Site-Specific GlcNAcylation of Human Erythrocyte Proteins

Potential Biomarker(s) for Diabetes

  1. Zihao Wang1,
  2. Kyoungsook Park1,
  3. Frank Comer1,
  4. Linda C. Hsieh-Wilson2,
  5. Christopher D. Saudek3 and
  6. Gerald W. Hart1
  1. 1Department of Biological Chemistry, School of Medicine, The Johns Hopkins University, Baltimore, Maryland
  2. 2Howard Hughes Medical Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California
  3. 3Department of Medicine, Division of Endocrinology and Metabolism, Johns Hopkins University School of Medicine, Baltimore, Maryland
  1. Corresponding author: Gerald W. Hart, gwhart{at}jhmi.edu

Abstract

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.

Footnotes

  • Published ahead of print at http://diabetes.diabetesjournals.org on 4 November 2008.

    F.C. is currently affiliated with the Cell and Molecular Biology Group, Wellstat Therapeutics, Gaithersburg, Maryland.

    Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

    The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

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  1. Diabetes vol. 58 no. 2 309-317
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