Article pubs.acs.org/jpr
MucoRice-cholera Toxin B‑subunit, a Rice-based Oral Cholera Vaccine, Down-regulates the Expression of α‑Amylase/trypsin Inhibitor-like Protein Family as Major Rice Allergens Shiho Kurokawa,†,‡ Rika Nakamura,§ Mio Mejima,† Hiroko Kozuka-Hata,∥ Masaharu Kuroda,⊥ Natsumi Takeyama,† Masaaki Oyama,∥ Shigeru Satoh,‡,# Hiroshi Kiyono,†,¶ Takehiro Masumura,‡,# Reiko Teshima,§ and Yoshikazu Yuki*,†,¶ †
Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 108-8639, Japan ‡ Laboratory of Genetic Engineering, Graduate School of Life and Environmental Science, Kyoto Prefectural University, 606-0823, Japan § Division of novel foods and immunochemistry, National Institute of Health Sciences, 158-8501, Japan ∥ Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, 108-8639 Japan ⊥ Crop Development Division, NARO Agriculture Research Center, 943-0193, Japan # Biotechnology Research Department, Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Research Center, 619-0244, Japan ¶ International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, 108-8639, Japan S Supporting Information *
ABSTRACT: To develop a cold chain- and needle/syringe-free rice-based cholera vaccine (MucoRice-CTB) for human use, we previously advanced the MucoRice system by introducing antisense genes specific for endogenous rice storage proteins and produced a molecularly uniform, human-applicable, high-yield MucoRice-CTB devoid of plant-associated sugar. To maintain the cold chain-free property of this vaccine for clinical application, we wanted to use a polished rice powder preparation of MucoRiceCTB without further purification but wondered whether this might cause an unexpected increase in rice allergen protein expression levels in MucoRice-CTB and prompt safety concerns. Therefore, we used two-dimensional fluorescence difference gel electrophoresis and shotgun MS/MS proteomics to compare rice allergen protein expression levels in MucoRice-CTB and wild-type (WT) rice. Both proteomics analyses showed that the only notable change in the expression levels of rice allergen protein in MucoRice-CTB, compared with those in WT rice, was a decrease in the expression levels of α-amylase/trypsin inhibitor-like protein family such as the seed allergen protein RAG2. Real-time PCR analysis showed mRNA of RAG2 reduced in MucoRice-CTB seed. These results demonstrate that no known rice allergens appear to be upreregulated by genetic modification of MucoRice-CTB, suggesting that MucoRice-CTB has potential as a safe oral cholera vaccine for clinical application. KEYWORDS: proteomics, 2D-DIGE, shotgun MS, rice allergens, rice-based vaccine, MucoRice
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INTRODUCTION
room temperature for three years without loss of immunogenicity.4 Accordingly, MucoRice-CTB, a rice-based vaccine expressing the cholera toxin B subunit (CTB), has attracted interest as a vaccine production, storage, and delivery system for mucosal immunization.1,2,4 The CTB of MucoRice-CTB is N-glycosylated with a plantderived N-glycan including xylose or fucose.5 Recently, we developed an advanced version of MucoRice-CTB that lacks
Transgenic rice expression systems offer some advantages over other systems designed to express foreign proteins, including prokaryotic and eukaryotic cell cultures.1−3 These advantages include cost-effective production, the ability to rapidly scale-up production to generate large quantities of the proteins, multiple, simultaneous gene expression, a low risk of contamination with human pathogens, and resistance to enzymatic digestion in the gastrointestinal tract.1 In addition, rice seed has been shown to provide a suitable environment for the stable expression and accumulation of a foreign protein at © 2013 American Chemical Society
Received: March 10, 2013 Published: June 13, 2013 3372
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family is down-regulated in the rice-based oral cholera vaccine MucoRice-CTB.
this plant-derived N-glycan by inducing the overexpression of an Asn to Gln substitution at the fourth amino acid residue of authentic CTB, CTB (N4Q).6 Carbohydrate-specific IgE antibodies (Abs) that predominantly bind “nonmammalian” xylose or fucose on plant N-glycans have been found in patients allergic to pollen allergens;7 however, the future clinical application of this MucoRice-CTB that lacks plant-derived Nglycan for human use clearly does not prompt potential safety concerns regarding the allergenicity of plant N-glycan. In addition, the MucoRice-CTB produced a single monomeric band on SDS-PAGE, allowing the quantification of the CTB expressed in rice seed by means of densitometry analysis of SDS-PAGE.6 The MucoRice-CTB was produced at a high level in rice seed by using a CTB overexpression system together with RNAi technology to suppress the production of both major endogenous storage proteins, 13 kDa prolamin and glutelin A;8−10 the amount of CTB reached only one-sixth of that of the MucoRice-CTB when expressed in rice seed without the RNAi trigger.6 Having confirmed that MucoRice-CTB is room-temperature stable (or cold chain-free) for three years in rice seed or in its nonpurification form,4 we now plan to use a polished rice powder preparation of MucoRice-CTB in a clinical study without further purification in an effort to maintain the coldchain-free property of MucoRice-CTB. The estimated dose of the MucoRice CTB powder (1−2 g) preparation for use in our clinical study is substantially lower than the amount of cooked rice that is generally consumed. Despite the wide consumption of rice, rice allergy is rare.11 There have been some reports of immediate hypersensitivity reactions after ingestion of rice, leading to rhinoconjunctivitis,12 bronchial asthma,13 and atopic dermatitis.14 The severity of allergic reactions to rice allergens is low; there are only a few reports of rice-induced anaphylaxis.11,15 Even though MucoRice-CTB is not a genetically modified (GM) food, but rather a nonpurified form of oral vaccine, to dispel safety concerns we plan to use the GM food safety assessment proposed by the Codex Alimentarius Commission, which investigated the potential of gene insertions to cause allergic reactions.16 In this paper, we investigated whether the expression level of rice allergen proteins is up- or down-regulated in MucoRiceCTB when a vaccine gene and RNAi triggers are introduced into the rice genome. Major rice allergens of 14−16 kDa in size were identified from a rice salt-soluble fraction as members of the α-amylase/trypsin inhibitor-like protein family, which includes RA17 (or RAG1), RA14 (or RAG2), and RA5.17,18 In addition, a 33 kDa major rice allergen from the same fraction was identified as a type of plant glyoxalase I.19 Globulin-like proteins with molecular weights of 19, 52, and 63 kDa were also identified as IgE-binding allergens.20 We, therefore, compared the protein levels in the salt-soluble fraction of MucoRice-CTB with that of wild-type (WT) rice, since many rice allergens have been found in the salt-soluble fraction of 1 M NaCl extraction preparations of rice seed.18 IgE-immunoblot analysis using sera from patients with rice allergies showed less IgE-binding activity in the salt-soluble fraction of MucoRice-CTB compared with that of WT rice. To examine the protein expression levels of the salt-soluble fraction, including rice allergen expression levels in detail, we investigated the protein expression levels of MucoRice-CTB and WT rice by using a proteomics approach involving both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and shotgun MS/MS. We show that expression of the α-amylase/trypsin inhibitor-like protein
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EXPERIMENTAL SECTION
Rice-expressed CTB (N4Q), MucoRice-CTB
A sequence encoding the CTB (N4Q) was synthesized with optimized codon usage for rice1 and inserted into a binary TDNA vector (pZ2028) with an overexpression cassette for CTB (N4Q) and a combination cassette for RNAi suppression of the major rice endogenous storage proteins 13 kDa prolamin and glutelin A as described previously.6 The RNAi trigger sequence for the gene encoding 13 kDa prolamin was a 45 bp fragment of the rice 13 kDa prolamin gene comprising coding sequence 1− 45.8−10 The RNAi trigger sequence for the glutelin gene was a 129 bp fragment of the rice glutelin A gene comprising coding sequence 142−270.8−10 The expression vectors were used to transform a japonica variety of rice, Nipponbare, by using an Agrobacterium-mediated method described previously.1 The MucoRice-CTB plant line with the highest levels of CTB antigen accumulated in the seed was selected and advanced to the T4 generation by self-crossing to obtain homozygous lines. Extraction of Rice Total Seed Protein and Rice Allergen Protein
Mature brown seeds of nontransgenic rice (Oryza sativa cv. Nipponbare) (WT) and transgenic rice lines (MucoRice-CTB) of the fourth generation onward were harvested for these experiments. Each grain was pulverized separately with a Multi Beads Shocker (Yasui Kikai Corp., Osaka, Japan). For total protein extraction, 10 mg of rice fine powder was homogenized with 500 μL of SDS sample buffer [500 mM Tris-HCl (pH 6.8), 5% (w/v) SDS, 6% (w/v) 2-mercaptoethanol, 10% (w/v) glycerol, 0.5% (w/v) Bromophenol Blue]. The homogenates were heated at 100 °C for 5 min and then centrifuged at 20400× g for 10 min at 4 °C. The supernatant was collected and then subjected to SDS-PAGE and immunoblot analysis. Because many rice allergens have been found in the salt-soluble fraction of 1 M NaCl extraction preparations of rice seed,18 we extracted the salt-soluble proteins from 0.2 g of the rice fine powder with 3 mL of 1 M NaCl by rotating for 3 h at 4 °C on a rotator (TAITEC Corp., Saitama, Japan) and then used these proteins for allergen analyses such as 1D-Immunoblotting, 2DDIGE, and Shotgun MS/MS, as described below. Extracted solutions containing salt-soluble proteins were centrifuged at 20400× g for 10 min at 4 °C, and the supernatant was then filtered through a 0.45 μm syringe filter and stored in aliquots at −80 °C until use. SDS-PAGE and 1D-Immunoblot Analysis
Rice total proteins and salt-soluble proteins were separated by SDS-PAGE (15% acrylamide) according to Laemmli’s method.21 After separation, the proteins were stained with Coomassie Brilliant Blue R-250. For 1D-Immunoblot analysis, the separated proteins were transferred onto PVDF membranes (Merck Millipore, MA, USA), which were then incubated overnight at 4 °C with 5% (w/v) skim milk-TBS containing 0.05% (w/v) Tween 20 (TBS-T). The membranes were then incubated for 1 h at room temperature with each antibody (monoclonal Ab to glutelin A raised in our lab from mice immunized with rice proteins; 0.5 μg/mL, rabbit polyclonal anti-CTB1 and anti-13 kDa-prolamin antisera22 raised in our lab from rabbits immunized with recombinant CTB and a peptide from 13 kDa prolamin, respectively; 4000−20000 times diluted 3373
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wavelengths using a Typhoon9400 variable image analyzer (GE Healthcare Biosciences) to generate Cy2, Cy3, and Cy5 images. The spots were matched by using Decyder software version 7 (GE Healthcare Biosciences). Because we previously used MALDI-TOF-MS/MS to identify major rice allergen spots as IgE-binding proteins in WT rice that could be separated by use of 2D-DIGE under the same conditions,25 we compared the expression levels of the salt-soluble fraction proteins of MucoRice-CTB seed with those of WT rice seed. The fluorescence intensity of each protein spot was normalized to that of the internal standard. Normalized fluorescence mean values of WT rice relative to those of MucoRice-CTB were expressed as a ratio. Differences between groups were assessed by Student’s t-test, and p < 0.05 was considered to indicate statistical significance. The 2D-DIGE analysis was carried out twice; because the results were consistent, only one of the two data sets is shown.
with TBS-T). After being washed three times with TBS-T, the membranes were incubated with a 20000-times dilution of peroxidase-labeled antimouse/rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., PA, USA) for 1 h at room temperature. After three more washes with TBS-T, the membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., IL, USA) according to the manufacturer’s protocol. The signal intensity of the bands was detected by using the ImageQuant LAS 4000 mini (GE Healthcare Biosciences, WI, USA). IgE-Immunoblot Analysis with Patient Sera
The salt-soluble proteins (3 μg) of WT rice and MucoRiceCTB were separated by SDS-PAGE through a 10−20% acrylamide gel (D.R.C., Tokyo, Japan), and the gel was stained with Coomassie Brilliant Blue R-250. For 1D-immunoblot analysis, the separated proteins were transferred onto a 0.2 μm PVDF membrane (Bio-Rad Laboratories, CA, USA). The membrane was incubated with blocking buffer [0.5% [w/v] casein-PBS] for 2 h at room temperature, and then incubated with rice-allergenic patient sera or control sera (Gemini BioProducts, West Sacramento, CA) (diluted 1: 2 with 0.1% [w/v] casein-PBS), for 1 h at room temperature and then overnight at 4 °C. After being washed three times with TBS-T, the membranes were incubated with horseradish-peroxidase (HRP)-linked antihuman IgE (1:500 dilution with 0.1% casein-PBS; Nordic Immunological Laboratories, Susteren, The Netherlands) for 90 min at room temperature. After three more washes with TBS-T, the color reaction was developed with Konica Immunostain (Konica Minolta, Tokyo, Japan) according to the manufacturer’s protocol. Informed consent was obtained from all rice allergic patients and volunteers. The study was approved by the Ethical Review Committee of the National Institute of Health Science, Japan.
2D-PAGE and Immunostaining with RAG2- or CTB-Specific Rabbit IgG
By using the above 2D-DIGE method, we purified the saltsoluble proteins (75 μg) with the 2D Clean-Up Kit (GE Healthcare Biosciences) and then dissolved them in 250 μL of lysis buffer containing 1% IPG buffer pH 3−10 nonlinear (GE Healthcare Biosciences). The protein mixture (250 μL) was applied to Immobiline Drystrips (pH 3−10 NL, 13 cm, GE Healthcare), which were rehydrated for 12 h at 20 °C. Firstdimensional IEF was then carried out using an Ettan IPGphor III (GE Healthcare Biosciences) under the following conditions: 500 V for 4 h; 1000 V for 1 h; 8000 V for 2.5 h; and 8000 V for 1.5 h. After being focused, the strips were equilibrated for 15 min in equilibration buffer (100 mM TrisHCl [pH 8.0], 6 M Urea, 30% [v/v] glycerol, 2% [w/v] SDS, and a small amount of BPB) containing 0.5% (w/v) dithiothreitol (DTT), and were then incubated again only this time in equilibration buffer containing 4.5% (w/v) iodoacetamide for 15 min. Second-dimensional SDS-PAGE was performed at 220 V for 3 h on 10−20% gradient acrylamide gels (14 × 14 cm gel length, DRC Co., Ltd., Tokyo, Japan). After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 (Invitrogen Life Technologies, CA, USA). For immunoblot analysis, the separated proteins were transferred to PVDF membranes (Merck Millipore), which were then incubated overnight at 4 °C with 5% (w/v) skim milk-TBS containing 0.05% (w/v) Tween 20 (TBS-T). The membranes were then incubated for 90 min at room temperature with each antibody (1 μg/mL of antirecombinant RAG2 rabbit IgG20 or antirecombinant CTB rabbit IgG26) diluted with TBS-T. After three 10 min washes with TBS-T, the membranes were incubated for 1 h at room temperature with peroxidase-labeled antimouse IgG (Jackson ImmunoResearch Laboratories, Inc.) or peroxidase-labeled antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) that was diluted 20000 times with TBS-T. Again, after three 10 min washes with TBS-T, peroxidase activity was measured by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer’s protocol. The signal intensity of the bands was detected by using the ImageQuant LAS 4000 mini (GE Healthcare Biosciences).
2D-DIGE Analysis of WT Rice and MucoRice-CTB
2D-DIGE analysis was carried out according to the standard method.23−25 The concentrations of the salt-soluble proteins were measured with a 2-D Quant kit (GE Healthcare Biosciences) and the proteins were then purified by using the 2-D Clean-Up Kit (GE Healthcare Biosciences) according to the manufacturer’s protocol. A mixture of equal quantities of purified proteins (25 μg) from WT rice and MucoRice-CTB served as an internal standard. The internal standard was labeled with Cy2 minimal fluorescent dye, whereas the protein samples were labeled with Cy3 and Cy5 minimal fluorescent dye according to the manufacturer’s protocol (GE Healthcare Biosciences). The Cy-labeled proteins (25 μg protein per sample) were mixed together and loaded onto an Immobilin Drystrip (pH 3−10 NL, 13 cm, GE Healthcare Biosciences), where they were rehydrated overnight at 20 °C. Firstdimensional isoelectric focusing (IEF) was conducted at 20 °C using an Ettan IPGphor (GE Healthcare Biosciences), programmed with voltages from 500 V for 4 h, 1000 V for 1 h, 8000 V for 2.5 h, and 8000 V for 1.5 h. After the firstdimensional IEF, the strips were incubated for 15 min in equilibration buffer (100 mM Tris-HCl [pH 8.0], 6 M Urea, 30% [v/v] glycerol, 2% [w/v] SDS, and a small amount of BPB) containing 0.5% [w/v] dithiothreitol (DTT). The strips were then incubated in equilibration buffer containing 4.5% [w/v] iodoacetamide for 15 min. Second-dimensional SDSPAGE was performed at 220 V for 3 h through 10−20% gradient acrylamide gels (14 × 14 cm gel length, DRC Co., Ltd., Tokyo, Japan). Each gel was scanned at three separate
Sample Preparation for Mass Spectrometry
For mass spectrometric analysis, we used the salt-soluble proteins that were extracted with 1 M NaCl from the MucoRice-CTB or WT rice fine powder as described above. 3374
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Figure 1. SDS-PAGE and immunoblot analysis of total or salt-soluble proteins from MucoRice-CTB. Total proteins and salt-soluble proteins from WT or MucoRice-CTB seeds were extracted in SDS-sample buffer and 1 M NaCl, respectively. SDS-PAGE of total proteins (A) and salt-soluble proteins (C) followed by immunoblot analysis with the indicated rabbit IgG antibodies (B) and rice allergic patient sera (D). (A, B) Expression levels of endogenous rice storage proteins, such as glutelins and 13 kDa prolamin, in MucoRice-CTB were inhibited by RNA interference induced in the rice genome and compared with those in WT rice. High level expression of CTB in MucoRice-CTB is shown. (C, D) Expression levels of αamylase/trypsin inhibitor-like proteins in the salt-soluble fraction of MucoRice-CTB were down-regulated, as shown by SDS-PAGE and IgE immunoblotting with patient 2’s serum (see arrowhead) compared with those in WT rice. A commercially available human serum pool served as the control serum and 0.1% casein-PBS as a dilution buffer served as the negative control.
spectrometer (LTQ-Orbitrap Velos, Thermo Fisher Scientific) coupled with a nanoflow LC system (Dina-2A, KYA Technologies, Tokyo, Japan). Samples were injected into a 75 μm reversed-phase C18 column at a flow rate of 10 μL/min and eluted with a linear gradient of solvent A (2% acetonitrile and 0.1% formic acid in H2O) to solvent B (40% acetonitrile and 0.1% formic acid in H2O) at 300 nL/min. The separated peptides were sequentially sprayed from the nanoelectrospray ion source (KYA Technologies) and analyzed by using collision-induced dissociation (CID).27 The analyses were carried out in data-dependent mode, switching automatically between MS and MS/MS acquisition. Full-scan MS spectra (from m/z 380 to 2000) were acquired in the orbitrap with a resolution of 100 000 at m/z 400 after ion count accumulation to the target value of 1 000 000. The 20 most-intense ions at a threshold above 2000 were fragmented in the linear ion trap with a normalized collision energy of 35% for an activation time of 10 ms. Dynamic exclusion was defined as follows: exclusion list size, 500; exclusion duration, 45 s. The orbitrap analyzer
Protein extracts were purified by using the 2D Clean-Up Kit (GE Healthcare Biosciences) and the purified proteins were then dissolved in urea buffer (8 M urea, 500 mM Tris-HCl [pH 8.2]). The dissolved proteins (10 μg, each) were reduced with 1 mM dithiothreitol (DTT) for 90 min, and subsequently alkylated with 5.5 mM iodoacetamide (IAA) for 30 min. After digestion with Lysyl Endopeptidase (Lys-C) (1:50 w/w) (Wako) at 37 °C for 3 h, the resulting peptide mixtures were diluted with 10 mM Tris-HCl (pH 8.2) to achieve a final urea concentration below 2 M and were subsequently digested with modified trypsin (1:50 w/w) (Sequencing grade, Promega) at 37 °C for 3 h. An equal amount of trypsin was then added for overnight digestion. The digested samples were desalted by using ZipTip C18 (Merck Millipore) and centrifuged by use of a vacuum concentrator. Shotgun MS/MS Analysis and Protein Identification
Shotgun proteomic analyses of the peptide mixtures were performed by using a linear ion trap-orbitrap mass 3375
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was operated with the “lock mass” option to perform shotgun detection with high accuracy.28 The ions at m/z 391.28429 and 445.12002 were used as lock masses for internal calibration. Protein identification was conducted by searching the MS and MS/MS data against the National Center for Biotechnology Information (NCBI) nonredundant rice protein database (135 203 protein sequences as of Jun 17, 2012) by using Mascot ver. 2.3.02 (Matrix Science). Carbamidomethylation of cysteine residues was set as a fixed modification, whereas methionine oxidation, protein N-terminal acetylation, and pyroglutamination of N-terminal glutamine were set as variable modifications. A maximum of two missed cleavages was allowed in our database search. The tolerance for mass deviation was set to 3 ppm (ppm) for peptide masses and 0.8 Da for MS/MS peaks. In the process of peptide identification, we conducted decoy database searching by using Mascot and applied a filter to satisfy a false-positive rate of less than 1%. The shotgun MS analysis was carried out twice; because the results were consistent, only one of the two data sets is shown.
suppress the production of the major endogenous storage proteins such as 13 kDa prolamin and glutelin A, we first analyzed the expression of the major endogenous rice proteins as well as CTB by using one-dimensional SDS-PAGE followed by Western blotting. As expected, the introduction of the RNAi system minimized the production of rice storage proteins such as prolamines and glutelins and thus expanded the space available for the expression and accumulation of CTB in MucoRice-CTB (Figure 1A, B). To compare the major allergen expression level in MucoRice-CTB with that in WT rice, we next analyzed the protein expression levels in 1 M NaCl extracts of MucoRice-CTB or WT rice seed by using onedimensional SDS-PAGE followed by IgE-Immunoblotting analysis with sera from two patients with rice allergies. A 10− 15 kDa protein from the 1 M NaCl fraction of MucoRice-CTB was expressed at a lower level than that of the WT rice (Figure 1C), and IgE-binding components from the salt-soluble extracts of both MucoRice-CTB and WT rice were detected as multiple bands (Figure 1D). When we used patient 2’s serum in the IgEImmunoblotting analysis, the IgE-binding band representing the 10−15 kDa protein were less intense in MucoRice-CTB than in WT rice seed (Figure 1D, arrow), suggesting the expression of a low molecular weight allergy protein such as RAG2 was reduced in MucoRice-CTB.
Quantitative Real-time PCR
Total RNA was extracted from 14 days after flowering (DAF) seeds of WT and MucoRice-CTB by using an RNeasy plant mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. After treatment with DNase (TaKaRa, Tokyo, Japan), cDNA was synthesized from 0.5 μg of total RNA by using the Prime Script reagent Kit gDNA Eraser (Takara, Otsu, Japan). The 20 μL of cDNA solution obtained was then diluted 50 times with distilled water for use as the PCR template. Quantitative real-time PCR (qRT-PCR) was performed in a volume of 20 μL using the FAST SYBER Green Master Mix (Applied Biosystems, CA, USA) on a StepOne Plus real-time PCR system (Applied Biosystems). The following primers were used: 13 kDa prolamin (λRM1) (RM1-F, 5′CAGGCTGGTAGCGCAACA-3′ and RM1-R, 5′-ACAATCGCCTGAACGCTACT-3′); Glutelin A (Glu A) (Glu A-F, 5′ACAAAGAGAAGGATGTGCTTAC-3′ and Glu A-R, 5′-ATTCTTTATCCGCATTGCCAAC-3′); RAG2 (RAG2-F, 5′CATGGCTTCCAACAAGGTAGTG-3′ and RAG2-R, 5′GAGCACGGAGACGATGATGA-3′); Glyoxalase I (Glyox) (Glyox-F, 5′-CGGCAGCCAGGTCCACTA-3′ and Glyox-R, 5′-GCCATCAGGGTCAAGGAAAG-3′); 19 kDa globulin (19k glob) (19k glob-F, 5′-CGGCCTGCGGATGCA-3′ and 19k glob-R, 5′-TCTCCTCGTAGCTCCTCACCAT-3′); 52 kDa globulin (52k glob) (52k glob-F, 5′-CGGGTTCTGAGTGGGAAATC-3′ and 52k glob-R, 5′-TTGTTGGAGAAGTACGGACTCTTG-3′); 63 kDa globulin (63k glob) (63k glob-F, 5′-GAGGAAGCTGATCGTTACCAAGA-3′ and 63k glob-R, 5′- GGCGAAGAAGAACTGGATCT-3′); and 17SrRNA (17SrRNA-F, 5′-TTCCGGTCCTATTGTGTTGG-3′ and 17SrRNA-R, 5′-ATGCTTTCGCAGTTGTTCGT-3′). Triplicate reactions were treated as follows: 20 s at 95 °C; 40 cycles of 3 s at 95 °C, 30 s at 60 °C, 15 s at 95 °C, and 1 min at 60 °C. The expression levels were normalized to the 17S RNA endogenous control. Differences of expression levels between WT and MucoRice-CTB were assessed by Student’s t-test, and p < 0.05 was considered to indicate statistical significance.
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2. 2D-DIGE of the Salt-soluble Fraction of Rice Seed Reveals Lower Expression of Major Rice Allergens Including RAG2 and 19 kDa Globulin in MucoRice-CTB than in WT Rice
Because we were previously used MALDI-TOF-MS/MS to successfully identify major rice allergen spots as IgE-binding proteins that could be separated by use of 2D-DIGE,25 we used the same conditions to compare the expression levels of the salt-soluble fraction proteins of MucoRice-CTB seed with those
Figure 2. Representative overlapping 2D-DIGE expression map of WT rice and MucoRice-CTB salt-soluble proteins. Salt-soluble proteins from WT or MucoRice-CTB seeds were extracted in 1 M NaCl and then labeled with Cy3 (green) or Cy5 (red), respectively. Cy-labeled proteins (25 μg of protein each) were mixed together and analyzed by two-dimensional PAGE. The gel was scanned at two separate wavelengths using a Typhoon 9400 variable image analyzer to generate Cy3 (green, WT) and Cy5 (red, MucoRice-CTB) images. Note the CTB bands (red, MucoRice-CTB) of 12 kDa on the alkaline pI and the α-amylase/trypsin inhibitor-like protein family bands (green, WT) of 14 kDa on the alkaline pI.
RESULTS
1. One-dimensional SDS-PAGE Reveals Less Major Allergen Expression in MucoRice-CTB than in WT-type Rice
Because MucoRice-CTB was produced by using a CTB overexpression system together with RNAi technology to 3376
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Figure 3. Annotation of rice allergen spots indentified as IgE-binding proteins by 2D-DIGE analysis. Salt-soluble proteins from WT and MucoRiceCTB seeds were prelabeled with Cy dyes and separated by 2D-PAGE. Representative fluorescence images of salt-soluble proteins from WT and MucoRice-CTB are shown. Normalized fluorescence values of the circled spots from the WT are compared to the IgE-binding proteins identified in our previous report19 and analyzed for significant differences (ratio > 2 or < −2, in Table 1) between WT rice and MucoRice-CTB by Student’s t test (p < 0.05).
of WT rice seed. The fluorescence intensities (FI) of each spot from four independent gels were normalized to the internal standard, and significant differences between those of MucoRice-CTB and those of WT rice (Nipponbare) were analyzed by using Student’s t test. From a total of 940 saltsoluble protein spots (Figure 2), 67 of the MucoRice-CTB spots had F.I. that were significantly different (p < 0.05) from those of WT rice with a ratio of >2 or < −2; CTB spots were included among these 67 spots (Supplementary Figure 1 and Supplementary Table 1, Supporting Information). Three to four of the CTB (N4Q) spots with dimeric one were confirmed to be charge isomers by use of a 2D-Western blot with antiCTB rabbit IgG, producing bands at 12 kDa in the MucoRiceCTB immunoblot but not in the WT rice immunoblot (Supplementary Figure 2, Supporting Information). The 32 spots that represented the known allergenic proteins 63 kDa globulin, 52 kDa globulin, glyoxalase I, 19 kDa globulin, and the α-amylase/trypsin inhibitor-like protein family19 in MucoRiceCTB rice seed were numbered on the 2D-DIGE gels (Figure 3 and Table 1). For all of the α-amylase/trypsin inhibitor-like protein spots and two of 19 kDa globulin spots, the ratio of the MucoRice spot FI to the WT spot FI (MucoRice-CTB/WT) was down-regulated to
