Site-specific GlcNAcylation of human erythrocyte proteins: potential biomarker(s) for diabetes.
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.


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.
Wang
Zihao
Z
Department of Biological Chemistry, School of Medicine, The Johns Hopkins University, Baltimore, Maryland, USA.
Park
Kyoungsook
K
Comer
Frank
F
Hsieh-Wilson
Linda C
LC
Saudek
Christopher D
CD
Hart
Gerald W
GW
D000117:Acetylglucosamine / Q000378:metabolism*; D015415:Biomarkers / Q000097:blood; D003920:Diabetes Mellitus / Q000097:blood / Q000175:diagnosis* / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008954:Models, Biological; D011506:Proteins / Q000378:metabolism*
D015415:Biomarkers; D011506:Proteins; D000117:Acetylglucosamine