Multiple Wheat Genomes Reveal Novel Gli-2 Sublocus Location and Variation of Celiac Disease Epitopes in Duplicated α-Gliadin Genes.
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SUBMITTER: Halstead-Nussloch G
PROVIDER: S-EPMC8446623 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC1368968 | biostudies-literature
Project description:Celiac disease (CD) is an enteropathy triggered by the ingestion of gluten proteins from wheat and similar proteins from barley and rye. The inflammatory reaction is controlled by T cells that recognize gluten peptides in the context of human leukocyte antigen (HLA) DQ2 or HLA-DQ8 molecules. The only available treatment for the disease is a lifelong gluten-exclusion diet. We have used RNAi to down-regulate the expression of gliadins in bread wheat. A set of hairpin constructs were designed and expressed in the endosperm of bread wheat. The expression of gliadins was strongly down-regulated in the transgenic lines. Total gluten protein was extracted from transgenic lines and tested for ability to stimulate four different T-cell clones derived from the intestinal lesion of CD patients and specific for the DQ2-α-II, DQ2-γ-VII, DQ8-α-I, and DQ8-γ-I epitopes. For five of the transgenic lines, there was a 1.5-2 log reduction in the amount of the DQ2-α-II and DQ2-γ-VII epitopes and at least 1 log reduction in the amount of the DQ8-α-I and DQ8-γ-I epitopes. Furthermore, transgenic lines were also tested with two T-cell lines that are reactive with ω-gliadin epitopes. The total gluten extracts were unable to elicit T-cell responses for three of the transgenic wheat lines, and there were reduced responses for six of the transgenic lines. This work shows that the down-regulation of gliadins by RNAi can be used to obtain wheat lines with very low levels of toxicity for CD patients.
| S-EPMC2947919 | biostudies-literature
Project description:Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars.
| S-EPMC7759465 | biostudies-literature
Project description:Celiac disease is a food-sensitive enteropathy triggered by the ingestion of wheat gluten proteins and related proteins from barley, rye, and some varieties of oat. There are no interventional therapies and the only solution is a lifelong gluten-free diet. The down-regulation of gliadins by RNAi provides wheat lines with all the gliadin fractions strongly down-regulated (low-gliadin). The technological properties of doughs prepared from the low-gliadin lines indicated a general weakening effect, although some of the lines displayed similar properties to that of the wild-type lines. In contrast, the stability was increased significantly in some of the transgenic lines, indicating better tolerance to over-mixing. Results reported here are the first analyses of the mixing and bread-making quality of the wheat lines with all gliadin fractions strongly down-regulated. Flour from these lines may be an important breakthrough in the development of new products for the celiac community. These lines might be used directly or blended with other non-toxic cereals, as raw material for developing food products that can be safely tolerated by CD patients and others with gluten intolerance or gluten sensitivity, incrementing the range of available food products and enhancing their diet.
| S-EPMC3954839 | biostudies-literature
Project description:BACKGROUND: A genome-wide assessment of nucleotide diversity in a polyploid species must minimize the inclusion of homoeologous sequences into diversity estimates and reliably allocate individual haplotypes into their respective genomes. The same requirements complicate the development and deployment of single nucleotide polymorphism (SNP) markers in polyploid species. We report here a strategy that satisfies these requirements and deploy it in the sequencing of genes in cultivated hexaploid wheat (Triticum aestivum, genomes AABBDD) and wild tetraploid wheat (Triticum turgidum ssp. dicoccoides, genomes AABB) from the putative site of wheat domestication in Turkey. Data are used to assess the distribution of diversity among and within wheat genomes and to develop a panel of SNP markers for polyploid wheat. RESULTS: Nucleotide diversity was estimated in 2114 wheat genes and was similar between the A and B genomes and reduced in the D genome. Within a genome, diversity was diminished on some chromosomes. Low diversity was always accompanied by an excess of rare alleles. A total of 5,471 SNPs was discovered in 1791 wheat genes. Totals of 1,271, 1,218, and 2,203 SNPs were discovered in 488, 463, and 641 genes of wheat putative diploid ancestors, T. urartu, Aegilops speltoides, and Ae. tauschii, respectively. A public database containing genome-specific primers, SNPs, and other information was constructed. A total of 987 genes with nucleotide diversity estimated in one or more of the wheat genomes was placed on an Ae. tauschii genetic map, and the map was superimposed on wheat deletion-bin maps. The agreement between the maps was assessed. CONCLUSIONS: In a young polyploid, exemplified by T. aestivum, ancestral species are the primary source of genetic diversity. Low effective recombination due to self-pollination and a genetic mechanism precluding homoeologous chromosome pairing during polyploid meiosis can lead to the loss of diversity from large chromosomal regions. The net effect of these factors in T. aestivum is large variation in diversity among genomes and chromosomes, which impacts the development of SNP markers and their practical utility. Accumulation of new mutations in older polyploid species, such as wild emmer, results in increased diversity and its more uniform distribution across the genome.
| S-EPMC3022916 | biostudies-literature
Project description:Rothia mucilaginosa, a natural microbial inhabitant of the oral cavity, cleaves gluten (gliadin) proteins at regions that are resistant to degradation by mammalian enzymes. The aim of this study was to investigate to what extent the R. mucilaginosa cell-associated enzymes abolish gliadin immunogenic properties. Degradation of total gliadins and highly immunogenic gliadin 33-mer or 26-mer peptides was monitored by SDS-PAGE and RP-HPLC, and fragments were sequenced by liquid chromatography and electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS). Peptide deamidation by tissue transglutaminase (TG2), a critical step in rendering the fragments more immunogenic, was assessed by TG2-mediated cross-linking to monodansyl cadaverine (MDC), and by a +1-Da mass difference by LC-ESI-MS. Survival of potential immunogenic gliadin epitopes was determined by use of the R5 antibody-based ELISA. R. mucilaginosa-associated enzymes cleaved gliadins, 33-mer and 26-mer peptides into smaller fragments. TG2-mediated cross-linking showed a perfect inverse relationship with intact 33-mer and 26-mer peptide levels, and major degradation fragments showed a slow rate of MDC cross-linking of 6.18 ± 2.20 AU/min compared with 97.75 ± 10.72 and 84.17 ± 3.25 AU/min for the intact 33-mer and 26-mer, respectively, which was confirmed by reduced TG2-mediated deamidation of the fragments in mass spectrometry. Incubation of gliadins with Rothia cells reduced R5 antibody binding by 20, 82, and 97% after 30 min, 2 h, and 5 h, respectively, which was paralleled by reduced reactivity of enzyme-treated 33-mer and 26-mer peptides in the R5 competitive ELISA. Our broad complementary approach to validate gluten degrading activities qualifies R. mucilaginosa-associated enzymes as promising tools to neutralize T cell immunogenic properties for treatment of celiac disease.
| S-EPMC4200315 | biostudies-literature
Project description:α-Gliadins are a major group of gluten proteins in wheat flour that contribute to the end-use properties for food processing and contain major immunogenic epitopes that can cause serious health-related issues including celiac disease (CD). α-Gliadins are also the youngest group of gluten proteins and are encoded by a large gene family. The majority of the gene family members evolved independently in the A, B, and D genomes of different wheat species after their separation from a common ancestral species. To gain insights into the origin and evolution of these complex genes, the genomic regions of the Gli-2 loci encoding α-gliadins were characterized from the tetraploid wild emmer, a progenitor of hexaploid bread wheat that contributed the AABB genomes. Genomic sequences of Gli-2 locus regions for the wild emmer A and B genomes were first reconstructed using the genome sequence scaffolds along with optical genome maps. A total of 24 and 16 α-gliadin genes were identified for the A and B genome regions, respectively. α-Gliadin pseudogene frequencies of 86% for the A genome and 69% for the B genome were primarily caused by C to T substitutions in the highly abundant glutamine codons, resulting in the generation of premature stop codons. Comparison with the homologous regions from the hexaploid wheat cv. Chinese Spring indicated considerable sequence divergence of the two A genomes at the genomic level. In comparison, conserved regions between the two B genomes were identified that included α-gliadin pseudogenes containing shared nested TE insertions. Analyses of the genomic organization and phylogenetic tree reconstruction indicate that although orthologous gene pairs derived from speciation were present, large portions of α-gliadin genes were likely derived from differential gene duplications or deletions after the separation of the homologous wheat genomes ~ 0.5 MYA. The higher number of full-length intact α-gliadin genes in hexaploid wheat than that in wild emmer suggests that human selection through domestication might have an impact on α-gliadin evolution. Our study provides insights into the rapid and dynamic evolution of genomic regions harboring the α-gliadin genes in wheat.
| S-EPMC6797660 | biostudies-literature
Project description:Gluten proteins from wheat can induce celiac disease (CD) in genetically susceptible individuals. Specific gluten peptides can be presented by antigen presenting cells to gluten-sensitive T-cell lymphocytes leading to CD. During the last decades, a significant increase has been observed in the prevalence of CD. This may partly be attributed to an increase in awareness and to improved diagnostic techniques, but increased wheat and gluten consumption is also considered a major cause. To analyze whether wheat breeding contributed to the increase of the prevalence of CD, we have compared the genetic diversity of gluten proteins for the presence of two CD epitopes (Glia-?9 and Glia-?20) in 36 modern European wheat varieties and in 50 landraces representing the wheat varieties grown up to around a century ago. Glia-?9 is a major (immunodominant) epitope that is recognized by the majority of CD patients. The minor Glia-?20 was included as a technical reference. Overall, the presence of the Glia-?9 epitope was higher in the modern varieties, whereas the presence of the Glia-?20 epitope was lower, as compared to the landraces. This suggests that modern wheat breeding practices may have led to an increased exposure to CD epitopes. On the other hand, some modern varieties and landraces have been identified that have relatively low contents of both epitopes. Such selected lines may serve as a start to breed wheat for the introduction of 'low CD toxic' as a new breeding trait. Large-scale culture and consumption of such varieties would considerably aid in decreasing the prevalence of CD.
| S-EPMC2963738 | biostudies-literature
Project description:BackgroundCertain immunotoxic peptides from gluten are resistant to gastrointestinal digestion and can interact with celiac-patient factors to trigger an immunologic response. A gluten-free diet (GFD) is the only effective treatment for celiac disease (CD), and its compliance should be monitored to avoid cumulative damage. However, practical methods to monitor diet compliance and to detect the origin of an outbreak of celiac clinical symptoms are not available.ObjectiveWe assessed the capacity to determine the gluten ingestion and monitor GFD compliance in celiac patients by the detection of gluten and gliadin 33-mer equivalent peptidic epitopes (33EPs) in human feces.DesignFecal samples were obtained from healthy subjects, celiac patients, and subjects with other intestinal pathologies with different diet conditions. Gluten and 33EPs were analyzed by using immunochromatography and competitive ELISA with a highly sensitive antigliadin 33-mer monoclonal antibody.ResultsThe resistance of a significant part of 33EPs to gastrointestinal digestion was shown in vitro and in vivo. We were able to detect gluten peptides in feces of healthy individuals after consumption of a normal gluten-containing diet, after consumption of a GFD combined with controlled ingestion of a fixed amount of gluten, and after ingestion of <100 mg gluten/d. These methods also allowed us to detect GFD infringement in CD patients.ConclusionsGluten-derived peptides could be sensitively detected in human feces in positive correlation with the amount of gluten intake. These techniques may serve to show GFD compliance or infringement and be used in clinical research in strategies to eliminate gluten immunotoxic peptides during digestion. This trial was registered at clinicaltrials.gov as NCT01478867.
| S-EPMC3278243 | biostudies-literature
Project description:Background & aimsCeliac disease could be treated, and potentially cured, by restoring T-cell tolerance to gliadin. We investigated the safety and efficacy of negatively charged 500-nm poly(lactide-co-glycolide) nanoparticles encapsulating gliadin protein (TIMP-GLIA) in 3 mouse models of celiac disease. Uptake of these nanoparticles by antigen-presenting cells was shown to induce immune tolerance in other animal models of autoimmune disease.MethodsWe performed studies with C57BL/6; RAG1-/- (C57BL/6); and HLA-DQ8, huCD4 transgenic Ab0 NOD mice. Mice were given 1 or 2 tail-vein injections of TIMP-GLIA or control nanoparticles. Some mice were given intradermal injections of gliadin in complete Freund's adjuvant (immunization) or of soluble gliadin or ovalbumin (ear challenge). RAG-/- mice were given intraperitoneal injections of CD4+CD62L-CD44hi T cells from gliadin-immunized C57BL/6 mice and were fed with an AIN-76A-based diet containing wheat gluten (oral challenge) or without gluten. Spleen or lymph node cells were analyzed in proliferation and cytokine secretion assays or by flow cytometry, RNA sequencing, or real-time quantitative polymerase chain reaction. Serum samples were analyzed by gliadin antibody enzyme-linked immunosorbent assay, and intestinal tissues were analyzed by histology. Human peripheral blood mononuclear cells, or immature dendritic cells derived from human peripheral blood mononuclear cells, were cultured in medium containing TIMP-GLIA, anti-CD3 antibody, or lipopolysaccharide (controls) and analyzed in proliferation and cytokine secretion assays or by flow cytometry. Whole blood or plasma from healthy volunteers was incubated with TIMP-GLIA, and hemolysis, platelet activation and aggregation, and complement activation or coagulation were analyzed.ResultsTIMP-GLIA did not increase markers of maturation on cultured human dendritic cells or induce activation of T cells from patients with active or treated celiac disease. In the delayed-type hypersensitivity (model 1), the HLA-DQ8 transgenic (model 2), and the gliadin memory T-cell enteropathy (model 3) models of celiac disease, intravenous injections of TIMP-GLIA significantly decreased gliadin-specific T-cell proliferation (in models 1 and 2), inflammatory cytokine secretion (in models 1, 2, and 3), circulating gliadin-specific IgG/IgG2c (in models 1 and 2), ear swelling (in model 1), gluten-dependent enteropathy (in model 3), and body weight loss (in model 3). In model 1, the effects were shown to be dose dependent. Splenocytes from HLA-DQ8 transgenic mice given TIMP-GLIA nanoparticles, but not control nanoparticles, had increased levels of FOXP3 and gene expression signatures associated with tolerance induction.ConclusionsIn mice with gliadin sensitivity, injection of TIMP-GLIA nanoparticles induced unresponsiveness to gliadin and reduced markers of inflammation and enteropathy. This strategy might be developed for the treatment of celiac disease.
| S-EPMC7198359 | biostudies-literature