title: Catalytic Promiscuity of O-GlcNAc Transferase Enables Unexpected Metabolic Engineering of Cytoplasmic Proteins with 2-Azido-2-deoxy-glucose.
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.
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.|Department of Molecular Biology and Biochemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: David L
authors.initials: DL
authors.lastname: Shen
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.|Department of Molecular Biology and Biochemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Ta-Wei
authors.initials: TW
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Wesley
authors.initials: W
authors.lastname: Zandberg
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Tom
authors.initials: T
authors.lastname: Clark
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Razieh
authors.initials: R
authors.lastname: Eskandari
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Alteen
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Hong Yee
authors.initials: HY
authors.lastname: Tan
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.|Department of Molecular Biology and Biochemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Samy
authors.initials: S
authors.lastname: Cecioni
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.|Department of Molecular Biology and Biochemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: David
authors.identifier: 0000-0001-6897-5558
authors.initials: D
authors.lastname: Vocadlo
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000737:chemistry* / Q000378:metabolism; D019556:COS Cells; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4 / Q000378:metabolism; D002522:Chlorocebus aethiops; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000737:chemistry; D005947:Glucose / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D060847:Metabolic Engineering; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014316:Tritium; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry; D016875:tau Proteins / Q000378:metabolism
chemical_list: C000622626:1,3,4,6-tetra-O-acetyl-5-thioglucopyranose; D048868:Adaptor Proteins, Signal Transducing; D001386:Azides; D008562:Membrane Glycoproteins; D028861:Nuclear Pore Complex Proteins; C486300:TAB1 protein, human; C049312:nuclear pore protein p62; D016875:tau Proteins; C000622625:uridine diphospho-2-azido-2-deoxyglucopyranose; D014316:Tritium; C070423:1,3,4,6-tetra-O-acetyl-2-azido-2-deoxyglucopyranose; D003847:Deoxyglucose; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; C110628:hexosaminidase C; D001619:beta-N-Acetylhexosaminidases; D005947:Glucose; D014532:Uridine Diphosphate Glucose