Celiac disease T-cell epitopes from gamma-gliadins: immunoreactivity depends on the genome of origin, transcript frequency, and flanking protein variation

Abstract

Background: Celiac disease (CD) is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins. The CD-toxicity of these proteins and their derived peptides is depending on the presence of specific T-cell epitopes (9-mer peptides; CD epitopes) that mediate the stimulation of HLA-DQ2/8 restricted T-cells. Next to the thoroughly characterized major T-cell epitopes derived from the α-gliadin fraction of gluten, γ-gliadin peptides are also known to stimulate T-cells of celiac disease patients. To pinpoint CD-toxic γ-gliadins in hexaploid bread wheat, we examined the variation of T-cell epitopes involved in CD in γ-gliadin transcripts of developing bread wheat grains.

Results: A detailed analysis of the genetic variation present in γ-gliadin transcripts of bread wheat (T. aestivum, allo-hexaploid, carrying the A, B and D genome), together with genomic γ-gliadin sequences from ancestrally related diploid wheat species, enabled the assignment of sequence variants to one of the three genomic γ-gliadin loci, Gli-A1, Gli-B1 or Gli-D1. Almost half of the γ-gliadin transcripts of bread wheat (49%) was assigned to locus Gli-D1. Transcripts from each locus differed in CD epitope content and composition. The Gli-D1 transcripts contained the highest frequency of canonical CD epitope cores (on average 10.1 per transcript) followed by the Gli-A1 transcripts (8.6) and the Gli-B1 transcripts (5.4). The natural variants of the major CD epitope from γ-gliadins, DQ2-γ-I, showed variation in their capacity to induce in vitro proliferation of a DQ2-γ-I specific and HLA-DQ2 restricted T-cell clone.

Conclusions: Evaluating the CD epitopes derived from γ-gliadins in their natural context of flanking protein variation, genome specificity and transcript frequency is a significant step towards accurate quantification of the CD toxicity of bread wheat. This approach can be used to predict relative levels of CD toxicity of individual wheat cultivars directly from their transcripts (cDNAs).

Figure 1

Schematic structure of a γ-gliadin protein. Structure of a γ-gliadin protein according to Anderson et al.[36] with the location of the CD epitopes. The protein consists of a short N-terminal signal peptide (S) followed by a unique N-terminal domain (I), two repetitive domains, R1 and R2 (II and IV) and two non-repetitive domains, NR1 and NR2 (III and V). In the first non-repetitive domain and in the second non-repetitive domain respectively six and two conserved cystein residues are present (indicated with vertical lines) that form four interchain disulfide bonds (indicated with arrows). The minimal T-cell epitopes are shown and their approximate position is indicated.

Figure 2

Neighbor-Joining relationships of γ-gliadin sequences. Neighbor-Joining relationship of γ-gliadin nucleotide sequences including transcript-contigs of bread wheat (T. aestivum, ABD genome, red circles) and genomic γ-gliadin sequences derived from diploid wheat species; T. monococcum (A^b, squares), Ae. speltoides (S, black circles), Ae. tauschii (D, triangles). In all cases, a region of the γ-gliadin gene, shared by all transcripts sequences and genomic sequences, encompassing domain III, IV and V was analyzed. Numbers on branch points indicate the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates).

Figure 3

CD-epitopes of γ-gliadin transcripts of T. aestivum in their natural context. The deduced aminoacid sequences of a T.aestivum γ-gliadin transcripts (N = 61) assigned to locus Gli-D, Neighbor Joining topology group 10, spanning a part of the first repetitive domain of the γ-gliadin sequence. CD T-cell epitopes are depicted: γ-I (PQQSFPQQQ), γ-III (QQPQQPYPQ), γ-IV (SQPQQQFPQ), γ-VI (QQPFPQQPQ), γ-VIIb (QQPQQPFPQ), 26-mer (FLQPQQPFPQQPQQPYPQQPQQPFPQ). Glutamine residues that are a primary targets for the enzyme tissue transglutaminase are underlined (Q) in QxP target sites whereas moderate target sites are depicted in italics (Q) [13,14]. Cleavage sites: in grey with white letters, chymotrypsin-high specificity; in grey with black letters, chymotrypsin-low specificity; cleavage occurs at the right side (C-terminal direction) of the marked amino acid.