A Chemoenzymatic Approach toward the Rapid and Sensitive Detection of <i>O</i>-GlcNAc Posttranslational Modifications

Khidekel, Nelly; Arndt, Sabine; Lamarre-Vincent, Nathan; Lippert, Alexander R.; Poulin-Kerstien, Katherine G.; Ramakrishnan, B.; Qasba, Pradman K.; Hsieh‐Wilson, Linda C.

doi:10.1021/ja038545r

Cited by 252 publications

(282 citation statements)

References 24 publications

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“…To validate our mass spectrometry findings, we used chemoenzymatic labeling (60) to transfer an azide-tagged galactose residue onto O-GlcNAc groups. Following biotinylation of the azide, O-GlcNAc proteins can be enriched with streptavidin beads as before, and the enriched fraction can be probed for proteins of interest by blotting.…”

Section: Blotting Techniques Verify Mass Spectrometry Resultsmentioning

confidence: 97%

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Lund

Elias

Davis

2016

The Journal of Immunology

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T cell activation in response to Ag is largely regulated by protein posttranslational modifications. Although phosphorylation has been extensively characterized in T cells, much less is known about the glycosylation of serine/threonine residues by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate cell signaling pathways and protein activity similarly to phosphorylation, we performed a comprehensive analysis of O-GlcNAc during T cell activation to address the functional importance of this modification and to identify the modified proteins. Activation of T cells through the TCR resulted in a global elevation of O-GlcNAc levels and in the absence of O-GlcNAc, IL-2 production and proliferation were compromised. T cell activation also led to changes in the relative expression of O-GlcNAc transferase (OGT) isoforms and accumulation of OGT at the immunological synapse of murine T cells. Using a glycoproteomics approach, we identified >200 O-GlcNAc proteins in human T cells. Many of the identified proteins had a functional relationship to RNA metabolism, and consistent with a connection between O-GlcNAc and RNA, inhibition of OGT impaired nascent RNA synthesis upon T cell activation. Overall, our studies provide a global analysis of O-GlcNAc dynamics during T cell activation and the first characterization, to our knowledge, of the O-GlcNAc glycoproteome in human T cells.

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Section: Blotting Techniques Verify Mass Spectrometry Resultsmentioning

confidence: 97%

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Lund

Elias

Davis

2016

The Journal of Immunology

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“…This technique has been adapted for the detection and enrichment of O-GlcNAc-modified proteins and peptides. Sugars containing either ketones or azido groups are incorporated into O-GlcNAc-modified proteins using one of two techniques: (1) a mutant GalT1 (Y289L) with an enlarged active site is used to add UDP-GalNAz to terminal GlcNAc residues similar to the GalT1 labeling discussed above (Khidekel et al 2003); and (2) in cells by metabolic labeling with a peracetylated azido-GlcNAc substrate (Vocadlo et al 2003;Sprung et al 2005), as many of the enzymes in the HBP will tolerate unnatural sugars. However, the kinetics of O-GlcNAz removal from proteins by OGlcNAcase is significantly reduced.…”

Section: Hek293t Cells Ion Trap Etd Ms/msmentioning

confidence: 99%

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

Groves

Lee

Yildirir

et al. 2013

Cell Stress and Chaperones

114

107

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O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) is a ubiquitous and dynamic post-translational modification known to modify over 3,000 nuclear, cytoplasmic, and mitochondrial eukaryotic proteins. Addition of O-GlcNAc to proteins is catalyzed by the O-GlcNAc transferase and is removed by a neutral-N-acetyl-β-glucosaminidase (OGlcNAcase). O-GlcNAc is thought to regulate proteins in a manner analogous to protein phosphorylation, and the cycling of this carbohydrate modification regulates many cellular functions such as the cellular stress response. Diverse forms of cellular stress and tissue injury result in enhanced O-GlcNAc modification, or O-GlcNAcylation, of numerous intracellular proteins. Stress-induced OGlcNAcylation appears to promote cell/tissue survival by regulating a multitude of biological processes including: the phosphoinositide 3-kinase/Akt pathway, heat shock protein expression, calcium homeostasis, levels of reactive oxygen species, ER stress, protein stability, mitochondrial dynamics, and inflammation. Here, we will discuss the regulation of these processes by O-GlcNAc and the impact of such regulation on survival in models of ischemia reperfusion injury and trauma hemorrhage. We will also discuss the misregulation of O-GlcNAc in diseases commonly associated with the stress response, namely Alzheimer's and Parkinson's diseases. Finally, we will highlight recent advancements in the tools and technologies used to study the O-GlcNAc modification.

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“…Khidekel and co-workers designed an unnatural substrate for GalT containing a ketone moiety at the C2 position of UDP-galactose 83 (Fig. 3a).…”

Section: Rapid Sensitive Detectionmentioning

confidence: 99%

“…General O-GlcNAc antibodies (CTD110.6 and RL-2) have been extremely valuable for measuring global changes in glycosylation in response to cellular stimuli 73,77 . However, a limitation of these antibodies is that they detect only a small subset of the OGlcNAc-modified proteins 19,83 . Moreover, it remains difficult to identify the specific proteins that undergo changes in glycosylation.…”

Section: Monitoring O-glcnac Dynamicsmentioning

confidence: 99%

Chemical approaches to understanding O-GlcNAc glycosylation in the brain

2008

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O -GlcNAc glycosylation is a unique, dynamic form of glycosylation found on intracellular proteins of all multicellular organisms. Studies suggest that O-GlcNAc represents a key regulatory modification in the brain, contributing to transcriptional regulation, neuronal communication and neurodegenerative disease. Recently, several new chemical tools have been developed to detect and study the modification, including chemoenzymatic tagging methods, quantitative proteomics strategies and small-molecule inhibitors of O-GlcNAc enzymes. Here we highlight some of the emerging roles for O-GlcNAc in the nervous system and describe how chemical tools have significantly advanced our understanding of the scope, functional significance and cellular dynamics of this modification.O-GlcNAc glycosylation, the covalent attachment of β-N-acetylglucosamine to serine or threonine residues of proteins, is an unusual form of protein glycosylation 1 (Fig. 1). Unlike other types of glycosylation, this single-sugar modification occurs on intracellular proteins and is not elaborated further into complex glycans. The O-GlcNAc transferase (OGT) enzyme is a soluble protein that is found in the cytosol, nucleus and mitochondria 2 , rather than in the endoplasmic reticulum or Golgi. The dynamics of O-GlcNAc are also unique among sugar modifications, being cycled on a shorter time scale than protein turnover 3 . Thus, in many respects O-GlcNAc is more akin to phosphorylation than to conventional forms of glycosylation. Several reviews have described the roles of O-GlcNAc in cellular processes, such as transcription 2,4 , the stress response 5,6 , apoptosis 7,8 , signal transduction 2,9 , glucose sensing 5,10 and proteasomal degradation 5 . Only a few reviews have highlighted the importance of O-GlcNAc glycosylation in the nervous system, and those reports have focused on its potential impact on neurodegenerative diseases 11,12 . However, several lines of evidence suggest that O-GlcNAc has crucial roles in both neuronal function and dysfunction. The enzymes responsible for the modification are most highly expressed in the brain 13,14 and are enriched at neuronal synapses 15,16 . Neuron-specific deletion of the OGT gene in mice leads to abnormal development and locomotor defects, resulting in neonatal death 17 . The O-GlcNAc modification is abundant in the brain and present on many proteins important for transcription, neuronal signaling and synaptic plasticity, such as cAMPresponsive element binding protein (CREB) 18 , synaptic Ras GTPase-activating protein (synGAP) 19 and β-amyloid precursor protein (APP) 20 . An intriguing interplay between OGlcNAc glycosylation and phosphorylation has been observed in cerebellar neurons, wherein activation of certain kinase pathways reduces O-GlcNAc levels on cytoskeleton-

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A Chemoenzymatic Approach toward the Rapid and Sensitive Detection of O-GlcNAc Posttranslational Modifications

Cited by 252 publications

References 24 publications

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

Chemical approaches to understanding O-GlcNAc glycosylation in the brain

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