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Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics

Abstract

The addition of the monosaccharide β-N-acetyl-D-glucosamine to proteins (O-GlcNAc glycosylation) is an intracellular, post-translational modification that shares features with phosphorylation. Understanding the cellular mechanisms and signaling pathways that regulate O-GlcNAc glycosylation has been challenging because of the difficulty of detecting and quantifying the modification. Here, we describe a new strategy for monitoring the dynamics of O-GlcNAc glycosylation using quantitative mass spectrometry-based proteomics. Our method, which we have termed quantitative isotopic and chemoenzymatic tagging (QUIC-Tag), combines selective, chemoenzymatic tagging of O-GlcNAc proteins with an efficient isotopic labeling strategy. Using the method, we detect changes in O-GlcNAc glycosylation on several proteins involved in the regulation of transcription and mRNA translocation. We also provide the first evidence that O-GlcNAc glycosylation is dynamically modulated by excitatory stimulation of the brain in vivo. Finally, we use electron-transfer dissociation mass spectrometry to identify exact sites of O-GlcNAc modification. Together, our studies suggest that O-GlcNAc glycosylation occurs reversibly in neurons and, akin to phosphorylation, may have important roles in mediating the communication between neurons.

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Figure 1: Accurate quantification of known O-GlcNAc peptides from complex mixtures using the QUIC-Tag approach.
Figure 2: O-GlcNAc glycosylation is reversible in cultured cortical neurons.
Figure 3: Sequencing of tagged O-GlcNAc peptides regulated by PUGNAc treatment using CAD.
Figure 4: Sequencing of tagged O-GlcNAc peptides regulated by PUGNAc treatment using ETD.
Figure 5: Quantification of O-GlcNAc glycosylation on intact proteins by immunoblotting and infrared imaging detection.
Figure 6: O-GlcNAc glycosylation is dynamically modulated by robust excitatory stimulation of the brain in vivo using kainic acid.

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Acknowledgements

We thank P. Qasba and B. Ramakrishnan for the generous gift of the GalT plasmid, S. Whiteheart for the OGA antibody, T.C. Neo for assistance with synthesis of ketogalactose probe 1, and A. Su for technical discussions. This work was supported by the US National Institutes of Health (RO1 NS045061), the National Science Foundation CAREER Award (CHE-0239861) and the Parson's Foundation (N.K.).

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Correspondence to Linda C Hsieh-Wilson.

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Supplementary information

Supplementary Fig. 1

Expression levels of EGR-1, GRASP55 and eIF4G following kainic acid treatment of rats. (PDF 22 kb)

Supplementary Fig. 2

Annotated CAD MS4 and ETD MS/MS mass spectra for all sequenced peptides. (PDF 1955 kb)

Supplementary Table 1

Mean ratios of individual peptides from α-crystallin and OGT, and mean ratios of all peptides. (PDF 85 kb)

Supplementary Table 2

Identification and quantification of changes in O-GlcNAc glycosylation induced by kainic acid. (PDF 9 kb)

Supplementary Table 3

O-GlcNAc glycosylated proteins identified from the cerebral cortex of kainic acid-stimulated rats. (PDF 10 kb)

Supplementary Methods (PDF 151 kb)

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Khidekel, N., Ficarro, S., Clark, P. et al. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat Chem Biol 3, 339–348 (2007). https://doi.org/10.1038/nchembio881

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