Abstract
Extracellular O-GlcNAc is a novel class of modification catalyzed by epidermal growth factor-like (EGF)-domain specific O-GlcNAc transferase (EOGT). In mammals, EOGT is required for ligand-mediated Notch signaling for vascular development. Previous studies have revealed that O-GlcNAc in mammalian cultured cells is subject to subsequent glycosylation, which may impose additional layers of regulation. This study aimed to analyze the O-GlcNAc glycans of Drosophila EGF20 as model substrates and mouse Notch1 EGF repeats by mass-spectrometry. The analysis of Drosophila EGF20 expressed in HEK293T cells revealed that the majority of the proteins are modified with an elongated form of O-GlcNAc glycan comprising terminal galactose or sialic acid residues. In contrast, recombinant Notch1 EGF repeats isolated from HEK293T cells revealed structural divergence of O-GlcNAc glycans among the different EGF domains. Although the majority of Notch1 EGF2 and EGF20 domains contained the extended forms of the glycan, the O-GlcNAc in many other domains mostly existed as a monosaccharide irrespective of the exogenous EOGT expression. Our results raised a hypothesis that an array of O-GlcNAc monosaccharides may impact the structure and function of Notch receptors.
Highlights
Extracellular O-GlcNAc fragments including the m/z 204.0865 (GlcNAc) modification was initially found on the epidermal growth factor-like (EGF)domain of Drosophila Notch receptor [1]
O-GlcNAc-Gal-Sia structure was observed in the mouse Notch1 isolated from HEK293T cells [12]
Structural difference in O-GlcNAc glycans might lead to functional differences of O-GlcNAc in Notch signaling in flies and mammals
Summary
Extracellular O-GlcNAc modification was initially found on the epidermal growth factor-like (EGF). Domain of Drosophila Notch receptor [1]. O-GlcNAc modification by O-GlcNAc transferase (OGT) occurs in the nucleus, cytoplasm, and mitochondria [2], the extracellular O-GlcNAc modification is catalyzed by the endoplasmic reticulum (ER)-localized EGF-domain specific O-GlcNAc transferase (EOGT) [3,4,5,6]. Notch signaling in mammals [7]. The EOGT knockout fly lacks an apparent Notch mutant phenotype [3,11]. The Eogt shows genetic interaction with dp that encodes extracellular matrix protein Dumpy. These results suggest that the function of O-GlcNAc is distinct in flies and mammals
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have