Site-Specific GlcNAcylation of Human Erythrocyte Proteins: Potential Biomarker(s) for Diabetes Mellitus

Abstract

Objective O-linked N-acetylglucosamine (O-GlcNAc) is up-regulated 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 that site specific O-GlcNAc levels not only reflect the glycemic status of an individual, but also that O-GlcNAc site occupancy on erythrocyte proteins may be eventually useful as diagnostic tool for the early detection of diabetes.