Abstract
The O-GlcNAc transferase Eogt modifies EGF repeats in proteins that transit the secretory pathway, including Dumpy and Notch. In this paper, we show that the Notch ligands Delta and Serrate are also substrates of Eogt, that mutation of a putative UDP-GlcNAc binding DXD motif greatly reduces enzyme activity, and that Eogt and the cytoplasmic O-GlcNAc transferase Ogt have distinct substrates in Drosophila larvae. Loss of Eogt is larval lethal and disrupts Dumpy functions, but does not obviously perturb Notch signaling. To identify novel genetic interactions with eogt, we investigated dominant modification of wing blister formation caused by knock-down of eogt. Unexpectedly, heterozygosity for several members of the canonical Notch signaling pathway suppressed wing blister formation. And importantly, extensive genetic interactions with mutants in pyrimidine metabolism were identified. Removal of pyrimidine synthesis alleles suppressed wing blister formation, while removal of uracil catabolism alleles was synthetic lethal with eogt knock-down. Therefore, Eogt may regulate protein functions by O-GlcNAc modification of their EGF repeats, and cellular metabolism by affecting pyrimidine synthesis and catabolism. We propose that eogt knock-down in the wing leads to metabolic and signaling perturbations that increase cytosolic uracil levels, thereby causing wing blister formation.
Highlights
Glycosylation is the most common post-translational modification of proteins
To establish that it has EOGT activity, Drosophila S2 cells or S2 cells incubated with a dsRNA designed to target endogenous eogt, were co-transfected with a soluble Drosophila Notch fragment tagged at the C-terminus with alkaline phosphatase (N(EGF1-20)-AP)
Each lysate contained a comparable amount of N(EGF1-20)-AP based on detection with anti-AP antibody (Ab), and EOGT and Ago61 were present in the appropriate samples (Fig. 1A)
Summary
Glycosylation is the most common post-translational modification of proteins. Different classes of glycans or individual sugars within a glycan have been shown to regulate cell-cell recognition, cell migration, cell proliferation, the binding of pathogenic viruses and bacteria, growth factor and cytokine signaling, and Notch signaling [1]. In Drosophila, Fringe adds a GlcNAc to O-Fuc on Notch (N) epidermal growth factor-like (EGF) repeats, thereby altering the binding of Notch ligands Delta (Dl) and Serrate (Ser), and up- or down-regulating Notch signaling, respectively [2,3]. This is critical for controling Notch signaling during boundary formation in the wing imaginal disc, in leg development, and in the eye [4,5]. Drosophila Ogt is encoded by the super sexcomb (sxc) gene, and sxc null mutants are late pupal lethal [9]
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