Tandem mass spectrometry identifies many mouse brain
            <i>O</i>
            -GlcNAcylated proteins including EGF domain-specific
            <i>O</i>
            -GlcNAc transferase targets

Alfaro, Joshua F.; Gong, Cheng Xin; Monroe, Matthew E.; Aldrich, Joshua T.; Clauss, Therese RW; Purvine, Samuel O.; Wang, Zihao; Camp, David G.; Shabanowitz, Jeffrey; Stanley, Pamela; Hart, Gerald W.; Hunt, Donald F.; Yang, Feng; Smith, Richard D.

doi:10.1073/pnas.1200425109

Cited by 272 publications

(280 citation statements)

References 55 publications

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“…We believe that this constitutes the first broad proteomic analysis of O-GlcNAc-modified proteins in primary human T cells. Many of the proteins that we identified appeared in prior proteomics studies (55,(76)(77)(78), supporting the validity of our results. However, we also identified novel O-GlcNAc substrates, including ARNT, HCLS1, ZAP-70, SHIP1, LCK, and PI3K.…”

Section: Discussionsupporting

confidence: 87%

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: Discussionsupporting

confidence: 87%

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

Lund

Elias

Davis

2016

The Journal of Immunology

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“…S3). Whereas previous studies have detected O-GlcNAc on EGF20 of Notch using mass spectral methods and other techniques (36,37,41,42), here we confirm the presence of a HexNAc on EGF20 and several other EGF repeats. Although we cannot differentiate GlcNAc from GalNAc using these methods, the localization of the HexNAc to peptides containing O-GlcNAcylation sites suggests a GlcNAc modification.…”

Section: Drosophila Notch Shows Glycosylation Consistent With the Presupporting

confidence: 73%

“…The current putative consensus sequence for O-GlcNAc exists on a serine or threonine between the fifth and sixth conserved cysteine (C 5 XXGX(T/S)GXXC 6 ) (41). Of note, whereas Matsuura et al and others have reported the presence of O-GlcNAc on Notch (36,37,41,42), like the other types of O-glycosylation, no data regarding how efficiently the sites on Notch are modified have been provided.…”

mentioning

confidence: 99%

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch

Harvey

Rana

Moss

et al. 2016

Journal of Biological Chemistry

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Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the ␤3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity.

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“…However, the recent development of lectin weak affinity chromatography (LWAC) and chemical/enzymatic tagging strategies has facilitated enrichment of these modified peptides from complex proteolytic digest mixtures (9,10), and the advent of electron transfer dissociation (ETD) mass spectrometry has facilitated the robust assignment of modification sites (11). These advances have been used effectively for studies in mammalian systems, and over 1,000 O-GlcNAc-modified proteins have been identified (9)(10)(11)(12)(13)(14). However, no similar study has been reported in plants, and thus it's unclear whether O-GlcNAc modification controls similar cellular process in plants and animals.…”

mentioning

confidence: 99%

Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis

Chalkley

Maynard

et al. 2017

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

113

161

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Genetic studies have shown essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana. Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.of proteins consisting of a single O-linked N-acetylglucosamine attached to serine and threonine residues. It has been extensively studied in animals, where it regulates a wide range of developmental and metabolic processes. O-GlcNAcylation is dynamically controlled by two enzymes: an O-GlcNAc transferase (OGT) and an O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation occurs in the cytoplasm, nucleus, and mitochondria and has been implicated in cellular processes, including transcription, translation, signal transduction, nuclear pore function, epigenetic regulation and proteasomal degradation (1). Altered levels of protein O-GlcNAcylation in animals have been associated with neurodegeneration, diabetes, cardiovascular diseases, and cancer (2) whereas knock out of OGT is embryonically lethal (3).The model plant Arabidopsis has two putative OGTs: SPINDLY (SPY) and SECRET AGENT (SEC). The spy mutant was identified based on its phenotypes that mimic gibberellin-treated plants, with elongated stems (4). The spy plants also show defects in light and cytokinin responses, leaf morphology and phyllotaxy, root growth, meristem activity, and circadian rhythms (5). The sec mutant displays defects in flower development (6). Although OGT enzymatic activity has been demonstrated in SEC, similar activity in SPY has not been confirmed (7). However, the spy;sec double mutants show severe defects in the development of gametes and are embryonically lethal (7), similar to the OGT knockout mutant in animals. Thus, genetic evidence indicates that O-GlcNAc modification is as important in plants as in animals. But little is known about its specific functions because few O-GlcNAc-modified proteins have been identified...

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Tandem mass spectrometry identifies many mouse brain O -GlcNAcylated proteins including EGF domain-specific O -GlcNAc transferase targets

Cited by 272 publications

References 55 publications

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

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

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch

Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis

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