Structural Divergence in O-GlcNAc Glycans Displayed on Epidermal Growth Factor-like Repeats of Mammalian Notch1

• Published in: Molecules (Basel, Switzerland) Vol. 23; no. 7; p. 1745
• Main Authors: Ogawa, Mitsutaka, Senoo, Yuya, Ikeda, Kazutaka, Takeuchi, Hideyuki, Okajima, Tetsuya
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 17.07.2018; MDPI
• Subjects: Acetylglucosamine - chemistry; Acetylglucosamine - metabolism; Animals; Communication; Drosophila melanogaster; EGF domain; EOGT; Epidermal growth factor; Glycosylation; HEK293 Cells; Humans; Insects; Mammals; Mice; Notch1; O-GlcNAc; O-GlcNAc glycan; Protein Domains; Receptor, Notch1 - biosynthesis; Receptor, Notch1 - chemistry; Repetitive Sequences, Amino Acid; O-GlcNAc glycan; EGF domain; Notch1; O-GlcNAc; EOGT
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules23071745

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.