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Hédou J, Bastide B, Page A, Michalski JC, Morelle W. Mapping of O-linked beta-N-acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle. Proteomics 2009 9(8) 19322778
O-linked beta-N-acetylglucosamine (O-GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O-GlcNAc modified. Moreover, it was demonstrated that O-GlcNAc moieties involved in contractile protein interactions could modulate Ca(2+) activation parameters of contraction. In order to better understand how O-GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O-GlcNAc modification in purified contractile protein homogenates. Using an MS-based method that relies on mild beta-elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O-GlcNAc site in the subdomain four of actin and four O-GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O-GlcNAc sites might modulate the actin-tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein-protein interactions but could also modulate the contractile properties of skeletal muscle.
Clark PM, Dweck JF, Mason DE, Hart CR, Buck SB, Peters EC, Agnew BJ, Hsieh-Wilson LC. Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. Journal of the American Chemical Society 2008 130(35) 18683930
We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc.
Khidekel N, Ficarro SB, Clark PM, Bryan MC, Swaney DL, Rexach JE, Sun YE, Coon JJ, Peters EC, Hsieh-Wilson LC. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nature chemical biology 2007 3(6) 17496889
The addition of the monosaccharide beta-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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