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

  1. Zihao Wang1,
  2. Kyoungsook Park1,
  3. Frank Comer1,2,
  4. Linda C. Hsieh-Wilson3,
  5. Christopher D. Saudek4 and
  6. Gerald W. Hart (gwhart{at}
  1. 1. Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, MD, 21205;
  2. 2. Current address: Cell and Molecular Biology Group, Wellstat Therapeutics Corporation, Gaithersburg, MD, 20878
  3. 3. Howard Hughes Medical Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, 91125
  4. 4. Department of Medicine, Division of Endocrinology and Metabolism, Johns Hopkins University School of Medicine, Baltimore, MD, 21205


    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.


      • Received July 22, 2008.
      • Accepted October 16, 2008.

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