacillus thuringiensis strain Bt185 toxic to the Asian cockchafer: Holotrichia parallela. Curr. Microbiol. 53, 13–17.
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Identification of a Bacillus thuringiensis Cry8Da toxin-binding glucosidase from the adult Japanese beetle, Popillia japonica Takuya Yamaguchi, Hisanori Bando, Shin-ichiro Asano ⇑ Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, N9 W9, Sapporo 060-8589, Japan
a r t i c l e
i n f o
Article history: Received 19 November 2012 Accepted 18 March 2013 Available online 28 March 2013 Keywords: Bacillus thuringiensis Receptor Japanese beetle Glucosidase Cry8
a b s t r a c t Cry8Da from Bacillus thuringiensis galleriae SDS-502 has insecticidal activity against both the larvae and adult Japanese beetle (Popillia japonica Newman). The receptor determines the specificity of the insecticidal activity of Cry proteins and hence, in order to reveal the mode of action of Cry toxin, receptor identification is a necessary step. However, a receptor for Cry8-type toxin has not been identified in the Scarabaeidae family of insects. Therefore, we aimed to identify the receptor of Cry8Da toxin in adult P. japonica BBMV. A ligand blot showed the Cry8Da toxin only bound to a 150 kDa protein in the BBMV of adult P. japonica. In order to identify the Cry8Da toxin binding protein, it was purified by column chromatography and three internal amino acid sequences were determined. Two of the three internal amino acid sequences shared homology with Coleopteran b-glucosidases. In addition, the fraction containing the Cry8Da toxin binding protein had b-glucosidase activity but no aminopeptidase N and alkaline phosphatase activity, both of which are commonly reported as receptors for Cry toxins in Lepidopteran and Dipteran insects. The b-glucosidase homologous genes could be amplified by PCR using degenerate oligonucleotide primers designed from a conserved sequence of Coleopteran b-glucosidases and an internal amino acid sequence of the Cry8Da toxin binding protein. Taken together, the b-glucosidase in adult P. japonica BBMV is the receptor for B. thuringiensis Cry8Da toxin. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Bacillus thuringiensis (Bt) is a rod shaped, Gram-positive, sporeforming bacterium. Bt produces parasporal crystal (Cry) proteins during sporulation. Since the crystal proteins often are insecticidal to specific species within the Lepidoptera, Diptera and Coleoptera orders, especially the larvae of these insects, Bt is widely used in pest control agents. Control of Coleopteran pests such as larvae of the Scarabaeidae family, which damage the roots of turf grass and other horticultural and agricultural plants, is difficult because they live in soil making it difficult for the sprayable Bt formulation to reach them. The Cry8-type proteins are insecticidal to the larvae of scarab beetles (Sato et al., 1994; Yu et al., 2006; Shu et al., 2009a, 2009b), but not insecticidal to adults. Therefore, it is necessary to identify a Bt Cry protein that effectively controls both the larvae and adults of the target beetles. We previously reported that Cry8Da and Cry8Db are toxic against not only larvae but also adults of Japanese beetle (Popillia japonica Newman) (Asano et al., 2003; Yamaguchi et al., 2008). It has also been reported that among the Coleopteran beetle order, few adult beetles were sensitive to Cry proteins like P. japonica. For instance, adult Batocera horsfieldi Hope is sensitive to Cry1Ac (Qi et al., 2011), Agrilus planipennis is sensitive ⇑ Corresponding author. Fax: +81 11 706 2423. E-mail address: [email protected] (S.-i. Asano). 0022-2011/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2013.03.006
to Cry8Da (Bauer and Londoño, 2010), and adult Agelastica coerulea Baly is sensitive to Cry8Da and Cry8Db (Yamaguchi et al., 2008). Generally Cry proteins, which are toxic to Lepidopteran larvae, are first solubilized in the larval midgut and are processed into active toxins by gut proteases such as trypsin and chymotrypsin. Activated Cry toxins bind specific receptors on the midgut epithelial cell brush border membrane vesicles (BBMVs). Following certain conformational changes and oligomerization, the toxins insert into the membrane to form cation-permeable pores, which cause swelling and death of epithelial cells by colloid-osmotic lysis (Schnepf et al., 1998; Soberón et al., 2009). Our previous study showed the 130 kDa Cry8Da protoxin is processed to 64 kDa, 54 kDa and 8 kDa fragments by the gut juice of larvae and adult P. japonica. The fragments of 54 kDa and 8 kDa are generated by intramolecular cleavage at the loop between the a3 and a4-helix of Domain I. Only the 54 kDa fragment binds to larvae and adult P. japonica BBMV, however it binds to different proteins in each (Yamaguchi et al., 2010). This indicates that the mode of action of Cry8Da against larvae and adult P. japonica is similar except the Cry8Da toxin utilizes different receptors. The binding of a toxin to a receptor is a critical step leading to cell death and is a key factor of toxin specificity (Bravo et al., 2011). In Lepidopteran insects and Dipteran insects, the cadherin like proteins, glycophosphatidiylinositol (GPI) anchored aminopeptidase N (APN) and GPI anchored alkaline phosphatase (ALP), have
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been reported as receptors of Cry toxins (Pigott and Ellar, 2007; Likitvivatanavong et al., 2011). In Coleopteran insects, cadherin like proteins have been identified as the receptor of Cry3A toxin in common with Lepidopteran and Dipteran insects (Fabrick et al., 2009; Park et al., 2009). In addition, Cry3A toxin interacts with a disintegrin and metalloprotease from Leptinotarsa decemlineata (Say) and a-amylase and vacuolar ATPase from Tenebrio molitor L. (Ochoa-Campuzano et al., 2007; Bulushova et al., 2011). In Scarabaeidae beetles, a Cry toxin binding protein has not been identified. Identification of a binding protein should prove useful in gaining a better understanding of how Cry8Da kills not only larval P. japonica but also adult. In this paper, the 150 kDa Cry8Da binding protein was purified from adult P. japonica BBMV and was identified as a b-glucosidase. The b-glucosidase genes were also partially cloned from the midgut of adult P. japonica. 2. Materials and methods 2.1. Insects and strains Adult beetles were collected around our University in Sapporo, Japan. Beetles were reared with Virginia creeper (Parthenocissus quinquefolia (L.) Planch.). BT51-8Da carrying the Cry8Da expression vector (Yamaguchi et al., 2010) was used for Cry8Da crystal protein expression. 2.2. Preparation and biotinylation of Cry8Da toxin The Cry8Da crystal protein and gut juice of adult P. japonica were prepared as described previously (Yamaguchi et al., 2008). The concentration of the Cry8Da protein was adjusted to 1 mg/ ml with 50 mM sodium carbonate buffer, pH 10.5 and activated with the gut juice of adult P. japonica (1:5, w/w) overnight at 25 °C. Activated Cry8Da toxin was purified by anion exchange chromatography followed by gel filtration chromatography as described previously (Yamaguchi et al., 2010). The purity of the Cry8Da toxin was determined by Tricine–SDS–PAGE. Purified Cry8Da toxin was biotinylated with ECL Protein Biotinylation Module (GE Healthcare, Chalfont St. Giles, UK) according to the manufacturer’s protocol. 2.3. Preparation of P. japonica BBMV BBMV were prepared from the dissected midguts of adult P. japonica by a differential magnesium precipitation method (Wolfersberger et al., 1987). Protein concentration was determined with a Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin (BSA) as the standard. 2.4. Ligand blot Proteins of adult P. japonica BBMV (20 lg) were separated by 8% SDS–PAGE and transferred to a PVDF membrane. The membranes were incubated in PBS containing 3% ECL Blocking Agent (GE Healthcare) and 0.1% Tween 20, pH 7.4 for 90 min at 25 °C. After washing three times with PBST for 10 min each, the membranes were incubated with PBST containing 3% ECL Blocking Agent and biotinylated Cry8Da toxin (1 lg/ml, final concentration) for 16 h at 4 °C. After washing three times with PBST for 10 min each, the membranes were incubated with a 1:1000 dilution of streptavidin-conjugated horseradish peroxidase (GE Healthcare) for 60 min at 25 °C. After another set of three washes, membranes were developed using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).
2.5. Solubilization of P. japonica BBMV A total of 25 mg of BBMV prepared from adult P. japonica was solubilized by gentle rotation at 4 °C for 1 h in 1% 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS) in 10 mM sodium phosphate buffer (SPB), pH 7.4. Insoluble materials were precipitated by ultracentrifugation (100,000g, 60 min, 4 °C) and CHAPS solubilized proteins were recovered. CHAPS soluble and insoluble fractions were subjected to ligand blot to evaluate the solubilization efficiency of the 150 kDa Cry8Da binding protein. 2.6. Purification of the Cry8Da binding protein on P. japonica BBMV Solubilized BBMV was fractionated by HiTrap Q HP (GE Healthcare) in 0.6% CHAPS containing 10 mM SPB, pH 7.4 using a NaCl step gradient elution (0.2, 0.4, 0.6, 1.0 M). The 150 kDa Cry8Da binding protein eluted fractions were identified by ligand blot and pooled followed by concentration using an Amicon Ultra concentrator (MWCO: 10 kDa). Concentrated fractions were loaded onto a Superdex 200 HR column (GE Healthcare) in 10 mM SPB containing 0.6% CHAPS and 0.1 M NaCl. The fractions containing the Cry8Da binding protein were identified by ligand blot, pooled and further purified on a UNO-Q column (Bio-Rad) in 10 mM SPB containing 0.6% CHAPS using a NaCl linear gradient (0.2–0.7 M). The Cry8Da binding protein containing fractions were identified by ligand blot and pooled. 2.7. N-terminal amino acid sequencing N-terminal amino acid sequencing of the 150 kDa Cry8Da binding protein was performed at the Center for Instrumental Analysis, Hokkaido University using Procise 491 cLC (Applied Biosystems, Foster City, CA, USA). Cry8Da binding protein was precipitated with 10% (final concentration) trichloroacetic acid. Precipitated protein was washed twice with ice cold acetone and solubilized with 5 M urea, 1.5 M thiourea, 2% CHAPS in 125 mM Tris–HCl, pH 6.5. The solubilized proteins were separated by 8% SDS–PAGE followed by transfer to a PVDF membrane. The membrane was stained by CBB-R250, the 150 kDa band was cut out from membrane and sequenced. 2.8. Peptide mapping V8 protease digestion of the Cry8Da binding protein was done by the method of Cleveland et al. (1977). Generated peptides were separated by 14% SDS–PAGE, transferred to a PVDF membrane and sequenced as described above. 2.9. Detection of b-glucosidase activity b-glucosidase activity was detected using the substrate pnitrophenyl-b-D-galactopyranoside (pNPG) purchased from Wako Chemicals. Adult P. japonica BBMV (2 lg) and the 150 kDa Cry8Da binding protein containing fraction (5 ll) eluted from the UNO-Q column were mixed with 100 ll of PBS containing pNPG for 10 min at room temperature. Color change resulting from the release of p-nitrophenol was quantified. 2.10. Amplification of b-glucosidase genes by DOP–PCR Midguts from adult P. japonica were obtained by dissection and maintained in RNAlater (Ambion) at 30 °C until total RNA preparation was carried out. Total RNA was prepared with Trizol (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using the PrimeScript RTreagent kit (Takara, Japan) according to manufacturer’s protocol. Two degenerated oligonucleotide primers (DOPs)
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BLAST X (http://blast.ncbi.nlm.nih.gov/) and Clustal X (Larkin et al., 2007).
Table 1 Degenerate oligonucleotide primes for PCR. Primers
Sequences (50 –30 )
Degeneracy
APj-bGlu-450FW APj-15060-RV
aWYcaYtgggaYYtRccNca ggagtNNNRtcRgaRaaNcc
x256 x2048
3. Results 3.1. Detection and partial purification of Cry8Da binding protein
200 116 97 66
200 116 97 66
45
45
To detect the Cry8Da binding protein, a ligand blot was performed. The results showed the Cry8Da toxin bound to a 150 kDa protein in adult P. japonica BBMV (Fig. 1). Adult P. japonica BBMV was solubilized with 1% CHAPS. Ligand blot and densitometry analysis showed more than 90% of the Cry8Da binding protein was contained in the CHAPS solubilized fraction (data not shown). CHAPS solubilized P. japonica BBMV was fractionated by anion exchange chromatography (Hitrap Q HP). Each column fraction was subjected to ligand blot for identification of Cry8Da toxin binding protein. Cry8Da toxin binding protein was eluted with 0.6 M NaCl (data not shown) and pooled. Pooled fractions were concentrated with an Amicon Ultra and further fractionated by gel filtration chromatography. The fractions containing the150 kDa Cry8Da toxin binding protein were pooled and were further fractionated by anion exchange chromatography using an UNO-Q column. The presence of the 150 kDa Cry8Da binding protein was confirmed by SDS–PAGE and ligand blot (Fig. 2). The 150 kDa Cry8Da binding protein eluted at around 0.5 M NaCl.
31
31
3.2. Peptide mapping and N-terminal amino acid sequencing of Cry8Da binding protein
21
21
14
14
were designed: APj-bGlu-450FW and APj-15060-RV (Table 1) from (F/Y)HWDLPQ, a conserved sequence of Coleopteran b-glucosidase and GFSDXTG, an internal sequence of the 150 kDa Cry8Da binding protein. Thirty cycles of stepwise amplification (94 °C for 1 min, 40 °C for 1 min, and 72 °C for 3 min) were carried out. After gel purification, the 900 bp of the PCR product was cloned into pCRII-TOPO (Invitrogen) and sequenced. Homology search and alignment of amino acid sequences were carried out using NCBI
(kDa)
(kDa)
Fig. 1. Ligand blot of biotinylated Cry8Da toxin to adult P. japonica BBMV. Twenty micro gram of adult P. japonica BBMV was subjected to 9% SDS–PAGE. And then, proteins were visualized by CBB G-250 stain (left panel) or transferred to PVDF membrane and probed with biotinylated Cry8Da toxin (right panel).
A
Partially purified 150 kDa Cry8Da binding protein was subjected to N-terminal amino acid sequencing but Edman degradation was unsuccessful. Instead, a peptide map of Cry8Da binding protein was prepared to allow identification of internal amino acid sequences. The 150 kDa Cry8Da binding protein was digested with V8 protease according to the method of Cleveland et al. (1977). Three generated peptides were subjected to N-terminal amino acid sequencing. The 60 kDa peptide named BG150-60 had the sequence XGFSDXTGXL, the 40 kDa peptide, BG150-40, had the sequence XAXNEDXKGE, and the 10 kDa peptide, BG150-10, had
B
0.15
(kDa) 250 150
absorbance
0.1
100 75 0.05
1
50 NaCl (M)
0
35
(kDa) 250 150 100 75
50
35
0
0
5
10
15
20
25
30
run volume (ml) Fig. 2. UNO-Q anion exchange chromatography. Pre-purified the 150 kDa Cry8Da binding protein from solubilized adult P. japonica BBMV using Hitrap Q HP and Superdex 200 HR were finally purified using UNO-Q. (A) Chromatogram of anion exchange chromatography. The absorbance at 280 nm (black line) is indicated at the left, the NaCl concentration (gray line) is indicated at the right and the run volume is indicated at the bottom. The 150 kDa Cry8Da binding protein was eluted at around 0.5 M NaCl and (B) detection of the 150 kDa Cry8Da binding protein. The Cry8Da binding protein eluted fraction was subjected to SDS–PAGE and visualized by CBB G-250 stain (left panel) or ligand blot (right panel).
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the sequence XRALVDGEAA. Homologous sequences were identified in the NCBI Coleopteran protein database using the BLAST P program. The sequence BG150-60 shared amino acid sequence homology with Caryedon serratus cytochrome b (AE072230), T. castaneum phospholipid transporting ATPase (XP_974455, XP_970033) and T. molitor b-glucosidase (AAG26008). The sequence 150-40 shared homology with T. castaneum and T. molitor b-glucosidase (XP_975665, XP_972082 AAG26008). The sequence of BG150-10 was homologous with various proteins however no homology was detected with a b-glucosidase. 3.3. Detection of b-glucosidase activity Two internal sequences of the 150 kDa Cry8Da binding protein shared homology with known b-glucosidase sequences. We attempted to detect b-glucosidase activity in adult P. japonica BBMV and the 150 kDa Cry8Da binding protein containing fraction from the UNO-Q column using the colorimetric substrate, pNPG. If a bglucosidase hydrolyzes pNPG, the reaction solution will turn yellow because of the release of p-nitrophenol. As shown in Fig. 4, BBMV and the 150 kDa Cry8Da binding protein containing fraction had b-glucosidase activity. 3.4. Amplification and sequencing of b-glucosidase genes To amplify the b-glucosidase, the cDNA was synthesized from the total RNA prepared from the midguts of adult P. japonica. Degenerated oligonucleotide primers (DOPs) were designed from an internal sequence of the 150 kDa Cry8Da binding protein and a conserved region of Coleopteran b-glucosidases. Around 900 bp of DNA was amplified, cloned into a vector and the sequence determined. We obtained four sequences homologous to b-glucosidase and named them APj-bGlu-1, APj-bGlu-2, APj-bGlu-3 and APj-bGlu4. The deduced amino acid sequences of these genes are shown in Fig. 5. APj-bGlu-1 had an amino acid sequence, RALVDGEAA, corresponding to 150-10, which is not similar to any known bglucosidase.
databases of Scarabaeidae beetle are still insufficient for this purpose. Proteins from adult P. japonica BBMV were solubilized in 1% CHAPS. The ligand blot showed that CHAPS treatment solubilized more than 90% of the total amount of 150 kDa Cry8Da binding protein (data not shown). CHAPS is a good detergent to solubilize Cry8Da binding protein in BBMV since after the purification, the Cry8Da binding protein was still remaining the b-glucosidase activity (Fig. 4). Cry8Da binding protein was purified to a sufficient level to attempt N-terminal amino acid sequencing as shown in Fig. 2. However, the N-terminal amino acid sequence of the 150 kDa Cry8Da binding protein could not be determined by automated Edman degradation using a protein sequencer likely because of post-translational modifications at the N-terminal amino acid such as acylation and/or polyglutamylation. Therefore, the 150 kDa Cry8Da binding protein was digested with V8 protease and three internal amino acid sequences were determined, BG150-60, BG150-40 and BG150-10 (Fig. 3). Homology searches revealed BG150-60 and BG150-40 shared amino acid sequence homology with Coleopteran b-glucosidases. APj-bGlu-1, one of the b-glucosidase genes amplified by PCR using DOP designed from BG150-60 and conserved region of Coleopteran glucosidase contained BG150-10, which had no homology with known glucosidases (Fig. 5). Taken together, these results strongly suggested receptor of Cry8Da is a b-glucosidase encoded by the APj-bGlu-1. The precise role of the receptor has been described for Manduca sexta and Cry1Ab toxin (Gómez et al., 2007). Activated Cry1Ab toxin binds first to the receptor, a cadherin like protein. This binding
(kDa) 97
45
150-40 XAXNEDXKGE
31
4. Discussion We previously reported Cry8Da showed insecticidal activity against not only the larvae of P. japonica but also the adult (Yamaguchi et al., 2008). Cry proteins are solubilized in the larval midgut and are processed into active toxins by gut juice proteases such as trypsin and chymotrypsin. Activated Cry toxins bind specific receptors on the midgut epithelial cell brush border membrane vesicles (BBMVs). Following certain conformational changes and oligomerization, the toxins insert into the membrane to form cation-permeable pores, which cause swelling and death of epithelial cells by colloid-osmotic lysis (Schnepf et al., 1998). The insect specificity of Cry toxins is largely determined by the specific binding of the toxins to receptors on the midgut epithelial cell BBMV (Bravo et al., 2011). In lepidopteran and dipteran insects, the cadherin like proteins, GPI anchored APN and ALP, have been reported as receptors of Cry toxins (Pigott and Ellar, 2007; Likitvivatanavong et al., 2011). Binding of these receptor proteins and Cry toxins can be demonstrated by ligand blot. In our previous study, the binding of Cry8Da toxin and the 150 kDa protein from adult P. japonica BBMV was demonstrated by ligand blot (Yamaguchi et al., 2010). To identify the 150 kDa Cry8Da binding protein from adult P. japonica BBMV, we partially purified the 150 kDa Cry8Da binding protein from BBMV and determined its internal amino acid sequence. Although peptide mass spectrometry finger printing is a powerful tool to identify proteins, it requires a comprehensive DNA and protein database. Unfortunately, the DNA and protein
150-60 XGFSDXTGXL
66
21 14
150-10 XRALVDGEAA
6.5
Fig. 3. Peptide mapping and N-terminal amino acid sequencing. The 150 kDa Cry8Da binding protein from adult P. japonica BBMV was digested with V8 proteinase according to Cleveland method. The peptides were subjected to Nterminal amino acid sequencing. Arrows indicate peptides, subjected to N-terminal amino acid sequencing and N-terminal amino acid sequences are shown in right side of gel picture.
1
2
3
Fig. 4. Detection of b-glucosidase activity. To detect b-glucosidase activity, PBS (1), BBMV (2) and the 150 kDa Cry8Da binding protein eluted fraction (3) were incubated in pNPG containing PBS. b-glucosidase hydrolyzes pNPG and then aliquot will turn to yellow because of released p-nitoroanilline.
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127
Fig. 5. Amino acid sequence alignment of DOP–PCR amplified b-glucosidases. Partial b-glucosidase genes were amplified using degenerated oligonucleotide primers (DOPs) designed from a conserved amino acid sequence of Coleopteran b-glucosidases and internal amino sequence (150-60) of the 150 kDa Cry8Da binding protein. Amino sequences responding to DOP were indicated by doted box. A sequence corresponding to a internal amino acid sequence, 150-10 were indicated by black box.
promotes the proteolytic cleavage of the a1-helix of Domain I, inducing oligomerization. Oligomerized Cry1Ab toxin binds to a second receptor, such as GPI anchored APN or ALP, and then inserts into the membrane to form pores that causes cell lysis. Although little is known specifically about toxin binding in Coleopteran insects, Cry3A seems to follow a similar mode of action to Cry8. In Coleopteran insects, Cry3A recognizes a cadherin like protein on BBMV (Fabrick et al., 2009; Park et al., 2009). The binding of Cry3A toxin and the cadherin like protein induces oligomerization of Cry3A (Fabrick et al., 2009). Cry3A is capable of creating pores on the BBMV of Cry3A sensitive insects such as L. decemlineata (Rausell et al., 2004). In contrast, chymotrypsin activated Cry8Ea, which is toxic to a Coleopteran chafer, only has pore-forming activity on artificial liposomes (Guo et al., 2009). b-glucosidase is a glycoside hydrolase, which hydrolyzes the bglycoside bond of a sugar. b-glucosidase is involved in the degradation of cellulose, which is a polysaccharide consisting of a linear chain of b-1, 4-linked D-glucose units and is a structural component of the cell walls of plant cells. b-glucosidases include APjbGlu-1 will be involved in cellulose digestion of adult P. japonica, since these glucosidases were found in the digestive system of adult P. japonica, voracious plant leaf eaters. Binding of glycohydrolase and Cry toxins have been previously reported. Cry4Ba and Cry11Aa bind to GPI anchored a-amylase of Anopheles albimanaus (Fernandez-Luna et al., 2010). In addition, binding of beetle specific Cry3A toxin and a-amylase of T. molitor is reported (Bulushova et al., 2011). Although precise role of these Cry toxins binding glycoside hydrolases is not described, APj-bGlu-1 is probably involved in oligomerization and pore formation of Cry8Da toxin. In this study receptor of Cry8Da toxin was identified as a b-glucosidase in adult P. japonica BBMV. Further research including complete gene cloning and studies into the interactions of Cry8Da toxin and b-glucosidase is needed to determine the role of this Cry8Da receptor in the mechanism of Cry8Da insecticidal activity. Acknowledgment This work was supported in part by the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (T.Y.).
References Asano, S.-I., Yamashita, C., Iizuka, T., Takeuchi, K., Yamanaka, S., Cerf, D., Yamamoto, T., 2003. A strain of Bacillus thuringiensis subsp. galleriae containing a novel cry8 gene highly toxic to Anomala cuprea (Coleoptera: Scarabaeidae). Biol. Control 28, 191–196. Bauer, L., Londoño, D., 2010. Effects of Bacillus thuringiensis SDS-502 on adult emerald ash borer. In: Proceedings, 21st U.S. Department of Agriculture Interagency Research Forum on Invasive Species 2010, 74–75. Bulushova, N., Zhuzhikov, D., Lyutikova, L., Kirillova, N., Zalunin, I., Chestukhina, G., 2011. Toxin-binding proteins isolated from yellow mealworm Tenebrio molitor and wax moth Galleria mellonella. Biochemistry Moscow 76, 202–208. Bravo, A., Likitvivatanavong, S., Gill, S.S., Soberón, M., 2011. Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem. Mol. Biol. 41, 423–431. Cleveland, D.W., Fischer, S.G., Kirschner, M.W., Laemmli, U.K., 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102–1106. Fabrick, J.A., Oppert, C., Lorenzen, M.D., Morris, K., Oppert, B., Jurat-Fuentes, J.L., 2009. A novel Tenebrio molitor cadherin is a functional receptor for Bacillus thuringiensis Cry3Aa toxin. J. Biol. Chem. 284, 18401–18410. Fernandez-Luna, M.T., Lanz-Mendoza, H., Gill, S.S., Bravo, A., Soberón, M., MirandaRios, J., 2010. An a-amylase is a novel receptor for Bacillus thuringiensis ssp. israelensis Cry4Ba and Cry11Aa toxins in the malaria vector mosquito Anopheles albimanus (Diptera: Culicidae). Environ. Microbiol. 12, 746–757. Gómez, I., Pardo-López, L., Muñoz-Garay, C., Fernandez, L.E., Pérez, C., Sánchez, J., Soberón, M., Bravo, A., 2007. Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis. Peptides 28, 169–173. Guo, S., Ye, S., Liu, Y., Wei, L., Xue, J., Wu, H., Song, F., Zhang, J., Wu, X., Huang, D., Rao, Z., 2009. Crystal structure of Bacillus thuringiensis Cry8Ea1: an insecticidal toxin toxic to underground pests, the larvae of Holotrichia parallela. J. Struct. Biol. 168, 259–266. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G., 2007. Clustal W and clustal X version 2.0. Bioinformatics 23, 2947–2948. Likitvivatanavong, S., Chen, J., Evans, A.M., Bravo, A., Soberón, M., Gill, S.S., 2011. Multiple receptors as targets of Cry toxins in mosquitoes. J. Agric. Food Chem. 59, 2829–2838. Ochoa-Campuzano, C., Real, M.D., Martínez-Ramírez, A.C., Bravo, A., Rausell, C., 2007. An ADAM metalloprotease is a Cry3Aa Bacillus thuringiensis toxin receptor. Biochem. Biophys. Res. Commun. 362, 437–442. Park, Y., Abdullah, M.A.F., Taylor, M.D., Rahman, K., Adang, M.J., 2009. Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb toxicities to Coleopteran larvae by a toxin-binding fragment of an insect cadherin. Appl. Environ. Microbiol. 75, 3086–3092. Pigott, C.R., Ellar, D.J., 2007. Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol. Mol. Biol. Rev. 71, 255–281. Qi, G., Lu, J., Zhang, P., Li, J., Zhu, F., Chen, J., Liu, Y., Yu, Z., Zhao, X., 2011. The cry1Ac gene of Bacillus thuringiensis ZQ-89 encodes a toxin against long-horned beetle adult. J. Appl. Microbiol. 110, 1224–1234.
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T. Yamaguchi et al. / Journal of Invertebrate Pathology 113 (2013) 123–128
Rausell, C., García-Robles, I., Sánchez, J., Muñoz-Garay, C., Martínez-Ramírez, A.C., Real, M.D., Bravo, A., 2004. Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata (Say). Biochim. Biophys. Acta 1660, 99–105. Sato, R., Takeuchi, K., Ogiwara, K., Minami, M., Kaji, Y., Suzuki, N., Hori, H., Asano, S., Ohba, M., Iwahana, H., 1994. Cloning, heterologous expression, and localization of a novel crystal protein gene from Bacillus thuringiensis serovar japonensis strain Buibui toxic to scarabaeid insects. Curr. Microbiol. 28, 15–19. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J.A., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775–806. Shu, C., Yan, G., Wang, R., Zhang, J., Feng, S., Huang, D., Song, F., 2009a. Characterization of a novel cry8 gene specific to Melolonthidae pests: Holotrichia oblita and Holotrichia parallela. Appl. Microbiol. Biotechnol. 84, 701–707. Shu, C., Yu, H., Wang, R., Fen, S., Su, X., Huang, D., Zhang, J., Song, F., 2009b. Characterization of two novel cry8 genes from Bacillus thuringiensis strain BT185. Curr. Microbiol. 58, 389–392.
Soberón, M., Gill, S.S., Bravo, A., 2009. Signaling versus punching hole: how do Bacillus thuringiensis toxins kill insect midgut cells? Cell Mol. Life Sci. 66, 1337– 1349. Wolfersberger, M., Luethy, P., Maurer, A., Parenti, P., Sacchi, F., Giordana, B., Hanozet, G., 1987. Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp. Biochem. Phys. A 86, 301–308. Yamaguchi, T., Sahara, K., Bando, H., Asano, S.-I., 2008. Discovery of a novel Bacillus thuringiensis Cry8D protein and the unique toxicity of the Cry8D-class proteins against scarab beetles. J. Invertebr. Pathol. 99, 257–262. Yamaguchi, T., Sahara, K., Bando, H., Asano, S.-I., 2010. Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle. J. Invertebr. Pathol. 105, 243–247. Yu, H., Zhang, J., Huang, D., Gao, J., Song, F., 2006. Characterization of Bacillus thuringiensis strain Bt185 toxic to the Asian cockchafer: Holotrichia parallela. Curr. Microbiol. 53, 13–17.