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A mutant O-GlcNAcase enriches Drosophila developmental regulators

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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Figure 1: A point mutant of CpOGA can be exploited as a substrate trap for the enrichment of O-GlcNAcylated proteins.
Figure 2: Pull down of O-GlcNAcylated proteins by CpOGAD298N.
Figure 3: Protein class grouping of proteins identified by CpOGAD298N and example ETD fragmentation spectra for HexNAc modified peptides from host cell factor and nucleoporin 153.
Figure 4: OGT catalytic activity potentiates the function of its substrates Grunge and myopic.

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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.

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Authors

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.

Corresponding author

Correspondence to Daan M F van Aalten.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–8 and Supplementary Figures 1–9 (PDF 1132 kb)

Supplementary Dataset 1

All proteins (from HeLa cells) identified in this study. (XLSX 134 kb)

Supplementary Dataset 2

HexNAc peptides identified (from HeLa cells) in this study. (XLSX 127 kb)

Supplementary Dataset 3

All proteins (from Drosophila embryos) identified in this study. (XLSX 501 kb)

Supplementary Dataset 4

HexNAc peptides identified (from Drosophila embryos) in this study. (XLSX 48 kb)

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Selvan, N., Williamson, R., Mariappa, D. et al. A mutant O-GlcNAcase enriches Drosophila developmental regulators. Nat Chem Biol 13, 882–887 (2017). https://doi.org/10.1038/nchembio.2404

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