Fringe benefits: Functional and structural impacts of O-glycosylation on the extracellular domain of Notch receptors
Highlights
► Notch is modified at multiple sites with O-fucose and O-glucose glycans ► Elongation of O-fucose glycans by Fringe regulates Notch-ligand binding. ► Rumi encodes the protein O-glucosyltransferase responsible for modifying Notch. ► O-Glucose may stabilize Notch to allow proper receptor activation. ► Multiple O-glycan sites contribute to Notch-ligand binding and activity ► O-Glycosylation may alter the flexibility of the Notch extracellular domain.
Introduction
The Notch protein plays an important role as a transmembrane signaling receptor in a wide variety of developmental pathways [1, 2]. Notch is conserved across all metazoans, and there are four mammalian homologs (Notch1–4). Loss of individual mouse Notch homologs 1 or 2 results in embryonic lethality, and mutations of Notch or downstream signaling components within the pathway have been implicated in a multitude of disease states in humans, including T-cell acute lymphoblastic leukemia, CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), Alagille syndrome, spondylocostal dysostosis, multiple sclerosis, several heart defects, and breast cancer (reviewed in Refs. [1, 2, 3, 4]). The Notch locus encodes a large (∼300 kDa) single-pass Type I transmembrane receptor, comprising a large extracellular domain (ECD) with multiple tandem epidermal growth factor-like (EGF) repeats and negative regulatory region (NRR), followed by a transmembrane region and a large intracellular domain (ICD) involved in downstream signaling events (Figure 1a) [1]. The Notch signaling pathway is activated upon binding of the Notch ECD to one of its ligands presented on an apposing cell (Figure 2).
Work in a number of laboratories over the past decade has demonstrated that O-fucosylation and O-glucosylation of the EGF repeats in the Notch ECD are essential for its function [3, 4, 5, 6, 7, 8, 9]. Elimination of the enzyme responsible for addition of O-fucose to EGF repeats (protein O-fucosyltransferase 1, Pofut1 in mice, Ofut1 in Drosophila) reveals that O-fucosylation is universally required for all Notch signaling [10, 11, 12]. Elongation at the O-fucose by the β1-3N-acetylglucosaminyltransferase, Fringe, on EGF repeats modulates Notch activity in a number of tissue-specific contexts and serves as a paradigm for how alterations in the glycosylation status of a receptor affect activity [13, 14]. Genetic studies on the biological role of O-glucosylation have not been as extensive as for O-fucosylation, but elimination of the enzyme responsible for addition of O-glucose to EGF repeats (protein O-glucosyltransferase, Poglut, gene name Rumi) also results in severe Notch-like phenotypes in flies or mice [15••, 16••]. In this review we will focus on advances in our understanding of Notch glycosylation since it was last reviewed in this series [7], with a focus on what we have learned about the biochemistry of Notch O-glycosylation and current models for how the glycans affect Notch function.
Section snippets
Biochemistry of Notch O-glycosylation
The O-fucose and O-glucose glycans on Notch occur at specific consensus sequences within the context of EGF repeats, which make up the majority of the Notch ECD (Figure 1). Recently, O-GlcNAc modification, a third form of O-glycosylation was identified on EGF repeats as well, occurring on hydroxy amino acids between the fifth and sixth conserved Cys of an EGF repeat [17]. This sequence context has been sufficient to identify predicted O-GlcNAc sites on other EGF repeat-containing proteins,
O-Fucose and Fringe modification affect ligand binding
Elimination of Pofut1 in mice has a profound effect on ligand binding in both embryonic stem cells [36] and lymphoid cells [37]. A chaperone-like activity has been reported for Drosophila Ofut1 that is required for cell-surface expression of Notch in flies [38], but this chaperone activity has not been clearly seen in the mouse system, as cells lacking Pofut1 have Notch proteins on their surfaces [36, 37]. This discrepancy may be explained by differences in species, or in cell-dependent
Conclusions
Just over 10 years ago, Notch activity was shown to be regulated by Fringe-mediated elongation of O-fucose on its ECD [13, 14]. Since then most of the enzymes responsible for the addition of O-fucose and O-glucose glycans have been identified, and their importance for Notch activity has been confirmed. We now know that the predicted consensus sites are modified, usually at high stoichiometries, at least in Notch protein overproduced in tissue culture systems. Future studies need to examine the
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
The authors would like to thank members of the Haltiwanger laboratory for critical reading of the manuscript and helpful discussions. Original work was supported by NIH grant GM061126 and training grant NCI T32 CA009176.
References (50)
- et al.
The canonical Notch signaling pathway: unfolding the activation mechanism
Cell
(2009) Notch signaling: the core pathway and its posttranslational regulation
Dev Cell
(2009)- et al.
Role of glycosylation of Notch in development
Semin Cell Dev Biol
(2010) Regulation of Notch signaling by glycosylation
Curr Opin Struct Biol
(2007)- et al.
Roles of glycosylation in Notch signaling
Curr Top Dev Biol
(2010) - et al.
Role of unusual O-glycans in intercellular signaling
Int J Biochem Cell Biol
(2009) - et al.
Regulation of notch signaling by O-linked fucose
Cell
(2002) - et al.
Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling
Cell
(2008) - et al.
O-Glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1
J Biol Chem
(2011) - et al.
Fringe modifies O-fucose on mouse Notch1 at epidermal growth factor-like repeats within the ligand-binding site and the Abruptex region
J Biol Chem
(2003)