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

Salentijn et al. BMC Genomics 2012, 13:277 http://www.biomedcentral.com/1471-2164/13/277 RESEARCH ARTICLE Open Access Celiac disease T-cell epitopes from gammagliadins: immunoreactivity depends on the genome of origin, transcript frequency, and flanking protein variation Elma MJ Salentijn1,2*, D Cristina Mitea3, Svetlana V Goryunova4, Ingrid M van der Meer1, Ismael Padioleau1, Luud JWJ Gilissen1, Frits Koning3 and Marinus JM Smulders1,2 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). Keywords: Wheat, Gluten, γ-gliadins, Celiac disease, T-cell epitopes * Correspondence: 1 Plant Research International, Wageningen UR, P.O. Box 16, NL-6700 AA Wageningen, The Netherlands 2 Wageningen UR Plant Breeding, Wageningen, The Netherlands Full list of author information is available at the end of the article © 2012 Salentijn et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Salentijn et al. BMC Genomics 2012, 13:277 http://www.biomedcentral.com/1471-2164/13/277 Background Gluten proteins present in wheat can cause celiac disease (CD) [1]. Wheat gluten consists of a complex mixture of α/β-, γ- and ω-gliadins, and high and low molecular weight (HMW; LMW) glutenins, which are all encoded by medium to large multigene families. In contrast to other dietary proteins, gluten proteins are minimally digested by gastrointestinal proteases, resulting in relatively large peptides that accumulate in the small intestine [2]. In CD patients, during passage through the mucosal tract these gluten fragments can bind to the HLA-DQ2/8 receptors present on antigen presenting cells (APCs) and trigger T-cell responses. This T-cell response to HLA-DQ2/8 restricted gluten peptides occurs only in celiac patients and not in healthy controls. The HLA-DQ2/8 receptor favors peptides that contain one or more amino acids with a negative charge [3-10]. These are not present in gluten peptides but can be introduced due to the activity of human tissue transglutaminase (TG2) [11-14] an enzyme that converts glutamine (Q) residues into negatively charged glutamic acid (E) residues. Gliadin peptides, with their high proline and glutamine content, are perfect substrates for the transglutaminase reaction of TG2, which is critical for the creation of active T-cell epitopes involved in CD (CD epitopes). Since target sites for TG2 may be determined by sequences flanking the core motif, the amino acid composition of the surrounding of the epitope cores can influence the immunogenic capacity of a peptide regarding CD [8,13,14]. Several HLA-DQ2 and -DQ8 restricted T-cell epitopes have been identified in wheat gluten and in homologous proteins from barley (hordeins) and rye (secalins) ([7,8,15-26]. A 33-mer peptide, resistant to the action of proteases, derived from α-gliadins is regarded as one of the most CD-immunodominant gluten peptides [2,27]. However, there is clear evidence that γ-gliadins also contain T-cell stimulatory peptides [7,8,19,22,24,27-29]. In several specific cohorts a high frequency of CD patients was observed that mainly reacted to γ-gliadin peptides. Vader et al. [22] and Camarca et al. [24] found that half of the CD patients (respectively 10 of 20 children and 7 of 14 adults from Southern Europe) did not respond to peptides derived from α-gliadins, but instead reacted to peptides derived from γ-gliadins and LMW glutenins. A major CD epitope derived from γ-gliadins is the DQ2-γ-I epitope (PQQSFPQQQ [17,19]), recognized by one third of the patients [22,24]. Among pasta and bread wheat varieties differences have been observed in the capacity to stimulate in vitro DQ2-γ-I specific T-cell clones [30,31]. These facts justify an elaborated investigation of the γ-gliadins with special regard to the natural variation in CD-epitope content. The γ-gliadins account for approximately 12% of the flour proteins of the hexaploid wheat cultivar Butte 86 Page 2 of 12 [32]. They are encoded by clusters of tightly linked genes present at the Gli-1 loci (Gli-A1, Gli-B1 or Gli-D1) that are located on the short arms of the respective homoeologous group 1 chromosomes (1AS, 1BS and 1DS) of hexaploid bread wheat (T. aestivum L.) [33] and are tightly linked to the Glu-3 (LMW glutenins) and Gli-3 (ω-gliadins) loci [[34], and references therein]. The number of different γ-gliadin genes in the genome of bread wheat was estimated at 15–40 [35,36] and these can be clustered into four up to eleven groups [36-41]. This diversity is qualitatively reflected at the protein level [42], but not all genes are expressed evenly. Epitope content of the genes varies [39], just as it varied among α-gliadins [31,43-45] according to the genome of origin of the loci. In the present study CD epitopes from γ-gliadins of bread wheat (...truncated)