2022
DOI: 10.1093/glycob/cwac015
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Glycoproteomics of NOTCH1 EGF repeat fragments overexpressed with different glycosyltransferases in HEK293T cells reveals insights into O-GlcNAcylation of NOTCH1

Abstract: The O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms of O-glycans on epidermal growth factor-like (EGF) repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specific O-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongated O-GlcNAc glycans have not been extensively explored. To extend our view of the specificity an… Show more

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Cited by 9 publications
(11 citation statements)
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“…O-glucose may be extended by xylose transferred from UDP-Xyl by xylosyltransferases GXYLT1 or GXYLT2, and a second Xyl may be added by XXYLT1 (Yu and Takeuchi, 2019). O-GlcNAc may be extended by Gal and Sia as well as Fuc (Tsukamoto et al, 2022). Several congenital human diseases arise from mutations in genes encoding the glycosyltransferases that modify EGF repeats in Notch receptors and ligands (Varshney and Stanley, 2018;Matsumoto et al, 2021).…”
Section: Introductionmentioning
confidence: 99%
“…O-glucose may be extended by xylose transferred from UDP-Xyl by xylosyltransferases GXYLT1 or GXYLT2, and a second Xyl may be added by XXYLT1 (Yu and Takeuchi, 2019). O-GlcNAc may be extended by Gal and Sia as well as Fuc (Tsukamoto et al, 2022). Several congenital human diseases arise from mutations in genes encoding the glycosyltransferases that modify EGF repeats in Notch receptors and ligands (Varshney and Stanley, 2018;Matsumoto et al, 2021).…”
Section: Introductionmentioning
confidence: 99%
“…To generate recombinant proteins bearing O -GlcNAc modifications, consecutive EGF14–16 domains derived from mouse Notch1 were selected because these domains possess a consensus sequence for O -GlcNAcylation. Furthermore, glycoproteomics data confirmed the O -GlcNAc modification [ 4 ]. To secrete the EGF domains, they were fused with an IL-2 signal sequence at the N-terminus and a hexa-histidine tag at the C-terminus (EGF14-16-His 6 ).…”
Section: Resultsmentioning
confidence: 91%
“…Notably, protein glycosylation of EGF domains includes O -fucosylation, O -glucosylation, and O -GlcNAcylation, which are atypical types of O -glycosylation, in contrast to the typical mucin-type O -glycosylation [ 2 ]. Although 22 of the 36 EGF repeats of mouse Notch1 contained a consensus sequence for O -GlcNAc modification, only 10 EGF repeats underwent O -GlcNAcylation in recombinantly expressed mouse Notch1 in HEK293T cells [ 4 ]. In addition, public scRNA-seq data indicate the tissue- or cell-type-specific expression of O -glycosyltransferases [ 22 ].…”
Section: Discussionmentioning
confidence: 99%
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