Metabolic cross-talk allows labeling of O-linked β-
            <i>N</i>
            -acetylglucosamine-modified proteins via the
            <i>N</i>
            -acetylgalactosamine salvage pathway

Boyce, Michael; Carrico, Isaac S.; Ganguli, Anjali S.; Yu, Seok‐Ho; Hangauer, Matthew J.; Hubbard, S C; Kohler, Jennifer J.; Bertozzi, Carolyn R.

doi:10.1073/pnas.1010045108

Cited by 296 publications

(339 citation statements)

References 43 publications

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“…To this end, we used a proteomics approach based on metabolic incorporation of Ac 4 GalNAz, which is processed intracellularly to UDPGlcNAz for use as the donor nucleotide sugar of OGT (53). O-GlcNAz groups are subsequently labeled with a biotin phosphine reagent through Staudinger ligation (54).…”

Section: Metabolic Labeling Identifies O-glcnac Proteins From Human Tmentioning

confidence: 99%

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: Metabolic Labeling Identifies O-glcnac Proteins From Human Tmentioning

confidence: 99%

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

Lund

Elias

Davis

2016

The Journal of Immunology

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“…Although these established methods provided great advancements for the detection of O-GlcNAc-modified proteins, the weak nature of metabolic labeling with peracetylated azido-GlcNAc has led to the development of better technologies. Subsequent studies have shown that GalNAz can be converted to UDP-GalNAz and epimerized to UDP-GlcNAz by mammalian biosynthetic enzymes, which is then be added to cells by OGT (Boyce et al 2011). Proof-of-principle experiments highlighting this metabolic labeling demonstrated that numerous proteins are OGlcNAc-modified, laying a solid framework for future studies to visualize and characterize dynamic O-GlcNAc-mediated signaling events (Boyce et al 2011).…”

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

116

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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“…Another approach, originally developed by Bertozzi and coworkers, relies on the promiscuity of the hexosamine biosynthetic pathway (HBP) to metabolically deliver azides into O-GlcNAcmodified proteins (44)(45)(46)(47)(48). Cells can be treated with the fully protected azide-bearing monosaccharide Ac 4 GlcNAz or Ac 4 GalNAz.…”

mentioning

confidence: 99%

Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1

Zaro¹,

Yang

Hang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

201

239

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The dynamic modification of nuclear and cytoplasmic proteins by the monosaccharide N-acetyl-glucosamine (GlcNAc) continues to emerge as an important regulator of many biological processes. Herein we describe the development of an alkynyl-modified GlcNAc analog (GlcNAlk) as a new chemical reporter of O-GlcNAc modification in living cells. This strategy is based on metabolic incorporation of reactive functionality into the GlcNAc biosynthetic pathway. When combined with the Cu(I)-catalyzed [3 þ 2] azidealkyne cycloaddition, this chemical reporter allowed for the robust in-gel fluorescent visualization of O-GlcNAc and affinity enrichment and identification of O-GlcNAc-modified proteins. Using ingel fluorescence detection, we characterized the metabolic fates of GlcNAlk and the previously reported azido analog, GlcNAz. We confirmed previous results that GlcNAz can be metabolically interconverted to GalNAz, whereas GlcNAlk does not, thereby yielding a more specific metabolic reporter of O-GlcNAc modification. We also used GlcNAlk, in combination with a biotin affinity tag, to identify 374 proteins, 279 of which were not previously reported, and we subsequently confirmed the enrichment of three previously uncharacterized proteins. Finally we confirmed the O-GlcNAc modification of the ubiquitin ligase NEDD4-1, the first reported glycosylation of this protein.click chemistry | bioorthogonal labeling | proteomics

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Metabolic cross-talk allows labeling of O-linked β- N -acetylglucosamine-modified proteins via the N -acetylgalactosamine salvage pathway

Cited by 296 publications

References 43 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 reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1

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