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

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

Zihao Wang et al. Diabetes. 2009 Feb.

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.

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Figures

FIG. 1.

FIG. 1.

Erythrocytic proteins are O-GlcNAc modified. Fifteen micrograms of erythrocyte proteins (hemoglobin depleted) were run on a 12.5% SDS-PAGE gel, transferred, and immunoblotted with antibodies against O-GlcNAc (A), O-GlcNAcase (B, top), or OGT (B, bottom). Different lanes represent samples from different individuals.

FIG. 2.

FIG. 2.

Enrichment and identification of O-GlcNAc–modified erythrocytic proteins. A: Scheme for enriching O-GlcNAc proteins. B: Negative control of the approach. C: Confirmation of the O-GlcNAc states on several proteins.

FIG. 3.

FIG. 3.

Mapping O-GlcNAc sites and site-specific quantitation. A: Scheme for enrichment of O-GlcNAc peptides. B: Structure (inset) and CAD fragmentation of fully tagged O-GlcNAc peptide (YSPgTSPSK). [M+GlcNAc+GalNAz+Biotin+3H]3+ = 614.6, [M+H]+ = 866.5, [M+GlcNAc+H]+ = 1069.6. C: Flow chart for comparing site-specific O-GlcNAc RORs. Inset: Scheme for solid-phase BEMAD. D: Protein expression level dynamics in diabetic erythrocytes compared with normal erythrocytes. E: Specificity control for the enrichment and site-mapping. Samples were untreated or treated with hexosaminidase at 37°C for 48 h before going through the work flow. Base peak chromatograms are shown. NL, intensity in counts normalized to 1 s; TBTA, Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; TCEP, Tris(2-carboxyethyl)phosphine.

FIG. 4.

FIG. 4.

O-GlcNAc as potential biomarkers for diabetes. Specific O-GlcNAc sites (underlined Ser) on ankyrin-1 (identified and quantified by QSTAR) and catalase (identified by LTQ-Orbitrap) were upregulated 2.7- and 3.9-fold, respectively. A: Extracted ion chromatogram (XIC). B: Averaged full-scan spectra during elution time of the ion pairs. C: MS/MS spectra that showed the peptide sequences and mapped DTT attachment sites.

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