In recent years, our lab developed a model describing the mechanism by which gluten-derived antigens trigger an inflammatory response in celiac disease (CeD). The enzyme transglutaminase 2 (TG2) deamidates glutamine residues in gluten peptides, allowing recognition by specific antigen-presenting cells including dendritic cells (DCs), and activating receptor-mediated endocytosis. This results in presentation of the deamidated gluten peptide on the disease-associated protein HLA-DQ2/8, triggering T-cell activation and ultimately causing mucosal injury. To study this pathway, our lab developed a gluten peptidomimetic small molecule, HB-230, featuring a sulfocyanine 5 (sulfo-Cy5) fluorophore, which forms a covalent complex with TG2 and can be traced using fluorescent microscopy. Using this tool, we demonstrated that intracellular staining of TG2 bound to HB-230 increases in a dose-dependent manner in wild type bone marrow-derived dendritic cells (BMDCs), but not in TG2 knockout cells. Our lab also demonstrated HB-230 can be used to visualize active TG2 in the small intestine in vivo.
Here, we utilize our fluorescently tagged active TG2 probe, HB-230, in cryo-correlative light and electron microscopy (cryo-CLEM) and in situ cryo-electron tomography (cryo-ET) approaches to provide direct evidence of active TG2-gluten peptide complex within the subcellular compartments of mouse BMDCs during endocytosis. We also use our probe in fluorescence-guided volume electron microscopy (vEM) methods. Specifically, using cryogenic focused ion beam scanning electron microscopy (cryo-FIB-SEM) of mouse BDMCs and small intestine tissue, we characterize the quantity and distribution of endolysosomal compartments in these samples. Overall, our findings demonstrate the ultrastructure-resolved localization of TG2 and provide an unprecedented insight into the variety and spatial organization of endolysosomal components within BMDCs and small intestine tissue.