A mutant O-GlcNAcase as a probe to reveal global dynamics of protein O-GlcNAcylation during Drosophila embryonic development
| Autor: | Daniel Mariappa, Jana Alonso, Nithya Selvan, Claire A. Shepherd, Iva Navratilova, Andrew T. Ferenbach, Daan M. F. van Aalten, Vladimir S. Borodkin |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
PNGase F
Embryo Nonmammalian Glycosylation Clostridium perfringens Acylation Mutant Embryonic Development Biology Blotting Far-Western Biochemistry Serine chemistry.chemical_compound Bacterial Proteins Animals Humans Threonine Protein kinase A Molecular Biology Adaptor Proteins Signal Transducing Binding protein HEK 293 cells Cell Biology beta-N-Acetylhexosaminidases HEK293 Cells chemistry Mutation Drosophila |
| Zdroj: | Biochemical Journal. 470:255-262 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20150610 |
| Popis: | O-GlcNAcylation is a reversible type of serine/threonine glycosylation on nucleocytoplasmic proteins in metazoa. Various genetic approaches in several animal models have revealed that O-GlcNAcylation is essential for embryogenesis. However, the dynamic changes in global O-GlcNAcylation and the underlying mechanistic biology linking them to embryonic development is not understood. One of the limiting factors towards characterizing changes in O-GlcNAcylation has been the limited specificity of currently available tools to detect this modification. In the present study, harnessing the unusual properties of an O-GlcNAcase (OGA) mutant that binds O-GlcNAc (O- N -acetylglucosamine) sites with nanomolar affinity, we uncover changes in protein O-GlcNAcylation as a function of Drosophila development. * ACN, : acetonitrile; ConA, : concanavalin A; Cp OGA, : Clostridium perfringens OGA orthologue; dHCF, : Drosophila host cell factor; Dm OGT, : Drosophila OGT; ETD, : electron transfer dissociation; GalNAz, : N-azidoacetylgalactosamine; GlcNAc, : N -acetylglucosamine; Fmoc, : 9-fluorenylmethyloxycarbonyl; HA, : haemagglutinin; HCF, : host cell factor; HEK, : human embryonic kidney; hOGA, : human OGA; LB-Amp, : LB medium containing 50 μg/ml ampicillin; OGA, : O-GlcNAcase; OGT, : O-GlcNAc transferase; Ph, : polyhomoeotic; PNGase F, : peptide: N-glycosylase F; PTM, : post-translational modification; SPR, : surface plasmon resonance; sxc , : super sex combs ; TAB1, : transforming growth factor-beta-activated protein kinase 1- binding protein 1; TFA, : trifluoroacetic acid |
| Databáze: | OpenAIRE |
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