Assessment of the Allergenic Potential of Transgenic Wheat

Article pubs.acs.org/JAFC

Assessment of the Allergenic Potential of Transgenic Wheat (Triticum aestivum) with Reduced Levels of ω5-Gliadins, the Major Sensitizing Allergen in Wheat-Dependent Exercise-Induced Anaphylaxis Susan B. Altenbach,*,† Charlene K. Tanaka,† Florence Pineau,‡ Roberta Lupi,‡ Martine Drouet,§ Etienne Beaudouin,∥ Martine Morisset,⊥ and Sandra Denery-Papini‡ †

Western Regional Research Center, Agricultural Research Services (ARS), United States Department of Agriculture (USDA), 800 Buchanan Street Albany, California 94710, United States ‡ UR1268 Biopolymers, Interactions, Assemblies, Institut National de la Recherche Agronomique (INRA), Rue de la Géraudière, F-44316 Nantes Cedex 03, France § Unité d’Allergologie Générale et de Pneumologie, Centre Hospitalier Universitaire (CHU) d’Angers, F-49933 Angers, France ∥ Service d’Allergologie, Centre Hospitalier (CH) Epinal, F-88021 Epinal, France ⊥ Immunologie-Allergologie, Centre Hospitalier (CH) de Luxembourg, L-1210 Luxembourg, Luxembourg S Supporting Information *

ABSTRACT: The ω5-gliadins are the major sensitizing allergens in wheat-dependent exercise-induced anaphylaxis (WDEIA). In this study, two-dimensional immunoblot analysis was used to assess the allergenic potential of two transgenic wheat lines in which ω5-gliadin genes were silenced by RNA interference. Sera from 7 of 11 WDEIA patients showed greatly reduced levels of immunoglobulin E (IgE) reactivity to ω5-gliadins in both transgenic lines. However, these sera also showed low levels of reactivity to other gluten proteins. Sera from three patients showed the greatest reactivity to proteins other than ω5-gliadins, either high-molecular-weight glutenin subunits (HMW-GSs), α-gliadins, or non-gluten proteins. The complexity of immunological responses among these patients suggests that flour from the transgenic lines would not be suitable for individuals already diagnosed with WDEIA. However, the introduction of wheat lacking ω5-gliadins could reduce the number of people sensitized to these proteins and thereby decrease the overall incidence of this serious food allergy. KEYWORDS: food allergy, gliadins, gene silencing, gluten proteins, immunoblots, RNA interference, Triticum aestivum, WDEIA, wheat

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INTRODUCTION Wheat flour contains a complex mixture of proteins, some of which are responsible for human health problems that include food allergies, inhalation allergies (baker’s asthma), and celiac disease. Understanding the immunogenic potential of specific flour proteins facilitates the development of improved diagnostic methods. This information also is essential for efforts to develop new wheat cultivars with reduced allergen contents that might be suitable for consumption by wheat allergy patients or prevent the sensitization of new individuals to specific wheat proteins. The gluten proteins comprise about 70−80% of the total flour protein and are insoluble in aqueous solutions. These proteins are rich in glutamine and proline, serve as storage proteins in the wheat grain, and are primarily responsible for the unique viscoelastic properties that make it possible to produce breads, noodles, and other products from wheat flour. The remaining flour proteins, the non-gluten proteins, consist of a wide variety of water- and salt-soluble proteins. These include many metabolic proteins as well as proteins involved in defense mechanisms in the grain. Among the gluten proteins are many individual proteins that are classified as either gliadins or glutenins, depending upon whether they are present as monomers or polymers in the flour (reviewed by Wieser1). The monomeric gliadins are separated into three groups on the basis of their sequences, referred to as α-, γ-, and ω-gliadins. α- and γ-gliadins contain N-terminal regions © 2015 American Chemical Society

with highly variable repetitive sequences and C-terminal regions containing either six or eight conserved cysteine residues, respectively. ω-gliadins consist almost entirely of repetitive sequences and do not contain cysteine. The polymeric glutenins consist of high-molecular-weight glutenin subunits (HMW-GSs) linked by disulfide bonds to low-molecular-weight glutenin subunits (LMW-GSs). HMW-GSs contain a central repetitive region flanked by small regions of unique sequence that include most of the cysteine residues. The LMW-GSs are grouped into three subclasses. The typical LMW-GSs, referred to as B-type LMW-GSs, have distinct N-terminal sequences followed by repetitive regions and conserved C-terminal domains. These proteins contain eight cysteine residues, six involved in intramolecular bonds and two available for interchain linkages that extend the glutenin polymer.2 The B-type LMW-GSs are further divided into s-, m-, and i-type LMW-GSs on the basis of their N-terminal amino acids, either serine, methionine, or isoleucine, respectively. The C- and D-type LMW-GSs have sequences like gliadins but contain extra cysteine residues that allow them to be linked into the glutenin polymer. In contrast to Received: Revised: Accepted: Published: 9323

July 21, 2015 October 6, 2015 October 8, 2015 October 8, 2015 DOI: 10.1021/acs.jafc.5b03557 J. Agric. Food Chem. 2015, 63, 9323−9332

Article

Journal of Agricultural and Food Chemistry

Table 1. Reactivity of IgE in Patient Sera (ng/mL) to Purified Protein Fractions or Extractsa from Non-transgenic Flour Determined by ELISA patient number 1 2 3 4 5 6 7 8 9 10 11 12

symptoms WDEIA WDEIA + rhinitis WDEIA + urticaria WDEIA WDEIA WDEIA WDEIA + urticaria WDEIA WDEIA WDEIA WDEIA none

serum number

ω5-gliadins

ω1,2-gliadins

b

1343 1175b

50 201

1310b

58

0.5

1174b 1363b 1592b 1305b

95 105 108 182

5 0.5 50 131

1183b 691b 1648b 1082c 1345b

145 80 0 0 0

31 0 5 0 0

0 111

albumin/globulin

0 70

0 0

0 5

0.5 141

398 7062

1/20 1/50

0

1

1

1

39

224

1/25

20 0.5 50 28

5 0 18 0.5

0 0 0 0

0 0 0 0

29 0.5 47 116

1809 2504 1178 4764

1/20 1/25 1/25 1/20

62 0 0 0 0

4.7 8 49 0 0

0 0 0 0 0

0 0 0 15 0

167 26 0 0 0

4872 2090 3254 306 1000

1/30 1/20 1/20 1/10 1/20

γ-gliadins

0 5

0 14

0 53

0

0 0 0 26 76

168 44 0 0 0

LMW-GSs

0 4 0 0 0

serum dilution for blots

LTP

α-gliadins

0 0 0 0.5

IgE total

total gliadin

HMW-GSs

a

Protein fractions and extracts were described by Battais et al.9 bSerum tested on immunoblots containing a total sodium dodecyl sulfate (SDS) protein extract from wheat flour. cSerum tested on immunoblots containing KCl-soluble proteins from wheat flour.

the B-type LMW-GSs, the C-type (similar to α- and γ-gliadins) and D-type (similar to ω-gliadins) LMW-GSs are thought to terminate the polymer chain. One of the most severe forms of wheat allergy, wheatdependent exercise-induced anaphylaxis (WDEIA), occurs when the ingestion of wheat is followed by physical exercise. This lifethreatening allergy has been documented mostly in adults. However, neither the ingestion of wheat nor exercise alone causes symptoms. In a study of 18 patients who experienced recurring episodes of WDEIA, Palosuo et al.3 found that individuals generally consumed wheat-containing food from 10 min to 4 h prior to exercise and exercised from 10 to 60 min before experiencing symptoms. Exercise could be as simple as light indoor activities and walking or as strenuous as jogging, dancing, and sports. Palosuo et al.3 further demonstrated that sera from all 18 WDEIA patients contained elevated levels of immunoglobulin E (IgE) to a 65 kDa flour protein that had a Nterminal sequence consistent with ω5-gliadins.4 The ω5-gliadins, sometimes referred to as the fast ω-gliadins because of their migration on acid gels, are a subgroup of ω-gliadins encoded at the Gli-B1 locus on chromosome 1 of hexaploid and tetraploid wheat.5 A number of subsequent studies also point to the ω5gliadins as the major sensitizing allergens in WDEIA.6−9 Dominant epitopes in ω5-gliadins that bind IgE from WDEIA patients have been identified using arrays of synthetic overlapping peptides based on the sequences of peptides from ω5gliadins10 or derived from the sequences of assembled expressed sequence tags (ESTs)7,11 or cloned genes.8 The consensus IgEbinding motif of ω5-gliadins was found to be QQX1PX2QQ, where X1 is I, L, F, S, or Y and X2 is Q or E. This motif is present numerous times in ω5-gliadins, likely contributing to their predominant reactivities with IgE antibodies from WDEIA patients. A number of approaches have been used to develop wheat with reduced immunogenic potential for WDEIA patients. DeneryPapini et al.12 surveyed a collection of wheat cultivars containing 13 different alleles at the Gli-B1 locus and found that there was little IgE reactivity to gliadins from a wheat line carrying a 1BL/ 1RS translocation from rye. However, lines carrying this translocation generally had poor bread-making quality. Lombardo et al.13 examined the immunogenicity of accessions of

Triticum monoccum, an ancestral diploid wheat lacking the B genome. While the IgE binding profiles from different WDEIA patients demonstrated the absence of 65 kDa proteins in some T. monoccum accessions, most showed at least some IgE reactivity with proteins of lower molecular weight. Waga and Skoczowski14 used traditional breeding methods to accumulate inactive ωgliadin gene variants in a single wheat genotype but found that the elimination of ω-gliadins was accompanied by changes in other gluten proteins, including decreases in certain γ-gliadins as well as increases in some α- and γ-gliadins. These lines showed an overall reduction of about 30% in immunoreactivity when assessed by an enzyme-linked immunosorbent assay (ELISA) with sera from a collection of 10 patients presenting a variety of different wheat allergy symptoms. Another recent study indicated that the deamidation of gliadins using cation exchangers might result in reduced immunogenicity of wheat flour for allergy patients.15 An alternate approach involves using gene-silencing. Recently, RNA interference (RNAi) was used to reduce the levels of ω5gliadins in transgenic wheat from the U.S. cultivar Butte 86.16 The precise effects of the genetic modifications on the wheat flour proteome were evaluated using quantitative two-dimensional gel electrophoresis (2DE), and two transgenic lines were selected for further study.17,18 In one line, the ω5-gliadins were substantially reduced with few changes in the levels of other proteins. In the second line, the complete elimination of the ω5gliadins was accompanied by partial reductions in the levels of the ω1,2-gliadins, a subgroup of ω-gliadins with repetitive motifs distinct from the ω5-gliadins, and the D-type LMW-GS (similar to ω1,2-gliadins in Butte 86). There were also small changes in several other proteins. In this study, the IgE reactivity of flour proteins from these transgenic lines is characterized on twodimensional (2D) immunoblots using sera from a collection of WDEIA patients. The availability of a proteomic map from the U.S. wheat cultivar Butte 86, in which most of the major flour proteins have been identified by tandem mass spectrometry (MS/MS),19 makes it possible to determine the specific flour proteins in both control and transgenic lines that react with sera from patients with WDEIA. 9324

DOI: 10.1021/acs.jafc.5b03557 J. Agric. Food Chem. 2015, 63, 9323−9332

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Figure 1. Immunoblot analysis of total flour proteins from Butte 86 with patient sera. Panel A shows a representative 2D gel of total wheat flour proteins stained with Coomassie. The positions of HMW-GSs, ω5-gliadins, ω1,2-gliadins, LMW-GSs, and α- and γ-gliadins are shown in light blue, red, dark blue, green, and yellow ovals, respectively. Blots were reacted with sera from WDEIA patients 1−10 (B−K), respectively, or with serum from a control patient (12) that did not have a wheat allergy. The positions of the ω5-gliadins are indicated with red ovals in panels B−J. The light blue oval in panel K shows the position of the y-type HMW-GSs. The black box in panel G shows the regions of the blots highlighted in Figure 2. The positions of molecular weight markers are shown on the right of each panel.

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wheat flour9 and using specific IgE assays to wheat flour, gluten, and ω5gliadin (Phadia ImmunoCAP). Wheat allergy was confirmed in one patient (9) by a positive challenge to wheat flour (70 g) after exercise and in the other patients by an evident effect of wheat avoidance as described11 (Supplementary Table 1). Serum from an adult with no food allergy (12) was used as a control (Table 1). Blood collection, SPTs, and

MATERIALS AND METHODS

Patient Sera. Sera were obtained from 11 adult patients diagnosed with WDEIA (Supplementary Table 1). Sensitization to wheat proteins was assessed in patients using skin prick tests (SPTs) to wheat flour (Moulins Soufflet), gluten (ALK-Abello), and ω5-gliadin purified from 9325


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Table 2. Reactivity of IgE in Sera from WDEIA Patients with Wheat Gluten Proteins in 2D Immunoblots from Non-transgenic Butte 86 and Transgenic Lines SA-8-35b-5 and SA-8-45a-2a patient number

plant type

ω5-gliadins

1

non-transgenic 35b 45a non-transgenic 35b 45a non-transgenic 35b 45a non-transgenic 35b 45a non-transgenic 35b 45a non-transgenic 35b 45a non-transgenic 35b 45a non-transgenic 35b 45a non-transgenic 35b 45a non-transgenic 35b 45a

+++ +

2

3

4

5

6

7

8

9

10

ω1,2-gliadins

α-gliadins

± +

+++ +++ + ++ +

γ-gliadins

LMW-GSs

+++ +++ +++ + + +

+ + + ++ +++ +++ + + + ++ ++ ++ + + + ++ ++ ++ +++ +++ +++ ++ ++ ++

+ ++ ++

+++ ++ +++ +

+ + + + + + +++ +++ +++ +++ +++ +++ +++ +++ +++

++ + +++ +

+++ +++ +

++ + +

HMW-GSs

++ ++ ++

Only patients that show reactivity to gluten proteins are included. ±, +, ++, and +++ indicate the relative amount of IgE reactivity. Blank denotes no reactivity. a

from 1:10 to 1:50, depending upon the level of specific IgE for ω5gliadins (patients 1−9), HMW-GS (patient 10), or albumins/globulins (patient 11) (Table 1). Revelation of IgE binding was performed using peroxidase-labeled rabbit anti-human IgE (9160-05 SouthernBiotech) diluted 1:500 000 and chemiluminescent substrate (WesternBright Quantum, Advansta K-12042-D20) according to the protocol of the manufacturer. Amounts of patient sera were limited; therefore, it was not possible to perform replicate blots. Each immunoblot image was aligned to the corresponding membrane stained with Ponceau Red using SameSpots version 4.5 software (TotalLab, Ltd., Newcastle upon Tyne, U.K.). Reactive proteins were identified by comparison to proteomic maps of Butte 86 flour or endosperm generated previously.19,21,23,24 Reactivities of individual protein spots from control and transgenic samples with serum from individual WDEIA patients are summarized in Supplementary Table 2.

challenges were performed with the informed consent of patients after receiving approval for the biomedical research from the Ethics Committee of Ile de France III and the French Agency for the Safety of Health Products (AFSSAPS) (authorization number 2008-A0156550). Sera from patients were characterized by immunological assays using gliadin and glutenin fractions, lipid transfer protein (LTP), albumin/ globulin, and total gliadin extracts described by Battais et al.9 (Table 1). Total and specific IgE concentrations were determined by fluorescent (F)-ELISA,20 using alkaline-phosphatase-conjugated goat anti-human IgE and 4-MUP as a substrate (Sigma A3525 and M3168, respectively, Saint-Quentin Fallavier, France). Proteins were coated at 5 μg/mL in carbonate buffer on white 384-well plates (NUNC 460372, Fisher Scientific), and patient sera were diluted 1:10. Flour Samples and Protein Extraction. Transgenic lines with reduced levels of ω5-gliadins were generated previously using RNAi.16 Flour samples from two of the resulting lines, SA-8-35b-5 and SA-8-45a2 (referred to here as 35b and 45a), and the Butte 86 non-transgenic control were produced from grain grown in a greenhouse under a 24/17 °C temperature regimen with post-anthesis fertilizer and are the same as those described by Altenbach et al.18 Total protein was extracted from flour as described by Dupont et al.19 For isolation of non-gluten proteins, the KCl-soluble proteins were extracted from wheat flour as described by Huebener et al.21 2DE and Immunoblot Analysis. 2DE and transfer of proteins onto nitrocellulose membranes was performed as described by Huebener et al.,21 except that 7.5 mg of protein was loaded on gels used for immunoblotting. Immunoblot analysis was performed as described by Lupi et al.22 The membranes were incubated with patient sera diluted

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RESULTS

Characterization of Patient Sera by ELISA. Sera were obtained from 11 adult patients diagnosed with WDEIA (Table 1). Several WDEIA patients also exhibited additional symptoms, either rhinitis (2) or urticaria (3 and 7). In addition, serum was obtained from a control patient without allergy to wheat (12). The reactivity of IgE in patient sera to purified protein fractions or extracts was evaluated by ELISA and revealed that sera from 9 of the 11 WDEIA patients (1−9) showed reactivity to ω5gliadins. Sera from eight patients also showed at least some reactivity to ω1,2-gliadins, four to α-gliadins, four to γ-gliadins, 9326


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Journal of Agricultural and Food Chemistry six to LMW-GSs, and eight to HMW-GSs. In addition, one WDEIA patient showed low levels of reactivity to a LTP, and three showed reactivity to an albumin/globulin protein fraction. Serum from the patient without wheat allergy did not react with any wheat proteins in ELISA. Reaction of Patient Sera with 2D Immunoblots of Total Flour Protein from Non-transgenic Plants. As expected from the ELISA results, most sera from WDEIA patients showed reactivity to ω5-gliadins on immunoblots of total flour protein from non-transgenic Butte 86 (panels B−J of Figure 1, Table 2, and Supplementary Table 2). Sera from eight patients (1−8) showed a strong reaction to multiple 2DE spots identified as ω5gliadins (panels B−I of Figure 1). In some patients, this was accompanied by weak reactivity to LMW-GSs (1−6) (panels B− G of Figure 1) and certain α-gliadins (2, 5, and 6) (panels C, F, and G of Figure 1). Serum from one patient (2) also showed weak reactivity to several ω1,2-gliadins (Table 2 and Figure 1C). In comparison, serum from patient 7 exhibited strong reactivity with ω5-gliadins as well as with ω1,2-gliadins, γ-gliadins, αgliadins, and LMW-GSs (Figure 1H), while serum from patient 8 showed strong reactivity with ω5-gliadins, most α-gliadins, and certain LMW-GSs and γ-gliadins (Figure 1I). Patient 9 showed a very different profile, reacting predominantly with α-gliadins (Figure 1J) and exhibiting only a faint reaction with the ω5gliadins. Although many sera showed reactivity to HMW-GSs in ELISA, only serum from patient 10 reacted with HMW-GSs in immunoblots, primarily with y-type HMW-GSs, Dy10 and By9, and very faintly with Bx7 (Supplementary Table 2). This serum did not show reactivity with any other gluten proteins (Figure 1K). In contrast to patients with WDEIA, serum from the patient free from wheat allergy (12) showed no IgE reactivity with wheat flour proteins (Figure 1L). Figure 2 highlights the LMW-GS and α- and γ-gliadin regions of five immunoblots. Of the eight sera that reacted with LMWGSs on immunoblots (Table 2), all showed reactivity with s-type LMW-GSs (red arrows in panels B−F of Figure 2 and Supplementary Table 2). A subset of spots containing m-type LMW-GSs also showed reactivity in most of the same samples (blue arrows in panels B−F of Figure 2). Only four patients showed strong reactivity with i-type LMW-GSs, 6 and 7 (green arrows in panels E and F of Figure 2) and 4 and 8 (Supplementary Table 2). A comparison of the immunoblots to a Coomassie-stained gel (Figure 2A) demonstrates that amounts of reactivity to specific LMW-GS spots are not directly related to protein abundances, suggesting differences in the affinity of sera from different patients for specific types of LMWGSs. For example, the normalized spot volume of spot 119, a stype LMW-GS, was 1.8-fold higher than that of spot 141, a i-type LMW-GS, in the non-transgenic flour.18 However, serum from patient 6 showed greater reactivity with spot 141 than spot 119 (Figure 2E). Reaction of Patient Sera with 2D Immunoblots of Total Flour Proteins from Transgenic Plants. Changes in the IgE reactivity between control and transgenic plants were similar for sera from six WDEIA patients (1, 3, 4, 5, 6, and 8) (Table 2). As exemplified by immunoblots for patient 1 (panels A−C of Figure 3), reactivity to ω5-gliadins was substantially reduced in transgenic line 35b relative to the non-transgenic control and absent in line 45a. Little change was observed in the reactivity to either LMW-GSs or other gluten proteins (Table 2). In patient 7 (panels D−F of Figure 3 and Table 2), the ω5-gliadins also showed a similar reduction in transgenic line 35b and were absent in line 45a. However, the ω1,2-gliadins (blue ovals) also showed

Figure 2. Reactivity of patient sera with LMW-GSs. Panel A shows an enlarged version of the region of a representative Coomassie-stained 2D gel containing the LMW-GSs and α- and γ-gliadins. Blots in panels B−F were reacted with sera from WDEIA patients 1 (B), 3 (C), 5 (D), 6 (E) or 7 (F). Reactive spots in each panel are indicated as s-type LMW-GSs by red arrows, m-type LMW-GSs by blue arrows, and i-type LMW-GSs by green arrows. Numbers correspond to several of the most abundant 2DE spots identified by MS/MS by Dupont et al.19 with N-terminal sequences SHIP (spots 119 and 314), METSRV (spot 144), METRCIP (spot 167), and ISQQQ (spot 141). Immunoblots contained flour proteins from the non-transgenic line (B−E) or transgenic line SA-845a-2 (F).

reduced reactivity in transgenic line 45a, essentially reflecting lower levels of these proteins that likely resulted from off-target gene silencing.18 Similar levels of IgE reactivity to LMW-GSs and α- and γ-gliadins were detected in the control and the two transgenic lines with this serum. Patient 9 exhibited only low levels of IgE reactivity to the ω5-gliadins in the control, and these proteins were not detected in either transgenic line (panels G−I of Figure 3). However, in this patient, levels of IgE reactivity to the α-gliadins were similar in the control and the transgenic lines. 9327


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Figure 3. Immunoblot analysis of total flour proteins from non-transgenic and transgenic wheat lines. Proteins from non-transgenic Butte 86 (A, D, G, J, and M), and transgenic lines 35b (B, E, H, K, and N) and 45a (C, F, I, L, and O) were reacted with sera from WDEIA patients 1 (A−C), 7 (D−F), 9 (G− I), 2 (J−L), or 10 (M−O). The positions of the ω5-gliadins are indicated with red ovals, and the positions of the ω1,2-gliadins are indicated with blue ovals. The inset in panel G shows a darker exposure of the same immunoblot to highlight the reactivity of serum with ω5-gliadins. ω5-gliadins were not detected on immunoblots of 35b or 45a with this serum.

In one patient (2), reactivity to ω5-gliadins in 35b was similar to that in the control (panels J and K of Figure 3). Serum from this patient also exhibited low levels of reactivity to ω5-gliadins in 45a (Figure 3L), even though these proteins were not evident on the Coomassie-stained 2D gels, even with three-dimensional (3D) enhancement.17 Additionally, there was a noticeable increase in the reactivity of several LMW-GSs and α-gliadins in both transgenic lines. It should be noted that this patient had very high IgE levels (Table 1) and showed symptoms of rhinitis in addition to WDEIA. This patient also had allergies to other foods, including shrimp and yeast. Not surprisingly, there was little difference in the reactivity of serum from patient 10 (panels

M−O of Figure 3) to immunoblots containing proteins from the control and the transgenic lines because this patient shows reactivity only to specific HMW-GSs. The control subject (12) did not show IgE reactivity to any flour proteins in either of the transgenic lines (data not shown). WDEIA Patient Sera That Reacts with Non-gluten Proteins. While sera from most WDEIA patients showed the greatest reactivity with gluten proteins by ELISA, serum from one patient (11) reacted only with non-gluten proteins. Because the total amount of IgE was low, this serum was reacted with immunoblots containing only the KCl-soluble proteins from Butte 86 flour (Figure 4). Among the proteins that reacted were 9328


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Figure 4. Immunoblot analysis of KCl-soluble flour proteins from non-transgenic and transgenic wheat lines. Proteins from non-transgenic Butte 86 (A) and transgenic lines 35b (B) and 45a (C) were reacted with serum from WDEIA patient 11. In panel A, red arrows point to proteins identified by MS/ MS as triticins, blue arrows point to proteins identified by MS/MS as peroxidases, and green arrows point to proteins identified by MS/MS as α-amylase inhibitors. The identities of other reactive proteins are shown in Supplementary Figure 1.

that these LMW-GSs contain some epitopes that are distinct from the ω5-gliadin epitopes. In contrast, a recombinant m-type LMW-GS showed weak IgE binding that was almost completely inhibited by the ω5-gliadin, indicating that the protein likely shared epitopes with the ω5-gliadins. Denery-Papini et al.11 used pepscan analysis to identify IgE-binding epitopes in a s-type LMW-GS and found two epitopes from the repetitive portion of the protein that were similar to epitopes found in ω5-gliadins and two epitopes from the C-terminal portion of the protein that were unique to LMW-GSs. Serum from patient 10 was the only one that showed reactivity to HMW-GSs on immunoblots. It is interesting that the y-type HMW-GSs By9 and Dy10 showed the strongest reaction to the sera. Of the x-type HMW-GSs, only Bx7 showed weak reactivity. Matsuo et al.26 identified three IgE-binding epitopes in HMWGSs for WDEIA patients. These epitopes are present in multiple copies in all of the Butte 86 HMW-GSs. Thus, it is likely that serum from this patient reacts with an epitope that is yet to be discovered and specific for the y-type HMW-GSs. Serum from one WDEIA patient (11) reacted only with nongluten proteins. Several of the IgE-reactive proteins have been previously shown to be immunogenic in patients with other wheat allergies, including the α-amylase trypsin inhibitors and peroxidases.22 Others, such as triticin, a protein similar to the 11S storage proteins of dicots, have not been implicated previously in food allergy to wheat. Comparison of Results Obtained by ELISA and Immunoblot Analysis. The study also revealed that results obtained with ELISAs and immunoblots are not necessarily the same. Sera from 9 of 11 WDEIA patients showed high levels of reactivity with ω5-gliadins by ELISA. Eight of these patients also exhibited strong reactivity to ω5-gliadins on immunoblots, while one (9) showed only weak reactivity. There was less consistency between the results obtained by ELISA and immunoblotting for some of the other gluten proteins. For the ω1,2-gliadins, 8 of 10 patients showed reactivity by ELISA but only two reacted on immunoblots; one patient showed strong reactivity to ω1,2gliadins (7), while the other reacted very weakly (2), despite the fact that IgE levels directed against ω1,2-gliadins were similar by ELISA. For the α-gliadins, patients 8 and 9 showed high levels of reactivity by ELISA, while patient 7 showed very low levels, but all exhibited strong reactivity on immunoblots. For the LMWGSs, the levels of IgE reactivity determined by ELISA did not necessarily correlate with those observed by immunoblots. Sera from two patients (1 and 3) that showed low levels of reactivity with LMW-GSs on immunoblots did not show reactivity by

several triticins (red arrows in Figure 4A), peroxidases (blue arrows in Figure 4A), and the tetrameric α-amylase inhibitors CM3, CM16, and CM17 (green arrows in Figure 4A). Other IgEreactive proteins included several peroxiredoxins and purinins as well as triosephosphate isomerase, purple acid phosphatase, alanine aminotransferase, and malate dehydrogenase (Supplementary Figure 1). There was little difference between the IgE reactivity of the control and transgenic lines (panels A−C of Figure 4).

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DISCUSSION Experiments using 2D immunoblotting highlight the complexity of the immunological responses to wheat flour in a group of patients that exhibit symptoms of WDEIA. Most but not all patients show reactivity to the ω5-gliadins, determined to be the primary sensitizing proteins in WDEIA in several studies.3,6−9 However, WDEIA patients with IgE directed against ω5-gliadins also show reactivity to other flour proteins. In some cases, the amount of reactivity with other gluten proteins is marginal and may be due to cross-reactions with similar epitopes in other protein types. Some cross-reactivity between gluten protein types would not be surprising because of the high abundance of glutamine and proline and the prevalence of repetitive sequences in all of the wheat gluten proteins.25 In a few cases, other types of gluten proteins were as reactive or more reactive than the ω5gliadins, supporting the notion that proteins other than the ω5gliadins may play a role in the elicitation of WDEIA. Indeed, a number of researchers have suggested a role for HMW-GSs,26 LMW-GSs,27,28 α-gliadins,29 α-amylase/trypsin inhibitors, and LTPs30 in WDEIA. Sera from eight WDEIA patients showed at least some reactivity with LMW-GSs. Immunoblot analysis revealed that the amounts of IgE reactivity to specific LMW-GS spots were not related to protein abundances, suggesting that there are differences in the affinity of sera for specific types of LMWGSs. In this study, all sera that reacted with LMW-GSs recognized s-type LMW-GSs and some m-type LMW-GSs, whereas only a few sera reacted with i-type LMW-GSs. This is perhaps not surprising because the repetitive portions of these LMW-GS types differ. Snégaroff et al.31 also concluded from IgE binding studies with recombinant forms of s- and m-type LMWGSs that all LMW-GSs do not have the same allergenic status in WDEIA. Additionally, in immunoblot inhibition studies using purified ω5-gliadins as inhibitors, Bouchez-Mahiout et al.27 found that IgE binding to a recombinant s-type LMW-GS was strong but only partly inhibited by the ω5-gliadins, suggesting 9329


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future acceptance of transgenic wheat by both regulatory agencies and consumers. Wheat with reduced allergen content might also have applications in new immunotherapy protocols designed to desensitize patients to specific food allergens. Important considerations in the development of reduced allergen wheat are the quality of the resulting flour and the fitness of the plant in different environments. A previous study demonstrated that flour from transgenic lines 35b and 45a had improved mixing properties relative to flour from non-transgenic Butte 86.18 Additionally, because the ω5-gliadins show large quantitative changes when plants are grown under different fertilizer or temperature regimens during grain development,33,34 a major source of environmental variation in flour composition has been eliminated in the transgenic lines. It is interesting that, with the exception of ω-gliadins, changes in flour protein composition in response to post-anthesis fertilizer were similar in non-transgenic and transgenic lines.18 Taken together, these data suggest that the transgenic lines may have more consistent quality when grown under different environmental conditions as well as reduced allergenicity.

ELISA. Serum from patient 7 showed strong reactivity with LMW-GSs on immunoblots, while serum from patient 2 showed weak reactivity, despite the fact that the reactivity of patient 2 was almost twice that of patient 7 in ELISA. The largest discrepancy between results obtained by ELISA and immunoblotting was for the HMW-GSs. While sera from eight patients showed some reactivity to HMW-GSs by ELISA, these proteins were only detected on immunoblots by serum from one patient (10). Overall, serum from patient 2 showed the largest discrepancy between ELISA and immunoblot results. This serum showed strong reactivities with ω1,2-gliadins and HMW-GSs by ELISA that were not detected by immunoblotting. Because ELISAs measure the amount of IgE that reacts with purified proteins or specific protein fractions, the specificity is dependent upon the purity of the proteins/fractions used for the analysis. Additionally, LMW-GS fractions generally contain Cand D-type LMW-GSs that have protein sequences very similar to α-, γ-, and ω-gliadins and cross-react with these protein types. Recombinant proteins can also be used in ELISAs. However, while offering greater specificity, a single recombinant gluten protein may not be representative of all proteins in the class. In comparison, immunoblotting provides a detailed look at the complement of all proteins in wheat flour and considers the relative abundance of the proteins in the flour sample. Additionally, C- and D-type LMW-GSs can be distinguished from B-type LMW-GSs provided that a detailed map of the flour proteome is available.19 Discrepancies between results obtained with ELISA and immunoblotting also may occur because conformations of the proteins in the two systems differ, thereby either allowing for better access to linear epitopes or destroying conformational epitopes. Indeed, a recent study by Mameri et al.32 emphasized the importance of the secondary structure of various wheat gluten proteins in IgE binding. For this reason, it can be valuable to consider results from both techniques when assessing the reactivity of antibodies directed against the wheat gluten proteins. Are Transgenic Approaches Feasible for Reducing the Allergenic Potential of Wheat? Transgenic lines in which genes encoding the ω5-gliadins were silenced showed reduced levels of IgE reactivity for 7 of 11 WDEIA patients. However, most patients still showed at least a low level of reactivity with other proteins in the transgenic lines. Some of this may be due to cross-reactivity between epitopes in ω5-gliadins and similar sequences in other gluten protein types. However, it is also possible that other flour proteins elicit WDEIA, particularly in those patients whose sera react primarily with α-gliadins (9) or HMW-GSs (10). The experiments highlight some of the difficulties faced in creating new wheat lines with reduced allergenic potential through either traditional breeding or biotechnology approaches for use in food products for allergy patients. Given the complexity of immunological responses in WDEIA patients and the severity of allergic responses, it would not be feasible for patients diagnosed with WDEIA to consume flour from wheat lacking ω5-gliadins without a thorough analysis of the IgE reactivity of the serum of each patient. Thus, better diagnostic methods should be developed in conjunction with reduced allergen wheat products. Nonetheless, the use of new wheat lines that lack ω5-gliadins in products for the general population may reduce the number of people that become sensitized to this group of flour proteins and to similar epitopes in other gluten proteins. In this way, it may be possible to reduce the number of people that become sensitized to wheat in the future. This would, of course, be subject to the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b03557. Serum identification and clinical characteristics of patients allergic to wheat (Supplementary Table 1) (PDF) Reactivity of sera to 2D spots identified by mass spectrometry (Supplementary Table 2) (PDF) Identification of some of the major wheat flour proteins in the KCl-soluble protein fraction that react with serum from WDEIA patient 11 (Supplementary Figure 1) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 510-559-5614. Fax: 510-559-5818. E-mail: susan. [email protected]. Funding

Research was funded by USDA−ARS Current Research Information System (CRIS) Project 5325-43000-028-00D. The USDA is an equal opportunity provider and employer. Mention of a specific product name by the USDA does not constitute an endorsement and does not imply a recommendation over other suitable products. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED 2DE, two-dimensional gel electrophoresis; ELISA, enzymelinked immunosorbent assay; HMW-GS, high-molecular-weight glutenin subunit; LTP, lipid transfer protein; LMW-GS, lowmolecular-weight glutenin subunit; MS/MS, tandem mass spectrometry; RNAi, RNA interference; WDEIA, wheat-dependent exercise-induced anaphylaxis

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REFERENCES

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Journal of Agricultural and Food Chemistry temperature and fertilizer on gliadin and glutenin accumulation in the endosperm of the developing wheat (Triticum aestivum L.) grain. Proteome Sci. 2013, 11, 8.

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Assessment of the Allergenic Potential of Transgenic Wheat

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