Live-cell imaging analysis of the intracellular behavior of C-type lectin receptor Mincle and its ligand using newly developed fluorescence-labeled molecular probes based on trehalose diester

Mincle, a member of the C-type lectin receptor (CLR) family, detects various glycolipids and glycerolipids, such as trehalose dimycolate (TDM) from Mycobacterium tuberculosis, leading to the activation of the innate immune system. In this study, we developed new fluorescence-labeled molecular probes, N-NBD-labeled TDE and O-NBD-labeled TDE, based on the structure of trehalose diester to elucidate the intracellular behavior of Mincle and its ligands. N-NBD-labeled TDE was prepared for the ligand analysis, and O-NBD-labeled TDE was specifically designed to label Mincle by Turn-ON fluorescent affinity labeling. Live-cell imaging analysis using these probes revealed that N-NBD-labeled TDE internalizes into the cell in a Mincle-dependent manner. Furthermore, imaging analysis using O-NBD-labeled TDE successfully detected Mincle in cells in a Mincle expression-dependent manner.