Dual functionality of O-GlcNAc transferase is required for Drosophila development.

Daniel Mariappa,Xiaowei Zheng,Daan M F Van Aalten,Olawale Raimi,Andrew T Ferenbach,H.-Arno J Müller,Marianne Schimpl

doi:10.1098/rsob.150234

Abstract

Post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) catalysed by O-GlcNAc transferase (OGT) has been linked to regulation of diverse cellular functions. OGT possesses a C-terminal glycosyltransferase catalytic domain and N-terminal tetratricopeptide repeats that are implicated in protein–protein interactions. Drosophila OGT (DmOGT) is encoded by super sex combs (sxc), mutants of which are pupal lethal. However, it is not clear if this phenotype is caused by reduction of O-GlcNAcylation. Here we use a genetic approach to demonstrate that post-pupal Drosophila development can proceed with negligible OGT catalysis, while early embryonic development is OGT activity-dependent. Structural and enzymatic comparison between human OGT (hOGT) and DmOGT informed the rational design of DmOGT point mutants with a range of reduced catalytic activities. Strikingly, a severely hypomorphic OGT mutant complements sxc pupal lethality. However, the hypomorphic OGT mutant-rescued progeny do not produce F2 adults, because a set of Hox genes is de-repressed in F2 embryos, resulting in homeotic phenotypes. Thus, OGT catalytic activity is required up to late pupal stages, while further development proceeds with severely reduced OGT activity.

Highlights

Post-translational modification of more than 1000 proteins with O-linked N-acetylglucosamine (O-GlcNAc) has been shown to affect a diverse array of cellular functions in metazoa, including protein stability, intracellular localization, protein–protein interaction, phosphorylation and ubiquitylation [1,2,3]
The enzyme that catalyses the addition of a single GlcNAc onto serine/threonine residues on intracellular proteins is O-GlcNAc transferase (OGT)
To determine how differences in sequence influence the overall structure of DmOGT and to provide a template for structure-guided mutagenesis, an N-terminally truncated construct starting at amino acid 353 in tetratricopeptide repeats (TPRs) 10 (D1–352) carrying a mutation (K872M) of a key catalytic lysine was expressed in Escherichia coli and crystallized in complex with the inhibitor/ substrate analogue UDP-5S-GlcNAc

Summary

Introduction

Post-translational modification of more than 1000 proteins with O-linked N-acetylglucosamine (O-GlcNAc) has been shown to affect a diverse array of cellular functions in metazoa, including protein stability, intracellular localization, protein–protein interaction, phosphorylation and ubiquitylation [1,2,3]. Recent studies on the structure of the catalytic domain of human OGT (hOGT) have provided an insight into the mechanism of catalysis and protein substrate recognition [4,5,6]. In hOGT, the TPRs adopt a right-hand super-helical conformation protruding away from the catalytic domain [5,8]. This TPR super-helix creates a large surface area that is thought to allow OGT to interact with a variety of protein substrates. It is plausible that proteins interacting only with the most N-terminal TPRs of OGT are non-substrate interactors

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