Combined Antibody/Lectin Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome upon Oxidative Stress

Abstract

O-Linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies and regulates over 3000 nuclear, cytoplasmic, and mitochondrial proteins. Upon exposure to stress and injury, cells and tissues increase the O-GlcNAc modification, or O-GlcNAcylation, of numerous proteins promoting the cellular stress response and thus survival. The aim of this study was to identify proteins that are differentially O-GlcNAcylated upon acute oxidative stress (HO) to provide insight into the mechanisms by which O-GlcNAc promotes survival. We achieved this goal by employing Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and a novel "G5-lectibody" immunoprecipitation strategy that combines four O-GlcNAc-specific antibodies (CTD110.6, RL2, HGAC39, and HGAC85) and the lectin WGA. Using the G5-lectibody column in combination with basic reversed phase chromatography and C RPLC-MS/MS, 990 proteins were identified and quantified. Hundreds of proteins that were identified demonstrated increased (>250) or decreased (>110) association with the G5-lectibody column upon oxidative stress, of which we validated the O-GlcNAcylation status of 24 proteins. Analysis of proteins with altered glycosylation suggests that stress-induced changes in O-GlcNAcylation cluster into pathways known to regulate the cell's response to injury and include protein folding, transcriptional regulation, epigenetics, and proteins involved in RNA biogenesis. Together, these data suggest that stress-induced O-GlcNAcylation regulates numerous and diverse cellular pathways to promote cell and tissue survival.

Fatty acid synthase inhibits the O-GlcNAcase during oxidative stress

“…Second, OGT interacts with p38 mitogen-activated protein kinase during glucose starvation, resulting in the targeting of OGT to glycosylate substrates such as neurofilament H (32). Third, we have recently reported that a subset of proteins exhibit decreased O-GlcNAcylation during oxidative stress, even when global O-GlcNAc levels are elevated (65). To define the regulation of OGA during oxidative stress, we have identified a subset of its interaction partners and determined how these interactions change with oxidative stress.…”

“…We set an arbitrary threshold of 25% above or below 1.32125 and 0.79275, respectively, for the HA dataset; and 1.635 and 0.981, respectively, for the Myc dataset (supplemental Tables 10 -12). Our rationale for this threshold was as follows: as signal-induced changes are diluted out by the BioID labeling method, we set a lower threshold than we have used previously (30 -50% change (42,65,92)). To validate this threshold, candidate proteins near the threshold (FAS and FLNA) were validated by co-immunoprecipitation.…”

A Sweet Embrace: Control of Protein–Protein Interactions by O-Linked β-N-Acetylglucosamine

“…Several groups, including our own, had observed previously that multiple core COPII proteins are reversibly O-GlcNAcylated, but the biochemical effects of these glycosylation events remained unknown. 67–71 Using GlcNDAz, we demonstrated that O-GlcNAc mediates multiple protein–protein interactions of COPII components. We mapped O-GlcNAc sites on several COPII proteins via mass spectrometry and used GlcNDAz and site-directed mutagenesis to pinpoint specific residues on the COPII protein Sec23A that are required for O-GlcNAc-mediated protein–protein interactions.…”

Effects of Acute Cold Stress on Liver O-GlcNAcylation and Glycometabolism in Mice

“…It has been shown previously that O -GlcNAc signaling is an essential stress and metabolic sensor, and that global O -GlcNAcylation levels and OGT activity are modulated by various stress stimuli [ 45 ]. Acute oxidative stress can induce both an increase and a decrease in global O -GlcNAcylation, thereby regulating many cellular pathways to promote cell and tissue survival [ 46 ]. Furthermore, enhanced global O -GlcNAc signaling is known to reduce endoplasmic reticulum (ER) stress-induced cell death [ 47 ].…”