Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

Proc Natl Acad Sci U S A. 2012 Mar 27;109(13):4834-9. doi: 10.1073/pnas.1114356109. Epub 2012 Mar 12.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / chemistry
  • Acetylglucosamine / metabolism*
  • Active Transport, Cell Nucleus / radiation effects
  • Cell Nucleus / metabolism
  • Cell Nucleus / radiation effects
  • Cross-Linking Reagents / metabolism*
  • Diazomethane / chemistry
  • Diazomethane / metabolism
  • HeLa Cells
  • Humans
  • Light*
  • Models, Biological
  • Mutagenesis / radiation effects
  • Nuclear Pore Complex Proteins / chemistry
  • Nuclear Pore Complex Proteins / metabolism*
  • Peptides / chemistry
  • Peptides / metabolism
  • Protein Binding / radiation effects
  • Protein Processing, Post-Translational / radiation effects*
  • Repetitive Sequences, Amino Acid
  • Staining and Labeling / methods*
  • Uridine Diphosphate / metabolism

Substances

  • Cross-Linking Reagents
  • Nuclear Pore Complex Proteins
  • Peptides
  • Uridine Diphosphate
  • Diazomethane
  • Acetylglucosamine