Article pubs.acs.org/JAFC
Study on the Immunoreactivity of Triticum monococcum (Einkorn) Wheat in Patients with Wheat-Dependent Exercise-Induced Anaphylaxis for the Production of Hypoallergenic Foods Carla Lombardo,†,‡ Michela Bolla,†,§ Roberto Chignola,§ Gianenrico Senna,‡ Giacomo Rossin,§ Beatrice Caruso,∥ Carlo Tomelleri,§ Daniela Cecconi,§ Andrea Brandolini,⊥ and Gianni Zoccatelli*,§ ‡
Allergy Unit, and ∥Clinical Chemistry and Haematology Unit, Azienda Ospedaliera Universitaria Integrata of Verona, 37126 Verona, Italy § Department of Biotechnology, University of Verona, 37134 Verona, Italy ⊥ CRA-SCV, Sant’Angelo Lodigiano, 26866 Lodi, Italy S Supporting Information *
ABSTRACT: Wheat [Triticum aestivum (T.a.)] ingestion can cause a specific allergic reaction, which is called wheat-dependent exercise-induced anaphylaxis (WDEIA). The major allergen involved is ω-5 gliadin, a gluten protein coded by genes located on the B genome. Our aim was to study the immunoreactivity of proteins in Triticum monococcum (einkorn, T.m.), a diploid ancestral wheat lacking B chromosomes, for possible use in the production of hypoallergenic foods. A total of 14 patients with a clear history of WDEIA and specific immunoglobulin E (IgE) to ω-5 gliadin were enrolled. Skin prick test (SPT) with a commercial wheat extract and an in-house T.a. gluten diagnostic solution tested positive for 43 and 100% of the cases, respectively. No reactivity in patients tested with solutions prepared from four T.m. accessions was observed. The immunoblotting of T.m. gluten proteins performed with the sera of patients showed different IgE-binding profiles with respect to T.a., confirming the absence of ω-5 gliadin. A general lower immunoreactivity of T.m. gluten proteins with scarce cross-reactivity to ω-5 gliadin epitopes was assessed by an enzyme-linked immunosorbent assay (ELISA). Given the absence of reactivity by SPT and the limited cross-reactivity with ω-5 gliadin, T.m. might represent a potential candidate in the production of hypoallergenic bakery products for patients sensitized to ω-5 gliadin. Further analyses need to be carried out regarding its safety. KEYWORDS: wheat, allergy, wheat-dependent exercise-induced anaphylaxis (WDEIA), Triticum monococcum, ω gliadin
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WDEIA is the best studied model of “co-factor-induced” anaphylaxis, where eliciting or favoring factors (e.g., physical exercise, acetylsalicylic acid, and alcohol) are required to provoke symptoms in patients who otherwise tolerate glutencontaining foods.5,6 Wheat-allergic patients must strictly avoid foods containing or that are suspected to contain wheat, whereas patients affected by WDEIA who usually tolerate wheat ingestion should avoid exercise or exposure to other co-factors to prevent symptom appearance. These conditions strongly affect their quality of life and, in young subjects, may generate psychological or growth problems. Different gluten-free products are available on the market, but they are often expensive. For this reason, the present law in Italy and in some other countries guarantees funding to celiac patients for the purchase of gluten-free products but not to people affected by other wheat-induced pathologies (i.e., food allergy). In addition, the taste and texture of gluten-free products is often not pleasant.7 These factors make the research of wheat substitutes
INTRODUCTION Wheat is one of the most consumed cereals around the world. One reason for its great success is the presence of gluten proteins in the caryopsis that allow for the formation of a unique viscoelastic dough, essential for the production of a broad range of bakery goods. Gluten is composed of two main kinds of proteins, the monomeric gliadins and the polymeric glutenins, that are coded by different genes on chromosomes 1 and 6 and present in three wheat genomes A, B, and D. Nowadays, the hexaploid AABBDD bread wheat Triticum aestivum (T.a.) and the tetraploid AABB durum wheat Triticum turgidum subsp. durum (T.d.) represent the most cultivated wheat species. Despite their widespread use, gluten proteins can cause adverse reactions after ingestion, such as celiac and immunoglobulin E (IgE)-mediated (allergic) diseases, including food allergy, wheat-dependent exercise-induced anaphylaxis (WDEIA), and atopic dermatitis.1 In patients with WDEIA, ingestion of wheat-containing products, followed by physical exercise, can induce anaphylactic symptoms, such as hypotension, dyspnea, and generalized urticaria. The great majority of patients usually have detectable IgE antibodies that bind the salt-insoluble protein ω-5 gliadin2,3 and, to a lesser extent, lowmolecular-weight (LMW) 4 and high-molecular-weight (HMW)5,6 glutenin subunits. © 2015 American Chemical Society
Received: Revised: Accepted: Published: 8299
May 28, 2015 August 19, 2015 September 2, 2015 September 2, 2015 DOI: 10.1021/acs.jafc.5b02648 J. Agric. Food Chem. 2015, 63, 8299−8306
Article
Journal of Agricultural and Food Chemistry
(62.5 mM Tris−HCl at pH 6.8, 1.3% (w/v) SDS, 10% (w/v) glycerol, and 4% β-mercaptoethanol) and heated at 100 °C for 5 min. Proteins (50 μg/cm of lane width) were separated in 16% SDS−PAGE at 20 mA current by means of a Mini Protean III apparatus (Bio-Rad, Hercules, CA) in 25 mM Tris−192 mM glycine and 0.1% SDS (pH 8.4). The gels were stained with Coomassie R250. Alternatively, proteins were electroblotted onto nitrocellulose (Millipore, Schwalbach, Germany) at 50 V for 150 min. The membrane was blocked with 1% BSA and 0.05% Tween 20 in PBS (PTB) and probed with the sera of patients at 1:10 dilution. Bound IgE was detected by a monoclonal horseradish peroxidase (HRP)conjugated anti-IgE antibody (Southern Biotech, Birmingham, AL) at 1:1500 dilution, followed by chemiluminescence reaction with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and image acquisition by a Chemidoc XRS apparatus (Bio-Rad). Purification of ω-5 Gliadin. A T.a. extract from 20 g of seeds was used to purify ω-5 gliadin. The ethanol-soluble protein fraction was concentrated by adding 2 volumes of 1.5 M NaCl and leaving the solution overnight at 4 °C. After the centrifugation at 10000g for 15 min, the supernatant was discarded and the gluten proteins in the pellet were solubilized in 6 M guanidine HCl, adjusted to pH 8 with Tris−HCl + 50 mM dithiothreitol (DTT). Purification was carried out by reversed-phase high-performance liquid chromatography (RPHPLC) on a Vydac 218TP C18 semi-preparative column (10 × 250 mm, 5 μm particle size) and separated at a 5 mL/min flow rate in 0.05% trifluoroacetic acid with a 10−70% acetonitrile gradient in 65 min and 70−90% acetonitrile gradient in 10 min as described previously.22 Peaks were collected, lyophilized, and analyzed by immunoblotting with the sera of patients to identify the immunoreactive ω-5 gliadin. HPLC−Tandem Mass Spectrometry (MS/MS) Analysis of the Purified ω-5 Gliadin. A protein amount of 3 μg of putative ω-5 gliadin isolated by HPLC was lyophilized and diluted in 50 μL of 0.084 N HCl. Pepsin from porcine pancreas (Sigma-Aldrich, St. Louis, MO) was added in a 1:60 enzyme/protein ratio. After 2 h, the digest underwent ZipTip cleaning (Millipore) following the procedure of the manufacturer and then lyophilized. Peptides were separated by reversed-phase nano-HPLC-Chip technology (Agilent Technologies, Palo Alto, CA) online coupled with a three-dimensional (3D) ion trap mass spectrometer (model Esquire 6000, Bruker Daltonics, Bremen, Germany). The chip was composed of a Zorbax 300SB-C18 (150 mm × 75 μm, with a 5 μm particle size) analytical column and a Zorbax 300SB-C18 (40 nL, 5 μm) enrichment column. The complete system was fully controlled by ChemStation (Agilent Technologies) and EsquireControl (Bruker Daltonics) softwares. The scan range used was from m/z 300 to 1800. For MS/MS experiments, the system was operated with automatic switching between MS and MS/MS modes. The three most abundant peptides of each m/z were selected to be further isolated and fragmented. The MS/MS scanning was performed in the normalresolution mode at a scan rate of m/z 13.000 per second. A total of five scans were averaged to obtain a MS/MS spectrum. Database searches were conducted using the MS/MS ion search of Mascot against all entries of the non-redundant National Center for Biotechnology Information (NCBI) database with the following parameters: specific pepsin digestion, up to two missed cleavage; fixed and variable modifications, carbamidomethyl-Cys and oxidated Met, respectively; peptide and fragment tolerances, ±0.9 and ±0.9 Da, respectively; and peptide charges, +1, +2, and +3. For positive identification, the score of the result of [−10 log(P)] had to be over the significance threshold level (p < 0.05). Patients and Diagnosis. A total of 14 patients [mean age of 42 years, with a standard deviation (SD) of 12.2; 10 male] attending the Verona Allergy Unit between January 2012 and January 2014 with an unequivocal clinical history of WDEIA (allergic symptoms after exercise within 4 h of ingestion of wheat-based products) and positive serum ω-5 gliadin-specific IgE were studied in the standard diagnostic setting. All of the participants were informed about the study by the medical team and signed an informed consent approved by the ethics
an important task to ameliorate the condition of allergic patients. The selection of wheat varieties expressing low or no concentrations of allergens is presently underway.8 This is favored by the wide biodiversity of the genus Triticum. Indeed, differences in the IgE-binding capacity of certain wheat varieties have been observed in patients affected by baker’s asthma9−11 and food allergy,10,12−14 including WDEIA. Although a certain degree of variability in the allergenic potency of the various cultivars was shown, previous works failed to find reliable hypoallergenic candidates. With regard to baker’s asthma, the polysensitization profile of patients is one of the limiting factors.15 Although sensitization is usually limited to ω-5 gliadins,16 as far as food allergy is concerned, no wheat varieties expressing this allergen have been identified thus far, except for a translocated wheat, in which part of the short arm of the 1B chromosome was replaced with a portion of the short arm of the 1R chromosome of rye.12 Finally, no study has specifically evaluated the immunoreactivity of different wheat varieties in vivo [i.e., by skin prick test (SPT)]. ω-5 gliadins, which are encoded at the Gli-B1 locus present in the B chromosome, are inevitably associated with durum and bread wheats. For this reason, the diploid wheat Triticum monococcum (T.m.), known also as “einkorn”, might represent a potential alternative to conventionally used wheat. Indeed, the genome of T.m. is AmAm, very close to the AuAu present in modern wheat; thus, the only ω gliadins expressed are those associated with the locus Gli-A1, never related to WDEIA thus far.12 Corbellini et al.17 demonstrated that some einkorn accessions possessing good bread-making characteristics do not present the typical electrophoretic bands of T.a. ω gliadins, which would support the possible use of this cereal as a hypoallergenic option for WDEIA patients. The aim of this study was to assess the immunoreactivity of T.m. proteins among patients suffering from WDEIA, by immunochemical techniques and SPT, and to evaluate the suitability of this cereal in the future development of hypoallergenic flours.
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MATERIALS AND METHODS
Wheat Extraction. Four einkorn wheat accessions cultivated at the Istituto Sperimentale della Cerealicoltura in Sant’Angelo Lodigiano, Lodi, Italy (ID109, ID358, ID1331, and Monlis) were selected. These accessions are characterized by good bread-making characteristics.17,19,20 T.a. cv. Bologna was used as a wheat reference. A total of 1 g of seeds of each sample was milled in a mortar and extracted in phosphate-buffered saline (PBS, pH 7.2, 1:10, w/v) as previously described21 for 2 h at room temperature (RT). After centrifugation at 12000g, the supernatants were collected and stored at −20 °C and the pellets were subsequently washed with PBS and distilled water. The supernatants obtained after these washing steps were discarded. The pellets were finally extracted with 40% ethanol (1:5) for 2 h and centrifuged. The supernatants containing the PBS-insoluble gliadins (mainly) and glutenins (to a lesser extent) were collected and stored at −20 °C. Preparation of Prick Test Diagnostic Solutions. Diagnostic solutions were produced by diluting the protein extracts (accessions ID109, ID358, ID1331, and Bologna) in a storage solution containing 40% glycerol and 0.4% phenol for commercial preparations. The protein concentrations were 1.5 mg/mL for the PBS-soluble proteins and 0.5 mg/mL for the PBS-insoluble proteins. The concentration of ω-5 gliadin was 0.1 mg/mL. Diagnostic solutions were filtered at 0.20 μm in sterile conditions. Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS−PAGE) and IgE Immunoblotting. The protein extracts from the different wheat samples were mixed with 2× loading buffer 8300
DOI: 10.1021/acs.jafc.5b02648 J. Agric. Food Chem. 2015, 63, 8299−8306
Article
Journal of Agricultural and Food Chemistry Table 1. Clinical Characteristics of Patients and Results of Skin Tests in-house SPTa soluble
patients
clinical symptoms
BT07 BT09 BT14 BT16 BT19 BT24 BT25
anaphylaxis urticaria anaphylaxis urticaria anaphylaxis anaphylaxis anaphylaxis
BT26 BT27 BT28 BT30 BT31 BT33 BT34
anaphylaxis urticaria anaphylaxis anaphylaxis anaphylaxis anaphylaxis anaphylaxis
a
other co-factors none NSAIDsc none NSAIDs none none NSAIDs and alcohol NSAIDs none none none alcohol alcohol alcohol
SPTa wheat (Lofarma)
insoluble
CAP wheat
CAP gluten
CAP ω-5 gliadin
T.a.
ID109
ID358
ID1331
T.a.
ID109
ID358
ID1331
purified ω-5 gliadin
0.73
