Title: Identification of the IgE-binding Epitope in ω-5 Gliadin, a Major Allergen in Wheat-dependent Exercise-induced Anaphylaxis
Abstract: Wheat-dependent exercise-induced anaphylaxis (WDEIA) is a severe IgE-mediated allergic reaction provoked by the combination of wheat-ingestion with intensive physical exercise over the next few hours. Among wheat proteins, ω-5 gliadin, which is one of the components of fast ω-gliadin, has been reported as a major allergen in the anaphylaxis. In this study, we detected IgE-binding epitopes within the primary sequence of ω-5 gliadin using arrays of overlapping peptides synthesized on derivatized cellulose membranes. Sera from four patients with WDEIA having specific IgE to the fast ω-gliadin were used to probe the membrane. Seven epitopes, QQIPQQQ, QQLPQQQ, QQFPQQQ, QQSPEQQ, QQSPQQQ, QQYPQQQ, and PYPP, were detected within the primary sequence of ω-5 gliadin. By using sera of 15 patients, 4 of them, QQIPQQQ, QQFPQQQ, QQSPEQQ, and QQSPQQQ, were found to be dominant epitopes. Mutational analysis of the QQIPQQQ and QQFPQQQ indicated that amino acids at positions Gln1, Pro4, Gln5, Gln6, and Gln7 were critical for IgE binding. These results will provide a useful tool for developing safer wheat products in addition to diagnostic and immunotherapy techniques for WDEIA. Wheat-dependent exercise-induced anaphylaxis (WDEIA) is a severe IgE-mediated allergic reaction provoked by the combination of wheat-ingestion with intensive physical exercise over the next few hours. Among wheat proteins, ω-5 gliadin, which is one of the components of fast ω-gliadin, has been reported as a major allergen in the anaphylaxis. In this study, we detected IgE-binding epitopes within the primary sequence of ω-5 gliadin using arrays of overlapping peptides synthesized on derivatized cellulose membranes. Sera from four patients with WDEIA having specific IgE to the fast ω-gliadin were used to probe the membrane. Seven epitopes, QQIPQQQ, QQLPQQQ, QQFPQQQ, QQSPEQQ, QQSPQQQ, QQYPQQQ, and PYPP, were detected within the primary sequence of ω-5 gliadin. By using sera of 15 patients, 4 of them, QQIPQQQ, QQFPQQQ, QQSPEQQ, and QQSPQQQ, were found to be dominant epitopes. Mutational analysis of the QQIPQQQ and QQFPQQQ indicated that amino acids at positions Gln1, Pro4, Gln5, Gln6, and Gln7 were critical for IgE binding. These results will provide a useful tool for developing safer wheat products in addition to diagnostic and immunotherapy techniques for WDEIA. Food-dependent exercise-induced anaphylaxis is a distinct form of food allergy induced by physical exercise (1Kidd J.M. Cohen S.H. Sosman A.J. Fink J.N. J. Allergy Clin. Immunol. 1983; 71: 407-411Abstract Full Text PDF PubMed Scopus (278) Google Scholar). Food items such as shrimp (2McNeil D. Strauss R.H. Ann. Allergy. 1988; 61: 440-442PubMed Google Scholar), hazelnut (3Martin Munoz F. Lopez Cazana J.M. Villas F. Contreras J.F. Diaz J.M. Ojeda J.A. Allergy. 1994; 49: 314-316Crossref PubMed Scopus (54) Google Scholar), buckwheat (4Noma T. Yoshizawa I. Ogawa N. Ito M. Aoki K. Kawano Y. Asian Pac. J. Allergy Immunol. 2001; 19: 283-286PubMed Google Scholar), corn (5Pauls J.D. Cross D. J. Allergy Clin. Immunol. 1998; 101: 853-854Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), and celery (6Silverstein S.R. Frommer D.A. Dobozin B. Rosen P. J. Emerg. Med. 1986; 4: 195-199Abstract Full Text PDF PubMed Scopus (49) Google Scholar) are responsible for the development of food-dependent exercise-induced anaphylaxis. However, of all of the various kinds of food, wheat is reported to be the allergen with the highest frequency in Japan (7Dohi M. Suko M. Sugiyama H. Yamashita N. Tadokoro K. Juji F. Okudaira H. Sano Y. Ito K. Miyamoto T. J. Allergy Clin. Immunol. 1991; 87: 34-40Abstract Full Text PDF PubMed Scopus (225) Google Scholar). Symptoms are typically generalized urticaria and severe allergic reactions such as shock or hypotension. Because of this serious reaction, it is important to determine the causative food to avoid the allergic reaction. A challenge test consisting of ingestion of the assumed food followed by intense physical exercise is the only reliable method to determine the causative food and to diagnose the disease. However, the challenge test is not always safe because in some cases the test induces an anaphylactic shock. In addition, the most reliable treatment for this disease is to avoid taking the causative food or, alternatively, to take a rest after meals. However, in the case of wheat allergy, elimination causes a decline in the quality of life for the patients. Thus, an in vitro diagnostic method as well as hypoallergenic wheat is necessary for patients with wheat-dependent exercise-induced anaphylaxis (WDEIA). 1The abbreviations used are: WDEIA, wheat-dependent exercise-induced anaphylaxis; LMW, low molecular weight; HMW, high molecular weight; TBST, 50 mm Tris-buffered saline, 1% Tween 20, pH 7.4; Fmoc, N-(9-fluorenyl)methoxycarbonyl; RAST, radioallergosorbent test. Recent studies have revealed the IgE-binding epitopes of several food allergens including egg (8Mine Y. Zhang J.W. Biochem. Biophys. Res. Commun. 2002; 292: 1070-1074Crossref PubMed Scopus (73) Google Scholar), milk (9Busse P.J. Jarvinen K.M. Vila L. Beyer K. Sampson H.A. Int. Arch. Allergy Immunol. 2002; 129: 93-96Crossref PubMed Scopus (81) Google Scholar, 10Cocco R.R. Jarvinen K.M. Sampson H.A. Beyer K.J. J. Allergy Clin. Immunol. 2003; 112: 433-437Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), soybean (11Xiang P. Beardslee T.A. Zeece M.G. Markwell J. Sarath G. Arch. Biochem. Biophys. 2002; 408: 51-57Crossref PubMed Scopus (51) Google Scholar), and peanut (12Rabjohn P. Helm E.M. Stanley J.S. West C.M. Sampson H.A. Burks A.W. Bannon G.A. J. Clin. Invest. 1999; 103: 535-542Crossref PubMed Scopus (345) Google Scholar), whereas the IgE-binding epitopes for wheat allergen are controversial. Wheat protein is composed of water/salt-soluble proteins and water/salt-insoluble proteins. Proteins in the water/salt-soluble fraction, such as α-amylase inhibitor, peroxidase, glycerinaldehyde-3-phosphate dehydrogenase, serpin, and triosephosphate isomerase, have been considered to be major allergens in patients with bakers' asthma (13Amano M. Ogawa H. Kojima K. Kamidaira T. Suetsugu S. Yoshihama M. Satoh T. Samejima T. Matsumoto I. Biochem. J. 1998; 330: 1229-1234Crossref PubMed Scopus (79) Google Scholar, 14Yamashita H. Nanba Y. Onishi M. Kimoto M. Hiemori M. Tsuji H. Biosci. Biotechnol. Biochem. 2002; 66: 2487-2490Crossref PubMed Scopus (28) Google Scholar, 15Sander I. Flagge A. Merget R. Halder T.M. Meyer H.E. Baur X. J. Allergy Clin. Immunol. 2001; 107: 907-913Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). On the other hand, Sandiford et al. (16Sandiford C.P. Tatham A.S. Fido R. Welch J.A. Jones M.G. Tee R.D. Shewry P.R. Newman Taylor A.J. Clin. Exp. Allergy. 1997; 27: 1120-1129Crossref PubMed Scopus (112) Google Scholar) showed that α-gliadin and fast ω-gliadin (water/salt-insoluble wheat proteins) are the allergens associated with bakers' asthma. Watanabe and co-workers (17Watanabe M. Tanabe S. Suzuki T. Ikezawa Z. Arai S. Biosci. Biotechnol. Biochem. 1995; 59: 1596-1597Crossref PubMed Scopus (62) Google Scholar, 18Tanabe S. Arai S. Yanagihara Y. Mita H. Takahashi K. Watanabe M. Biochem. Biophys. Res. Commun. 1996; 219: 290-293Crossref PubMed Scopus (123) Google Scholar) found that low molecular weight (LMW) glutenin, one of the water/salt-insoluble proteins, is the major allergen for patients allergic to wheat, and identified QQQPP motif as an IgE-binding epitope. Maruyama et al. (19Maruyama N. Ichise K. Katsube T. Kishimoto T. Kawase S. Matsumura Y. Takeuchi Y. Sawada T. Utsumi S. Eur. J. Biochem. 1998; 255: 739-745Crossref PubMed Scopus (88) Google Scholar) reported that α-gliadin and γ-gliadin in addition to LMW glutenin are the allergens for patients with wheat allergies (19Maruyama N. Ichise K. Katsube T. Kishimoto T. Kawase S. Matsumura Y. Takeuchi Y. Sawada T. Utsumi S. Eur. J. Biochem. 1998; 255: 739-745Crossref PubMed Scopus (88) Google Scholar). These observations indicate that a variety of wheat proteins are allergenic. In terms of WDEIA, in our previous studies (20Morita E. Matsuo H. Mihara S. Morimoto K. Savage A.W.J. Tatham A.S. J. Dermatol. Sci. 2003; 33: 99-104Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 21Morita E. Yamamura Y. Mihara S. Kameyoshi Y. Yamamoto S. Br. J. Dermatol. 2000; 143: 1059-1063Crossref PubMed Scopus (48) Google Scholar, 22Morita E. Kameyoshi Y. Mihara S. Hiragun T. Yamamoto S. Br. J. Dermatol. 2001; 145: 182-184Crossref PubMed Scopus (35) Google Scholar), we identified wheat fast ω-gliadin as a major allergen and found that the specific IgE against fast ω-gliadin cross-reacts to γ-gliadin and slow ω-gliadin (20Morita E. Matsuo H. Mihara S. Morimoto K. Savage A.W.J. Tatham A.S. J. Dermatol. Sci. 2003; 33: 99-104Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 21Morita E. Yamamura Y. Mihara S. Kameyoshi Y. Yamamoto S. Br. J. Dermatol. 2000; 143: 1059-1063Crossref PubMed Scopus (48) Google Scholar, 22Morita E. Kameyoshi Y. Mihara S. Hiragun T. Yamamoto S. Br. J. Dermatol. 2001; 145: 182-184Crossref PubMed Scopus (35) Google Scholar). Fast ω-gliadin is also called 1B-type ω-gliadin (23DuPont F.M. Vensel W.H. Chan R. Kasarda D.D. Cereal Chem. 2000; 77: 607-614Crossref Scopus (76) Google Scholar) or ω-5-type gliadin (24Seilmeier W. Valdez I. Mendez E. Wieser H. Eur. Food Res. Technol. 2001; 212: 355-363Crossref Scopus (35) Google Scholar, 25Kasarda D.D. Autran J.C. Lew E.J.L. Nimmo C.C. Shewry P.R. Biochim. Biophys. Acta. 1983; 747: 138-150Crossref Scopus (205) Google Scholar) and can be divided into several peaks by reversed-phase high performance liquid chromatography (24Seilmeier W. Valdez I. Mendez E. Wieser H. Eur. Food Res. Technol. 2001; 212: 355-363Crossref Scopus (35) Google Scholar). Palosuo et al. (26Palosuo K. Alenius H. Varjonen E. Koivuluhta M. Mikkola J. Keskinen H. Kalkkinen N. Reunala T. J. Allergy Clin. Immunol. 1999; 103: 912-917Abstract Full Text Full Text PDF PubMed Google Scholar, 27Palosuo K. Varjonen E. Kekki O.M. Klemola T. Kalkkinen N. Alenius H. Reunala T. J. Allergy Clin. Immunol. 2001; 108: 634-638Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) identified ω-5 gliadin, a component of fast ω-gliadin, as a major allergen in WDEIA. They revealed that the ω-5 gliadin is also an allergen in children with an immediate-type allergy to wheat (27Palosuo K. Varjonen E. Kekki O.M. Klemola T. Kalkkinen N. Alenius H. Reunala T. J. Allergy Clin. Immunol. 2001; 108: 634-638Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) and that transglutaminase-mediated cross-linking of a pepsin-trypsin-digested ω-5 gliadin causes a marked increase in IgE-binding both in vitro and in vivo (28Palosuo K. Varjonen E. Nurkkala J. Kalkkinen N. Harvima R. Reunala T. Alenius H.J. J. Allergy Clin. Immunol. 2003; 111: 1386-1392Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). In this study, we analyzed the linear IgE-binding epitopes of ω-5 gliadin by using synthetic peptides. Subjects—Sera were collected from 15 patients with WDEIA who had recurrent episodes of anaphylaxis and a positive-provocation test to exercise after wheat ingestion, 15 healthy subjects without episodes of food allergy and 6 patients with atopic dermatitis without episodes of WDEIA. The sera were stored at –80 °C until use. Specific IgE antibodies for gluten in the sera were determined by the radioallergosorbent test system (CAP-RAST, Amersham Biosciences) (29Sigurs N. Hattevig G. Kjellman B. Kjellman N.I. Nilsson L. Bjorksten B. J. Allergy Clin. Immunol. 1994; 94: 757-763Abstract Full Text PDF PubMed Scopus (152) Google Scholar). Dot-blotting—Wheat α-gliadin, β-gliadin, γ-gliadin, fast ω-gliadin, slow ω-gliadin, high molecular weight (HMW) glutenin, and LMW glutenin were prepared as described previously (16Sandiford C.P. Tatham A.S. Fido R. Welch J.A. Jones M.G. Tee R.D. Shewry P.R. Newman Taylor A.J. Clin. Exp. Allergy. 1997; 27: 1120-1129Crossref PubMed Scopus (112) Google Scholar, 30Tatham A.S. Shewry P.R. J. Cereal Sci. 1985; 3: 103-113Crossref Scopus (154) Google Scholar). Specific IgE antibodies in the sera for α-gliadin, β-gliadin, γ-gliadin, fast ω-gliadin, slow ω-gliadin, HMW glutenin, and LMW glutenin were detected using a method described previously with a slight modification (20Morita E. Matsuo H. Mihara S. Morimoto K. Savage A.W.J. Tatham A.S. J. Dermatol. Sci. 2003; 33: 99-104Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). 2 μg of each gliadin, glutenin, and gluten (NAKARAI TESQUE, Kyoto, Japan) were spotted on polyvinylidene fluoride membrane (Immobilon, Millipore) and blocked with 5% skim milk in TBST. The membrane was washed three times with TBST for 10 min and then incubated with 10% of the patient's serum overnight at 4 °C. After washing with TBST, the membrane was incubated with goat anti-human IgE horseradish peroxidase-conjugate (BIOSOURCE) for 1 h at room temperature. The membrane was washed three times with TBST for 10 min, and the IgE antibodies reacting to the wheat proteins were visualized using ECL Plus Western blotting detection reagents (Amersham Biosciences). The resulting light was detected on autoradiography film (Hyperfilm ECL, Amersham Biosciences). After scanning the film, the spot intensities were measured using the Gel-Pro Analyzer software (Media Cybernetics, Inc). Peptide Synthesis and IgE-binding Assay—Four DNA sequences of ω-5 gliadin fragments (GenBank™ accession numbers BE590673, BQ608902, BQ805830, and BQ245835) were selected from the wheat-expressed sequence tags data base. These sequences were aligned, and the assumed amino acid sequence of ω-5 gliadin was constructed (Fig. 1). Based on the assumed amino acid sequence of the ω-5 gliadin, the individual peptides were synthesized on a membrane using a SPOTs kit (Sigma), which contains a derivatized cellulose membrane (SPOTs membrane). Fmoc amino acids, by coupling Fmoc amino acids according to the manufacturer's instructions. After washing with methanol, the SPOTs membrane was blocked overnight at 4 °C with blocking buffer (Sigma). The membrane was washed with TBST for 10 min at room temperature and then incubated overnight at 4 °C with 10% patient's serum. The bound IgE antibodies were detected as described above with goat anti-human IgE horseradish peroxidase conjugate by using ECL Plus Western blotting detection reagents. The SPOTs membrane was used repeatedly after the following procedure. The membrane was washed in order with water, N,N-dimethylformamide (WAKO Chemical, Osaka, Japan), Regeneration buffer A (8 m urea, 10% sodium dodecyl sulfate), and Regeneration buffer B (10% (v/v) acetic acid, 50% (v/v) ethanol), rinsed with methanol twice for 10 min at room temperature, and dried. Dot-blotting for Purified Gliadins and Glutenins—Specific IgE reacting with the purified gliadins and glutenins in the sera of 15 patients with WDEIA and 21 control subjects were determined by dot-blotting. Table I shows the serum IgE levels, the CAP-RAST value for gluten, and the result of the dot-blotting analysis. In the data of CAP-RAST for gluten, sensitivity and specificity for diagnosis of WDEIA were 80 (12/15) and 90% (19/21), respectively, when the cutoff value for a positive result was set at 0.35 units of allergen-specific IgE per ml. In the data of dot-blotting for gluten, when a cutoff value was set at 100 based on the data of control subjects, sensitivity and specificity were 60 (9/15) and 85% (18/21), respectively, both of which were worse than those of CAP-RAST for gluten. Additionally, the values of spot intensity for gluten were also not always parallel to that of CAP-RAST. This may be attributed to the unstable technique of dot-blotting.Table IResults of dot blot analysisTotal IgEGluten RASTSpot intensityαβγFωSωHMWLMWGlutenIU/mlUa/mlWDEIA patients 1146<0.3511013326847 22281.3299536053125111238 321.90.510012169922 417795.3463961767715861541724 544.51.55500696339161290 61761.22200911137846166 71842.3510471243655 8169<0.350001818341428 91361.33104230823611119 103973.83438233226681147 11aPatient with atopic dermatitis.14,7233.3346011706469817612271831331 1243414.06111345524719158 13150<0.35331126717114438 141041.790001535043839 151711.543114140203736Non-WDEIA controls 1657<0.352006130234 17110<0.3551362009382 18100<0.351832241033 19500<0.3581551912298 2031<0.35000210153 211600<0.351710163972327142 227.2<0.35012115017184 2326<0.35235112472972 249.8<0.3518127781741794 25290<0.354177232285133 2641<0.3571491448396853 27310<0.3500072417116 28310<0.35310343610823 2947<0.35000837883 3021<0.35154458218915818961 31aPatient with atopic dermatitis.16,1001.64102033816177113 32aPatient with atopic dermatitis.10,9900.8102316576100126 33aPatient with atopic dermatitis.656<0.3504210213021124 34aPatient with atopic dermatitis.1900<0.35976461236292104 35aPatient with atopic dermatitis.580<0.351301047132541 36aPatient with atopic dermatitis.370<0.3523352911638a Patient with atopic dermatitis. Open table in a new tab Among the individual data of spot intensity for purified wheat proteins, the strongest reaction was observed with the fast ω-gliadin in 12 of 15 patients with WDEIA (patients 1, 2, 3, 5, 6, 7, 8, 9, 10, 12, 14, and 15), confirming the data reported by Palosuo et al. (26Palosuo K. Alenius H. Varjonen E. Koivuluhta M. Mikkola J. Keskinen H. Kalkkinen N. Reunala T. J. Allergy Clin. Immunol. 1999; 103: 912-917Abstract Full Text Full Text PDF PubMed Google Scholar, 27Palosuo K. Varjonen E. Kekki O.M. Klemola T. Kalkkinen N. Alenius H. Reunala T. J. Allergy Clin. Immunol. 2001; 108: 634-638Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). The remaining three patients (patients 4, 11, and 13) had specific IgE reacting predominantly to HMW glutenin. In contrast, the strongest reaction was observed with slow ω-gliadin in 10 of 21 control subjects and LMW glutenin was the second (6 of 21 patients). In 4 of 21 control subjects, the strongest reaction was also observed with fast ω-gliadin as in the patients with WDEIA; however, the values of spot intensities were relatively low compared with those of patients with WDEIA. Multiple IgE-binding Epitopes in the ω-5 Gliadin—Because no cDNA and genomic DNA sequence of ω-5-type gliadins has been reported, we collected four DNA fragment sequences of ω-5 type gliadins from the wheat-expressed sequence tags data base based on the N-terminal and internal protein sequences (23DuPont F.M. Vensel W.H. Chan R. Kasarda D.D. Cereal Chem. 2000; 77: 607-614Crossref Scopus (76) Google Scholar, 24Seilmeier W. Valdez I. Mendez E. Wieser H. Eur. Food Res. Technol. 2001; 212: 355-363Crossref Scopus (35) Google Scholar). We then aligned these sequences and constructed an assumed amino acid sequence of ω-5 gliadin (Fig. 1). The molecular mass of the assumed ω-5 gliadin was calculated at 48-kDa, suggesting that it was almost full-length because the determination of molecular mass by matrix-assisted laser desorption ionization time-of-flight revealed a range of 44–55 kDa for the ω-5-type gliadins (24Seilmeier W. Valdez I. Mendez E. Wieser H. Eur. Food Res. Technol. 2001; 212: 355-363Crossref Scopus (35) Google Scholar). 66 overlapping peptides were synthesized by the SPOTs method to determine which regions of the assumed ω-5 gliadin were recognized by the serum IgE of the patients with WDEIA. Each peptide was 13 amino acids long and offset by six amino acids. Individual sera from four patients (patients 1, 2, 3, and 5) whose serum had specific IgE to fast ω-gliadin predominantly were selected and used to map the IgE-binding region in the assumed amino acid sequence of ω-5 gliadin. Fig. 2 summarizes the IgE-binding peptides of each of the four patients. Many IgE-binding peptides were detected; however, most of these positive sequences were overlapped among the tested patients. From the results of the alignment of the IgE-bound peptide sequences, we expected that common epitope sequences are QQIPQQQ (peptides 10, 26, and 43), QQFPQQQ (peptides 5, 8, 21, 32, 36, 37, 39, 40, 45, 46, 47, 49, 51, 52, 53, 54, 58, and 60), and QQLPQQQ (peptides 13, 18, 23, 24, 28, 34, 41, and 50). Peptides 38, 57, and 64 also reacted to the sera commonly in four patients, respectively. The sera of patient 3 reacted with two other peptides (peptides 61 and 62), indicating proper epitopes to this patient. Determination of the Epitope Sequence and Critical Amino Acids—For determination of the exact amino acid sequence of IgE-binding regions, synthetic peptides comprising 10 amino acids (offset by one amino acid) were further synthesized with respect to the IgE-bound peptides (peptides 26, 28, 38, 47, 57, 61, 62, and 64, respectively) derived from the results of Fig. 2. These peptides then were probed with the sera of randomly selected patients with WDEIA (patients 1, 2, 3, and 5). The immunoblotting result of peptide 26 with the serum of patient 1 was shown in Fig. 3. The boxed area indicates the core epitope sequence. The IgE-binding epitopes on ω-5 gliadin obtained from all four patients identified in the same manner were summarized in Table II. All of the sera reacted to QQIPQQQ, QQLPQQQ, QQFPQQQ, QQSPEQQ, QQSPQQQ, and QQYPQQQ with the exception of patient 5 whose serum failed to react to QQLPQQQ. Additionally, the serum of patient 3 had specific IgE to PYPP.Table IIIgE-binding epitope sequences for patients with WDEIAPatients1235QQIPQQQQQIPQQQQQIPQQQQQIPQQQQQLPQQQQQLPQQQQQLPQQQQQFPQQQQQFPQQQQQFPQQQQQFPQQQQQSPEQQQQSPEQQQQSPEQQQQSPEQQQQSPQQQQQSPQQQQQSPQQQQQSPQQQQQYPQQQQQYPQQQQQYPQQQQQYPQQQPYPP Open table in a new tab The critical amino acids for IgE-binding in two epitopes, QQIPQQQ and QQFPQQQ, were determined by synthesizing epitope peptides with single amino acid mutation at each position followed by probing with individual sera from four patients (patient 1, 2, 3, and 5). The data of the immunoblot strip in patient 1 were representatively shown in Fig. 4. No binding of the serum IgE was observed when the alanine was substituted at the amino acid positions Gln1, Pro4, Gln5, Gln6, and Gln7, respectively, indicating that these amino acids are critical for IgE binding in both epitopes. The remaining sera of the three patients were analyzed in the same manner, and the critical amino acids for IgE-binding were found to be the same as in patient 1 (data not shown). Immunodominance of the ω-5 Gliadin Epitopes—To determine whether any of the seven epitopes of ω-5 gliadin found in this study were immunodominant, each peptide was synthesized and probed with sera of 15 patients. The results are summarized in Fig. 5. The QQIPQQQ and QQFPQQQ peptides were recognized by 13 of 15 WDEIA patients (87%). The QQSPEQQ and QQSPQQQ peptides were recognized by 12 of 15 patients (80%), and the QQLPQQQ and QQYPQQQ epitopes were recognized by 9 of 15 patients and 8 of 15 patients (60 and 53%), respectively. These data indicate that the QQIPQQQ, QQFPQQQ, QQSPQQQ, and QQSPEQQ epitopes are immunodominant and that the IgE antibodies of the patients probably cross-react to these epitopes. The PYPP epitope was unique for patient 3. Patients 11 and 13 had no IgE antibody bound to these tested epitopes, although their sera had IgE bound to fast ω-gliadin (Table I). The water/salt-insoluble wheat proteins, termed gluten, have been considered to be causative allergens for WDEIA. Gluten is composed of two classes of proteins, the 70% ethanol-soluble gliadins (α-, β-, γ-, and ω-gliadins) and the 70% ethanol-insoluble glutenins (HMW and LMW glutenins). Among these proteins, fast ω-gliadin, especially ω-5 gliadin, is considered to be a major allergen for WDEIA (20Morita E. Matsuo H. Mihara S. Morimoto K. Savage A.W.J. Tatham A.S. J. Dermatol. Sci. 2003; 33: 99-104Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 26Palosuo K. Alenius H. Varjonen E. Koivuluhta M. Mikkola J. Keskinen H. Kalkkinen N. Reunala T. J. Allergy Clin. Immunol. 1999; 103: 912-917Abstract Full Text Full Text PDF PubMed Google Scholar, 27Palosuo K. Varjonen E. Kekki O.M. Klemola T. Kalkkinen N. Alenius H. Reunala T. J. Allergy Clin. Immunol. 2001; 108: 634-638Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). In this study, we determined IgE binding against a panel of purified gluten proteins by using sera obtained from 15 patients defined as having WDEIA by a challenge test and found that ∼80% of the patients reacted to fast ω-gliadin most strongly, confirming that the fast ω-gliadin is a predominant allergen for WDEIA (Table I). We then analyzed the epitope sequences by means of peptide array of assumed ω-5 gliadin with the sera of four. The epitope sequences of ω-5 gliadin were found to be QQXPQQQ (X = I, F, S, L, or Y), QQSPEQQ, and PYPP (Table II), four of which, QQXPQQQ (X = I, F, or S) and QQSPEQQ, were immunodominant. These epitopes appear frequently in the amino acid sequence of ω-5 gliadin as shown in Fig. 2. This is compatible with the data reported by DuPont et al. (23DuPont F.M. Vensel W.H. Chan R. Kasarda D.D. Cereal Chem. 2000; 77: 607-614Crossref Scopus (76) Google Scholar) in which repeats of QQXP, QQQXP, or QQQQXP (X = F, I, and L in order of predominance) are characteristic for the ω-5-type gliadins. We further determined that the critical amino acids in the QQIPQQQ and QQFPQQQ epitopes were positions Gln1, Pro4, Gln5, Gln6, and Gln7, suggesting the epitope sequence QXXPQQQ. In addition, it was speculated that the glutamine residue in the epitope sequence could be replaced by glutamic acid without loss of allergenic ability because both QQSPQQQ and QQSPEQQ epitopes were recognized equally. These data suggest that there are many IgE-binding sites in the ω-5 gliadin. This may account for the serious allergic reactions such as shock, which is characteristic of WDEIA, despite a low proportion of ω-5-type gliadins in gluten (24Seilmeier W. Valdez I. Mendez E. Wieser H. Eur. Food Res. Technol. 2001; 212: 355-363Crossref Scopus (35) Google Scholar, 31Wieser H. Eur. Food Res. Technol. 2000; 211: 262-268Crossref Scopus (89) Google Scholar). 2 of the 15 patients with WDEIA (patients 11 and 13) had no specific IgE bound to these epitopes (Fig. 5), although their sera had IgE bound to fast ω-gliadin. These results suggest that the two patients recognize other epitopes in the ω-5 gliadin or that there was nonspecific binding. Interestingly, the sera of these patients in addition to patient 4 reacted to HMW glutenin dominantly among purified water/salt-insoluble wheat proteins (Table I). Taking into consider the relatively low reaction to HMW glutenin in control subjects, these results might suggest that there is another minor subgroup in WDEIA, which reacts to other epitopes in HMW glutenin as a cause of the disease. This study also showed that the sera of all 15 patients with WDEIA reacted to slow ω-gliadin, LMW, and HMW glutenin apart from the strong reactivity to fast ω-gliadin (Table I). Previously, we reported (20Morita E. Matsuo H. Mihara S. Morimoto K. Savage A.W.J. Tatham A.S. J. Dermatol. Sci. 2003; 33: 99-104Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) that fast ω-gliadin cross-reacts to γ-gliadin and slow ω-gliadin, suggesting high sequence homology in these proteins. Palouso et al. (32Palosuo K. Alenius H. Varjonen E. Kalkkinen N. Reunala T. Clin. Exp. Allergy. 2001; 31: 466-473Crossref PubMed Scopus (101) Google Scholar) also revealed that γ-70 and γ-35 secalins in rye and γ-3 hordein in barley cross-react with ω-5 gliadin (32Palosuo K. Alenius H. Varjonen E. Kalkkinen N. Reunala T. Clin. Exp. Allergy. 2001; 31: 466-473Crossref PubMed Scopus (101) Google Scholar). This is also supported by amino acid sequences of the several gliadins thus far reported. The amino acid sequence of slow ω-gliadin is homologous to the C-hordein of barley and ω-secalins of rye and has PFPQ1–2PQQ repeat motif in repetitive domain (33Hsia C.C. Anderson O.D. Theor. Appl. Genet. 2001; 103: 37-44Crossref Scopus (65) Google Scholar). The sequence of γ-gliadin also has PFPQI1–2(PQQ)1–2 motif that is similar to slow ω-gliadin in the repeat domain (34Anderson O.D. Hsia C.C. Torres V. Theor. Appl. Genet. 2001; 103: 323-330Crossref Scopus (73) Google Scholar), and there is a QPYPQQQ sequence in slow ω-gliadin and QSFPQQQ and QPFPQQQ sequences in γ-gliadin. Because these sequences are very similar to the epitopes of ω-5 gliadin and include critical amino acids for IgE binding, it is likely that specific IgE to ω-5 gliadin cross-reacts with γ-gliadin and slow ω-gliadin. In addition, the sera of some patients had specific IgE to α-gliadin, which had been reported as a major allergen for WDEIA by Palosuo et al. (26Palosuo K. Alenius H. Varjonen E. Koivuluhta M. Mikkola J. Keskinen H. Kalkkinen N. Reunala T. J. Allergy Clin. Immunol. 1999; 103: 912-917Abstract Full Text Full Text PDF PubMed Google Scholar). The α-gliadin has QQLPQEQ, QQQPQEQ, and QPYPQQQ sequences (35Anderson O.D. Greene F.C. Theor. Appl. Genet. 1997; 95: 59-65Crossref Scopus (119) Google Scholar); therefore, this may cross-react with ω-5 gliadin as well as γ-gliadin and slow ω-gliadin. Moreover, the IgE-binding epitope of ω-5 gliadin QQLPQQQ was seen in the VolckA3 and LMW21 and QQFPQQQ was seen in the VolckB3, respectively, all of which belong to the LMW glutenins (36Cassidy B.G. Dvorak J. Anderson O.D. Theor. Appl. Genet. 1998; 96: 743-750Crossref Scopus (141) Google Scholar), suggesting that ω-5 gliadin also cross-reacted with LMW glutenin. These results might account partly for the diversity of the reactivity in the dot-blot analysis with the sera of the patients. These IgE-binding epitopes are different from QQQPP previously identified by Tanabe et al. (18Tanabe S. Arai S. Yanagihara Y. Mita H. Takahashi K. Watanabe M. Biochem. Biophys. Res. Commun. 1996; 219: 290-293Crossref PubMed Scopus (123) Google Scholar) as the major allergen for wheat allergy. In this study, we synthesized the peptide SQQQPPF and probed with the sera of the WDEIA patients; however, only a weak IgE-bound signal to this peptide was observed in 10 of 15 patients (67%) (data not shown). The result indicates that the IgE-binding epitope sequences, QQXPQQQ (X = I, F, S, L, or Y), QQSPEQQ, and PYPP, are distinct and responsible for WDEIA. These motifs do not exist in the proteins, which are major allergens for bakers' asthma, such as α-amylase inhibitors peroxidase and serpin, supporting the idea that these epitopes are characteristic for WDEIA. The CAP-RAST system is now widely used for the diagnosis of allergy, whereas as found in this study, some patients with definite WDEIA were negative in CAP-RAST for gluten (Table I), suggesting unreliable sensitivity of the test for the diagnosis of WDEIA. In addition, a number of the patients with atopic dermatitis reacted positively to CAP-RAST for gluten, although the patients did not have an anaphylactic reaction to gluten such as control subjects 31 and 32 in Table I. These findings suggest that the measurement of specific IgE for gluten is not always satisfactory for the screening as well as diagnosis of WDEIA. It is conceivable that the simultaneous measurement of specific IgE antibodies to both fast-ω gliadin and the epitope peptides was more sensitive and specific to diagnose WDEIA than that to gluten. In fact, to diagnose WDEIA, the clinical efficacy of the measurement of specific IgE to ω-5 gliadin has already been reported (26Palosuo K. Alenius H. Varjonen E. Koivuluhta M. Mikkola J. Keskinen H. Kalkkinen N. Reunala T. J. Allergy Clin. Immunol. 1999; 103: 912-917Abstract Full Text Full Text PDF PubMed Google Scholar, 27Palosuo K. Varjonen E. Kekki O.M. Klemola T. Kalkkinen N. Alenius H. Reunala T. J. Allergy Clin. Immunol. 2001; 108: 634-638Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Food-dependent exercise-induced anaphylaxis is a life-threatening disease, but the only reliable therapy is strict elimination of allergen from dietary foods. However, it is not easy to eliminate wheat from foodstuff for a long time. Mutated allergen protein immunotherapy or peptide immunotherapy for food allergy was successful in animal models such as peanut anaphylaxis by means of the allergens or peptides lacking the ability to bind to IgE but retaining the ability to activate T-cells (37Nowak-Wegrzyn A. Pediatrics. 2003; 111: 1672-1680Crossref PubMed Scopus (298) Google Scholar). The elucidation of the major IgE-binding epitopes on ω-5 gliadin in this study may provide a useful tool for developing hypoallergenic foods as well as a new diagnostic technique and immunotherapy for patients with WDEIA.