Abstract

Protein O-GlcNAcylation is a reversible post-translational modification of serines and threonines on nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is essential for early embryogenesis. The Drosophila melanogaster gene supersex combs (sxc), which encodes OGT, is a polycomb gene, whose null mutants display homeotic transformations and die at the pharate adult stage. However, the identities of the O-GlcNAcylated proteins involved and the underlying mechanisms linking these phenotypes to embryonic development are poorly understood. Identification of O-GlcNAcylated proteins from biological samples is hampered by the low stoichiometry of this modification and by limited enrichment tools. Using a catalytically inactive bacterial O-GlcNAcase mutant as a substrate trap, we have enriched the O-GlcNAc proteome of the developing Drosophila embryo, identifying, among others, known regulators of Hox genes as candidate conveyors of OGT function during embryonic development.

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References

  1. 1.

    , , & Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease. Annu. Rev. Biochem. 80, 825–858 (2011).

  2. 2.

    et al. O-GlcNAc-modification of SNAP-29 regulates autophagosome maturation. Nat. Cell Biol. 16, 1215–1226 (2014).

  3. 3.

    A gene that regulates the bithorax complex differentially in larval and adult cells of Drosophila. Cell 37, 815–823 (1984).

  4. 4.

    Genetic control of the spatial pattern of selector gene expression in Drosophila. Cold Spring Harb. Symp. Quant. Biol. 50, 201–208 (1985).

  5. 5.

    et al. O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development. BMC Dev. Biol. 9, 28 (2009).

  6. 6.

    , & Pilot morpholino screen in Xenopus tropicalis identifies a novel gene involved in head development. Dev. Dyn. 229, 289–299 (2004).

  7. 7.

    , & Essential role of the glycosyltransferase sxc/Ogt in polycomb repression. Science 325, 93–96 (2009).

  8. 8.

    et al. Drosophila O-GlcNAc transferase (OGT) is encoded by the Polycomb group (PcG) gene, super sex combs (sxc). Proc. Natl. Acad. Sci. USA 106, 13427–13432 (2009).

  9. 9.

    & O-GlcNAcylation prevents aggregation of the Polycomb group repressor polyhomeotic. Dev. Cell 31, 629–639 (2014).

  10. 10.

    & A critical perspective of the diverse roles of O-GlcNAc transferase in chromatin. Chromosoma 124, 429–442 (2015).

  11. 11.

    , , & Blocking O-linked GlcNAc cycling in Drosophila insulin-producing cells perturbs glucose-insulin homeostasis. J. Biol. Chem. 285, 38684–38691 (2010).

  12. 12.

    & O-GlcNAcylation of a circadian clock protein: dPER taking its sweet time. Genes Dev. 26, 415–416 (2012).

  13. 13.

    et al. O-GlcNAc reports ambient temperature and confers heat resistance on ectotherm development. Proc. Natl. Acad. Sci. USA 111, 5592–5597 (2014).

  14. 14.

    et al. Protein O-GlcNAcylation is required for fibroblast growth factor signaling in Drosophila. Sci. Signal. 4, ra89 (2011).

  15. 15.

    et al. O-GlcNAc modification is essential for the regulation of autophagy in Drosophila melanogaster. Cell. Mol. Life Sci. 72, 3173–3183 (2015).

  16. 16.

    et al. A mutant O-GlcNAcase as a probe to reveal global dynamics of protein O-GlcNAcylation during Drosophila embryonic development. Biochem. J. 470, 255–262 (2015).

  17. 17.

    et al. Tagging-via-substrate strategy for probing O-GlcNAc modified proteins. J. Proteome Res. 4, 950–957 (2005).

  18. 18.

    & O-GlcNAc profiling: from proteins to proteomes. Clin. Proteomics 11, 8 (2014).

  19. 19.

    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).

  20. 20.

    , , , & 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).

  21. 21.

    et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol. Cell. Proteomics 11, 215–229 (2012).

  22. 22.

    et al. GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody. Biochem. Biophys. Res. Commun. 440, 88–93 (2013).

  23. 23.

    et al. Structural insights into the mechanism and inhibition of eukaryotic O-GlcNAc hydrolysis. EMBO J. 25, 1569–1578 (2006).

  24. 24.

    et al. O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release. EMBO J. 31, 1394–1404 (2012).

  25. 25.

    , , & Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol. 21, 660–666 (2003).

  26. 26.

    et al. Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry. J. Proteome Res. 12, 927–936 (2013).

  27. 27.

    et al. Global identification of O-GlcNAc-modified proteins. Anal. Chem. 78, 452–458 (2006).

  28. 28.

    et al. A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo. Nat. Chem. Biol. 4, 483–490 (2008).

  29. 29.

    , & Beta-N-acetylglucosamine (O-GlcNAc) is part of the histone code. Proc. Natl. Acad. Sci. USA 107, 19915–19920 (2010).

  30. 30.

    et al. Activation of the transcriptional function of the NF-κB protein c-Rel by O-GlcNAc glycosylation. Sci. Signal. 6, ra75 (2013).

  31. 31.

    et al. Dynamic O-GlcNAc modification regulates CREB-mediated gene expression and memory formation. Nat. Chem. Biol. 8, 253–261 (2012).

  32. 32.

    , , , & Structure of human O-GlcNAc transferase and its complex with a peptide substrate. Nature 469, 564–567 (2011).

  33. 33.

    et al. Mutant huntingtin represses CBP, but not p300, by binding and protein degradation. Mol. Cell. Neurosci. 30, 560–571 (2005).

  34. 34.

    & Glycosylation of the nuclear pore. Traffic 15, 347–361 (2014).

  35. 35.

    et al. Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc. Nat. Chem. Biol. 6, 338–343 (2010).

  36. 36.

    et al. Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis. Sci. Signal. 3, ra2 (2010).

  37. 37.

    , , & Electron transfer dissociation (ETD): the mass spectrometric breakthrough essential for O-GlcNAc protein site assignments-a study of the O-GlcNAcylated protein host cell factor C1. Proteomics 13, 982–991 (2013).

  38. 38.

    et al. Grunge, related to human Atrophin-like proteins, has multiple functions in Drosophila development. Development 129, 1119–1129 (2002).

  39. 39.

    , , & Atrophin contributes to the negative regulation of epidermal growth factor receptor signaling in Drosophila. Dev. Biol. 291, 278–290 (2006).

  40. 40.

    , , & Drosophila atrophin homolog functions as a transcriptional corepressor in multiple developmental processes. Cell 108, 45–56 (2002).

  41. 41.

    , , , & Histone deacetylase-associating Atrophin proteins are nuclear receptor corepressors. Genes Dev. 20, 525–530 (2006).

  42. 42.

    et al. Atrophin-Rpd3 complex represses Hedgehog signaling by acting as a corepressor of CiR. J. Cell Biol. 203, 575–583 (2013).

  43. 43.

    , , & Myopic acts in the endocytic pathway to enhance signaling by the Drosophila EGF receptor. Development 135, 1913–1922 (2008).

  44. 44.

    et al. The Bro1-domain-containing protein Myopic/HDPTP coordinates with Rab4 to regulate cell adhesion and migration. J. Cell Sci. 125, 4841–4852 (2012).

  45. 45.

    , , , & Endocytic pathway is required for Drosophila Toll innate immune signaling. Proc. Natl. Acad. Sci. USA 107, 8322–8327 (2010).

  46. 46.

    & The role of Bro1- domain-containing protein Myopic in endosomal trafficking of Wnt/Wingless. Dev. Biol. 392, 93–107 (2014).

  47. 47.

    & The Myopic-Ubpy-Hrs nexus enables endosomal recycling of Frizzled. Mol. Biol. Cell 26, 3329–3342 (2015).

  48. 48.

    , , & A screen for conditional growth suppressor genes identifies the Drosophila homolog of HD-PTP as a regulator of the oncoprotein Yorkie. Dev. Cell 20, 700–712 (2011).

  49. 49.

    et al. Genome-wide chemical mapping of O-GlcNAcylated proteins in Drosophila melanogaster. Nat. Chem. Biol. 13, 161–167 (2017).

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Acknowledgements

This work is funded by a Wellcome Trust Senior Investigator Award (110061) to D.M.F.v.A. M.T. is funded by a MRC grant (MC_UU_12016/5). R.W. is funded by a Royal Society Research Grant. We thank J. Peltier for help with mass spectrometry and O. Raimi for help with protein purification.

Author information

Author notes

    • Nithya Selvan
    •  & Ritchie Williamson

    Present addresses: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA (N.S.); School of Pharmacy and Medical Sciences, Faculty of Life Sciences, University of Bradford, Bradford, UK (R.W.).

    • Nithya Selvan
    • , Ritchie Williamson
    •  & Daniel Mariappa

    These authors contributed equally to this work.

Affiliations

  1. MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.

    • Nithya Selvan
    • , Ritchie Williamson
    • , Daniel Mariappa
    • , David G Campbell
    • , Robert Gourlay
    • , Andrew T Ferenbach
    • , Matthias Trost
    •  & Daan M F van Aalten
  2. Division of Gene Regulation and Expression, University of Dundee, Dundee, UK.

    • Daniel Mariappa
    • , Andrew T Ferenbach
    •  & Daan M F van Aalten
  3. Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, UK.

    • Tonia Aristotelous
    •  & Iva Hopkins-Navratilova
  4. Institute for Cell and Molecular Biosciences (ICaMB), Newcastle University, Newcastle-upon-Tyne, UK.

    • Matthias Trost

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Contributions

N.S., R.W. and D.M.F.v.A. conceived the study; N.S., R.W., and D.M. performed experiments; D.G.C., R.G. and M.T. performed mass spectrometry; A.T.F. performed molecular biology; T.A. and I.H.-N. performed SPR; N.S., D.G.C., and M.T. analyzed MS data; D.M. analyzed genetics data; and N.S., R.W., D.M., and D.M.F.v.A. interpreted the data and wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Daan M F van Aalten.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1–8 and Supplementary Figures 1–9

Excel files

  1. 1.

    Supplementary Dataset 1

    All proteins (from HeLa cells) identified in this study.

  2. 2.

    Supplementary Dataset 2

    HexNAc peptides identified (from HeLa cells) in this study.

  3. 3.

    Supplementary Dataset 3

    All proteins (from Drosophila embryos) identified in this study.

  4. 4.

    Supplementary Dataset 4

    HexNAc peptides identified (from Drosophila embryos) in this study.

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DOI

https://doi.org/10.1038/nchembio.2404

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