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.
MeSH terms
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Adaptor Proteins, Signal Transducing / metabolism
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Animals
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Azides / chemistry*
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Azides / metabolism
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COS Cells
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Calcium-Calmodulin-Dependent Protein Kinase Type 4 / metabolism
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Chlorocebus aethiops
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Deoxyglucose / analogs & derivatives*
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Deoxyglucose / chemistry
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Glucose / analogs & derivatives
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Glucose / chemistry*
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Glucose / metabolism
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Glycosylation
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Humans
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Membrane Glycoproteins / metabolism
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Metabolic Engineering
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Mice
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N-Acetylglucosaminyltransferases / genetics
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N-Acetylglucosaminyltransferases / metabolism*
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Nuclear Pore Complex Proteins / metabolism
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Substrate Specificity
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Tritium
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Uridine Diphosphate Glucose / analogs & derivatives
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Uridine Diphosphate Glucose / chemistry
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Uridine Diphosphate Glucose / metabolism
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beta-N-Acetylhexosaminidases / chemistry
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tau Proteins / metabolism
Substances
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1,3,4,6-tetra-O-acetyl-5-thioglucopyranose
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Adaptor Proteins, Signal Transducing
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Azides
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Membrane Glycoproteins
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Nuclear Pore Complex Proteins
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TAB1 protein, human
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nuclear pore protein p62
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tau Proteins
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uridine diphospho-2-azido-2-deoxyglucopyranose
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Tritium
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1,3,4,6-tetra-O-acetyl-2-azido-2-deoxyglucopyranose
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Deoxyglucose
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N-Acetylglucosaminyltransferases
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O-GlcNAc transferase
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Calcium-Calmodulin-Dependent Protein Kinase Type 4
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hexosaminidase C
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beta-N-Acetylhexosaminidases
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Glucose
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Uridine Diphosphate Glucose