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

(3), O-GlcNAc modification is now appreciated as a nutrient-responsive mechanism for modulating signal transduction (4, 5) and transcriptional regulation (6, 7). In contrast to N-and O-linked glycosylation taking place in the endoplasmic reticulum and Golgi apparatus, reversible O-GlcNAcylation occurs in the cytoplasm and nucleus and is catalyzed by O-GlcNAc transferase (OGT), which transfers GlcNAc from UDP-GlcNAc to Ser/Thr residues, and O-GlcNAcase (OGA), which removes it. These highly conserved enzymes are expressed in all mammalian tissues (8 -11).The OGT gene encodes three splice variants differing in the number of N-terminal tetratricopeptide repeats that mediate protein-protein interactions and subcellular localization (8,12). The full-length nucleocytoplasmic form of OGT is OGlcNAc-modified, tyrosine-phosphorylated, and dynamically redistributed within the cell upon insulin stimulation (2,(13)(14)(15). Ablation of OGT is embryonically lethal or developmentally limiting in animal models (10,16,17) with the noted exception of Caenorhabditis elegans. In C. elegans, the phenotypes of oga-1 and ogt-1 null mutants suggest the enzymes are involved in macronutrient storage and life span (18 -20), and subsequent studies revealed the presence of O-GlcNAc modFrom the

Effects of adding resorbable chitosan microspheres to calcium phosphate cements for bone regeneration

“…3B, the chitosan microspheres dissolved and the sample formed a porous surface. Studies have found that the chitosan degradation product, N-acetyl glucosamine, could promote osteoblast activity, hematoma and fibrous callus formation, and significantly shorten the healing time of bone defects [33]. Gregoire et al thought the reason for these outcomes was that Nacetyl glucosamine could promote osteoblast activity and increase bone morphogenetic protein expression in the bone defects [34].…”

Relative Quantification of Sites of Peptide and Protein Modification Using Size Exclusion Chromatography Coupled with Electron Transfer Dissociation

“…Mass spectrometry has been successfully used to identify even labile protein modifications, including protein phosphorylation 1–2 , and O-GlcNAc modifications 3–4 , as well as identifying chemical modifications including covalent labeling for protein surface mapping 5–6 , crosslinking 7–8 , deamidation of asparagine 9–10 , and protein oxidation 11–12 . In all of these systems, the amount of modification that occurs at each amino acid of a peptide or protein can have serious consequences to the interpretation of the data, whether for understanding chemical or enzymatic modification mechanisms and consequences 13–16 or for interpreting data from covalent labeling or chemical cross-linking studies 7, 17–20 .…”

Intracellular and extracellular O-linked N-acetylglucosamine in the nervous system

“…During osteogenesis in mouse MC3T3-E1 cells, differentiation from preosteoblasts to osteoblasts gives rise to a global increase in the level of OGlcNAc modification (Kim et al, 2007). The activity of Runx2, a critical transcription factor for osteogenesis, was shown to be regulated by O-GlcNAc modification using the reporter assay system (Nagel and Ball, 2014;Nagel et al, 2013). In addition to osteogenesis, mouse 3T3-L1 preadipocytes also exhibit a drastic increase in global OGlcNAc levels during adipogenesis (Ishihara et al, 2010).…”