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    Area of Science:

    • Biochemistry and Molecular Biology
    • Cellular Physiology
    • Chemical Biology

    Background:

    • Protein O-linked N-acetylglucosamine (O-GlcNAc) modification is a crucial cellular regulator.
    • Existing metabolic labeling strategies for O-GlcNAc are often two-step, limiting experimental scope.
    • Understanding O-GlcNAc dynamics and O-GlcNAc transferase (OGT) regulation is vital.

    Purpose of the Study:

    • To develop a simplified, one-step method for monitoring O-GlcNAcylation in live cells.
    • To create fluorescent UDP-N-acetylglucosamine analogues for direct OGT activity assessment.
    • To enable real-time studies of O-GlcNAc modification and OGT regulation.

    Main Methods:

    • Synthesized fluorescent uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) analogues.
    • Tested fluorescent analogues with human O-GlcNAc transferase (OGT) in vitro.
    • Administered fluorescent metabolic precursors to live cells for in-cell labeling.

    Main Results:

    • Fluorescent UDP-GlcNAc analogues directly monitored OGT activity in vitro.
    • A one-step metabolic feeding strategy successfully labeled O-GlcNAcylated proteins in live cells.
    • The glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate showed time- and dose-dependent accumulation.

    Conclusions:

    • A novel one-step in-cell labeling strategy for O-GlcNAc was established.
    • This method simplifies the study of O-GlcNAcylation and OGT regulation without genetic manipulation.
    • The approach facilitates advanced experiments like two-color pulse chases and real-time OGT monitoring.