Abstract
BackgroundWheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD.ResultsA 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 ‘unique deduced protein fragments’ of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes.ConclusionsThe dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains.
Highlights
Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.)
RNA-amplicon sequencing and sequence analysis pipeline To assess a large, diverse set of durum wheat landraces and genebank accessions for their celiac disease (CD) epitope content, a deep 454 RNA-amplicon sequencing pipeline was developed to target the genetic variation in the first repetitive domain of alpha-gliadins (Figure 1, underlined in blue), which contains the major CD epitopes DQ2.5-glia-α1, DQ2.5-glia-α2 and DQ2.5-glia-α3
In this way an estimate of the alpha-gliadin protein composition and CD epitope composition was made for the 77 durum wheat plants from 61 different durum wheat cultivars and accessions, including landraces and breeders material (Additional file 1: Table S1)
Summary
Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and to accurately quantify the overall CD epitope load of a given wheat variety. Several specific bioactive gluten peptides have been identified that survive proteolysis in the human intestine and that can stimulate T cells [2,3,4,5] and trigger celiac disease (CD) in genetically susceptible individuals. A P to S substitution at the epitope core position 8 was shown to be sufficient to abolish T cell stimulation [11]
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