O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals.

Shogo Sawaguchi,Hirokazu Yagi,Tetsuya Okajima,Kyosuke Takeshita,Toyoaki Murohara,Pamela Stanley,Mitsutaka Ogawa,Subha Sundaram,Yuta Sakaidani,Koichi Kato,Shweta Varshney

doi:10.7554/elife.24419

Abstract

The glycosyltransferase EOGT transfers O-GlcNAc to a consensus site in epidermal growth factor-like (EGF) repeats of a limited number of secreted and membrane proteins, including Notch receptors. In EOGT-deficient cells, the binding of DLL1 and DLL4, but not JAG1, canonical Notch ligands was reduced, and ligand-induced Notch signaling was impaired. Mutagenesis of O-GlcNAc sites on NOTCH1 also resulted in decreased binding of DLL4. EOGT functions were investigated in retinal angiogenesis that depends on Notch signaling. Global or endothelial cell-specific deletion of Eogt resulted in defective retinal angiogenesis, with a mild phenotype similar to that caused by reduced Notch signaling in retina. Combined deficiency of different Notch1 mutant alleles exacerbated the abnormalities in Eogt-/- retina, and Notch target gene expression was decreased in Eogt-/-endothelial cells. Thus, O-GlcNAc on EGF repeats of Notch receptors mediates ligand-induced Notch signaling required in endothelial cells for optimal vascular development.

Highlights

N-acetylglucosamine linked to Ser or Thr (O-GlcNAc) is a rare form of post-translational modification modifying epidermal growth factor-like (EGF) domains in secreted or membrane proteins (Alfaro et al, 2012; Matsuura et al, 2008; Stanley and Okajima, 2010)
To address whether EOGT regulates physical interactions between Notch receptors and ligands, Notch ligand binding assays were performed on control and Eogt–siRNA Lec1 Chinese hamster ovary (CHO) cells
Notch receptors in Eogt-null cells responded to controls to JAG1 sending cells. These results suggest that EOGT is dispensable for DLL4 and JAG1 as inducers of Notch signaling, and that O-GlcNAc on Notch receptors is required for optimal DLL- but not JAG-induced Notch activation

Summary

Introduction

N-acetylglucosamine linked to Ser or Thr (O-GlcNAc) is a rare form of post-translational modification modifying epidermal growth factor-like (EGF) domains in secreted or membrane proteins (Alfaro et al, 2012; Matsuura et al, 2008; Stanley and Okajima, 2010). Only a small number of secreted or membrane proteins have been shown to be O-GlcNAcylated, including Notch receptors and their ligands (Alfaro et al, 2012; Muller et al, 2013; Tashima and Stanley, 2014). Loss of O-fucose, but not O-glucose glycans reduces binding of canonical Notch ligands to Notch receptors, and Notch signaling. Removing one class of O-glycan will affect many EGF repeats but is not expected to affect modification of EGF repeats by unrelated

Methods

Results

Conclusion