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Microarray Discovery of New OGT Substrates: The Medulloblastoma Oncogene OTX2 Is O-GlcNAcylated

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† †Department of Microbiology and Immunobiology, and ‡Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, United States
[email protected]
Cite this: J. Am. Chem. Soc. 2014, 136, 13, 4845–4848
Publication Date (Web):February 28, 2014
https://doi.org/10.1021/ja500451w

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Abstract

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O-GlcNAc transferase (OGT) is a serine/threonine glycosyltransferase that is essential for development and continues to be critically important throughout life. Understanding OGT’s complex biology requires identifying its substrates. Here we demonstrate the utility of a microarray approach for discovering novel OGT substrates. We also report a rapid method to validate OGT substrates that combines in vitro transcription-translation with O-GlcNAc mass tagging. Among the validated new OGT targets is Orthodenticle homeobox 2 (OTX2), a transcription factor critical for brain development, which is primarily expressed only during early embryogenesis and in medulloblastomas, where it functions as an oncogene. We show that endogenous OTX2 from a medulloblastoma cell line is O-GlcNAcylated at several sites. Our results demonstrate that protein microarray technology, combined with the target validation strategy we report, is useful for identifying biologically important OGT substrates, including substrates not present in most tissue types or cell lines.

Protein post-translational modifications (PTMs) are crucial for the proper metabolic control of protein activity. One of the most common PTMs in eukaryotes is O-GlcNAcylation, the attachment of N-acetyl glucosamine (GlcNAc) to serine and threonine side chains of intracellular proteins. This modification is effected by O-GlcNAc transferase (OGT), an enzyme essential for development that has been implicated in a variety of different signaling pathways. (1) OGT targets include kinases, phosphatases, transcription factors, and metabolic enzymes, and OGT has been implicated in insulin signaling, stress response pathways, and cell-cycle regulation, among other processes. Dysregulated O-GlcNAcylation has been linked to cancer, diabetic complications, and other pathologies. (2) Deconvoluting OGT’s myriad functions requires identifying its cellular substrates and considerable effort has been devoted to this endeavor. The vast majority of known substrates were identified using proteomic approaches based on liquid chromatography tandem mass spectrometry (LC-MS/MS). These methods require that O-GlcNAcylated cellular proteins be enriched from the general protein population prior to LC-MS/MS analysis. (3) A number of enrichment approaches have been developed based on lectin affinity capture or bioorthogonal labeling with biotin followed by streptavidin affinity capture. (4) While proteomic approaches are powerful, they have limitations. Enrichment methods can isolate proteins that are not O-GlcNAcylated as well as ones that are, resulting in false positives, and some low abundance OGT substrates may still be missed. Furthermore, O-GlcNAcylated proteins expressed only during development or in specialized cell types will not be identified in standard cell lines.

Figure 1

Figure 1. Known and novel OGT targets can be identified on microarrays. (a) Schematic of the microarray approach. A fluorescent antibody reports on O-GlcNAcylated proteins. (b) Normalized signal intensities plotted against Array ID. Selected known (red) and novel (blue) targets are indicated. Complete list of 230 targets can be found in Tables S1 and S2.

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Human protein microarrays can circumvent common pitfalls of methods that rely on substrate enrichment from complex mixtures. Thousands of purified human proteins, including rare and disease-relevant proteins, are present in known, roughly equivalent concentrations on these arrays with their identities spatially encoded. (5) Large microarrays have been used to identify substrates for phosphorylation, acetylation and ubiquitylation, but have not been applied to O-GlcNAcylation. (6) Here we demonstrate the utility of large microarrays for discovering novel OGT substrates. Among a dozen new substrates confirmed using an efficient, orthogonal validation method is OTX2, a master regulator of neural development and a known oncogene for medulloblastoma, the most common malignant brain tumor in children. (7)

Human OGT contains a C-terminal catalytic domain fused to an N-terminal tetratricopeptide repeat (TPR) domain, which is involved in protein–protein interactions. (8) OGT is expressed as three different isoforms that contain identical catalytic domains but different numbers of TPRs: nucleocytoplasmic OGT (ncOGT) contains 12.5 TPRs, mitochondrially targeted OGT (mOGT) contains 9.5 TPRs, and short OGT (sOGT) contains 2.5 TPRs. (9) We purified each isoform from a bacterial overexpression system and tested them for glycosylation of protein substrates in HeLa cell extracts. (10) ncOGT and mOGT, but not sOGT, showed robust glycosylation activity in these extracts (Figure S1), consistent with previous reports. (9) Therefore, we used ncOGT and mOGT to probe a commercially available microarray containing ∼8000 unique human proteins. Microarrays were incubated with 40 μM UDP-GlcNAc and 3 μM purified ncOGT or mOGT at room temperature for one hour, and O-GlcNAc-modified proteins were then detected using a monoclonal anti-O-GlcNAc primary antibody (CTD110.6, Sigma) and a fluorescent secondary antibody (anti-mouse IgG, Alexa-fluor 646 conjugate, Cellsignal) (Figure 1a). (11) Because the anti-O-GlcNAc antibody CTD110.6 can react with pre-existing O-GlcNAc residues on the arrayed proteins, and has also been shown to bind to both O- and N-linked glycans, (12) we incubated a control microarray with UDP-GlcNAc, but without exogenous OGT, and subsequently treated it with the antibodies. The signal intensities from this control array were subtracted from the data for the test arrays to ensure that any observed signal increase in the test arrays was due to glycosylation by exogenously added OGT. The top 2% of normalized signal intensities for each OGT isoform, encompassing a total of 230 proteins, were identified as possible targets of OGT (Figure 1b). This is a stringent cutoff and there are likely many other OGT targets in the array, our first goal was not to comprehensively assess all substrates, but to establish whether a microarray approach would be useful for identifying new substrates. Among the 230 proteins were several known targets of human OGT, including IRS1 and AKT1, which are involved in insulin signaling, CRTC2, a central regulator of gluconeogenic gene expression, and CKII, a well-studied kinase involved in numerous cellular processes. (13) However, the majority of the top hits were not previously identified human OGT targets. Ingenuity Pathway Analysis showed that the hits fall into several functional classes, with the major categories including kinases, transcription factors and apoptosis-related proteins (Table S3).

Figure 2

Figure 2. A rapid, orthogonal validation method confirms new OGT substrates. (a) Schematic of the validation method. (b) Radiolabeled HGS, E2F8, and OTX2 bands shift to higher molecular weight bands. Nine other OGT substrates were validated (Figure S4 and Table S4).

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Validation is a major bottleneck for all OGT substrate discovery efforts. Putative OGT substrates identified by proteomic methods are typically validated by transfecting mammalian cell lines with an overexpression vector, immunoprecipitating the expressed protein with a specific antibody, and immunoblotting for the O-GlcNAc modification using an anti-O-GlcNAc antibody. (6b) Alternatively, chemoenzymatic methods can be used to install a biotin handle on all O-GlcNAcylated proteins in a cell lysate, and specific proteins of interest can be identified after streptavidin immunoprecipitation by Western blotting using protein-specific antibodies. (14) These approaches work, but are time-intensive, and screening multiple targets requires a collection of protein-specific antibodies or mammalian overexpression vectors. To accelerate substrate validation, we developed an approach based on a method used to confirm ubiquitylation substrates. (15) In this approach, a radiolabeled protein is expressed in vitro from a commercially available cDNA clone and the O-GlcNAc post-translational modification is then detected following gel electrophoresis (Figure 2a). Unlike ubiquitylation, O-GlcNAc modifications are usually not detectable by mass shifts unless many sites are modified. Therefore, we employed a mass-tagging strategy to overcome this limitation. (16) Candidate proteins were expressed from their respective cDNA clones in an in vitro transcription/translation system (IVT, TnT SP6, Promega) supplemented with 35S-methionine (35S-Met), which radiolabels the expressed protein. Reactions were then incubated with OGT and a UDP-GlcNAc analogue containing an N-acyl azide (UDP-GlcNAz; Figure 2), (17) followed by azadibenzocyclooctyne-PEG, which adds a 5 kDa tag to each GlcNAz. (18) Prior to using the method to validate new substrates, we confirmed that OGT efficiently transfers GlcNAz to IVT-expressed nucleoporin62 (Nup62), a well-characterized OGT target that has ten glycosylation sites and thus undergoes a significant mass shift even without a PEG tag (Figure S2). (19) We then verified the mass-tagging method using IVT-expressed Nup62 and CRTC2, which also has multiple O-GlcNAcylation sites (Figure S3). (13c) For both proteins, gel analysis of IVT reactions showed a single band that shifted to a set of higher molecular weight bands only in reactions containing OGT as well as UDP-GlcNAz and azadibenzocyclooctyne-PEG. Twenty IVT-expressed proteins were then analyzed using the method outlined in Figure 2a, and 12 of these were mass shifted, confirming the microarray identification of them as substrates (Figure 2, Table S4, Figure S4). The remainder either are not OGT substrates or failed to confirm for technical reasons; i.e., the IVT-expressed protein is not correctly folded or is not identical to the form on the microarray, which was expressed in cells and may contain other PTMs. To distinguish between these possibilities, other validation methods can be used. (6b) Nevertheless, the confirmed substrates highlight the utility of the approach. The IVT, OGT, and mass-tagging reactions can be accomplished in under four hours, making it possible to validate a large number of possible substrates in a day once in vitro expression from a commercially available cDNA clone has been verified. This validation procedure should be generally useful for confirming new OGT substrates. Several of the twelve validated OGT substrates are involved in gene transcription, (20) including SSBP2, SSBP3, the glucocorticoid receptor, MEF2A, E2F8, and OTX2. Some of these proteins showed a single 5 kDa shift, consistent with one glycosylation event per protein (HGS, Figure 2b), while others showed multiple bands, indicating several O-GlcNAc modifications per protein (OTX2 and E2F8, Figure 2b). (21)

Figure 3

Figure 3. Endogenous OTX2 is O-GlcNAcylated in a medulloblastoma cell line. (a) Schematic of a validation method adapted from ref 21 with data shown for SP1 (positive control) and OTX2. (b) Immunoblot analysis of inhibitor-treated D283 Med cells. O-GlcNAc antibody shows decreased global O-GlcNAc, Nup62 and OTX2 show mass shifts when OGT is inhibited.

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OTX2, a previously unknown OGT substrate, is a transcription factor that acts as a master regulator of brain development. (7) Although otx2 expression is switched off in most tissues after early embryogenesis, it is highly expressed in ∼75% of medulloblastomas, responsible for the majority of fatal childhood brain cancers. (22) Along with c-Myc, OTX2 has been identified as an important oncogene in these tumors. (23) After verifying that OTX2 is expressed in the medulloblastoma cell line D283 Med (Figure S5), we adapted a previously developed mass-tagging approach to assess its endogenous O-GlcNAcylation status (Figure 3a). (21) In the adaptation of this approach, a β-1,4-galactosyltransferase variant, GalTY289, is used to transfer azido-GalNAc (GalNAz) to existing O-GlcNAcs on proteins obtained from a cell lysate. Lysates are then treated with ADIBO-PEG to install a mass tag on each GalNAz. Following SDS-PAGE of total cell lysate, proteins of interest are detected by immunoblotting. SP1, a ubiquitous human transcription factor known to be highly O-GlcNAcylated, (24) shifted to higher molecular weight upon mass-tagging while actin, which is not O-GlcNAcylated, did not. As with SP1, the OTX2 bands shifted to higher molecular weight (Figure 3a), confirming that this protein is O-GlcNAcylated. Several shifted bands were observed, implying that OTX2 from medulloblastoma cells is O-GlcNAcylated at multiple sites, as it is when expressed via IVT (Figure 2b). We confirmed this finding by treating D283 Med cells with 50 μM Ac-5S-GlcNAc, which is metabolized to the validated OGT inhibitor UDP-5S-GlcNAc. (25) Using anti-Nup62 and anti-OTX2 antibodies, we found that inhibitor treatment resulted in a detectable shift to lower molecular weight for both proteins, consistent with the loss of multiple O-GlcNAc modifications due to OGT inhibition (Figure 3b). The roles of O-GlcNAc modification in the oncogenic functions of OTX2 are under investigation.

We have shown that human protein microarrays can detect novel OGT substrates. We note that antibody bias and subtraction of the control array mean that we are not detecting all of the O-GlcNAcylated substrates on the array. The development of alternative detection methods should overcome this limitation. While these microarrays do not yet contain all human proteins, they contain many thousands, including substrates of low abundance, temporally limited expression or glycosylation patterns, or highly restricted cell type specificity. Hence, microarray substrate profiling, combined with the IVT validation method described, can powerfully complement existing proteomic approaches. Moreover, microarray methods are uniquely well suited to exploring the effects of changes in OGT structure on substrate selection.

Supporting Information

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Experimental procedures, microarray protocol and data analysis, IVT validation data, and list of OGT targets. This material is available free of charge via the Internet at http://pubs.acs.org.

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  • Corresponding Author
    • Suzanne Walker - †Department of Microbiology and Immunobiology, and ‡Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, United States
  • Authors
    • Rodrigo F. Ortiz-Meoz - †Department of Microbiology and Immunobiology, and ‡Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, United States
    • Yifat Merbl - †Department of Microbiology and Immunobiology, and ‡Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, United States
    • Marc W. Kirschner - †Department of Microbiology and Immunobiology, and ‡Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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The OGT inhibitor was a gift from David Vocadlo (Simon Fraser University). This research was supported by the National Institutes of Health (GM094263, GM26875, and GM100539).

References

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This article references 25 other publications.

  1. 1
    (a) Hart, G. W.; Slawson, C.; Ramirez-Correa, G.; Lagerlof, O. Annu. Rev. Biochem. 2011, 80, 825
    [Crossref], [PubMed], [CAS], Google Scholar
    1a
    Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease
    Hart, Gerald W.; Slawson, Chad; Ramirez-Correa, Genaro; Lagerlof, Olof
    Annual Review of Biochemistry (2011), 80 (), 825-858CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
    A review. O-GlcNAcylation is the addn. of β-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1980s and still remains difficult to detect and quantify. Nonetheless, O-GlcNAc is highly abundant and cycles on proteins with a timescale similar to protein phosphorylation. O-GlcNAc occurs in organisms ranging from some bacteria to protozoans and metazoans, including plants and nematodes up the evolutionary tree to man. O-GlcNAcylation is mostly on nuclear proteins, but it occurs in all intracellular compartments, including mitochondria. Recent glycomic analyses have shown that O-GlcNAcylation has surprisingly extensive cross talk with phosphorylation, where it serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amts. of O-GlcNAcylation underlie the etiol. of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Current data justify extensive efforts toward a better understanding of this invisible, yet abundant, modification. As tools for the study of O-GlcNAc become more facile and available, exponential growth in this area of research will eventually take place.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptVCntr4%253D&md5=58f3a0d39238d6fffa91641b2db9252e
    (b) Hanover, J. A.; Krause, M. W.; Love, D. C. Nat. Rev. Mol. Cell. Biol. 2012, 13 (5) 312
    [Crossref], [PubMed], [CAS], Google Scholar
    1b
    Post-translational modifications Bittersweet memories: linking metabolism to epigenetics through O-GlcNAcylation
    Hanover, John A.; Krause, Michael W.; Love, Dona C.
    Nature Reviews Molecular Cell Biology (2012), 13 (5), 312-321CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review. O-GlcNAcylation, which is a nutrient-sensitive sugar modification, participates in the epigenetic regulation of gene expression. The enzymes involved in O-linked β-D-N-acetylglucosamine (O-GlcNAc) cycling - O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) - target key transcriptional and epigenetic regulators including RNA polymerase II, histones, histone deacetylase complexes and members of the Polycomb and Trithorax groups. Thus, O-GlcNAc cycling may serve as a homeostatic mechanism linking nutrient availability to higher-order chromatin organization. In response to nutrient availability, O-GlcNAcylation is poised to influence X chromosome inactivation and genetic imprinting, as well as embryonic development. The wide range of physiol. functions regulated by O-GlcNAc cycling suggests an unexplored nexus between epigenetic regulation in disease and nutrient availability.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XlvVKmsL8%253D&md5=072d75b0082cfe3b138af45f7acb4f5f
  2. 2
    (a) Wells, L.; Kreppel, L. K.; Comer, F. I.; Wadzinski, B. E.; Hart, G. W. J. Biol. Chem. 2004, 279 (37) 38466
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
    (b) Yang, X. Y.; Ongusaha, P. P.; Miles, P. D.; Havstad, J. C.; Zhang, F. X.; So, W. V.; Kudlow, J. E.; Michell, R. H.; Olefsky, J. M.; Field, S. J.; Evans, R. M. Nature 2008, 451 (7181) 964
    [Crossref], [PubMed], [CAS], Google Scholar
    2b
    Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance
    Yang, Xiaoyong; Ongusaha, Pat P.; Miles, Philip D.; Havstad, Joyce C.; Zhang, Fengxue; So, W. Venus; Kudlow, Jeffrey E.; Michell, Robert H.; Olefsky, Jerrold M.; Field, Seth J.; Evans, Ronald M.
    Nature (London, United Kingdom) (2008), 451 (7181), 964-969CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degrdn. Here we show that O-GlcNAc transferase (OGT) harbors a previously unrecognized type of phosphoinositide-binding domain. After induction with insulin, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where the enzyme catalyzes dynamic modification of the insulin signaling pathway by O-GlcNAc. This results in the alteration in phosphorylation of key signaling mols. and the attenuation of insulin signal transduction. Hepatic overexpression of OGT impairs the expression of insulin-responsive genes and causes insulin resistance and dyslipidemia. These findings identify a mol. mechanism by which nutritional cues regulate insulin signaling through O-GlcNAc, and underscore the contribution of this modification to the etiol. of insulin resistance and type 2 diabetes.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXit1yns74%253D&md5=b4c33d036aea5d0ecd44978dfa823164
    (c) Lehman, D. M.; Fu, D. J.; Freeman, A. B.; Hunt, K. J.; Leach, R. J.; Johnson-Pais, T.; Hamlington, J.; Dyer, T. D.; Arya, R.; Abboud, H.; Goring, H. H. H.; Duggirala, R.; Blangero, J.; Konrad, R. J.; Stern, M. P. Diabetes 2005, 54 (4) 1214
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
    (d) Zachara, N. E.; O’Donnell, N.; Cheung, W. D.; Mercer, J. J.; Marth, J. D.; Hart, G. W. J. Biol. Chem. 2004, 279 (29) 30133
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
    (e) Slawson, C.; Zachara, N. E.; Vosseller, K.; Cheung, W. D.; Lane, M. D.; Hart, G. W. J. Biol. Chem. 2005, 280 (38) 32944
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
    (f) Lazarus, B. D.; Love, D. C.; Hanover, J. A. Int. J. Biochem. Cell Biol. 2009, 41 (11) 2134
    [Crossref], [PubMed], [CAS], Google Scholar
    2f
    O-GlcNAc cycling: Implications for neurodegenerative disorders
    Lazarus, Brooke D.; Love, Dona C.; Hanover, John A.
    International Journal of Biochemistry & Cell Biology (2009), 41 (11), 2134-2146CODEN: IJBBFU; ISSN:1357-2725. (Elsevier Ltd.)
    A review. The dynamic post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), termed O-GlcNAcylation, is an important mechanism for modulating cellular signaling pathways. O-GlcNAcylation impacts transcription, translation, organelle trafficking, proteasomal degrdn. and apoptosis. O-GlcNAcylation has been implicated in the etiol. of several human diseases including type-2 diabetes and neurodegeneration. This review describes the pair of enzymes responsible for the cycling of this post-translational modification: O-GlcNAc transferase (OGT) and β-N-acetylglucosaminidase (OGA), with a focus on the function of their structural domains. We will also highlight the important processes and substrates regulated by these enzymes, with an emphasis on the role of O-GlcNAc as a nutrient sensor impacting insulin signaling and the cellular stress response. Finally, we will focus attention on the many ways by which O-GlcNAc cycling may affect the cellular machinery in the neuroendocrine and central nervous systems.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFyntL3J&md5=70b2a12840567017d8e8a7b3b7fc0686
    (g) Caldwell, S. A.; Jackson, S. R.; Shahriari, K. S.; Lynch, T. P.; Sethi, G.; Walker, S.; Vosseller, K.; Reginato, M. J. Oncogene 2010, 29 (19) 2831
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
    (h) Ma, Z.; Vocadlo, D. J.; Vosseller, K. J. Biol. Chem. 2013, 288 (21) 15121
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
    (i) Lynch, T. P.; Ferrer, C. M.; Jackson, S. R.; Shahriari, K. S.; Vosseller, K.; Reginato, M. J. J. Biol. Chem. 2012, 287 (14) 11070
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Hahne, H.; Sobotzki, N.; Nyberg, T.; Helm, D.; Borodkin, V. S.; van Aalten, D. M.; Agnew, B.; Kuster, B. J. Proteome. Res. 2013, 12 (2) 927
    [ACS Full Text ACS Full Text], [CAS], Google Scholar
    3
    Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry
    Hahne, Hannes; Sobotzki, Nadine; Nyberg, Tamara; Helm, Dominic; Borodkin, Vladimir S.; van Aalten, Daan M. F.; Agnew, Brian; Kuster, Bernhard
    Journal of Proteome Research (2013), 12 (2), 927-936CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    The post-translational modification of proteins with N-acetylglucosamine (O-GlcNAc) is involved in the regulation of a wide variety of cellular processes and assocd. with a no. of chronic diseases. Despite its emerging biol. significance, the systematic identification of O-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved O-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copper-mediated Click chem. for purifn. of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC-MS/MS afforded the identification of around 1500 O-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound O-GlcNAc peptides using selective β-elimination enabled the identification of 185 O-GlcNAc modification sites on 80 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the O-GlcNAcase inhibitor GlcNAcstatin G on the level of O-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signaling pathway showed significantly increased O-GlcNAcylation levels in response to the drug, which further strengthens the link of O-GlcNAc protein modification to cellular nutrient sensing and response.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXls1CgtA%253D%253D&md5=3ceafc8dcfb4ae70fab6c2239ffa09b5
  4. 4
    (a) Sprung, R.; Nandi, A.; Chen, Y.; Kim, S. C.; Barma, D.; Falck, J. R.; Zhao, Y. J. Proteome. Res. 2005, 4 (3) 950
    [ACS Full Text ACS Full Text], [CAS], Google Scholar
    4a
    Tagging-via-Substrate Strategy for Probing O-GlcNAc Modified Proteins
    Sprung, Robert; Nandi, Animesh; Chen, Yue; Kim, Sung Chan; Barma, Deb; Falck, John R.; Zhao, Yingming
    Journal of Proteome Research (2005), 4 (3), 950-957CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Identification of proteins bearing a specific post-translational modification would imply functions of the modification. Proteomic anal. of post-translationally modified proteins is usually challenging due to high complexity and wide dynamic range, as well as unavailability of efficient methods to enrich the proteins of interest. Here, the authors report a strategy for the detection, isolation, and profiling of O-linked N-acetylglucosamine (O-GlcNAc) modified proteins, which involves 3 steps: metabolic labeling of cells with an unnatural GlcNAc analog, peracetylated azido-GlcNAc; chemoselective conjugation of azido-GlcNAc modified proteins via the Staudinger ligation, which is specific between phosphine and azide, using a biotinylated phosphine capture reagent; and detection and affinity purifn. of the resulting conjugated O-GlcNAc modified proteins. Since the approach relies on a tag (azide) in the substrate, we designated it the tagging-via-substrate (TAS) strategy. A similar strategy was used previously for protein farnesylation, phosphorylation, and sumoylation. Using this approach, we were able to specifically label and subsequently detect azido-GlcNAc modified proteins from the cytosolic lysates of HeLa, 3T3, COS-1, and S2 cell lines, suggesting the azido-substrate could be tolerated by the enzymic systems among these cells from diverse biol. species. We isolated azido-GlcNAc modified proteins from the cytosolic ext. of S2 cells and identified 10 previously reported and 41 putative O-GlcNAc modified proteins, by nano-HPLC-MS/MS. Our study demonstrates that the TAS approach is a useful tool for the detection and proteomic anal. of O-GlcNAc modified proteins.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXkt1Kltrw%253D&md5=4942a1764cb55fb23d9fdadd57e9670e
    (b) Nandi, A.; Sprung, R.; Barma, D. K.; Zhao, Y.; Kim, S. C.; Falck, J. R.; Zhao, Y. Anal. Chem. 2006, 78 (2) 452
    [ACS Full Text ACS Full Text], Google Scholar
    There is no corresponding record for this reference.
    (c) Gurcel, C.; Vercoutter-Edouart, A. S.; Fonbonne, C.; Mortuaire, M.; Salvador, A.; Michalski, J. C.; Lemoine, J. Anal. Bioanal. Chem. 2008, 390 (8) 2089
    [Crossref], [PubMed], [CAS], Google Scholar
    4c
    Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging
    Gurcel, Caroline; Vercoutter-Edouart, Anne-Sophie; Fonbonne, Catherine; Mortuaire, Marlene; Salvador, Arnaud; Michalski, Jean-Claude; Lemoine, Jerome
    Analytical and Bioanalytical Chemistry (2008), 390 (8), 2089-2097CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells. This dynamic glycosylation plays a fundamental role in the activity of many nuclear and cytoplasmic proteins and is assocd. with pathologies like type II diabetes, Alzheimer's disease or some cancers. However the exact link between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Here the authors report a strategy based on the 1,3-dipolar cycloaddn., called click chem., between unnatural N-acetylglucosamine (GlcNAc) analogs (substituted with an azido or alkyne group) and the corresponding biotinylated probe to specifically detect, enrich and identify O-GlcNAc-modified proteins. This bio-orthogonal conjugation confirms that only azido analog of GlcNAc is metabolized by the cell. Thanks to the biotin probe, affinity purifn. on streptavidin beads allowed the authors to identify 32 O-GlcNAc-azido-tagged proteins by LC-MS/MS anal. in an MCF-7 cellular model, 14 of which were previously unreported. This work illustrates the use of the click-chem.-based strategy combined with a proteomic approach to get further insight into the pattern of O-GlcNAc-modified proteins and the biol. significance of this post-translational modification.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXktlags7w%253D&md5=a52d65f5f856d97608426010c96f5670
    (d) Zaro, B. W.; Yang, Y. Y.; Hang, H. C.; Pratt, M. R. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (20) 8146
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
    (e) Alfaro, J. F.; Gong, C. X.; Monroe, M. E.; Aldrich, J. T.; Clauss, T. R.; Purvine, S. O.; Wang, Z.; Camp, D. G., II; Shabanowitz, J.; Stanley, P.; Hart, G. W.; Hunt, D. F.; Yang, F.; Smith, R. D. Proc. Natl. Acad. Sci. U.S.A. 2012, 109 (19) 7280
    [Crossref], [PubMed], [CAS], Google Scholar
    4e
    Tandem mass spectrometry identifies many-mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets
    Alfaro, Joshua F.; Gong, Cheng-Xin; Monroe, Matthew E.; Aldrich, Joshua T.; Clauss, Therese R. W.; Purvine, Samuel O.; Wang, Zihao; Camp, David G., II; Shabanowitz, Jeffrey; Stanley, Pamela; Hart, Gerald W.; Hunt, Donald F.; Yang, Feng; Smith, Richard D.
    Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (19), 7280-7285, S7280/1-S7280/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chem./enzymic photochem. cleavage approach for enriching O-GlcNAcylated peptides in samples contg. ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we obsd. O-β-GIcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N-acetylglucosaminyltransferases.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnsVWksLw%253D&md5=ca88d9e5172172a3d69e81b789ae03b9
    (f) Khidekel, N.; Ficarro, S. B.; Peters, E. C.; Hsieh-Wilson, L. C. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (36) 13132
    [Crossref], [PubMed], [CAS], Google Scholar
    4f
    Exploring the O-GlcNAc proteome: Direct identification of O-GlcNAc-modified proteins from the brain
    Khidekel, Nelly; Ficarro, Scott B.; Peters, Eric C.; Hsieh-Wilson, Linda C.
    Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (36), 13132-13137CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The covalent modification of intracellular proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is emerging as a crucial regulatory posttranslational modification akin to phosphorylation. Numerous studies point to the significance of O-GlcNAc in cellular processes such as nutrient sensing, protein degrdn., and gene expression. Despite its importance, the breadth and functional roles of O-GlcNAc are only beginning to be elucidated. Advances in our understanding will require the development of new strategies for the detection and study of O-GlcNAc-modified proteins in vivo. Herein we report the direct, high-throughput anal. of O-GlcNAc-glycosylated proteins from the mammalian brain. The proteins were identified by using a chemoenzymic approach that exploits an engineered galactosyltransferase enzyme to selectively label O-GlcNAc proteins with a ketone-biotin tag. The tag permits enrichment of low-abundance O-GlcNAc species from complex mixts. and localization of the modification to short amino acid sequences. Using this approach, we discovered 25 O-GlcNAc-glycosylated proteins from the brain, including regulatory proteins assocd. with gene expression, neuronal signaling, and synaptic plasticity. The functional diversity represented by this set of proteins suggests an expanded role for O-GlcNAc in regulating neuronal function. Moreover, the chemoenzymic strategy described here should prove valuable for identifying O-GlcNAc-modified proteins in various tissues and facilitate studies of the physiol. significance of O-GlcNAc across the proteome.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXnvFenurw%253D&md5=1cedf76e59c92f1b907c584f2e80e82c
  5. 5
    MacBeath, G.; Schreiber, S. L. Science 2000, 289 (5485) 1760
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  6. 6
    (a)

    A small kinase microarray in which 3H-GlcNAc was detected was used to identify kinase substrates of OGT. See:

    Google Scholar
    There is no corresponding record for this reference.
    (b) Dias, W. B.; Cheung, W. D.; Hart, G. W. Biochem. Biophys. Res. Commun. 2012, 422 (2) 224
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  7. 7
    Beby, F.; Lamonerie, T. Exp. Eye. Res. 2013, 111, 9
    [Crossref], [PubMed], [CAS], Google Scholar
    7
    The homeobox gene Otx2 in development and disease
    Beby, Francis; Lamonerie, Thomas
    Experimental Eye Research (2013), 111 (), 9-16CODEN: EXERA6; ISSN:0014-4835. (Elsevier Ltd.)
    A review. The Otx2 gene encodes a transcription factor essential for the normal development of brain, cerebellum, pineal gland, and eye. In the retina, Otx2 has essential functions from early embryogenesis to adulthood. As soon as the optic vesicle is formed, the gene is required for retinal pigment epithelium specification. Otx2 is also a key regulator of photoreceptor genesis and differentiation, and is required after birth for bipolar cells terminal maturation. Otx2 expression is maintained in the differentiated retina wherein the gene is crit. for the outer retina maintenance. In the visual cortex, the gene modulates the neuronal plasticity through a paracrine mechanism. OTX2 heterozygous mutations in humans have been linked to severe ocular malformations assocd. with brain abnormalities and pituitary dysfunction. Recent studies have also established the OTX2 gene as an oncogene for medulloblastoma, a malignant brain tumor originating in the cerebellum.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXot1Kkurg%253D&md5=e9374d523dfa130f8e17ef4897fa63fd
  8. 8
    Jinek, M.; Rehwinkel, J.; Lazarus, B. D.; Izaurralde, E.; Hanover, J. A.; Conti, E. Nat. Struct. Mol. Biol. 2004, 11 (10) 1001
    [Crossref], [PubMed], [CAS], Google Scholar
    8
    The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin α
    Jinek, Martin; Rehwinkel, Jan; Lazarus, Brooke D.; Izaurralde, Elisa; Hanover, John A.; Conti, Elena
    Nature Structural & Molecular Biology (2004), 11 (10), 1001-1007CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
    Addn. of N-acetylglucosamine (GlcNAc) is a ubiquitous form of intracellular glycosylation catalyzed by the conserved O-linked GlcNAc transferase (OGT). OGT contains an N-terminal domain of tetratricopeptide (TPR) repeats that mediates the recognition of a broad range of target proteins. Components of the nuclear pore complex are major OGT targets, as OGT depletion by RNA interference (RNAi) results in the loss of GlcNAc modification at the nuclear envelope. To gain insight into the mechanism of target recognition, we solved the crystal structure of the homodimeric TPR domain of human OGT, which contains 11.5 TPR repeats. The repeats form an elongated superhelix. The concave surface of the superhelix is lined by absolutely conserved asparagines, in a manner reminiscent of the peptide-binding site of importin α. Based on this structural similarity, we propose that OGT uses an analogous mol. mechanism to recognize its targets.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXnvVOnsLw%253D&md5=aff0a8c9b455734b3e94da8d1a58cbbb
  9. 9
    Lazarus, B. D.; Love, D. C.; Hanover, J. A. Glycobiology 2006, 16 (5) 415
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  10. 10
    (a) Merbl, Y.; Kirschner, M. W. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (8) 2543
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
    (b) Gross, B. J.; Kraybill, B. C.; Walker, S. J. Am. Chem. Soc. 2005, 127 (42) 14588
    [ACS Full Text ACS Full Text], Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Comer, F. I.; Vosseller, K.; Wells, L.; Accavitti, M. A.; Hart, G. W. Anal. Biochem. 2001, 293 (2) 169
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  12. 12
    Isono, T. PLoS One 2011, 6 (4) e18959
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  13. 13
    (a) Whelan, S. A.; Dias, W. B.; Thiruneelakantapillai, L.; Lane, M. D.; Hart, G. W. J. Biol. Chem. 2010, 285 (8) 5204
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
    (b) Wang, S.; Huang, X.; Sun, D.; Xin, X.; Pan, Q.; Peng, S.; Liang, Z.; Luo, C.; Yang, Y.; Jiang, H.; Huang, M.; Chai, W.; Ding, J.; Geng, M. PLoS One 2012, 7 (5) e37427
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
    (c) Dentin, R.; Hedrick, S.; Xie, J.; Yates, J., III; Montminy, M. Science 2008, 319 (5868) 1402
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
    (d) Tarrant, M. K.; Rho, H. S.; Xie, Z.; Jiang, Y. L.; Gross, C.; Culhane, J. C.; Yan, G.; Qian, J.; Ichikawa, Y.; Matsuoka, T.; Zachara, N.; Etzkorn, F. A.; Hart, G. W.; Jeong, J. S.; Blackshaw, S.; Zhu, H.; Cole, P. A. Nat. Chem. Biol. 2012, 8 (3) 262
    [Crossref], [PubMed], [CAS], Google Scholar
    13d
    Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis
    Tarrant, Mary Katherine; Rho, Hee-Sool; Xie, Zhi; Jiang, Yu Lin; Gross, Christopher; Culhane, Jeffrey C.; Yan, Gai; Qian, Jiang; Ichikawa, Yoshitaka; Matsuoka, Tatsuji; Zachara, Natasha; Etzkorn, Felicia A.; Hart, Gerald W.; Jeong, Jun Seop; Blackshaw, Seth; Zhu, Heng; Cole, Philip A.
    Nature Chemical Biology (2012), 8 (3), 262-269CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked β-N-acetylglucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2α by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degrdn. By performing kinase assays with site-specifically phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biol. outputs.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVKjtbY%253D&md5=6854c5234c1087ff123c580496eeb2c2
    (e)

    11 out of 80 validated O-GlcNAc proteins discovered in ref 3 were also present on the protein microarray; 5 were in our top 2% of hits, and 7 were in the top 6%.

    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Tai, H. C.; Khidekel, N.; Ficarro, S. B.; Peters, E. C.; Hsieh-Wilson, L. C. J. Am. Chem. Soc. 2004, 126 (34) 10500
    [ACS Full Text ACS Full Text], Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Merbl, Y.; Refour, P.; Patel, H.; Springer, M.; Kirschner, M. W. Cell 2013, 152 (5) 1160
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  16. 16
    Boyce, M.; Bertozzi, C. R. Nat. Methods 2011, 8 (8) 638
    [Crossref], [PubMed], [CAS], Google Scholar
    16
    Bringing chemistry to life
    Boyce, Michael; Bertozzi, Carolyn R.
    Nature Methods (2011), 8 (8), 638-642CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Bioorthogonal chem. allows a wide variety of biomols. to be specifically labeled and probed in living cells and whole organisms. Here we discuss the history of bioorthogonal reactions and some of the most interesting and important advances in the field.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXpsFaht70%253D&md5=8ad08fe14193afffdc99fca2d7c92d06
  17. 17
    Vocadlo, D. J.; Hang, H. C.; Kim, E. J.; Hanover, J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (16) 9116
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  18. 18
    Yao, J. Z.; Uttamapinant, C.; Poloukhtine, A.; Baskin, J. M.; Codelli, J. A.; Sletten, E. M.; Bertozzi, C. R.; Popik, V. V.; Ting, A. Y. J. Am. Chem. Soc. 2012, 134 (8) 3720
    [ACS Full Text ACS Full Text], Google Scholar
    There is no corresponding record for this reference.
  19. 19
    Holt, G. D.; Snow, C. M.; Senior, A.; Haltiwanger, R. S.; Gerace, L.; Hart, G. W. J. Cell. Biol. 1987, 104 (5) 1157
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  20. 20
    Slawson, C.; Hart, G. W. Nat. Rev. Cancer 2011, 11 (9) 678
    [Crossref], [PubMed], [CAS], Google Scholar
    20
    O-GlcNAc signalling: implications for cancer cell biology
    Slawson, Chad; Hart, Gerald W.
    Nature Reviews Cancer (2011), 11 (9), 678-684CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
    A review. O-GlcNAcylation is the covalent attachment of β-D-N-acetylglucosamine (GlcNAc) sugars to serine or threonine residues of nuclear and cytoplasmic proteins, and it is involved in extensive crosstalk with other post-translational modifications, such as phosphorylation. O-GlcNAcylation is becoming increasing realized as having important roles in cancer-relevant processes, such as cell signalling, transcription, cell division, metab. and cytoskeletal regulation. However, currently little is known about the specific roles of aberrant O-GlcNAcylation in cancer. In this Opinion article, we summarize the current understanding of O-GlcNAcylation in cancer and its emerging functions in transcriptional regulation at the level of chromatin and transcription factors.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVehs73E&md5=b0daec25f9b979d512af2282054b9115
  21. 21
    Rexach, J. E.; Rogers, C. J.; Yu, S. H.; Tao, J.; Sun, Y. E.; Hsieh-Wilson, L. C. Nat. Chem. Biol. 2010, 6 (9) 645
    [Crossref], [PubMed], [CAS], Google Scholar
    21
    Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags
    Rexach, Jessica E.; Rogers, Claude J.; Yu, Seok-Ho; Tao, Jifang; Sun, Yi E.; Hsieh-Wilson, Linda C.
    Nature Chemical Biology (2010), 6 (9), 645-651CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here the authors describe a powerful method to visualize the O-GlcNAc-modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purifn., advanced instrumentation or expensive radiolabels. In addn., it establishes the glycosylation state (for example, mono-, di-, tri-) of proteins, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, the authors apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation and discover an unexpected reverse 'yin-yang' relation on the transcriptional repressor MeCP2 that was undetectable by traditional methods. The authors anticipate that this mass-tagging strategy will advance the authors' understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXpt1ars7k%253D&md5=1250b849843a3526bb0d6cfb1996aabe
  22. 22
    Adamson, D. C.; Shi, Q.; Wortham, M.; Northcott, P. A.; Di, C.; Duncan, C. G.; Li, J.; McLendon, R. E.; Bigner, D. D.; Taylor, M. D.; Yan, H. Cancer Res. 2010, 70 (1) 181
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  23. 23
    Bai, R. Y.; Staedtke, V.; Lidov, H. G.; Eberhart, C. G.; Riggins, G. J. Cancer Res. 2012, 72 (22) 5988
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  24. 24
    Jackson, S. P.; Tjian, R. Cell 1988, 55 (1) 125
    [Crossref], Google Scholar
    There is no corresponding record for this reference.
  25. 25
    Gloster, T. M.; Zandberg, W. F.; Heinonen, J. E.; Shen, D. L.; Deng, L.; Vocadlo, D. J. Nat. Chem. Biol. 2011, 7 (3) 174
    [Crossref], [PubMed], [CAS], Google Scholar
    25
    Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells
    Gloster, Tracey M.; Zandberg, Wesley F.; Heinonen, Julia E.; Shen, David L.; Deng, Le-Hua; Vocadlo, David J.
    Nature Chemical Biology (2011), 7 (3), 174-181CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Glycosyltransferases are ubiquitous enzymes that catalyze the assembly of glycoconjugates throughout all kingdoms of nature. A long-standing problem is the rational design of probes that can be used to manipulate glycosyltransferase activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analog that inhibits, with high potency both in vitro and in cells, the human glycosyltransferase responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show that the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analog. Treatment of cells with the precursor lowers O-GlcNAc in a targeted manner with a single-digit micromolar EC50. This approach to inhibition of glycosyltransferases should be applicable to other members of this superfamily of enzymes and enable their manipulation in a biol. setting.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXps1ahsw%253D%253D&md5=907a1c87e4e14057548a6290d34dbbd3

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  3. Weston Kightlinger, Katherine F. Warfel, Matthew P. DeLisa, Michael C. Jewett. Synthetic Glycobiology: Parts, Systems, and Applications. ACS Synthetic Biology 2020, 9 (7) , 1534-1562. https://doi.org/10.1021/acssynbio.0c00210
  4. Cassandra M. Joiner, Zebulon G. Levine, Chanat Aonbangkhen, Christina M. Woo, Suzanne Walker. Aspartate Residues Far from the Active Site Drive O-GlcNAc Transferase Substrate Selection. Journal of the American Chemical Society 2019, 141 (33) , 12974-12978. https://doi.org/10.1021/jacs.9b06061
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  13. Daniel H. Ramirez, Bo Yang, Alexandria K. D’Souza, Dacheng Shen, Christina M. Woo. Truncation of the TPR domain of OGT alters substrate and glycosite selection. Analytical and Bioanalytical Chemistry 2021, 413 (30) , 7385-7399. https://doi.org/10.1007/s00216-021-03731-8
  14. Logan J Massman, Michael Pereckas, Nathan T Zwagerman, Stephanie Olivier-Van Stichelen. O -GlcNAcylation is essential for rapid Pomc expression and cell proliferation in corticotropic tumor cells. Endocrinology 2021, https://doi.org/10.1210/endocr/bqab178
  15. Arielis Estevez, Dongsheng Zhu, Connor Blankenship, Jiaoyang Jiang. Molecular Interrogation to Crack the Case of O‐GlcNAc. Chemistry – A European Journal 2020, 26 (53) , 12086-12100. https://doi.org/10.1002/chem.202000155
  16. Chen-chen Li, Ying Li, Yan Zhang, Chun-yang Zhang. Single-molecule fluorescence resonance energy transfer and its biomedical applications. TrAC Trends in Analytical Chemistry 2020, 122 , 115753. https://doi.org/10.1016/j.trac.2019.115753
  17. Chia‐Wei Hu, Matthew Worth, Hao Li, Jiaoyang Jiang. Chemical and Biochemical Strategies To Explore the Substrate Recognition of O ‐GlcNAc‐Cycling Enzymes. ChemBioChem 2019, 20 (3) , 312-318. https://doi.org/10.1002/cbic.201800481
  18. Jie Shi, Rob Ruijtenbeek, Roland J Pieters. Demystifying O-GlcNAcylation: hints from peptide substrates. Glycobiology 2018, 28 (11) , 814-824. https://doi.org/10.1093/glycob/cwy031
  19. Saar A. M. van der Laarse, Aneika C. Leney, Albert J. R. Heck. Crosstalk between phosphorylation and O‐Glc NA cylation: friend or foe. The FEBS Journal 2018, 285 (17) , 3152-3167. https://doi.org/10.1111/febs.14491
  20. Weston Kightlinger, Liang Lin, Madisen Rosztoczy, Wenhao Li, Matthew P. DeLisa, Milan Mrksich, Michael C. Jewett. Design of glycosylation sites by rapid synthesis and analysis of glycosyltransferases. Nature Chemical Biology 2018, 14 (6) , 627-635. https://doi.org/10.1038/s41589-018-0051-2
  21. Zafer Gurel, Nader Sheibani. O-Linked β- N -acetylglucosamine (O-GlcNAc) modification: a new pathway to decode pathogenesis of diabetic retinopathy. Clinical Science 2018, 132 (2) , 185-198. https://doi.org/10.1042/CS20171454
  22. Eun Ju Kim. In Vitro Biochemical Assays for O-GlcNAc-Processing Enzymes. ChemBioChem 2017, 18 (15) , 1462-1472. https://doi.org/10.1002/cbic.201700138
  23. Kristýna Slámová, Pavla Bojarová. Engineered N-acetylhexosamine-active enzymes in glycoscience. Biochimica et Biophysica Acta (BBA) - General Subjects 2017, 1861 (8) , 2070-2087. https://doi.org/10.1016/j.bbagen.2017.03.019
  24. An-Qi Yang, Daoyuan Li, Lianli Chi, Xin-Shan Ye. Validation, Identification, and Biological Consequences of the Site-specific O-GlcNAcylation Dynamics of Carbohydrate-responsive Element-binding Protein (ChREBP). Molecular & Cellular Proteomics 2017, 16 (7) , 1233-1243. https://doi.org/10.1074/mcp.M116.061416
  25. Nathan J. Cox, Thomas R. Meister, Michael Boyce. Chemical Biology of O -GlcNAc Glycosylation. 2017, 94-149. https://doi.org/10.1039/9781782623823-00094
  26. Zhoumin Li, Zhonghui Li, Qiqi Niu, Hui Li, Maika Vuki, Danke Xu. Visual microarray detection for human IgE based on silver nanoparticles. Sensors and Actuators B: Chemical 2017, 239 , 45-51. https://doi.org/10.1016/j.snb.2016.07.142
  27. Zebulon G. Levine, Suzanne Walker. The Biochemistry of O -GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells?. Annual Review of Biochemistry 2016, 85 (1) , 631-657. https://doi.org/10.1146/annurev-biochem-060713-035344
  28. Avital Percher, Srinivasan Ramakrishnan, Emmanuelle Thinon, Xiaoqiu Yuan, Jacob S. Yount, Howard C. Hang. Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation. Proceedings of the National Academy of Sciences 2016, 113 (16) , 4302-4307. https://doi.org/10.1073/pnas.1602244113
  29. Jie Shi, Suhela Sharif, Rob Ruijtenbeek, Roland J. Pieters, . Activity Based High-Throughput Screening for Novel O-GlcNAc Transferase Substrates Using a Dynamic Peptide Microarray. PLOS ONE 2016, 11 (3) , e0151085. https://doi.org/10.1371/journal.pone.0151085
  30. Kelly N. Chuh, Anna R. Batt, Matthew R. Pratt. Chemical Methods for Encoding and Decoding of Posttranslational Modifications. Cell Chemical Biology 2016, 23 (1) , 86-107. https://doi.org/10.1016/j.chembiol.2015.11.006
  31. Yingshuai Liu, Jin Xie, Zeying Zhang, Zhisong Lu. An ultrasensitive colorimetric strategy for protein O-GlcNAcylation detection via copper deposition-enabled nonenzymatic signal amplification. RSC Advances 2016, 6 (92) , 89484-89491. https://doi.org/10.1039/C6RA17119B
  32. Stéphanie Olivier-Van Stichelen, John A. Hanover. You are what you eat. Current Opinion in Clinical Nutrition and Metabolic Care 2015, 18 (4) , 339-345. https://doi.org/10.1097/MCO.0000000000000188
  33. Anne‐Sophie Vercoutter‐Edouart, Ikram El Yazidi‐Belkoura, Céline Guinez, Steffi Baldini, Maïté Leturcq, Marlène Mortuaire, Anne‐Marie Mir, Agata Steenackers, Vanessa Dehennaut, Annick Pierce, Tony Lefebvre. Detection and identification of O ‐GlcNAcylated proteins by proteomic approaches. PROTEOMICS 2015, 15 (5-6) , 1039-1050. https://doi.org/10.1002/pmic.201400326
  34. Alexis K. Nagel, Lauren E. Ball. Intracellular Protein O-GlcNAc Modification Integrates Nutrient Status with Transcriptional and Metabolic Regulation. 2015, 137-166. https://doi.org/10.1016/bs.acr.2014.12.003
  35. John Janetzko, Suzanne Walker. The Making of a Sweet Modification: Structure and Function of O-GlcNAc Transferase. Journal of Biological Chemistry 2014, 289 (50) , 34424-34432. https://doi.org/10.1074/jbc.R114.604405
  36. Alexis K. Nagel, Lauren E. Ball. O-GlcNAc transferase and O-GlcNAcase: achieving target substrate specificity. Amino Acids 2014, 46 (10) , 2305-2316. https://doi.org/10.1007/s00726-014-1827-7
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  • Abstract

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    Figure 1

    Figure 1. Known and novel OGT targets can be identified on microarrays. (a) Schematic of the microarray approach. A fluorescent antibody reports on O-GlcNAcylated proteins. (b) Normalized signal intensities plotted against Array ID. Selected known (red) and novel (blue) targets are indicated. Complete list of 230 targets can be found in Tables S1 and S2.

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    Figure 2

    Figure 2. A rapid, orthogonal validation method confirms new OGT substrates. (a) Schematic of the validation method. (b) Radiolabeled HGS, E2F8, and OTX2 bands shift to higher molecular weight bands. Nine other OGT substrates were validated (Figure S4 and Table S4).

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    Figure 3

    Figure 3. Endogenous OTX2 is O-GlcNAcylated in a medulloblastoma cell line. (a) Schematic of a validation method adapted from ref 21 with data shown for SP1 (positive control) and OTX2. (b) Immunoblot analysis of inhibitor-treated D283 Med cells. O-GlcNAc antibody shows decreased global O-GlcNAc, Nup62 and OTX2 show mass shifts when OGT is inhibited.

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  • References

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    This article references 25 other publications.

    1. 1
      (a) Hart, G. W.; Slawson, C.; Ramirez-Correa, G.; Lagerlof, O. Annu. Rev. Biochem. 2011, 80, 825
      [Crossref], [PubMed], [CAS], Google Scholar
      1a
      Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease
      Hart, Gerald W.; Slawson, Chad; Ramirez-Correa, Genaro; Lagerlof, Olof
      Annual Review of Biochemistry (2011), 80 (), 825-858CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
      A review. O-GlcNAcylation is the addn. of β-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1980s and still remains difficult to detect and quantify. Nonetheless, O-GlcNAc is highly abundant and cycles on proteins with a timescale similar to protein phosphorylation. O-GlcNAc occurs in organisms ranging from some bacteria to protozoans and metazoans, including plants and nematodes up the evolutionary tree to man. O-GlcNAcylation is mostly on nuclear proteins, but it occurs in all intracellular compartments, including mitochondria. Recent glycomic analyses have shown that O-GlcNAcylation has surprisingly extensive cross talk with phosphorylation, where it serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amts. of O-GlcNAcylation underlie the etiol. of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Current data justify extensive efforts toward a better understanding of this invisible, yet abundant, modification. As tools for the study of O-GlcNAc become more facile and available, exponential growth in this area of research will eventually take place.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptVCntr4%253D&md5=58f3a0d39238d6fffa91641b2db9252e
      (b) Hanover, J. A.; Krause, M. W.; Love, D. C. Nat. Rev. Mol. Cell. Biol. 2012, 13 (5) 312
      [Crossref], [PubMed], [CAS], Google Scholar
      1b
      Post-translational modifications Bittersweet memories: linking metabolism to epigenetics through O-GlcNAcylation
      Hanover, John A.; Krause, Michael W.; Love, Dona C.
      Nature Reviews Molecular Cell Biology (2012), 13 (5), 312-321CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. O-GlcNAcylation, which is a nutrient-sensitive sugar modification, participates in the epigenetic regulation of gene expression. The enzymes involved in O-linked β-D-N-acetylglucosamine (O-GlcNAc) cycling - O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) - target key transcriptional and epigenetic regulators including RNA polymerase II, histones, histone deacetylase complexes and members of the Polycomb and Trithorax groups. Thus, O-GlcNAc cycling may serve as a homeostatic mechanism linking nutrient availability to higher-order chromatin organization. In response to nutrient availability, O-GlcNAcylation is poised to influence X chromosome inactivation and genetic imprinting, as well as embryonic development. The wide range of physiol. functions regulated by O-GlcNAc cycling suggests an unexplored nexus between epigenetic regulation in disease and nutrient availability.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XlvVKmsL8%253D&md5=072d75b0082cfe3b138af45f7acb4f5f
    2. 2
      (a) Wells, L.; Kreppel, L. K.; Comer, F. I.; Wadzinski, B. E.; Hart, G. W. J. Biol. Chem. 2004, 279 (37) 38466
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
      (b) Yang, X. Y.; Ongusaha, P. P.; Miles, P. D.; Havstad, J. C.; Zhang, F. X.; So, W. V.; Kudlow, J. E.; Michell, R. H.; Olefsky, J. M.; Field, S. J.; Evans, R. M. Nature 2008, 451 (7181) 964
      [Crossref], [PubMed], [CAS], Google Scholar
      2b
      Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance
      Yang, Xiaoyong; Ongusaha, Pat P.; Miles, Philip D.; Havstad, Joyce C.; Zhang, Fengxue; So, W. Venus; Kudlow, Jeffrey E.; Michell, Robert H.; Olefsky, Jerrold M.; Field, Seth J.; Evans, Ronald M.
      Nature (London, United Kingdom) (2008), 451 (7181), 964-969CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degrdn. Here we show that O-GlcNAc transferase (OGT) harbors a previously unrecognized type of phosphoinositide-binding domain. After induction with insulin, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where the enzyme catalyzes dynamic modification of the insulin signaling pathway by O-GlcNAc. This results in the alteration in phosphorylation of key signaling mols. and the attenuation of insulin signal transduction. Hepatic overexpression of OGT impairs the expression of insulin-responsive genes and causes insulin resistance and dyslipidemia. These findings identify a mol. mechanism by which nutritional cues regulate insulin signaling through O-GlcNAc, and underscore the contribution of this modification to the etiol. of insulin resistance and type 2 diabetes.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXit1yns74%253D&md5=b4c33d036aea5d0ecd44978dfa823164
      (c) Lehman, D. M.; Fu, D. J.; Freeman, A. B.; Hunt, K. J.; Leach, R. J.; Johnson-Pais, T.; Hamlington, J.; Dyer, T. D.; Arya, R.; Abboud, H.; Goring, H. H. H.; Duggirala, R.; Blangero, J.; Konrad, R. J.; Stern, M. P. Diabetes 2005, 54 (4) 1214
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
      (d) Zachara, N. E.; O’Donnell, N.; Cheung, W. D.; Mercer, J. J.; Marth, J. D.; Hart, G. W. J. Biol. Chem. 2004, 279 (29) 30133
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
      (e) Slawson, C.; Zachara, N. E.; Vosseller, K.; Cheung, W. D.; Lane, M. D.; Hart, G. W. J. Biol. Chem. 2005, 280 (38) 32944
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
      (f) Lazarus, B. D.; Love, D. C.; Hanover, J. A. Int. J. Biochem. Cell Biol. 2009, 41 (11) 2134
      [Crossref], [PubMed], [CAS], Google Scholar
      2f
      O-GlcNAc cycling: Implications for neurodegenerative disorders
      Lazarus, Brooke D.; Love, Dona C.; Hanover, John A.
      International Journal of Biochemistry & Cell Biology (2009), 41 (11), 2134-2146CODEN: IJBBFU; ISSN:1357-2725. (Elsevier Ltd.)
      A review. The dynamic post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), termed O-GlcNAcylation, is an important mechanism for modulating cellular signaling pathways. O-GlcNAcylation impacts transcription, translation, organelle trafficking, proteasomal degrdn. and apoptosis. O-GlcNAcylation has been implicated in the etiol. of several human diseases including type-2 diabetes and neurodegeneration. This review describes the pair of enzymes responsible for the cycling of this post-translational modification: O-GlcNAc transferase (OGT) and β-N-acetylglucosaminidase (OGA), with a focus on the function of their structural domains. We will also highlight the important processes and substrates regulated by these enzymes, with an emphasis on the role of O-GlcNAc as a nutrient sensor impacting insulin signaling and the cellular stress response. Finally, we will focus attention on the many ways by which O-GlcNAc cycling may affect the cellular machinery in the neuroendocrine and central nervous systems.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFyntL3J&md5=70b2a12840567017d8e8a7b3b7fc0686
      (g) Caldwell, S. A.; Jackson, S. R.; Shahriari, K. S.; Lynch, T. P.; Sethi, G.; Walker, S.; Vosseller, K.; Reginato, M. J. Oncogene 2010, 29 (19) 2831
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
      (h) Ma, Z.; Vocadlo, D. J.; Vosseller, K. J. Biol. Chem. 2013, 288 (21) 15121
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
      (i) Lynch, T. P.; Ferrer, C. M.; Jackson, S. R.; Shahriari, K. S.; Vosseller, K.; Reginato, M. J. J. Biol. Chem. 2012, 287 (14) 11070
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    3. 3
      Hahne, H.; Sobotzki, N.; Nyberg, T.; Helm, D.; Borodkin, V. S.; van Aalten, D. M.; Agnew, B.; Kuster, B. J. Proteome. Res. 2013, 12 (2) 927
      [ACS Full Text ACS Full Text], [CAS], Google Scholar
      3
      Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry
      Hahne, Hannes; Sobotzki, Nadine; Nyberg, Tamara; Helm, Dominic; Borodkin, Vladimir S.; van Aalten, Daan M. F.; Agnew, Brian; Kuster, Bernhard
      Journal of Proteome Research (2013), 12 (2), 927-936CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      The post-translational modification of proteins with N-acetylglucosamine (O-GlcNAc) is involved in the regulation of a wide variety of cellular processes and assocd. with a no. of chronic diseases. Despite its emerging biol. significance, the systematic identification of O-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved O-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copper-mediated Click chem. for purifn. of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC-MS/MS afforded the identification of around 1500 O-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound O-GlcNAc peptides using selective β-elimination enabled the identification of 185 O-GlcNAc modification sites on 80 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the O-GlcNAcase inhibitor GlcNAcstatin G on the level of O-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signaling pathway showed significantly increased O-GlcNAcylation levels in response to the drug, which further strengthens the link of O-GlcNAc protein modification to cellular nutrient sensing and response.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXls1CgtA%253D%253D&md5=3ceafc8dcfb4ae70fab6c2239ffa09b5
    4. 4
      (a) Sprung, R.; Nandi, A.; Chen, Y.; Kim, S. C.; Barma, D.; Falck, J. R.; Zhao, Y. J. Proteome. Res. 2005, 4 (3) 950
      [ACS Full Text ACS Full Text], [CAS], Google Scholar
      4a
      Tagging-via-Substrate Strategy for Probing O-GlcNAc Modified Proteins
      Sprung, Robert; Nandi, Animesh; Chen, Yue; Kim, Sung Chan; Barma, Deb; Falck, John R.; Zhao, Yingming
      Journal of Proteome Research (2005), 4 (3), 950-957CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Identification of proteins bearing a specific post-translational modification would imply functions of the modification. Proteomic anal. of post-translationally modified proteins is usually challenging due to high complexity and wide dynamic range, as well as unavailability of efficient methods to enrich the proteins of interest. Here, the authors report a strategy for the detection, isolation, and profiling of O-linked N-acetylglucosamine (O-GlcNAc) modified proteins, which involves 3 steps: metabolic labeling of cells with an unnatural GlcNAc analog, peracetylated azido-GlcNAc; chemoselective conjugation of azido-GlcNAc modified proteins via the Staudinger ligation, which is specific between phosphine and azide, using a biotinylated phosphine capture reagent; and detection and affinity purifn. of the resulting conjugated O-GlcNAc modified proteins. Since the approach relies on a tag (azide) in the substrate, we designated it the tagging-via-substrate (TAS) strategy. A similar strategy was used previously for protein farnesylation, phosphorylation, and sumoylation. Using this approach, we were able to specifically label and subsequently detect azido-GlcNAc modified proteins from the cytosolic lysates of HeLa, 3T3, COS-1, and S2 cell lines, suggesting the azido-substrate could be tolerated by the enzymic systems among these cells from diverse biol. species. We isolated azido-GlcNAc modified proteins from the cytosolic ext. of S2 cells and identified 10 previously reported and 41 putative O-GlcNAc modified proteins, by nano-HPLC-MS/MS. Our study demonstrates that the TAS approach is a useful tool for the detection and proteomic anal. of O-GlcNAc modified proteins.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXkt1Kltrw%253D&md5=4942a1764cb55fb23d9fdadd57e9670e
      (b) Nandi, A.; Sprung, R.; Barma, D. K.; Zhao, Y.; Kim, S. C.; Falck, J. R.; Zhao, Y. Anal. Chem. 2006, 78 (2) 452
      [ACS Full Text ACS Full Text], Google Scholar
      There is no corresponding record for this reference.
      (c) Gurcel, C.; Vercoutter-Edouart, A. S.; Fonbonne, C.; Mortuaire, M.; Salvador, A.; Michalski, J. C.; Lemoine, J. Anal. Bioanal. Chem. 2008, 390 (8) 2089
      [Crossref], [PubMed], [CAS], Google Scholar
      4c
      Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging
      Gurcel, Caroline; Vercoutter-Edouart, Anne-Sophie; Fonbonne, Catherine; Mortuaire, Marlene; Salvador, Arnaud; Michalski, Jean-Claude; Lemoine, Jerome
      Analytical and Bioanalytical Chemistry (2008), 390 (8), 2089-2097CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells. This dynamic glycosylation plays a fundamental role in the activity of many nuclear and cytoplasmic proteins and is assocd. with pathologies like type II diabetes, Alzheimer's disease or some cancers. However the exact link between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Here the authors report a strategy based on the 1,3-dipolar cycloaddn., called click chem., between unnatural N-acetylglucosamine (GlcNAc) analogs (substituted with an azido or alkyne group) and the corresponding biotinylated probe to specifically detect, enrich and identify O-GlcNAc-modified proteins. This bio-orthogonal conjugation confirms that only azido analog of GlcNAc is metabolized by the cell. Thanks to the biotin probe, affinity purifn. on streptavidin beads allowed the authors to identify 32 O-GlcNAc-azido-tagged proteins by LC-MS/MS anal. in an MCF-7 cellular model, 14 of which were previously unreported. This work illustrates the use of the click-chem.-based strategy combined with a proteomic approach to get further insight into the pattern of O-GlcNAc-modified proteins and the biol. significance of this post-translational modification.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXktlags7w%253D&md5=a52d65f5f856d97608426010c96f5670
      (d) Zaro, B. W.; Yang, Y. Y.; Hang, H. C.; Pratt, M. R. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (20) 8146
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
      (e) Alfaro, J. F.; Gong, C. X.; Monroe, M. E.; Aldrich, J. T.; Clauss, T. R.; Purvine, S. O.; Wang, Z.; Camp, D. G., II; Shabanowitz, J.; Stanley, P.; Hart, G. W.; Hunt, D. F.; Yang, F.; Smith, R. D. Proc. Natl. Acad. Sci. U.S.A. 2012, 109 (19) 7280
      [Crossref], [PubMed], [CAS], Google Scholar
      4e
      Tandem mass spectrometry identifies many-mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets
      Alfaro, Joshua F.; Gong, Cheng-Xin; Monroe, Matthew E.; Aldrich, Joshua T.; Clauss, Therese R. W.; Purvine, Samuel O.; Wang, Zihao; Camp, David G., II; Shabanowitz, Jeffrey; Stanley, Pamela; Hart, Gerald W.; Hunt, Donald F.; Yang, Feng; Smith, Richard D.
      Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (19), 7280-7285, S7280/1-S7280/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chem./enzymic photochem. cleavage approach for enriching O-GlcNAcylated peptides in samples contg. ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we obsd. O-β-GIcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N-acetylglucosaminyltransferases.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnsVWksLw%253D&md5=ca88d9e5172172a3d69e81b789ae03b9
      (f) Khidekel, N.; Ficarro, S. B.; Peters, E. C.; Hsieh-Wilson, L. C. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (36) 13132
      [Crossref], [PubMed], [CAS], Google Scholar
      4f
      Exploring the O-GlcNAc proteome: Direct identification of O-GlcNAc-modified proteins from the brain
      Khidekel, Nelly; Ficarro, Scott B.; Peters, Eric C.; Hsieh-Wilson, Linda C.
      Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (36), 13132-13137CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The covalent modification of intracellular proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is emerging as a crucial regulatory posttranslational modification akin to phosphorylation. Numerous studies point to the significance of O-GlcNAc in cellular processes such as nutrient sensing, protein degrdn., and gene expression. Despite its importance, the breadth and functional roles of O-GlcNAc are only beginning to be elucidated. Advances in our understanding will require the development of new strategies for the detection and study of O-GlcNAc-modified proteins in vivo. Herein we report the direct, high-throughput anal. of O-GlcNAc-glycosylated proteins from the mammalian brain. The proteins were identified by using a chemoenzymic approach that exploits an engineered galactosyltransferase enzyme to selectively label O-GlcNAc proteins with a ketone-biotin tag. The tag permits enrichment of low-abundance O-GlcNAc species from complex mixts. and localization of the modification to short amino acid sequences. Using this approach, we discovered 25 O-GlcNAc-glycosylated proteins from the brain, including regulatory proteins assocd. with gene expression, neuronal signaling, and synaptic plasticity. The functional diversity represented by this set of proteins suggests an expanded role for O-GlcNAc in regulating neuronal function. Moreover, the chemoenzymic strategy described here should prove valuable for identifying O-GlcNAc-modified proteins in various tissues and facilitate studies of the physiol. significance of O-GlcNAc across the proteome.
      >> More from SciFinder ®
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    5. 5
      MacBeath, G.; Schreiber, S. L. Science 2000, 289 (5485) 1760
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    6. 6
      (a)

      A small kinase microarray in which 3H-GlcNAc was detected was used to identify kinase substrates of OGT. See:

      Google Scholar
      There is no corresponding record for this reference.
      (b) Dias, W. B.; Cheung, W. D.; Hart, G. W. Biochem. Biophys. Res. Commun. 2012, 422 (2) 224
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    7. 7
      Beby, F.; Lamonerie, T. Exp. Eye. Res. 2013, 111, 9
      [Crossref], [PubMed], [CAS], Google Scholar
      7
      The homeobox gene Otx2 in development and disease
      Beby, Francis; Lamonerie, Thomas
      Experimental Eye Research (2013), 111 (), 9-16CODEN: EXERA6; ISSN:0014-4835. (Elsevier Ltd.)
      A review. The Otx2 gene encodes a transcription factor essential for the normal development of brain, cerebellum, pineal gland, and eye. In the retina, Otx2 has essential functions from early embryogenesis to adulthood. As soon as the optic vesicle is formed, the gene is required for retinal pigment epithelium specification. Otx2 is also a key regulator of photoreceptor genesis and differentiation, and is required after birth for bipolar cells terminal maturation. Otx2 expression is maintained in the differentiated retina wherein the gene is crit. for the outer retina maintenance. In the visual cortex, the gene modulates the neuronal plasticity through a paracrine mechanism. OTX2 heterozygous mutations in humans have been linked to severe ocular malformations assocd. with brain abnormalities and pituitary dysfunction. Recent studies have also established the OTX2 gene as an oncogene for medulloblastoma, a malignant brain tumor originating in the cerebellum.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXot1Kkurg%253D&md5=e9374d523dfa130f8e17ef4897fa63fd
    8. 8
      Jinek, M.; Rehwinkel, J.; Lazarus, B. D.; Izaurralde, E.; Hanover, J. A.; Conti, E. Nat. Struct. Mol. Biol. 2004, 11 (10) 1001
      [Crossref], [PubMed], [CAS], Google Scholar
      8
      The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin α
      Jinek, Martin; Rehwinkel, Jan; Lazarus, Brooke D.; Izaurralde, Elisa; Hanover, John A.; Conti, Elena
      Nature Structural & Molecular Biology (2004), 11 (10), 1001-1007CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      Addn. of N-acetylglucosamine (GlcNAc) is a ubiquitous form of intracellular glycosylation catalyzed by the conserved O-linked GlcNAc transferase (OGT). OGT contains an N-terminal domain of tetratricopeptide (TPR) repeats that mediates the recognition of a broad range of target proteins. Components of the nuclear pore complex are major OGT targets, as OGT depletion by RNA interference (RNAi) results in the loss of GlcNAc modification at the nuclear envelope. To gain insight into the mechanism of target recognition, we solved the crystal structure of the homodimeric TPR domain of human OGT, which contains 11.5 TPR repeats. The repeats form an elongated superhelix. The concave surface of the superhelix is lined by absolutely conserved asparagines, in a manner reminiscent of the peptide-binding site of importin α. Based on this structural similarity, we propose that OGT uses an analogous mol. mechanism to recognize its targets.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXnvVOnsLw%253D&md5=aff0a8c9b455734b3e94da8d1a58cbbb
    9. 9
      Lazarus, B. D.; Love, D. C.; Hanover, J. A. Glycobiology 2006, 16 (5) 415
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    10. 10
      (a) Merbl, Y.; Kirschner, M. W. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (8) 2543
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
      (b) Gross, B. J.; Kraybill, B. C.; Walker, S. J. Am. Chem. Soc. 2005, 127 (42) 14588
      [ACS Full Text ACS Full Text], Google Scholar
      There is no corresponding record for this reference.
    11. 11
      Comer, F. I.; Vosseller, K.; Wells, L.; Accavitti, M. A.; Hart, G. W. Anal. Biochem. 2001, 293 (2) 169
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    12. 12
      Isono, T. PLoS One 2011, 6 (4) e18959
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    13. 13
      (a) Whelan, S. A.; Dias, W. B.; Thiruneelakantapillai, L.; Lane, M. D.; Hart, G. W. J. Biol. Chem. 2010, 285 (8) 5204
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
      (b) Wang, S.; Huang, X.; Sun, D.; Xin, X.; Pan, Q.; Peng, S.; Liang, Z.; Luo, C.; Yang, Y.; Jiang, H.; Huang, M.; Chai, W.; Ding, J.; Geng, M. PLoS One 2012, 7 (5) e37427
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
      (c) Dentin, R.; Hedrick, S.; Xie, J.; Yates, J., III; Montminy, M. Science 2008, 319 (5868) 1402
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
      (d) Tarrant, M. K.; Rho, H. S.; Xie, Z.; Jiang, Y. L.; Gross, C.; Culhane, J. C.; Yan, G.; Qian, J.; Ichikawa, Y.; Matsuoka, T.; Zachara, N.; Etzkorn, F. A.; Hart, G. W.; Jeong, J. S.; Blackshaw, S.; Zhu, H.; Cole, P. A. Nat. Chem. Biol. 2012, 8 (3) 262
      [Crossref], [PubMed], [CAS], Google Scholar
      13d
      Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis
      Tarrant, Mary Katherine; Rho, Hee-Sool; Xie, Zhi; Jiang, Yu Lin; Gross, Christopher; Culhane, Jeffrey C.; Yan, Gai; Qian, Jiang; Ichikawa, Yoshitaka; Matsuoka, Tatsuji; Zachara, Natasha; Etzkorn, Felicia A.; Hart, Gerald W.; Jeong, Jun Seop; Blackshaw, Seth; Zhu, Heng; Cole, Philip A.
      Nature Chemical Biology (2012), 8 (3), 262-269CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked β-N-acetylglucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2α by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degrdn. By performing kinase assays with site-specifically phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biol. outputs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVKjtbY%253D&md5=6854c5234c1087ff123c580496eeb2c2
      (e)

      11 out of 80 validated O-GlcNAc proteins discovered in ref 3 were also present on the protein microarray; 5 were in our top 2% of hits, and 7 were in the top 6%.

      Google Scholar
      There is no corresponding record for this reference.
    14. 14
      Tai, H. C.; Khidekel, N.; Ficarro, S. B.; Peters, E. C.; Hsieh-Wilson, L. C. J. Am. Chem. Soc. 2004, 126 (34) 10500
      [ACS Full Text ACS Full Text], Google Scholar
      There is no corresponding record for this reference.
    15. 15
      Merbl, Y.; Refour, P.; Patel, H.; Springer, M.; Kirschner, M. W. Cell 2013, 152 (5) 1160
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    16. 16
      Boyce, M.; Bertozzi, C. R. Nat. Methods 2011, 8 (8) 638
      [Crossref], [PubMed], [CAS], Google Scholar
      16
      Bringing chemistry to life
      Boyce, Michael; Bertozzi, Carolyn R.
      Nature Methods (2011), 8 (8), 638-642CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Bioorthogonal chem. allows a wide variety of biomols. to be specifically labeled and probed in living cells and whole organisms. Here we discuss the history of bioorthogonal reactions and some of the most interesting and important advances in the field.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXpsFaht70%253D&md5=8ad08fe14193afffdc99fca2d7c92d06
    17. 17
      Vocadlo, D. J.; Hang, H. C.; Kim, E. J.; Hanover, J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (16) 9116
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    18. 18
      Yao, J. Z.; Uttamapinant, C.; Poloukhtine, A.; Baskin, J. M.; Codelli, J. A.; Sletten, E. M.; Bertozzi, C. R.; Popik, V. V.; Ting, A. Y. J. Am. Chem. Soc. 2012, 134 (8) 3720
      [ACS Full Text ACS Full Text], Google Scholar
      There is no corresponding record for this reference.
    19. 19
      Holt, G. D.; Snow, C. M.; Senior, A.; Haltiwanger, R. S.; Gerace, L.; Hart, G. W. J. Cell. Biol. 1987, 104 (5) 1157
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    20. 20
      Slawson, C.; Hart, G. W. Nat. Rev. Cancer 2011, 11 (9) 678
      [Crossref], [PubMed], [CAS], Google Scholar
      20
      O-GlcNAc signalling: implications for cancer cell biology
      Slawson, Chad; Hart, Gerald W.
      Nature Reviews Cancer (2011), 11 (9), 678-684CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
      A review. O-GlcNAcylation is the covalent attachment of β-D-N-acetylglucosamine (GlcNAc) sugars to serine or threonine residues of nuclear and cytoplasmic proteins, and it is involved in extensive crosstalk with other post-translational modifications, such as phosphorylation. O-GlcNAcylation is becoming increasing realized as having important roles in cancer-relevant processes, such as cell signalling, transcription, cell division, metab. and cytoskeletal regulation. However, currently little is known about the specific roles of aberrant O-GlcNAcylation in cancer. In this Opinion article, we summarize the current understanding of O-GlcNAcylation in cancer and its emerging functions in transcriptional regulation at the level of chromatin and transcription factors.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVehs73E&md5=b0daec25f9b979d512af2282054b9115
    21. 21
      Rexach, J. E.; Rogers, C. J.; Yu, S. H.; Tao, J.; Sun, Y. E.; Hsieh-Wilson, L. C. Nat. Chem. Biol. 2010, 6 (9) 645
      [Crossref], [PubMed], [CAS], Google Scholar
      21
      Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags
      Rexach, Jessica E.; Rogers, Claude J.; Yu, Seok-Ho; Tao, Jifang; Sun, Yi E.; Hsieh-Wilson, Linda C.
      Nature Chemical Biology (2010), 6 (9), 645-651CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here the authors describe a powerful method to visualize the O-GlcNAc-modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purifn., advanced instrumentation or expensive radiolabels. In addn., it establishes the glycosylation state (for example, mono-, di-, tri-) of proteins, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, the authors apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation and discover an unexpected reverse 'yin-yang' relation on the transcriptional repressor MeCP2 that was undetectable by traditional methods. The authors anticipate that this mass-tagging strategy will advance the authors' understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXpt1ars7k%253D&md5=1250b849843a3526bb0d6cfb1996aabe
    22. 22
      Adamson, D. C.; Shi, Q.; Wortham, M.; Northcott, P. A.; Di, C.; Duncan, C. G.; Li, J.; McLendon, R. E.; Bigner, D. D.; Taylor, M. D.; Yan, H. Cancer Res. 2010, 70 (1) 181
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    23. 23
      Bai, R. Y.; Staedtke, V.; Lidov, H. G.; Eberhart, C. G.; Riggins, G. J. Cancer Res. 2012, 72 (22) 5988
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    24. 24
      Jackson, S. P.; Tjian, R. Cell 1988, 55 (1) 125
      [Crossref], Google Scholar
      There is no corresponding record for this reference.
    25. 25
      Gloster, T. M.; Zandberg, W. F.; Heinonen, J. E.; Shen, D. L.; Deng, L.; Vocadlo, D. J. Nat. Chem. Biol. 2011, 7 (3) 174
      [Crossref], [PubMed], [CAS], Google Scholar
      25
      Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells
      Gloster, Tracey M.; Zandberg, Wesley F.; Heinonen, Julia E.; Shen, David L.; Deng, Le-Hua; Vocadlo, David J.
      Nature Chemical Biology (2011), 7 (3), 174-181CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Glycosyltransferases are ubiquitous enzymes that catalyze the assembly of glycoconjugates throughout all kingdoms of nature. A long-standing problem is the rational design of probes that can be used to manipulate glycosyltransferase activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analog that inhibits, with high potency both in vitro and in cells, the human glycosyltransferase responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show that the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analog. Treatment of cells with the precursor lowers O-GlcNAc in a targeted manner with a single-digit micromolar EC50. This approach to inhibition of glycosyltransferases should be applicable to other members of this superfamily of enzymes and enable their manipulation in a biol. setting.
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    Experimental procedures, microarray protocol and data analysis, IVT validation data, and list of OGT targets. This material is available free of charge via the Internet at http://pubs.acs.org.


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