Catalytic Promiscuity of O-GlcNAc Transferase Enables Unexpected Metabolic Engineering of Cytoplasmic Proteins with 2-Azido-2-deoxy-glucose

  • ACS Chem Biol. 2017 Jan 20;12(1):206-213. doi: 10.1021/acschembio.6b00876.
David L Shen  1  2 ,  Ta-Wei Liu  1  2 ,  Wesley Zandberg  1 ,  Tom Clark  1 ,  Razieh Eskandari  1 ,  Matthew G Alteen  1 ,  Hong Yee Tan  1 ,  Yanping Zhu  1  2 ,  Samy Cecioni  1 ,  David Vocadlo  1  2
Affiliations
  • 1. Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
  • 2. Department of Molecular Biology and Biochemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
PMID: 27935279 DOI: 10.1021/acschembio.6b00876
Abstract

O-GlcNAc transferase (OGT) catalyzes the installation of N-acetylglucosamine (GlcNAc) O-linked to nucleocytoplasmic proteins (O-GlcNAc) within multicellular eukaryotes. OGT shows surprising tolerance for structural changes in the sugar component of its nucleotide sugar donor substrate, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Here, we find that OGT uses UDP-glucose to install O-linked glucose (O-Glc) onto proteins only 25-fold less efficiently than O-GlcNAc. Spurred by this observation, we show that OGT transfers 2-azido-2-deoxy-d-glucose (GlcAz) in vitro from UDP-GlcAz to proteins. Further, feeding cells with per-O-acetyl GlcAz (AcGlcAz), in combination with inhibition or inducible knockout of OGT, shows OGT-dependent modification of nuclear and cytoplasmic proteins with O-GlcAz as detected using microscopy, immunoblot, and proteomics. We find that O-GlcAz is reversible within cells, and an unidentified cellular enzyme exists to cleave O-Glc that can also process O-GlcAz. We anticipate that AcGlcAz will prove to be a useful tool to study the O-GlcNAc modification. We also speculate that, given the high concentration of UDP-Glc within certain mammalian tissues, O-Glc may exist within mammals and serve as a physiologically relevant modification.

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