Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes

Abstract

Background: Bread wheat (Triticum aestivum) is an important staple food. However, wheat gluten proteins cause celiac disease (CD) in 0.5 to 1% of the general population. Among these proteins, the alpha-gliadins contain several peptides that are associated to the disease.

Results: We obtained 230 distinct alpha-gliadin gene sequences from severaldiploid wheat species representing the ancestral A, B, and D genomes of the hexaploid bread wheat. The large majority of these sequences (87%) contained an internal stop codon. All alpha-gliadin sequences could be distinguished according to the genome of origin on the basis of sequence similarity, of the average length of the polyglutamine repeats, and of the differences in the presence of four peptides that have been identified as T cell stimulatory epitopes in CD patients through binding to HLA-DQ2/8. By sequence similarity, alpha-gliadins from the public database of hexaploid T. aestivum could be assigned directly to chromosome 6A, 6B, or 6D. T. monococcum (A genome) sequences, as well as those from chromosome 6A of bread wheat, almost invariably contained epitope glia-alpha9 and glia-alpha20, but never the intact epitopes glia-alpha and glia-alpha2. A number of sequences from T. speltoides, as well as a number of sequences fromchromosome 6B of bread wheat, did not contain any of the four T cell epitopes screened for. The sequences from T. tauschii (D genome), as well as those from chromosome 6D of bread wheat, were found to contain all of these T cell epitopes in variable combinations per gene. The differences in epitope composition resulted mainly from point mutations. These substitutions appeared to be genome specific.

Conclusion: Our analysis shows that alpha-gliadin sequences from the three genomes of bread wheat form distinct groups. The four known T cell stimulatory epitopes are distributed non-randomly across the sequences, indicating that the three genomes contribute differently to epitope content. A systematic analysis of all known epitopes in gliadins and glutenins will lead to better understanding of the differences in toxicity among wheat varieties. On the basis of such insight, breeding strategies can be designed to generate less toxic varieties of wheat which may be tolerated by at least part of the CD patient population.

Figure 1

Schematic structure of an α-type gliadin protein. The protein consists of a short N-terminal signal peptide (S) followed by a repetitive domain (R) and a longer non-repetitive domain (NR1 and NR2), separated by two polyglutamine repeats (Q1 and Q2). In the non-repetitive domains five conserved cystein residues are present which are indicated with vertical lines. The T cell epitopes are shown and their approximate position is indicated.

Figure 2

Dendrogram of a ClustalX alignment of the obtained full-ORF α-gliadin deduced proteins, which are indicated by their accession numbers (see Table 1). A PAM350 matrix and the neighbor joining method were used. Bootstrap values (of 1000 replications) are given for nodes only if they were 950 or higher.

Figure 3

Analysis of the two glutamine repeats in the 31 obtained full-ORF α-gliadin proteins from diploid wheat species, according to the genome of origin. The average number of the glutamine residues in the first (Q1) and second repeat (Q2) are shown according to the genome of origin. The A genome (T. monococcum) sequences possessed a significantly higher average number of glutamine residues in the first glutamine repeat (27.7 +/- 1.7) than the B (20.0 +/- 3.4) and D (20.7 +/- 1.1) genomes did. For the second glutamine repeat, the B genome sequences demonstrated a significantly higher number of glutamine residues (18.8 +/- 1.9) than those of the other two genomes (A, 10.2 +/- 0.6; D, 9.7 +/- 1.4).

Figure 4

Distribution of stop codons in the pseudogenes according to the amino acid position in the sequences. The positions of the stop codons are not distributed evenly across the various diploid species. The A genome sequences have a high percentage of stop codons at positions 24, 42, 145, 199 and these four stop codons may occur jointly in one pseudogene sequence. The B genome sequences also contain a high percentage of the jointly occurring stop codons at position 24, 145 and 199 but do not contain the stop codon at position 42. The jointly occurring stop codons 24, 145 and 199 are present in a few pseudogenes originating from the D genome. Pseudogenes from the A genome may contain another pair of jointly occurring stop codons at position 113 and 184 whereas the pair at positions 64 and 121 occurs in B genome pseudogenes, and pairs of stop codons at positions 65 and 83 and at the positions 99 and 252 occur in D genome pseudogenes.

Figure 5

The relation of the relative numbers of synonymous substitutions (K_a) and non-synonymous substitutions (K_s) per site for pairwise comparisons among full-ORF α-gliadins and pseudogene sequences. The dotted line represents a K_a/K_s ratio of 1. Linear trendlines with the intercept set to zero are shown both for full-ORF sequences and pseudogene sequences.

Figure 6

Partial detailed alignment of the obtained full-ORF α-gliadin proteins. The figure shows the disruption of epitope glia-α (QGSFQPSQQ) by a single amino acid change in all T. monococcum (A genome) sequences andthree of the T. speltoides (B genome) sequences.

Figure 7

Partial detailed alignment of the obtained full-ORF α-gliadin proteins, showing the disruption of epitope glia-α2 (PQPQLPYPQ) in all T. speltoides (B genome), T. monococcum (A genome) and three T. tauschii (D genome) sequences. Secondly the figure shows the disruption of epitope glia-α9 (PFPQPQLPY) in the T. speltoides (B genome) sequences and finally the disruption of epitope glia-α20 (FRPQQPYPQ) in all T. speltoides (B genome) and in one T. tauschii (D genome) sequence.