Celiac disease is a chronic immune-mediated disorder of the small intestine that is triggered by the ingestion of wheat, rye and barley gluten proteins in genetically predisposed individuals. With a worldwide prevalence of about 1%, celiac disease is one of the most frequent food-related disorders. Due to their glutamine- and proline-rich amino acid sequences gluten proteins are only partially degraded by human digestive enzymes so that gluten peptides with nine amino acid residues and more persistent and elicit the celiac disease-specific immune cascade in the gut-associated lymphoid tissue. Endogenous transglutaminase 2 (TG2) plays a key role in adaptive immunity, because it serves as antigen for the production of autoantibodies against TG2. The TG2-catalyzed formation of complexes, including TG2 multimers and TG2-gluten peptide complexes, is deemed essential for activating T and B cells. However, the substrate specificity of TG2 and the regulation of its activity in terms of TG2-gluten peptide crosslink formation is still mostly unknown.

The aim of this study is to identify the parameters regulating TG2 activity towards deamidation and crosslinking to elucidate the molecular structures of TG2-gluten peptide complexes. First, non-targeted nLC-MS/MS data of model experiments with TG2 and all wheat, rye and barley gluten protein types will be used to identify further TG2-gluten isopeptides. To verify them in experiments, further nLC-MS/MS measurements will be carried out. The substrate specificity of TG2 related to the formation of TG2-gluten peptides crosslinks will be determined in detail using synthetic peptide libraries. By systematic variation of the reaction conditions, we aim to derive the important factors that regulate TG2 activity towards preferred deamidation or formation of crosslinks. The findings on the molecular structures of TG2-gluten peptide complexes shall help refine current models to explain the interaction of T and B cells involved in triggering the celiac disease-specific autoimmune reaction. An in-depth fundamental understanding of the mechanisms behind the formation of TG2-complexes will also help further our understanding of similar processes in other autoimmune or TG-dependent disorders.