Towards reducing the immunogenic potential of wheat flour: omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA

Abstract

Background: Omega-5 gliadins are a group of highly repetitive gluten proteins in wheat flour encoded on the 1B chromosome of hexaploid wheat. These proteins are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). The elimination of omega-5 gliadins from wheat flour through biotechnology or breeding approaches could reduce the immunogenic potential and adverse health effects of the flour.

Results: A mutant line missing low-molecular weight glutenin subunits encoded at the Glu-B3 locus was selected previously from a doubled haploid population generated from two Korean wheat cultivars. Analysis of flour from the mutant line by 2-dimensional gel electrophoresis coupled with tandem mass spectrometry revealed that the omega-5 gliadins and several gamma gliadins encoded by the closely linked Gli-B1 locus were also missing as a result of a deletion of at least 5.8 Mb of chromosome 1B. Two-dimensional immunoblot analysis of flour proteins using sera from WDEIA patients showed reduced IgE reactivity in the mutant relative to the parental lines due to the absence of the major omega-5 gliadins. However, two minor proteins showed strong reactivity to patient sera in both the parental and the mutant lines and also reacted with a monoclonal antibody against omega-5 gliadin. Analysis of the two minor reactive proteins by mass spectrometry revealed that both proteins correspond to omega-5 gliadin genes encoded on chromosome 1D that were thought previously to be pseudogenes.

Conclusions: While breeding approaches can be used to reduce the levels of the highly immunogenic omega-5 gliadins in wheat flour, these approaches are complicated by the genetic linkage of different classes of gluten protein genes and the finding that omega-5 gliadins may be encoded on more than one chromosome. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.

Keywords: Food allergy; Omega-5 gliadins; Proteomics; Reduced immunogenic potential; Wheat flour.

Fig. 1

Total flour proteins from parental lines Keumkang (a, d) and Olgeuru (b, e) and mutant line DH20 (c, f). Protein spots within the black boxes in panels (a, b and c) were identified by MS/MS. Proteins circled in red, blue, yellow, purple, green and black in panels (d, e and f) were identified as omega-5 gliadins, s-type LMW-GS, i-type LMW-GS, m-type LMW-GS, gamma gliadins and triticins, respectively. Dashed circles denote proteins identified in at least one of the parental lines but absent in the mutant line. MS/MS data are summarized in Table 1 and Additional file 1

Fig. 2

PCR analysis with primers specific for LMW-GS encoded at the Glu-B3 locus (a), omega-5 gliadins (b), and repeat junction primers 19S-1.3-2 (c) and 143E-1-600 (d) located at the ends of a 5.8 Mb region of chromosome 1B in Chinese Spring. In each panel, genomic DNA from Keumkang (1), Olgeuru (2), DH20 (3), N1BT1A (4) and N1BT1D (5) nullisomic tetrasomic lines of Chinese Spring or Chinese Spring (6) was amplified. The sizes of molecular weight markers in kb are shown in lane M in each panel. Primer sequences are shown in Table 2

Fig. 3

Reactivity of serum from WDEIA patients with total flour proteins from the parental cultivars Keumkang (a, d), Olgeuru (b, e) or the mutant DH20 (c, f). Serum was from patient #4 (a-c) and patient #5 (d-f) described in [16]. Positions of protein spots identified as omega-5 gliadins in the parental lines that are absent in the mutant line are shown with an oval. Arrows in panels (c and f) point to unidentified protein spots that are reactive in both the mutant and the parental lines

Fig. 4

Reactivity of a monoclonal antibody made to the N-terminal sequence of omega-5 gliadin with total flour proteins from (a) Keumkang (b) Olgeuru or (c) DH20. Positions of protein spots identified as omega-5 gliadins in the parental lines that are absent in the mutant line are shown with an oval. Arrows in the mutant line in panel (c) point to unidentified protein spots that are reactive in both the mutant and the parental lines

Fig. 5

Genomic arrangement of omega gliadin genes on chromosomes 1A (a), 1B (b) and 1D (c) of Chinese Spring summarized from [11]. The positions of ancestral genes found on all three chromosomes are indicated with black dots. Genes with repetitive motifs characteristic of omega-5 and omega-1,2 gliadins are shown in red and blue ovals, respectively. Predicted N-terminal sequences of the encoded proteins are shown above each group of genes. Genes that encode full length omega gliadins are denoted by solid ovals, pseudogenes by open ovals and truncated genes by hatched ovals. If expressed, truncated genes are predicted to encode proteins missing a portion of the COOH terminus. The truncated gene on chromosome 1A is located outside the 5.3 Mb region that was annotated

Fig. 6

Sequence comparison of omega-5 gliadins encoded by the B and D genomes in Chinese Spring with the full-length omega-5 gliadin BAE20328. N- and C-terminal sequences are shown in black and blue boxes, respectively. Within each protein sequence, the dominant WDEIA epitopes QQFPQQQ and QQIPQQQ are shown in red and blue, respectively. Lines above the protein sequence highlight peptides unique to omega-D4 that were identified by MS/MS in protein spots from DH20 reacting with sera from WDEIA patients. Red lines highlight peptides identified in the more basic protein while blue lines highlight peptides identified in the more acidic protein. MS/MS data are shown in Additional file 1

Fig. 7

PCR analysis with primers specific for the omega-D4 gene from Chinese Spring. Genomic DNA from Keumkang (1), Olgeuru (2), DH20 (3), Chinese Spring (4) and nullisomic tetrasomic lines of Chinese Spring N1AT1B (5), N1AT1D (6), N1BT1A (7), N1BT1D (8), N1DT1A (9) and N1DT1B (10) was amplified. The sizes of molecular weight markers in kb are shown in lane M