Xylosyl Extension of O-Glucose Glycans on the Extracellular Domain of NOTCH1 and NOTCH2 Regulates Notch Cell Surface Trafficking.

Yusuke Urata,Hiroaki Sago,Hideyuki Takeuchi,Hideharu Hibi,Yohei Tsukamoto,Tetsuya Okajima,Wataru Saiki

doi:10.3390/cells9051220

Yusuke Urata, Hiroaki Sago + Show 5 more

Open Access

https://doi.org/10.3390/cells9051220

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Journal: Cells	Publication Date: May 14, 2020
Citations: 21	License type: CC BY 4.0

Affiliation: Nagoya University

Abstract

Biochemical and genetic studies have indicated that O-linked glycosylation such as O-glucose (Glc), fucose (Fuc), and N-acetylglucosamine (GlcNAc) is critical for Notch signaling; however, it is not fully understood how O-glycans regulate the Notch receptor function. Notch receptors are type-I transmembrane proteins with large extracellular domains (ECD), containing 29–36 epidermal growth factor-like (EGF) repeats. Here, we analyzed O-Glc glycans on NOTCH1 and NOTCH2 expressed in HEK293T cells using an Orbitrap Fusion mass spectrometer and successfully revealed the structures and stoichiometries of all 17 EGF repeats of NOTCH1 with the O-Glc consensus sequence (C1-X-S-X-(P/A)-C2), and 16 out of 17 EGF repeats of NOTCH2 with the same consensus sequence. High levels of O-Glc attachment and xylosyl elongation were detected on most NOTCH1 and NOTCH2 EGF repeats. When both glucoside xylosyltransferases, GXYLT1 and GXYLT2, responsible for the xylosyl elongation of O-glucose, were genetically deleted, the expression of endogenous NOTCH1 and NOTCH2 on the surface of HEK293T cells did not change, but the cell surface expression of overexpressed NOTCH1 and NOTCH2 decreased compared with that in the wild type cells. In vitro secretion assays consistently showed a reduced secretion of both the NOTCH1 and NOTCH2 ECDs in GXYLT1 and GXYLT2 double knockout cells compared with the wild type cells, suggesting a significant role of the elongation of O-Glc glycans on the Notch ECDs in the quality control of Notch receptors.

Highlights

Notch receptors initiate the Notch signaling pathway, an evolutionarily well-conserved pathway essential for development in metazoans [1,2,3,4]
The selection of specific proteases and buffers and the optimization of modes with a single level of collision energy (CE) or with mixed CEs during the mass spectral analysis helped us accomplish the semi-quantitative characterization of O-Glc glycoforms on almost all of the epidermal growth factor-like (EGF) repeats from NOTCH1 and NOTCH2 (Figures 2 and 3; Supplementary Materials, Figures S1 and S2, Tables S1 and S2)
Cells or with the XXYLT1 expression vector in the XXYLT1 KO cells rescued the xylosylation of O-Glc glycans on NOTCH1 (Figure S7). These results indicate that GXYLT1 and/or GXYLT2 are responsible for the addition of the first xylose, while XXYLT1 is responsible for the addition of the second xylose on O-Glc glycans of the NOTCH1 and NOTCH2 EGF repeats in HEK293T cells

Summary

Introduction

Notch receptors initiate the Notch signaling pathway, an evolutionarily well-conserved pathway essential for development in metazoans [1,2,3,4]. Multiple components are known to regulate the functions of Notch receptors, the glycosylation of Notch receptors has emerged as a critical mechanism for their folding/trafficking, ligand binding, and subsequent signaling [6,7,8,9,10]. Notch receptors function as heterodimers on the surface of cells. They have large extracellular domains (ECD) which contain up to 36 tandemly repeated epidermal growth factor-like (EGF) repeats, and are involved in ligand binding that triggers the activation of the receptors [11].

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