Reductionist animal models to probe protein O-GlcNAcylation

Abstract

Protein O-GlcNAcylation is a reversible type of glycosylation of serine and threonine residues of nucleocytoplasmic proteins occurring in all animals examined to date. Its installation on protein substrates is carried out by O-GlcNAc transferase (OGT), and its removal by O-GlcNAc hydrolase (O-GlcNAcase or OGA). A range of proteomics studies have suggested that over a thousand intracellular proteins are O-GlcNAc modified. However, little is known of the role of O-GlcNAc on these proteins. In a search for a reductionist model with a small proteome to dissect the general global mechanisms of protein O-GlcNAcylation, I discovered the presence of functional OGT, OGA and nucleocytoplasmic protein O-GlcNAcylation in the most basal extant animal, the placozoan Trichoplax adhaerens. I show via enzymatic characterization of Trichoplax OGT/OGA and genetic rescue experiments in Drosophila melanogaster that these proteins possess activities/functions similar to their bilaterian counterparts. While Trichoplax is not currently amenable to genetic manipulation, studying protein O-GlcNAcylation in this organism has revealed the presence of OGT, OGA and O-GlcNAc in non-bilaterian animals. Together with the absence of O-GlcNAcylation in lower organisms, this suggests that OGT-dependent reversible protein O-GlcNAcylation is a metazoan innovation, which may have facilitated the rapid and complex signaling mechanisms required for the evolution of multicellular organisms.

Various genetic approaches in several animal models have revealed that protein O-GlcNAcylation is required for embryogenesis. Embryonic development in a genetically tractable organism is thus a model in which the mechanisms of protein O-GlcNAcylation can be investigated. Drosophila melanogaster OGT is a polycomb gene, null mutants of which display homeotic transformations and die at the pharate adult stage. However, the identities of the modified proteins involved, and the underlying biology linking these to embryonic development are poorly understood. One of the limiting factors towards characterizing O-GlcNAcylation has been the limited specificity of currently available tools to detect this modification. Harnessing the unusual properties of a catalytically inactive bacterial O-GlcNAcase mutant that binds O-GlcNAc sites with sub-micromolar affinity, I show that protein O-GlcNAcylation is dynamic along Drosophila embryonic development. In addition to immunoprecipitation using the anti-O-GlcNAc antibody RL2, I have used the mutant OGA probe to enrich for O-GlcNAcylated proteins from samples of embryos at various developmental stages, and using mass spectrometry, identified novel conserved O-GlcNAcylated proteins. There is evidence in the literature for some of these O-GlcNAc proteins being involved in the regulation of hox gene expression, suggesting that the lack of O-GlcNAcylation of these proteins may contribute to the homeotic phenotypes observed in ogt null Drosophila mutants. This thesis therefore lays the foundation for the investigation of the mechanisms by which protein O-GlcNAcylation of specific substrates would affect a process such as embryonic development.

Date of Award	2015
Original language	English
Awarding Institution	University of Dundee
Supervisor	Daan van Aalten (Supervisor)