Protein-Specific Imaging of O-GlcNAcylation in Single Cells.
Thousands of intracellular proteins are post-translationally modified with O-GlcNAc, and O-GlcNAcylation impacts the function of modified proteins and mediates diverse biological processes. However, the ubiquity of this important glycosylation makes it highly challenging to probe the O-GlcNAcylation state of a specific protein at the cellular level. Herein, we report the development of a FLIM-FRET-based strategy, which exploits the spatial proximity of the O-GlcNAc moiety and the attaching protein, for protein-specific imaging of O-GlcNAcylation in single cells. We demonstrated this strategy by imaging the O-GlcNAcylation state of tau and β-catenin inside the cells. Furthermore, the changes in tau O-GlcNAcylation were monitored when the overall cellular O-GlcNAc was pharmacologically altered by using the OGT and OGA inhibitors. We envision that the FLIM-FRET strategy will be broadly applicable to probe the O-GlcNAcylation state of various proteins in the cells.
Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, P. R. China.
Wei
W
Lin
Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, P. R. China.
Ling
L
Gao
Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, P. R. China. xingchen@pku.edu.cn.|Synthetic and Functional Biomolecules Center, Key Laboratory of Bioorganic Chemistry, and Molecular Engineering of Ministry of Education, Peking University, Beijing, 100871, P. R. China. xingchen@pku.edu.cn.
Xing
X
Chen
D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D036681:Fluorescence Recovery After Photobleaching; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008856:Microscopy, Fluorescence; D011499:Protein Processing, Post-Translational; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism; D016875:tau Proteins / Q000235:genetics / Q000378:metabolism*
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D012235:Rhodamines; D051176:beta Catenin; C485184:enhanced green fluorescent protein; D016875:tau Proteins; D049452:Green Fluorescent Proteins; D001619:beta-N-Acetylhexosaminidases; D000117:Acetylglucosamine
FLIM; FRET; O-GlcNAc; glycan imaging; protein labeling