The vast array of cell types of multicellular organisms must individually fine-tune their internal metabolism. One important metabolic and stress regulatory mechanism is the dynamic attachment/removal of glucose-derived sugar N-acetylglucosamine on proteins (O-GlcNAcylation). The number of proteins modified by O-GlcNAc is bewildering, with at least 7,000 sites in human cells. The outstanding challenge is determining how key O-GlcNAc sites regulate a target pathway amidst thousands of potential global sites. Innovative solutions are required to address this challenge in cell models and disease therapy. This Perspective shares critical suggestions for the O-GlcNAc field gleaned from the international O-GlcNAc community. Further, we summarize critical tools and tactics to enable newcomers to O-GlcNAc biology to drive innovation at the interface of metabolism and disease. The growing pace of O-GlcNAc research makes this a timely juncture to involve a wide array of scientists and new toolmakers to selectively approach the regulatory roles of O-GlcNAc in disease.
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Molecular Cancer Open Access 30 June 2023
Hyperglycemia and O-GlcNAc transferase activity drive a cancer stem cell pathway in triple-negative breast cancer
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Hart, G. W. Nutrient regulation of signaling and transcription. J. Biol. Chem. 294, 2211–2231 (2019).
Yang, X. & Qian, K. Protein O-GlcNAcylation: emerging mechanisms and functions. Nat. Rev. Mol. Cell Biol. 18, 452–465 (2017).
Ferrer, C. M., Sodi, V. L. & Reginato, M. J. O-GlcNAcylation in cancer biology: linking metabolism and signaling. J. Mol. Biol. 428, 3282–3294 (2016).
Hanover, J. A., Chen, W. & Bond, M. R. O-GlcNAc in cancer: an oncometabolism-fueled vicious cycle. J. Bioenerg. Biomembr. 50, 155–173 (2018).
Ma, J. & Hart, G. W. Protein O-GlcNAcylation in diabetes and diabetic complications. Expert Rev. Proteom. 10, 365–380 (2013).
Wang, A. C., Jensen, E. H., Rexach, J. E., Vinters, H. V. & Hsieh-Wilson, L. C. Loss of O-GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration. Proc. Natl Acad. Sci. USA 113, 15120–15125 (2016). This paper used conditional knockout of OGT to precisely determine hippocampal neuron-specific roles for O-GlcNAc, especially neurodegeneration versus neuroprotection effects.
Wheatley, E. G. et al. Neuronal O-GlcNAcylation improves cognitive function in the aged mouse brain. Curr. Biol. 29, 3359–3369 (2019).
Martinez, M. R., Dias, T. B., Natov, P. S. & Zachara, N. E. Stress-induced O-GlcNAcylation: an adaptive process of injured cells. Biochem. Soc. Trans. 45, 237–249 (2017).
Yang, Y. R. & Suh, P. G. O-GlcNAcylation in cellular functions and human diseases. Adv. Biol. Regul. 54, 68–73 (2014).
Slawson, C., Copeland, R. J. & Hart, G. W. O-GlcNAc signaling: a metabolic link between diabetes and cancer? Trends Biochem. Sci. 35, 547–555 (2010).
Wulff-Fuentes, E. et al. The human O-GlcNAcome database and meta-analysis. Sci. Data 8, 25 (2021).
Ma, J., Li, Y., Hou, C. & Wu, C. O-GlcNAcAtlas: a database of experimentally identified O-GlcNAc sites and proteins. Glycobiology 31, 719–723 (2021).
Shafi, R. et al. The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny. Proc. Natl Acad. Sci. USA 97, 5735–5739 (2000).
O’Donnell, N., Zachara, N. E., Hart, G. W. & Marth, J. D. OGT-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability. Mol. Cell. Biol. 24, 1680–1690 (2004).
Yang, Y. R. et al. O-GlcNAcase is essential for embryonic development and maintenance of genomic stability. Aging Cell 11, 439–448 (2012).
Marshall, S., Nadeau, O. & Yamasaki, K. Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes. J. Biol. Chem. 279, 35313–35319 (2004).
Zachara, N. E., Molina, H., Wong, K. Y., Pandey, A. & Hart, G. W. The dynamic stress-induced “O-GlcNAc-ome” highlights functions for O-GlcNAc in regulating DNA damage/repair and other cellular pathways. Amino Acids 40, 793–808 (2011).
Lu, L. et al. Distributive O-GlcNAcylation on the highly repetitive C-terminal domain of RNA polymerase II. Biochemistry 55, 1149–1158 (2016).
Tan, Z.-W. et al. O-GlcNAc regulates gene expression by controlling detained intron splicing. Nucleic Acids Res. 48, 5656–5669 (2020). This paper identifies an elegant mechanism for rapid re-balancing of OGT/OGA activity after O-GlcNAc disruption via O-GlcNAc-induced alternative splicing.
Miller, M. W., Caracciolo, M. R., Berlin, W. K. & Hanover, J. A. Phosphorylation and glycosylation of nucleoporins. Arch. Biochem. Biophys. 367, 51–60 (1999).
Eustice, M., Bond, M. R. & Hanover, J. A. O-GlcNAc cycling and the regulation of nucleocytoplasmic dynamics. Biochem. Soc. Trans. 45, 427–436 (2017).
Groenevelt, J. M., Corey, D. J. & Fehl, C. Chemical synthesis and biological applications of O-GlcNAcylated peptides and proteins. ChemBioChem 22, 1854–1870 (2021). This paper collects known synthetic methods for site-specific O-GlcNAc installation. Also see refs. 23 and 25 for thorough collections of O-GlcNAc detection tools and assays.
Gorelik, A. & van Aalten, D. M. F. Tools for functional dissection of site-specific O-GlcNAcylation. RSC Chem. Biol. 1, 98–109 (2020).
Stoevesandt, O. & Taussig, M. J. Phospho-specific antibodies by design. Nat. Biotechnol. 31, 889–891 (2013).
Alteen, M. G., Tan, H. Y. & Vocadlo, D. J. Monitoring and modulating O-GlcNAcylation: assays and inhibitors of O-GlcNAc processing enzymes. Curr. Opin. Struct. Biol. 68, 157–165 (2021).
Martin, S. E. S. et al. Structure-based evolution of low nanomolar O-GlcNAc transferase inhibitors. J. Am. Chem. Soc. 140, 13542–13545 (2018).
Gloster, T. M. et al. Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells. Nat. Chem. Biol. 7, 174–181 (2011).
Ju Kim, E. O-GlcNAc transferase: structural characteristics, catalytic mechanism and small-molecule inhibitors. ChemBioChem 21, 3026–3035 (2020).
Liu, T. W. et al. Metabolic inhibitors of O-GlcNAc transferase that act in vivo implicate decreased O-GlcNAc levels in leptin-mediated nutrient sensing. Angew. Chem. Int. Ed. Engl. 57, 7644–7648 (2018).
Elbatrawy, A. A., Kim, E. J. & Nam, G. O-GlcNAcase: emerging mechanism, substrate recognition and small-molecule inhibitors. ChemMedChem 15, 1244–1257 (2020).
Selnick, H. G. et al. Discovery of MK-8719, a potent O-GlcNAcase inhibitor as a potential treatment for tauopathies. J. Med. Chem. 62, 10062–10097 (2019). This paper reports the medicinal chemistry efforts that led to the first FDA-sanctioned inhibitor of OGA in human disease.
Carrillo, L. D., Krishnamoorthy, L. & Mahal, L. K. A cellular FRET-based sensor for β-O-GlcNAc, a dynamic carbohydrate modification involved in signaling. J. Am. Chem. Soc. 128, 14768–14769 (2006).
Carrillo, L. D., Froemming, J. A. & Mahal, L. K. Targeted in vivo O-GlcNAc sensors reveal discrete compartment-specific dynamics during signal transduction. J. Biol. Chem. 286, 6650–6658 (2011).
Cecioni, S. & Vocadlo, D. J. Carbohydrate bis-acetal-based substrates as tunable fluorescence-quenched probes for monitoring exo-glycosidase activity. J. Am. Chem. Soc. 139, 8392–8395 (2017).
Lee, J.-H. et al. PET quantification of brain O-GlcNAcase with [18F]LSN3316612 in healthy human volunteers. EJNMMI Res. 10, 20 (2020).
Paul, S. et al. Evaluation of a PET radioligand to image O-GlcNAcase in brain and periphery of rhesus monkey and knock-out mouse. J. Nucl. Med. 60, 129–134 (2019).
Aguilar, A. L., Hou, X., Wen, L., Wang, P. G. & Wu, P. A chemoenzymatic histology method for O-GlcNAc detection. ChemBioChem 18, 2416–2421 (2017).
Haynes, P. A. & Aebersold, R. Simultaneous detection and identification of O-GlcNAc-modified glycoproteins using liquid chromatography−tandem mass spectrometry. Anal. Chem. 72, 5402–5410 (2000).
Vosseller, K. et al. Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol. Proteomics 5, 388–398 (2005).
Vosseller, K. et al. O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry. Mol. Cell. Proteomics 5, 923–934 (2006).
Chalkley, R. J., Thalhammer, A., Schoepfer, R. & Burlingame, A. L. Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides. Proc. Natl Acad. Sci. USA 106, 8894–8899 (2009).
Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc. Natl Acad. Sci. USA 109, 7280–7285 (2012).
Darabedian, N. & Pratt, M. R. Identifying potentially O-GlcNAcylated proteins using metabolic labeling, bioorthogonal enrichment, and Western blotting. Methods Enzymol. 622, 293–307 (2019).
Qin, W. et al. Artificial cysteine S-glycosylation induced by per-O-acetylated unnatural monosaccharides during metabolic glycan labeling. Angew. Chem. Int. Ed. Engl. 57, 1817–1820 (2018). This paper reports off-target S-GlcNAcylated ‘artifacts’ in metabolic labeling, suggesting that stringent validation of O-GlcNAc proteomic studies is required.
Pedowitz, N. J. et al. Anomeric fatty acid functionalization prevents nonenzymatic S-glycosylation by monosaccharide metabolic chemical reporters. ACS Chem. Biol. https://doi.org/10.1021/acschembio.1c00470 (2021).
Hao, Y. et al. Next-generation unnatural monosaccharides reveal that ESRRB O-GlcNAcylation regulates pluripotency of mouse embryonic stem cells. Nat. Commun. 10, 4065 (2019).
Khidekel, N. et al. A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. J. Am. Chem. Soc. 125, 16162–16163 (2003).
Woo, C. M. et al. Mapping and quantification of over 2000 O-linked glycopeptides in activated human T cells with isotope-targeted glycoproteomics (Isotag). Mol. Cell. Proteomics 17, 764–775 (2018). This paper reports exquisitely sensitive O-GlcNAc labeling tools with isotope tagging to enable global characterization of O-GlcNAc sites during T cell activation events.
Li, J. et al. An isotope-coded photocleavable probe for quantitative profiling of protein O-GlcNAcylation. ACS Chem. Biol. 14, 4–10 (2019).
Levine, P. M. et al. α-Synuclein O-GlcNAcylation alters aggregation and toxicity, revealing certain residues as potential inhibitors of Parkinson’s disease. Proc. Natl Acad. Sci. USA 116, 1511–1519 (2019).
Galesic, A. et al. Comparison of N-acetyl-glucosamine to other monosaccharides reveals structural differences for the inhibition of α-synuclein aggregation. ACS Chem. Biol. 16, 14–19 (2021).
Schwagerus, S., Reimann, O., Despres, C., Smet-Nocca, C. & Hackenberger, C. P. Semi-synthesis of a tag-free O-GlcNAcylated tau protein by sequential chemoselective ligation. J. Pept. Sci. 22, 327–333 (2016).
Lin, W., Gao, L. & Chen, X. Protein-specific imaging of O-GlcNAcylation in single cells. ChemBioChem 16, 2571–2575 (2015).
Ramirez, D. H. et al. Engineering a proximity-directed O-GlcNAc transferase for selective protein O-GlcNAcylation in cells. ACS Chem. Biol. 15, 1059–1066 (2020).
Ge, Y. et al. Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase. Nat. Chem. Biol. 17, 593–600 (2021).
Boulard, M., Rucli, S., Edwards, J. R. & Bestor, T. H. Methylation-directed glycosylation of chromatin factors represses retrotransposon promoters. Proc. Natl Acad. Sci. USA 117, 14292–14298 (2020).
Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018).
Gorelik, A. et al. Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation. Nat. Struct. Mol. Biol. 26, 1071–1077 (2019).
Macauley, M. S., Stubbs, K. A. & Vocadlo, D. J. O-GlcNAcase catalyzes cleavage of thioglycosides without general acid catalysis. J. Am. Chem. Soc. 127, 17202–17203 (2005).
Kim, E. Y. et al. A role for O-GlcNAcylation in setting circadian clock speed. Genes Dev. 26, 490–502 (2012).
Keembiyehetty, C. et al. Conditional knock-out reveals a requirement for O-linked N-acetylglucosaminase (O-GlcNAcase) in metabolic homeostasis. J. Biol. Chem. 290, 7097–7113 (2015).
Okuyama, R. & Marshall, S. UDP-N-acetylglucosaminyl transferase (OGT) in brain tissue: temperature sensitivity and subcellular distribution of cytosolic and nuclear enzyme. J. Neurochem. 86, 1271–1280 (2003).
Levine, Z. G. et al. Mammalian cell proliferation requires noncatalytic functions of O-GlcNAc transferase. Proc. Natl Acad. Sci. USA 118, e2016778118 (2021). OGT has distinct protein-regulatory roles through three mechanisms: O-GlcNAc catalytic modification of proteins, O-GlcNAc-driven proteolysis, and non-catalytic functions. Rapid OGT regulatory tools are also developed herein.
Konzman, D. et al. O-GlcNAc: regulator of signaling and epigenetics linked to X-linked intellectual disability. Front. Genet. 11, 605263 (2020).
Pravata, V. M. et al. An intellectual disability syndrome with single-nucleotide variants in O-GlcNAc transferase. Eur. J. Hum. Genet 28, 706–714 (2020). X-linked intellectual disability is the first human disease implicated with OGT single-nucleotide polymorphisms, indicating a genetic basis (also see ref. 64).
Yuzwa, S. A. et al. A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo. Nat. Chem. Biol. 4, 483–490 (2008).
Stewart, L. T. et al. Acute increases in protein O-GlcNAcylation dampen epileptiform activity in hippocampus. J. Neurosci. 37, 8207–8215 (2017).
Lagerlöf, O. et al. The nutrient sensor OGT in PVN neurons regulates feeding. Science 351, 1293–1296 (2016). This paper reveals that organismal feeding behavior is controlled by O-GlcNAc, which is revealed to influence appetite at the molecular level in PVN neurons.
Vaidyanathan, K. & Wells, L. Multiple tissue-specific roles for the O-GlcNAc post-translational modification in the induction of and complications arising from type II diabetes. J. Biol. Chem. 289, 34466–34471 (2014).
Chen, P.-H. et al. Gigaxonin glycosylation regulates intermediate filament turnover and may impact giant axonal neuropathy etiology or treatment. JCI Insight 5, e127751 (2019).
Abramowitz, L. K., Harly, C., Das, A., Bhandoola, A. & Hanover, J. A. Blocked O-GlcNAc cycling disrupts mouse hematopoeitic stem cell maintenance and early T cell development. Sci. Rep. 9, 12569 (2019).
Baumann, D. et al. Role of nutrient-driven O-GlcNAc-post-translational modification in pancreatic exocrine and endocrine islet development. Development 147, dev186643 (2020).
Hruby, A. & Hu, F. B. The epidemiology of obesity: a big picture. Pharmacoeconomics 33, 673–689 (2015).
Wang, Z. et al. Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis. Sci. Signal. 3, ra2 (2010).
Leney, A. C., El Atmioui, D., Wu, W., Ovaa, H. & Heck, A. J. R. Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation. Proc. Natl Acad. Sci. USA 114, E7255–E7261 (2017).
Yang, X. et al. Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance. Nature 451, 964–969 (2008).
Lambert, M., Bastide, B. & Cieniewski-Bernard, C. Involvement of O-GlcNAcylation in the skeletal muscle physiology and physiopathology: focus on muscle metabolism. Front. Endocrinol. (Lausanne) 9, 578 (2018).
Yang, Y. et al. O-GlcNAc transferase inhibits visceral fat lipolysis and promotes diet-induced obesity. Nat. Commun. 11, 181 (2020).
Parker, M. P., Peterson, K. R. & Slawson, C. O-GlcNAcylation and O-GlcNAc cycling regulate gene transcription: emerging roles in cancer. Cancers 13, 1666 (2021).
Ozcan, S., Andrali, S. S. & Cantrell, J. E. Modulation of transcription factor function by O-GlcNAc modification. Biochim. Biophys. Acta 1799, 353–364 (2010).
Akella, N. M. et al. O-GlcNAc transferase regulates cancer stem-like potential of breast cancer cells. Mol. Cancer Res. 18, 585–598 (2020).
Hrit, J. et al. OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development. eLife 7, e34870 (2018).
Hornbeck, P. V. et al. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 43, D512–D520 (2015).
York, W. S. et al. GlyGen: computational and informatics resources for glycoscience. Glycobiology 30, 72–73 (2020).
Darabedian, N., Thompson, J. W., Chuh, K. N., Hsieh-Wilson, L. C. & Pratt, M. R. Optimization of chemoenzymatic mass tagging by strain-promoted cycloaddition (SPAAC) for the determination of O-GlcNAc stoichiometry by Western blotting. Biochemistry 57, 5769–5774 (2018).
Wells, L. et al. Mapping sites of <em>O</em>-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol. Cell. Proteomics 1, 791–804 (2002).
Gupta, R. & Brunak, S. Prediction of glycosylation across the human proteome and the correlation to protein function. Pac. Symp. Biocomput. 310–322 (2002).
Jia, C., Zuo, Y. & Zou, Q. O-GlcNAcPRED-II: an integrated classification algorithm for identifying O-GlcNAcylation sites based on fuzzy undersampling and a K-means PCA oversampling technique. Bioinformatics 34, 2029–2036 (2018).
Jochmann, R., Holz, P., Sticht, H. & Sturzl, M. Validation of the reliability of computational O-GlcNAc prediction. Biochim. Biophys. Acta 1844, 416–421 (2014).
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
McKitrick, T. R. et al. Development of smart anti-glycan reagents using immunized lampreys. Commun. Biol. 3, 91 (2020).
Zichel, R., Chearwae, W., Pandey, G. S., Golding, B. & Sauna, Z. E. Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins. PLoS One 7, e31948 (2012).
Levine, Z. G. & Walker, S. The biochemistry of O-GlcNAc transferase: which functions make it essential in mammalian cells? Annu. Rev. Biochem. 85, 631–657 (2016).
Shen, D. L., Gloster, T. M., Yuzwa, S. A. & Vocadlo, D. J. Insights into O-linked N-acetylglucosamine ([0-9]O-GlcNAc) processing and dynamics through kinetic analysis of O-GlcNAc transferase and O-GlcNAcase activity on protein substrates. J. Biol. Chem. 287, 15395–15408 (2012). Detailed in vitro kinetic studies of OGT and OGA reveal mechanistic roles in nutrient sensing, adding to a rich body of OGT and OGA mechanistic studies.
Levine, Z. G. et al. O-GlcNAc transferase recognizes protein substrates using an asparagine ladder in the tetratricopeptide repeat (TPR) superhelix. J. Am. Chem. Soc. 140, 3510–3513 (2018).
Kositzke, A. et al. Elucidating the protein substrate recognition of O-GlcNAc transferase (OGT) toward O-GlcNAcase (OGA) using a GlcNAc electrophilic probe. Int. J. Biol. Macromol. 169, 51–59 (2021).
Toleman, C. A. et al. Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins. Proc. Natl Acad. Sci. USA 115, 5956–5961 (2018). O-GlcNAcylation was long thought to prevent protein-protein interactions, but this report shows O-GlcNAc-driven interactions for the first time, confirmed by structural biology.
Myers, S. A. et al. SOX2 O-GlcNAcylation alters its protein-protein interactions and genomic occupancy to modulate gene expression in pluripotent cells. eLife 5, e10647 (2016).
Yu, S.-H. et al. Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners. Proc. Natl Acad. Sci. USA 109, 4834–4839 (2012).
Balana, A. T. et al. O-GlcNAc modification of small heat shock proteins enhances their anti-amyloid chaperone activity. Nat. Chem. 13, 441–450 (2021).
We thank P. Marino, Program Director at the US National Institute of General Medical Sciences (NIGMS) for her commitment and vision in expanding glycoscience as a national priority. We thank M. Bond and K. Krueger, Program Officers, for their support with glycobiology tools and for discussions and notes. We thank all participants of the international O-GlcNAc workshop, held in March 2020, who contributed to discussion, both during and after the meeting. We thank NIGMS for grant no. 1R35GM142637-01 (to C.F.) and the National Institutes of Diabetes and Digestive and Kidney Diseases for financial support (to J.A.H.).
The authors declare no competing interests.
Peer review information Nature Chemical Biology thanks Matthew Pratt, Xiaoyong Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Fehl, C., Hanover, J.A. Tools, tactics and objectives to interrogate cellular roles of O-GlcNAc in disease. Nat Chem Biol 18, 8–17 (2022). https://doi.org/10.1038/s41589-021-00903-6
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