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OBJECTIVE-O-linked N-acetylglucosamine (O-GlcNAc) is upregulated in diabetic tissues and plays a role in insulin resistance and glucose toxicity. Here, we investigated the extent of GlcNAcylation on human erythrocyte proteins and compared site-specific GlcNAcylation on erythrocyte proteins from diabetic and normal individuals.
RESEARCH DESIGN AND METHODS-GlcNAcylated erythrocyte proteins or GlcNAcylated peptides were tagged and selectively enriched by a chemoenzymatic approach and identified by mass spectrometry. The enrichment approach was combined with solid-phase chemical derivatization and isotopic labeling to detect O-GlcNAc modification sites and to compare site-specific O-GlcNAc occupancy levels between normal and diabetic erythrocyte proteins.
RESULTS-The enzymes that catalyze the cycling (addition and removal) of O-GlcNAc were detected in human erythrocytes. Twenty-five GlcNAcylated erythrocyte proteins were identified. Protein expression levels were compared between diabetic and normal erythrocytes. Thirty-five O-GlcNAc sites were reproducibly identified, and their site-specific O-GlcNAc occupancy ratios were calculated.
CONCLUSIONS-GlcNAcylation is differentially regulated at individual sites on erythrocyte proteins in response to glycemic status. These data suggest not only that site-specific O-GlcNAc levels reflect the glycemic status of an individual but also that O-GlcNAc site occupancy on erythrocyte proteins may be eventually useful as a diagnostic tool for the early detection of diabetes. Diabetes 58:309-317, 2009
The dynamic, enzyme-catalyzed modification of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) has extensive cross talk with phosphorylation (1) and serves as a nutrient sensor to regulate signaling, transcription, proteasomal activity, and stress responses (2-4). GlcNAcylation is highly sensitive to nutrients and to cellular stress (5-9). Therefore, we hypothesize that the extent of GlcNAcylation can be used to evaluate the glucoregulatory status of people with both subtle and overt glucose dysregulation, perhaps to identify normal, pre-diabetic individuals and overtly diabetic individuals (10,11). GlcNAcylation is nearly as ubiquitous as phosphorylation in all multicellular eukaryotes and, in many cases, competes with phosphorylation for the same or adjacent hydroxyl groups on serine or threonine residues (1,5). The donor substrate for GlcNAcylation, uridine diphosphate (UDP)-GIcNAc, occurs within cells at up to millimolar concentrations-levels approaching that for ATP. In fact, between 2 and 5% of all of the glucose used by cells is consumed by the hexosamine biosynthetic pathway (HBP) with UDP-GlcNAc as the major end product (7). Studies from many laboratories have shown that the HBP, and O-GlcNAc in particular, plays a...