Detection and Analysis of Proteins Modified by O‐Linked <i>N</i>‐Acetylglucosamine

Zachara, Natasha E.; Vosseller, Keith; Hart, Gerald W.

doi:10.1002/0471142727.mb1706s95

Cited by 40 publications

(49 citation statements)

References 89 publications

(121 reference statements)

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“…Wheat germ agglutinin (WGA) is a commonly used plant lectin for enriching and detecting O-GlcNAc-modified proteins (Zachara et al 2004a(Zachara et al , 2011bVosseller et al 2006). WGA has weak affinity for single GlcNAc residues, but its affinity is dramatically increased for GlcNAcs that are clustered (Finlay et al 1987;Lundquist and Toone 2002;Lee and Lee 2000).…”

Section: Lectin Affinity Chromatographymentioning

confidence: 99%

“…The drawback of WGA as a tool for OGlcNAc enrichment and detection is its additional recognition of sialic acid (NeuAcα(2-3)) residues (Monsigny et al 1980). Treating samples initially with PNGase F to remove N-glycans, and by performing nuclear and cytoplasmic protein extractions (Zachara et al 2011b) can resolve these shortcomings. Succinylation of WGA (sWGA) increases its specificity for GlcNAc as it ablates its reactivity to sialic acid (Monsigny et al 1980).…”

Section: Lectin Affinity Chromatographymentioning

confidence: 99%

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

Groves

Lee

Yildirir

et al. 2013

Cell Stress and Chaperones

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120

112

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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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Section: Lectin Affinity Chromatographymentioning

confidence: 99%

Section: Lectin Affinity Chromatographymentioning

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

Self Cite

120

112

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“…O ‐GlcNAcylation on serine/threonine is one of the most abundant PTMs in mammalian cells, where it plays key roles in numerous BPs and is involved in many challenging diseases. There are more than 1000 proteins that could be O ‐GlcNAcylated in human cells and many more remain to be discovered. However, there is only one enzyme, OGT, that is responsible for O ‐GlcNAcylation in human cells.…”

Section: Discussionmentioning

confidence: 99%

“…Since the discovery of O ‐GlcNAcylation, there are more than 3000 proteins that can be O ‐GlcNAcylated that have been identified , and it is believed that many more remain to be discovered. Although O ‐GlcNAcylation has many properties similar to that of phosphorylation, its regulation is expected to be drastically different since there are 518 protein kinases and only one OGT coding gene in human .…”

Section: Introductionmentioning

confidence: 99%

Global identification of O‐GlcNAc transferase (OGT) interactors by a human proteome microarray and the construction of an OGT interactome

Deng

Guo

et al. 2014

Proteomics

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O-Linked β-N-acetylglucosamine (O-GlcNAcylation) is an important protein PTM, which is very abundant in mammalian cells. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT), whose substrate specificity is believed to be regulated through interactions with other proteins. There are a handful of known human OGT interactors, which is far from enough for fully elucidating the substrate specificity of OGT. To address this challenge, we used a human proteome microarray containing ~17,000 affinity-purified human proteins to globally identify OGT interactors and identified 25 OGT-binding proteins. Bioinformatics analysis showed that these interacting proteins play a variety of roles in a wide range of cellular functions and are highly enriched in intra-Golgi vesicle-mediated transport and vitamin biosynthetic processes. Combining newly identified OGT interactors with the interactors identified prior to this study, we have constructed the first OGT interactome. Bioinformatics analysis suggests that the OGT interactome plays important roles in protein transportation/localization and transcriptional regulation. The novel OGT interactors that we identified in this study could serve as a starting point for further functional analysis. Because of its high-throughput and parallel analysis capability, we strongly believe that protein microarrays could be easily applied for the global identification of regulators for other key enzymes.

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“…The utility of lectins and antibodies for the detection and analysis of O‐GlcNAcylated proteins is dependent on controls. Suggested controls include competition with free GlcNAc, removal of N‐linked glycans with Peptide‐ N ‐Glycosidase F (PNGase F), on‐blot β‐elimination, or modulation of O‐GlcNAc cycling enzyme expression or activity . While useful, treatment of proteins with a β‐ N ‐hexosaminidase does not typically discriminate between O‐GlcNAc and terminal GlcNAc residues displayed on other glycans.…”

Section: : Identification Of the First O‐glcnac‐reactive Antibodymentioning

confidence: 99%

Critical observations that shaped our understanding of the function(s) of intracellular glycosylation (O‐GlcNAc)

Zachara

2018

FEBS Letters

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Almost 100 years after the first descriptions of proteins conjugated to carbohydrates (mucins), several studies suggested that glycoproteins were not restricted to the serum, extracellular matrix, cell surface, or endomembrane system. In the 1980’s, key data emerged demonstrating that intracellular proteins were modified by monosaccharides of O-linked β-N-acetylglucosamine (O-GlcNAc). Subsequently, this modification was identified on thousands of proteins that regulate cellular processes as diverse as protein aggregation, localization, post-translational modifications, activity, and interactions. In this Review, we will highlight critical discoveries that shaped our understanding of the molecular events underpinning the impact of O-GlcNAc on protein function, the role that O-GlcNAc plays in maintaining cellular homeostasis, and our understanding of the mechanisms that regulate O-GlcNAc-cycling.

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Detection and Analysis of Proteins Modified by O‐Linked N‐Acetylglucosamine

Cited by 40 publications

References 89 publications

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

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

Global identification of O‐GlcNAc transferase (OGT) interactors by a human proteome microarray and the construction of an OGT interactome

Critical observations that shaped our understanding of the function(s) of intracellular glycosylation (O‐GlcNAc)

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