title: Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1.
abstract: The dynamic modification of nuclear and cytoplasmic proteins by the monosaccharide N-acetyl-glucosamine (GlcNAc) continues to emerge as an important regulator of many biological processes. Herein we describe the development of an alkynyl-modified GlcNAc analog (GlcNAlk) as a new chemical reporter of O-GlcNAc modification in living cells. This strategy is based on metabolic incorporation of reactive functionality into the GlcNAc biosynthetic pathway. When combined with the Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition, this chemical reporter allowed for the robust in-gel fluorescent visualization of O-GlcNAc and affinity enrichment and identification of O-GlcNAc-modified proteins. Using in-gel fluorescence detection, we characterized the metabolic fates of GlcNAlk and the previously reported azido analog, GlcNAz. We confirmed previous results that GlcNAz can be metabolically interconverted to GalNAz, whereas GlcNAlk does not, thereby yielding a more specific metabolic reporter of O-GlcNAc modification. We also used GlcNAlk, in combination with a biotin affinity tag, to identify 374 proteins, 279 of which were not previously reported, and we subsequently confirmed the enrichment of three previously uncharacterized proteins. Finally we confirmed the O-GlcNAc modification of the ubiquitin ligase NEDD4-1, the first reported glycosylation of this protein.
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Balyn W
authors.initials: BW
authors.lastname: Zaro
authors.forename: Yu-Ying
authors.initials: YY
authors.lastname: Yang
authors.forename: Howard C
authors.initials: HC
authors.lastname: Hang
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Pratt
mesh_terms: D016475:3T3 Cells; D000818:Animals; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis; D006031:Glycosylation; D051379:Mice; D000075702:Nedd4 Ubiquitin Protein Ligases; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D005456:Fluorescent Dyes; D011506:Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases