Small Molecule-Activated <i>O</i>-GlcNAcase for Spatiotemporal Removal of <i>O</i>-GlcNAc in Live Cells
Yun Ge, Hailin Lu, Bo Yang, Christina M. Woo
Abstract
The nutrient sensor O-linked N -acetylglucosamine ( O -GlcNAc) is a post-translational modification found on thousands of nucleocytoplasmic proteins. O -GlcNAc levels in cells dynamically respond to environmental cues in a temporal and spatial manner, leading to altered signal transduction and functional effects. The spatiotemporal regulation of O -GlcNAc levels would accelerate functional interrogation of O -GlcNAc and manipulation of cell behaviors for desired outcomes. Here, we report a strategy for spatiotemporal reduction of O -GlcNAc in live cells by designing an O -GlcNAcase (OGA) fused to an intein triggered by 4-hydroxytamoxifen (4-HT). After rational protein engineering and optimization, we identified an OGA–intein variant whose deglycosidase activity can be triggered in the desired subcellular compartments by 4-HT in a time- and dose-dependent manner. Finally, we demonstrated that 4-HT activation of the OGA–intein fusion can likewise potentiate inhibitory effects in breast cancer cells by virtue of the reduction of O -GlcNAc. The spatiotemporal control of O -GlcNAc through the chemically activatable OGA–intein fusion will facilitate the manipulation and functional understanding of O -GlcNAc in live cells.