Article

O-Glucose Trisaccharide Is Present at High but Variable Stoichiometry at Multiple Sites on Mouse Notch1

Department of Biochemistry and Cell Biology, Institute of Cell and Developmental Biology, Stony Brook University, Stony Brook, New York 11794-5215, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2011; 286(36):31623-37. DOI: 10.1074/jbc.M111.268243
Source: PubMed

ABSTRACT

Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted
O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C1XSXPC2. We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site- and cell type-dependent. For instance, although most sites are modified at high stoichiometries, the
site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type. O-Glucose is also found at a novel, non-traditional consensus site at EGF 9. Based on this finding, we propose a revision of
the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2: C1XSX(A/P)C2. We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with
O-glucose on EGF repeats. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of
individual O-glucose sites in mN1, we used a cell-based signaling assay. Elimination of most individual sites shows little or no effect
on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1, and they play important roles in Notch function.

    • "Interestingly, overexpression of human GXYLT1 in Drosophila wing results in Notch loss-of-function phenotypes but overexpression of Shams does not (Lee et al. 2013), suggesting that human GXYLT1 is a more efficient xylosyltransferase. This might explain the mass spectrometry data indicating that all the O-glucosylated EGF repeats of the mouse Notch1 are xylosylated, albeit at variable stoichiometry (Rana et al. 2011). However, the increased xylosylation of mouse Notch may also be, at least in part, due to the fact that mammals have two GXYLT enzymes (Sethi et al. 2010). "
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    ABSTRACT: The epidermal growth factor-like (EGF) repeat is a common, evolutionarily conserved motif found in secreted proteins and the extracellular domain of transmembrane proteins. EGF repeats harbor six cysteine residues which form three disulfide bonds and help generate the three-dimensional structure of the EGF repeat. A subset of EGF repeats harbor consensus sequences for the addition of one or more specific O-glycans, which are initiated by O-glucose, O-fucose or O-GlcNAc (N-acetylglucosamine). These glycans are relatively rare compared to mucin-type O-glycans. However, genetic experiments in model organisms and cell-based assays indicate that at least some of the glycosyltransferases involved in the addition of O-glycans to EGF repeats play important roles in animal development. These studies, combined with state-of-the-art biochemical and structural biology experiments have started to provide an in-depth picture of how these glycans regulate the function of the proteins to which they are linked. In this review, we will discuss the biological roles assigned to EGF repeat O-glycans and the corresponding glycosyltransferases. Since Notch receptors are the best studied proteins with biologically-relevant O-glycans on EGF repeats, a significant part of this review is devoted to the role of these glycans in the regulation of the Notch signaling pathway. We also discuss recently identified proteins other than Notch which depend on EGF repeat glycans to function properly. Several glycosyltransferases involved in the addition or elongation of O-glycans on EGF repeats are mutated in human diseases. Therefore, mechanistic understanding of the functional roles of these carbohydrate modifications is of interest from both basic science and translational perspectives. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
    No preview · Article · Jul 2015 · Glycobiology
    • "Interestingly, overexpression of human GXYLT1 in Drosophila wing results in Notch loss-of-function phenotypes but overexpression of Shams does not (Lee et al. 2013), suggesting that human GXYLT1 is a more efficient xylosyltransferase. This might explain the mass spectrometry data indicating that all the O-glucosylated EGF repeats of the mouse Notch1 are xylosylated, albeit at variable stoichiometry (Rana et al. 2011). However, the increased xylosylation of mouse Notch may also be, at least in part, due to the fact that mammals have two GXYLT enzymes (Sethi et al. 2010). "
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    ABSTRACT: O-linked glycosylation is the addition of carbohydrate residues to serine or threonine on target proteins. Identification of mutations in the enzymes responsible for O-glycosylation in several human diseases and studies in model organisms have shown that O-glycans play important roles in animal development and physiology. Drosophila is an important genetic model system to study the developmental roles of various glycans. After providing a brief overview of the types of O-glycans identified on Drosophila proteins, this chapter describes the function of two conserved forms of O-glycan, namely, O-mannose and mucin-type O-GalNAc glycans, in Drosophila development. O-mannose modifications are found on the transmembrane protein dystroglycan and are critical for its function, as mutations in the protein O-mannosyltransferases cause human dystroglycanopathies. Mucin-type O-glycosylation, defects in which are implicated in a number of human diseases, also plays critical roles in the development of several fly organ systems such as gut, wing, respiratory system, and the embryonic mesoderm. Future studies in Drosophila are likely to further elucidate the role of O-glycosylation in animal development and might provide insight into the pathophysiology of human diseases caused by alteration in O-glycosylation.
    No preview · Article · Jan 2015
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    • "We recently revised this to C 1 -X-S-X(P/A )-C 2 based on our biochemical and mass spectral analyses of O-glucose glycan modification sites on mouse Notch1 and Drosophila Notch [67]. Database searches for this consensus sequence identify over 40 proteins predicted to be O-glucosylated, although like O-fucose, the Notch family of receptors has more consensus sites than any other protein (Fig. 1) [67]. O-Glucose glycans are also essential for Notch activity in both mice and flies [66] [68]. "
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    ABSTRACT: Notch signaling is essential for cell-fate specification in metazoans, and dysregulation of the pathway leads to a variety of human diseases including heart and vascular defects as well as cancer. Glycosylation of the Notch extracellular domain has emerged as an elegant means for regulating Notch activity, especially since the discovery that Fringe is a glycosyltransferase that modifies O-fucose in 2000. Since then, several other O-glycans on the extracellular domain have been demonstrated to modulate Notch activity. Here we will describe recent results on the molecular mechanisms by which Fringe modulates Notch activity, summarize recent work on how O-glucose, O-GlcNAc, and O-GalNAc glycans affect Notch, and discuss several human genetic disorders resulting from defects in Notch glycosylation.
    Preview · Article · Jun 2014 · Biochemical and Biophysical Research Communications
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