SUMMARY Celiac disease (CeD) is a gluten-induced inflammatory disorder of the small intestine for which no non-dietary therapy has been approved for clinical use. In this ongoing collaborative project, we study the pathogenic role of transglutaminase 2 (TG2), a validated drug target for CeD, through a synergistic combination of tools and concepts from chemical biology and mucosal immunology. During the past grant cycle, we have uncovered a novel pathway by which antigenic gluten peptides are potently escorted into the endosomes of antigen presenting cells through the formation of complexes involving TG2, 2-macroglobulin (2M), and low density lipoprotein receptor-related protein 1 (LRP1). In the next cycle, we will characterize this mechanism for TG2- dependent inflammatory T cell activation through the following Specific Aims: Aim 1: Structure-function analysis of molecular interactions involved in LRP1-mediated gluten antigen presentation: We will biochemically characterize how a representative intermediate formed between TG2 and a gluten peptide is recognized by 2M. Our goal is to define the molecular features facilitating 2M-TG2 recognition and the role of the peptide in this three-component interaction. Separately, we will also study how the resulting peptide-TG2-2M complex is recognized and endocytosed by the LRP1 receptor. Aim 2: Defining the pathogenic role of the LRP1 pathway for gluten peptide uptake in CeD pathogenesis: While LRP1-mediated gluten antigen endocytosis has been studied in cultured cells, we do not know the identities of small intestinal cells that deamidate and take up gluten peptides and present them to CeD-specific T cells. Using fluorogenic peptides and peptidomimetics that undergo LRP1-mediated endocytosis, we will identify intestinal cells that exhibit this property in selected strains of mice. In turn, we will leverage mouse models developed through the support of this grant to examine the role of the TG2-2M-LRP1 pathway in the loss of oral tolerance to dietary gluten as well as the onset of gluten-induced villous atrophy. Not only does our proposed research offer the promise of breaking fundamentally new ground in our understanding of CeD pathogenesis, but the resulting tools and methods could be leveraged to investigate the relevance of the TG2-2M-LRP1 endocytic pathway in other patho-physiological situations.