A Universal Approach to Eliminate Antigenic Properties of Alpha-Gliadin Peptides in Celiac Disease

et al. (2010) A Universal Approach to Eliminate Antigenic Properties of Alpha- Gliadin Peptides in Celiac Disease. PLoS ONE 5(12): e15637. doi:10.1371/journal.pone.0015637 A Universal Approach to Eliminate Antigenic Properties of Alpha-Gliadin Peptides in Celiac Disease Cristina Mitea 0 Elma M. J. Salentijn 0 Peter van Veelen 0 Svetlana V. Goryunova 0 Ingrid M. van der Meer 0 Hetty C. van den Broeck 0 Jorge R. Mujico 0 Veronica Monserrat 0 Luud J. W. J. Gilissen 0 Jan Wouter Drijfhout 0 Liesbeth Dekking 0 Frits Koning 0 Marinus J. M. Smulders 0 Vladimir N. Uversky, University of South Florida College of Medicine, United States of America 0 1 Department of Immunohematology and Blood Transfusion, Leiden University Medical Center , Leiden , The Netherlands, 2 Plant Research International, Wageningen UR, Wageningen , The Netherlands, 3 Allergy Centre Wageningen, Wageningen , The Netherlands Celiac disease is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins, including the a-gliadins. It has been shown that a-gliadins harbor several major epitopes involved in the disease pathogenesis. A major step towards elimination of gluten toxicity for celiac disease patients would thus be the elimination of such epitopes from a-gliadins. We have analyzed over 3,000 expressed a-gliadin sequences from 11 bread wheat cultivars to determine whether they encode for peptides potentially involved in celiac disease. All identified epitope variants were synthesized as peptides and tested for binding to the disease-associated HLA-DQ2 and HLA-DQ8 molecules and for recognition by patient-derived a-gliadin specific T cell clones. Several specific naturally occurring amino acid substitutions were identified for each of the a-gliadin derived peptides involved in celiac disease that eliminate the antigenic properties of the epitope variants. Finally, we provide proof of principle at the peptide level that through the systematic introduction of such naturally occurring variations a-gliadins genes can be generated that no longer encode antigenic peptides. This forms a crucial step in the development of strategies to modify gluten genes in wheat so that it becomes safe for celiac disease patients. It also provides the information to design and introduce safe gluten genes in other cereals, which would exhibit improved quality while remaining safe for consumption by celiac disease patients. - Funding: This study was supported by the Celiac Disease Consortium, an Innovative Cluster approved by The Netherlands Genomics Initiative and partially funded by the Dutch Government (BSIK03009), by the Center for Medical Systems Biology (CMSB), a center of excellence approved by The Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NWO), and by The Netherlands Ministry of Agriculture, Nature, and Food Safety (project KB-05-001019). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors equally contributed to this work. Current address: Department of Metabolism and Nutrition, Institute of Food Science and Technology and Nutrition (CSIC), Madrid, Spain Celiac Disease (CD) is an intestinal T cell-mediated disease caused by the gluten fraction of wheat or the homologous proteins from barley or rye. CD has prevalence between 0.5 and 2% in human populations [1] and is characterized by a chronic intestinal inflammation upon ingestion of gluten proteins. Recently, the molecular aspects have been comprehensively addressed in several review papers [25]. In short, in CD patients CD4+ T cells are present in the lamina propria that secrete interferon-gamma upon recognition of gluten-derived peptides bound to HLA-DQ2 or HLA-DQ8 molecules present on antigen presenting cells. Strikingly, most of the gluten peptides implicated in CD require modification by the enzyme tissue transglutaminase before they can bind to the disease-predisposing HLA-DQ molecules and trigger T cell responses [25]. In addition to the adaptive CD4+ T cell response to gluten, CD is characterized by the upregulation of IL-15, an intraepithelial T cell infiltrate expressing the NKG2D receptor, and the overexpression of a ligand for NKG2D (MICA) [6,7]. Many gluten peptides with T cell stimulatory properties have now been identified. Such peptides have been found in wheat a-, c-, and v-gliadins as well as in low molecular weight (LMW) and high molecular weight (HMW) glutenins [814]. Several studies have demonstrated that peptides derived from a-gliadins induce strong T cell responses in the large majority of patients, while responses to the other peptides are less frequently found [810,13]. An a-gliadin derived 33-mer peptide (amino acid sequence LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) was identified that encodes six partially overlapping T cell epitopes and has very potent T cell stimulatory properties [13]. It harbours the p5675 peptide (LQLQPFPQPQLPYPQPQLPY) that has been identified as the dominant gluten epitope [9,10]. Furthermore, agliadins are the only gluten molecules that harbor the p31-43/49 peptide that has been implicated in the innate immune response induced by gluten [7]. The a-gliadins are a gene family encoded by the Gli-2 loci, GliA2, Gli-B2 and Gli-D2, located on the short arm of three homoeologous chromosomes (6AS, 6BS and 6DS) of hexaploid bread wheat (Triticum aestivum L.). These loci may contain from 25 35 to even 150 a-gliadin genes per haploid genome [1517], although most of these (7295%) are presumably pseudogenes [16,17]. Sequencing of genomic a-gliadin clones from hexaploid bread wheat enabled to differentiate the sequences according to their loci Gli-A2, Gli-B2 and Gli-D2 based on genome-specific SNPs [16,17]. Relevant for CD, the occurrence and frequency of the HLA-DQ2 epitopes DQ2-Glia-a1, DQ2-Glia-a2 and DQ2Glia-a3 (previously designated Glia-a20, ref. 12] and the HLADQ8 T-cell epitope DQ8-Glia-a1 also differs between the loci [17]. This was corroborated by the observation that T cell clones specific for the DQ2-Glia-a2 epitope did not recognise gluten derived from diploid species carrying the S-genome, ancestrally related to the B genome of bread wheat, while gluten derived from diploid A- and D-genome species was recognized [18]. Variation in T cell stimulatory capacity of cereal-derived gluten was observed with other T cell clones as well [1921]. Indeed, differences have been observed in the T cell stimulatory capacity of pasta and bread wheat varieties [22,23], but none were safe for CD patients. Given the overall importance of the a-gliadins in CD we set out to determine the naturally existing sequence variation in CD epitopes as deduced from a-gliadin transcripts from developing wheat grains. The immunogenic potential of these epitope variants was subsequently tested in (...truncated)