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.

Fig. 1.

Changes in global protein O-GlcNAc modification during differentiation of MC3T3E1 osteoblasts. MC3T3E1 cells were differentiated for up to 21 days under osteogenic conditions. A, Protein lysates (30 μg) were probed for protein O-GlcNAc modification and OGT, OGA, and α-tubulin as a loading control. Three biological replicates are shown. B, Elevated expression of osteoblast markers was confirmed by quantitative PCR analysis of OCN transcript (left panel) and staining for ALP activity (right panel). OCN transcript levels were normalized against GAPDH (mean ± S.E., n = 3). Significant differences between day 0 and any given time point were determined by Student's unpaired t test: p < 0.01 < (**), or p < 0.001 (***). C, Immunoblot analysis of cytosolic (C) and nuclear (N) fractions prepared from cells differentiated in the presence of osteogenic factors (5 mm β-glycerol phosphate, 50 μg/ml ascorbate) for 3 days with 10% FCS or 1% FCS in the absence or presence of the OGA inhibitor PUGNAc. GAPDH, histone H1 (HisH1), and RUNX2 served as controls for subcellular fractionation and loading.

Fig. 2.

O-GlcNAc modification of TGF-β-activated kinase 1/MAP3K7-binding protein 2 (TAB2), in differentiating MC3T3E1 osteoblasts. The ETD tandem mass spectrum of TAB2, residues 453–465, confirms a 203.2-Da mass shift at residue Thr-456 (top panel). The peaks labeled with asterisks correspond to unfragmented precursor ions that have undergone a neutral loss of m/z 203.2. Immunoblot analyses of TAB2 immunoprecipitated from nontransfected (NT) or 3×FLAG-TAB2-transfected HEK293 cells cultured in the presence of PUGNAc (50 μm) or Thiamet G (20 μm) further confirm the 203.2-Da mass shift is due to O-GlcNAc modification and reveal the presence of O-GlcNAc-modified TAB2 in both the nuclear and cytoplasmic compartments.

Fig. 3.

O-GlcNAc modification of CBP in differentiating MC3T3E1 osteoblasts. The ETD tandem mass spectrum of HexNAc-modified CBP, residues 2344–2365, indicates O-GlcNAc modification at Ser-2360. Immunoblot analysis of CBP immunoprecipitated from cytosolic and nuclear fractions of HEK293 treated with Thiamet G (20 μm) indicates the presence of O-GlcNAc modification of CBP localized in the nucleus. Immunoprecipitation with IgG was performed as a control for nonspecific binding.