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Wheatω-5 gliadin has been identified as a major allergen in wheat-dependent exercise-induced anaphylaxis. We have detected seven IgE-binding epitopes in primary sequence of the protein. We newly identified four additional IgE-binding epitope sequences, QQFHQQQ, QSPEQQQ, YQQYPQQ and QQPPQQ, in three patients with wheat-dependent exercise-induced anaphylaxis in this study. Diagnosis and therapy of food allergy would benefit from the availability of defined recombinant allergens. However, because ω-5 gliadin gene has not been cloned, recombinant protein is currently unavailable. We sought to clone the ω-5 gliadin gene and produce the homogeneous recombinant protein for use in an in vitro diagnostic tool. Using a PCR-based strategy we isolated two full-length ω-5 gliadin genes, designated ω-5 and ω-5b, from wheat genomic DNA and determined the nucleotide sequences. The protein encoded by ω-5a was predicted to be 439 amino acids long with a calculated mass of 53 kDa; the ω-5b gene would encode a 393 amino acid, but it contains two stop codons indicating that ω-5b is pseudogene. The C-terminal half (178 amino acids) of the ω-5a gliadin protein, including all 11 IgE-binding epitope sequences, was expressed in Escherichia coli by means of the pET system and purified using RP-HPLC. Western blot analysis and dot blot inhibition assay of recombinant and native ω-5 gliadin purified from wheat flour demonstrated that recombinant protein had IgE-binding ability. Our results suggest that the recombinant protein can be a useful tool for identifying patients with wheat-dependent exercise-induced anaphylaxis in vitro.
Wheat is one of the most widely cultivated staple foods for western people. Patient with wheat allergy, especially wheat-dependent exercise-induced anaphylaxis (WDEIA) has increased recently, as there is now a higher consumption of western style food in Japan [1,2]. WDEIA is a distinct form of wheat allergy in which the patient experiences a very severe allergic reaction in response to intense exercise after ingestion of wheat [3,4]. Our previous study demonstrated that exercise and aspirin intake facilitate absorption of the wheat allergens from the gastrointestinal tract in patients with WDEIA . It follows that the allergens transferred into circulating blood cross-link receptor-bound IgE on mast cells and cause degranulation followed by release of chemical mediators such as histamine. They induce immediate inflammatory reactions similar to those of common food allergies such as urticaria, angioedema, hypotension, and shock.
To diagnose WDEIA, we typically perform an exercise challenge test combined with wheat ingestion for patients who have episodes of anaphylaxis after wheat intake. However, the challenge test is unsafe for patients because an anaphylactic shock is sometimes provoked in the test. An radioallergosorbent test to wheat protein or wheat gluten is commercially available for diagnosis of wheat allergy, but this test is not always satisfactory to diagnose WDEIA because of low sensitivity or the occurrence of false-positive results . The heterogeneity of antigens used in the test is considered to be a major cause of these problems. It has been reported that ω-5 gliadin is a major allergen in patients with WDEIA; the skin prick test and radioallergosorbent test with ω-5 gliadin is considered to be useful to diagnose WDEIA [6–8].
Common wheat (Triticum aestivum) is a hexaploid species, in which each cell contains six sets of chromosomes and is estimated to have several copies of the ω-5 gliadin gene . In wheat there are at least six different ω-5 gliadin proteins; the primary structures of these is very similar but the contents vary according to growing districts or cultivated variety . Hence it is difficult to prepare a homogeneous ω-5 gliadin protein from wheat flour.
In the present study we analyzed IgE-binding epitopes in an extra three patients with WDEIA and cloned the ω-5 gliadin gene to obtain the IgE-reactive homogeneous recombinant ω-5 gliadin protein that can be used for diagnosis and possibly treatment of WDEIA.
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In this study we identified new linear IgE-binding epitopes in ω-5 gliadin and described the gene cloning, expression in E. coli, purification, and immunological characterization of the recombinant ω-5 gliadin.
In our previous study we showed that QQIPQQQ, QQFPQQQ, QQLPQQQ, QQSPQQQ, QQSPEQQ, QQYPQQQ and PYPP sequences in ω-5 gliadin are IgE-binding epitopes in patients with WDEIA and that four of these sequences, QQIPQQQ, QQFPQQQ, QQSPQQQ and QQSPEQQ, are dominant . In the present study we carried out an additional IgE epitope analysis in three patients with WDEIA. Two of the three patients have IgE antibodies that react with the four dominant epitope sequences but IgE antibodies in the serum of patient three reacted only with peptide QQFPQQQ (Table 1). In addition, IgE antibodies of patients two and three did not react with QQYPQQQ but did react with YQQYPQQ. The two epitopes, QSPEQQQ and QQFHQQQ, were detected only in patient three, and similarly QQPPQQ was detected only in patient one. These results indicate that the four newly detected IgE-binding epitopes, QQPPQQ, YQQYPQQ, QSPEQQQ and QQFHQQQ, are not common but might be important epitopes for the development of allergic symptoms in WDEIA.
We cloned and determined the nucleotide sequence of two ω-5 gliadin genes, ω-5a and ω-5b, from genomic DNA of wheat cultivar Norin 61, a Japanese soft wheat variety. Neither of the isolated genes contains introns, like other genes encoding gliadins such as α-gliadin, γ-gliadin, ω-1,2 gliadin. The ω-5a gene has an ORF which may encode the protein but the ω-5b gene is assumed to be a pseudogene because it has two stop codons in the putative ORF (Fig. 2). Some gliadin genes are unstable in the E. coli vector and deletion of the repetitive domain usually occurred during DNA cloning . The nucleotide sequences of ω-5a determined from five clones in this study were identical and the 1413 bp DNA of the sequenced ω-5a gene is the same length as the PCR products indicating that the cloned ω-5a gene has no artificial deletion. The existence of repeat sequences of QQXP, QQQXP and QQQQXP where X is F, I or L and the lack of a cysteine residue in ω-5a gliadin are compatible with the structural features of ω-5 gliadin. Kasarda et al. reported that the N-terminal amino acid sequence of ω-5 gliadin from wheat (T. aestivum‘Justin’) is SRLLSPRGKELHTPQQQFPQQ . DuPont et al. showed that ω-5 gliadin from wheat (T. aestivum‘Butte’) was separated into two fractions, 1B1 and 1B2, and the N-terminal amino acid sequences are SRLLSPRGKELHTPQEFQFPQQQ and SRLLSPRGKELHTPQEQFPQQQ, respectively . The deduced N-terminal amino acid sequence of ω-5a is identical with 1B2 ω-5 gliadin. The 1B2 ω-5 gliadin fraction from T. aestuvum Butte was resolved into three peaks of molecular mass 49 085, 50 300, and 51 500 by MALDI-TOF MS . However the calculated molecular mass (50 900) of ω-5a gliadin did not coincide with any of these molecular masses. The differences in mass between the three 1B2 ω-5 gliadins and ω-5a gliadin may be accounted for by a difference of the number of repeat sequences.
In wheat allergy, sensitization to inhaled wheat flour leads to baker's asthma , whereas sensitization to ingested wheat develops into a common food allergy or WDEIA. In addition, the causative allergen is different in various clinical manifestations, for instance the major allergen for baker's asthma is α-amylase inhibitor whereas that for WDEIA is ω-5 gliadin . Recent studies have shown that ω-5 gliadin is a good candidate as a diagnostic tool not only for WDEIA but also for immediate allergy to wheat [20–22]. Accurate diagnosis of food allergy requires standardization of the food antigen used in the skin test and allergen specific-IgE RAST. However, it is difficult to prepare homogeneous allergen by direct extraction from food because the allergen content depends on the cultivated variety and place. Therefore identification and characterization of major allergens for each clinical manifestation is important and the use of standardized recombinant proteins might reduce inaccurate diagnosis. Some recombinant food allergens have been produced and the advantages of recombinant proteins have been clearly demonstrated for diagnosis [23,24]. One type of recombinant wheat gliadin has been produced in E. coli using a pET vector and applied to the identification of major allergens in patients with wheat allergy . In the present study we tried to produce full-length ω-5a gliadin in E. coli but the entire DNA of ω-5a coding region could not be inserted into several types of E. coli expression vectors because of plasmid instability. Thus the C-terminal half of ω-5a gliadin, designated rOG5C and containing all detected IgE-binding epitopes, was overproduced using pET-21a vector. The calculated mass (21.7 kDa) of the purified rOG5C was approximately 20% lower than the apparent molecular mass (27.2 kDa) determined by SDS/PAGE. This difference in mass is accounted for by the behaviour of native ω-5 gliadin as published previously .
It is vital to compare immunological properties of a recombinant protein with those of the native form before using the recombinant for diagnosis or treatment of food allergies. Western blot analysis of nOG5 and rOG5C showed that the IgE antibodies in sera of patients with WDEIA react to nOG5 rather than to rOG5C. Dot blot inhibition assays indicate that the IgE-binding capacity of nOG5 is larger than that of rOG5C due to a lack of N-terminal half of rOG5C. However, rOG5C had sufficient ability to detect the specific IgE to ω-5 gliadin because rOG5C completely inhibited the IgE binding to nOG5. Thus the recombinant ω-5 gliadin produced in this study provides reagent quantities of protein that would be useful in the serologic diagnosis of WDEIA.