Peptide binding to HLA-DQ2 and development of blocking agents for the treatment of celiac disease

Celiac disease is a chronic inflammatory disorder of the small intestine with a strong association to HLA-DQ2. This thesis focused on peptide binding to HLA-DQ2 and the development of peptide blockers for the treatment of celiac disease. We have investigated the relative contribution of each hydrogen bond interaction between the peptide main chain and MHC residues in DQ2 and our results demonstrated that hydrogen bonds at positions P4 and P2 are most important for peptide binding. In addition, no evidence of a hydrogen bond in position P1 was found. Detailed characterization of gluten T cell epitopes gave knowledge on the importance of proline spacing for the binding to DQ2. Cyclic and dimeric peptides were investigated as peptide blockers, and we demonstrated proof-of principle for inhibition of DQ2 mediated presentation of gluten T cell epitopes. Finally, a study was performed to elucidate peptide determinant selection by HLA-DQ2 as a factor to explain the strong HLA association in celiac disease. The results demonstrated that the gluten T cell epitopes mainly bind to HLA-DQ2 and not to other HLA class II molecules

Different binding motifs of the celiac disease-associated HLA molecules DQ2.5, DQ2.2, and DQ7.5 revealed by relative quantitative proteomics of endogenous peptide repertoires

Celiac disease is caused by intolerance to cereal gluten proteins, and HLA-DQ molecules are involved in the disease pathogenesis by presentation of gluten peptides to CD4+ T cells. The α- or β-chain sharing HLA molecules DQ2.5, DQ2.2, and DQ7.5 display different risks for the disease. It was recently demonstrated that T cells of DQ2.5 and DQ2.2 patients recognize distinct sets of gluten epitopes, suggesting that these two DQ2 variants select different peptides for display. To explore whether this is the case, we performed a comprehensive comparison of the endogenous self-peptides bound to HLA-DQ molecules of B-lymphoblastoid cell lines. Peptides were eluted from affinity-purified HLA molecules of nine cell lines and subjected to quadrupole orbitrap mass spectrometry and MaxQuant software analysis. Altogether, 12,712 endogenous peptides were identified at very different relative abundances. Hierarchical clustering of normalized quantitative data demonstrated significant differences in repertoires of peptides between the three DQ variant molecules. The neural network-based method, NNAlign, was used to identify peptide-binding motifs. The binding motifs of DQ2.5 and DQ7.5 concurred with previously established binding motifs. The binding motif of DQ2.2 was strikingly different from that of DQ2.5 with position P3 being a major anchor having a preference for threonine and serine. This is notable as three recently identified epitopes of gluten recognized by T cells of DQ2.2 celiac patients harbor serine at position P3. This study demonstrates that relative quantitative comparison of endogenous peptides sampled from our protein metabolism by HLA molecules provides clues to understand HLA association with disease.Fil: Bergseng, Elin. University of Oslo; NoruegaFil: Dørum, Siri. University of Oslo; NoruegaFil: Arntzen, Magnus Ø.. University of Oslo; NoruegaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Nygård, Ståle. University of Oslo; NoruegaFil: Buus, Søren. Universidad de Copenhagen; DinamarcaFil: de Souza, Gustavo A.. University of Oslo; NoruegaFil: Sollid, Ludvig M.. University of Oslo; Norueg

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells