title: Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners.
abstract: O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038, USA.
authors.forename: Seok-Ho
authors.initials: SH
authors.lastname: Yu
authors.forename: Michael
authors.initials: M
authors.lastname: Boyce
authors.forename: Amberlyn M
authors.initials: AM
authors.lastname: Wands
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Bond
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Kohler
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*; D021581:Active Transport, Cell Nucleus / Q000528:radiation effects; D002467:Cell Nucleus / Q000378:metabolism / Q000528:radiation effects; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003978:Diazomethane / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008954:Models, Biological; D016296:Mutagenesis / Q000528:radiation effects; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000528:radiation effects; D011499:Protein Processing, Post-Translational / Q000528:radiation effects*; D020449:Repetitive Sequences, Amino Acid; D013194:Staining and Labeling / Q000379:methods*; D014530:Uridine Diphosphate / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D028861:Nuclear Pore Complex Proteins; D010455:Peptides; D014530:Uridine Diphosphate; D003978:Diazomethane; D000117:Acetylglucosamine