Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins in Live Cells Using Metabolic Intermediates

Hong Yee Tan, Razieh Eskandari, David Shen, Yanping Zhu, Ta-wei Liu, Lianne Irene Willems, Matthew Alteen, Zarina Madden, David J. Vocadlo

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The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells has proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here we report on the creation of fluorescent uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time and dose dependent manner. Since no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular phys-iology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-colour pulse chase ex-periments and monitoring OGT activity on specific protein substrate in live cells.

Original languageEnglish
Pages (from-to)15300-15308
Number of pages9
JournalJournal of the American Chemical Society
Issue number45
Early online date8 Oct 2018
Publication statusPublished - 14 Nov 2018

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