title: Identification of O-linked N-acetylglucosamine (O-GlcNAc)-modified osteoblast proteins by electron transfer dissociation tandem mass spectrometry reveals proteins critical for bone formation.
abstract: The nutrient-responsive β-O-linked N-acetylglucosamine (O-GlcNAc) modification of critical effector proteins modulates signaling and transcriptional pathways contributing to cellular development and survival. An elevation in global protein O-GlcNAc modification occurs during the early stages of osteoblast differentiation and correlates with enhanced transcriptional activity of RUNX2, a key regulator of osteogenesis. To identify other substrates of O-GlcNAc transferase in differentiating MC3T3E1 osteoblasts, O-GlcNAc-modified peptides were enriched by wheat germ agglutinin lectin weak affinity chromatography and identified by tandem mass spectrometry using electron transfer dissociation. This peptide fragmentation approach leaves the labile O-linkage intact permitting direct identification of O-GlcNAc-modified peptides. O-GlcNAc modification was observed on enzymes involved in post-translational regulation, including MAST4 and WNK1 kinases, a ubiquitin-associated protein (UBAP2l), and the histone acetyltransferase CREB-binding protein. CREB-binding protein, a transcriptional co-activator that associates with CREB and RUNX2, is O-GlcNAcylated at Ser-147 and Ser-2360, the latter of which is a known site of phosphorylation. Additionally, O-GlcNAcylation of components of the TGFβ-activated kinase 1 (TAK1) signaling complex, TAB1 and TAB2, occurred in close proximity to known sites of Ser/Thr phosphorylation and a putative nuclear localization sequence within TAB2. These findings demonstrate the presence of O-GlcNAc modification on proteins critical to bone formation, remodeling, and fracture healing and will enable evaluation of this modification on protein function and regulation.
authors: Department of Craniofacial Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
authors: Alexis K
authors: AK
authors: Nagel
authors: Michael
authors: M
authors: Schilling
authors: Susana
authors: S
authors: Comte-Walters
authors: Mary N
authors: MN
authors: Berkaw
authors: Lauren E
authors: LE
authors: Ball
mesh_terms: D000116:Acetylgalactosamine / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism*; D010012:Osteogenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006023:Glycoproteins; D010446:Peptide Fragments; D020543:Proteome; D000116:Acetylgalactosamine