A mutant O-GlcNAcase enriches Drosophila developmental regulators

• Published in: Nature chemical biology Vol. 13; no. 8; pp. 882 - 887
• Main Authors: Selvan, Nithya, Williamson, Ritchie, Mariappa, Daniel, Campbell, David G, Gourlay, Robert, Ferenbach, Andrew T, Aristotelous, Tonia, Hopkins-Navratilova, Iva, Trost, Matthias, van Aalten, Daan M F
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.08.2017; Nature Publishing Group
• Subjects: 631/92/1268; 631/92/221; 631/92/458; 631/92/96; 64; 64/24; 82/103; 82/58; 82/80; 82/83; Animals; Bacteria; beta-N-Acetylhexosaminidases - genetics; beta-N-Acetylhexosaminidases - metabolism; Biochemical Engineering; Biochemistry; Biological properties; Biological samples; Bioorganic Chemistry; Cell Biology; Chemistry; Chemistry/Food Science; Conveyors; Drosophila; Drosophila melanogaster - embryology; Drosophila melanogaster - enzymology; Drosophila melanogaster - genetics; Embryogenesis; Embryonic growth stage; Enrichment; Hydrolase; Insects; Mutation; O-GlcNAcylation; Polycomb gene; Polycomb group proteins; Post-translation; Proteins; Proteomes; Regulators
• ISSN: 1552-4450; 1552-4469
• DOI: 10.1038/nchembio.2404

An inactive mutant of a bacterial O-GlcNAc hydrolase was used as an affinity reagent to enrich O -GlcNAc-modified proteins from Drosophila embryos and led to the identification, by MS–proteomics, of O-GlcNAcylated proteins involved in embryogenesis. 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.