Review
Role of glycosylation of Notch in development

https://doi.org/10.1016/j.semcdb.2010.03.003Get rights and content

Abstract

The Notch pathway is one of the major signaling pathways required for proper development in metazoans. Notch activity is regulated at numerous levels, and increasing evidence reveals the importance of “protein glycosylation” (modification of Notch receptors with sugars) for its regulation. In this review we summarize the significance of the Notch pathway in development and the players responsible for its glycosylation, and then discuss the molecular mechanisms by which protein glycosylation may regulate Notch function.

Introduction

Notch signaling is essential for proper development in metazoans, and defects in this pathway result in a number of human diseases [1], [2]. Notch is regulated at numerous overlapping levels, including endocytosis, ubiquitination, intracellular trafficking, degradation, and glycosylation [2], [3], [4], [5], [6]. Many genes impinge on this pathway, and the number of these genes continues to increase with the improved techniques for genome-wide analysis [7]. This review focuses on regulation of the Notch pathway by glycosylation.

The Notch phenotype was originally described in Drosophila nearly 100 years ago as an X-linked, dominant mutation which showed irregular “notches” at the tips of the wings [8]. Subsequent work demonstrated that Notch plays key roles in development of many tissues in flies, including formation of neurons and glial cells, leg segments, eyes, heart, muscles, and blood lineages [2], [9], [10]. Drosophila has a single Notch receptor, while mammals have four [1]. Targeted disruption of the four mouse Notch genes demonstrated that these genes play important roles in development of many tissues. Loss of mouse Notch1 results in an embryonic lethal phenotype with severe defects in somitogenesis [11], [12]. Subsequent studies showed that Notch1 is also involved in neurogenesis and vasculogenesis [13], [14]. Deletion of mouse Notch2 also results in an embryonic lethal phenotype with apoptotic cell death in a wide variety of tissues, especially neural tissues, from embryonic day 9.5 [15]. Notch3−/− mice are viable and fertile, but have defects in arterial differentiation and maturation of vascular smooth muscle [16]. Although Notch4−/− mice are viable and fertile [14], loss of Notch4 exacerbates the vascular remodeling defects observed in Notch1−/− embryo [14], suggesting partially overlapping function of Notch1 and 4 during embryogenesis. Aberrant Notch signaling leads to multiple human disorders [1], [17]. Mutations of Notch and the components of this pathway are implicated in human developmental disorders such as Alagille Syndrome and Spondylocostal Dysostosis, adult onset diseases such as CADASIL and Multiple Scleorosis, and cancers such as T cell acute lymphoblastic leukemia (T-ALL) and colon cancer.

Notch receptors are large type I transmembrane proteins [2]. Their basic molecular structure is evolutionarily conserved and consists of three domains: an extracellular domain (ECD) with 29–36 tandem epidermal growth factor-like (EGF) repeats and a unique negative regulatory region (NRR) which consists of three Lin-12/Notch repeats and a heterodimerization domain; a single transmembrane domain; and an intracellular domain with an RBP-Jκ (recombination signal sequence-binding protein-Jκ) association module domain, several nuclear localization sequences, seven ankyrin repeats, and a transactivation domain that harbors proline/glutamic acid/serine/threonine-rich motifs responsible for rapid degradation. The mature receptor is a heterodimer with the ECD tethered to the transmembrane/intracellular domain (T/ICD) through non-covalent, calcium dependent interactions. The heterodimer is formed by cleavage of the nascent polypeptide at site 1 by a furin-like protease in the Golgi [18], [19].

Notch ligands are also type I transmembrane proteins with a similar overall architecture: an ECD containing an N-terminal DSL (Delta/Serrate/LAG-2) motif, specialized tandem EGF repeats termed the DOS (Delta and OSM-11-like proteins) domain, and several tandem EGF repeats; a single transmembrane domain; and a small intracellular domain [20]. Drosophila has two ligands, Delta and Serrate, while mammals have three Delta-like ligands (Dll1, 3, and 4) and two Serrate homologues (Jagged1 and 2).

Notch activation is initiated by ligand binding, and accomplished through a proteolytic mechanism [21]. The first cleavage occurs at site 2 (S2), just outside the membrane on the T/ICD, and is catalyzed by a metalloprotease of the ADAM family. In the absence of ligand, S2 appears to be covered by the NRR, sterically blocking access of the ADAM protease to the site. Ligand binding results in a conformational change in the NRR, exposing the site and allowing cleavage [22], [23], [24]. Subsequently, cleavage at site 3 (S3) in the Notch transmembrane domain by the γ-secretase complex results in the release of the Notch intracellular domain (NICD), and translocation of the NICD into the nucleus [25]. Interaction between NICD and DNA binding proteins such as RBP-Jκ, activate target gene transcription [26].

Section snippets

Regulation of Notch function with glycosylation

The discovery that Fringe, a known modulator of Notch activity, is a glycosyltransferase modifying O-fucose glycans on Notch EGF repeats [27], [28], brought the study of Notch into the field of Glycobiology [29]. The EGF repeats of Notch are modified with three different types of O-linked glycosylation: O-fucosylation, O-glucosylation, and O-GlcNAc’ylation (Fig. 1) [30], [31], [32]. Addition of O-fucose to Ser/Thr occurs within the consensus sequence C2-X-X-X-X-(S/T)-C3 (C, cysteine; X, any

Conclusions

Evidence for the importance of carbohydrate modifications on Notch for signaling is largely based on genetic studies. While unidentified sugars may yet exist on Notch, most of the genes encoding the enzymes responsible for the synthesis of the known structures have been identified. The potential sites for O-fucosylation and O-glucosylation on the ECD of Notch are well conserved among species (Fig. 2), suggesting a distinct pattern of each modification on the entire ECD of Notch. Such

Acknowledgements

We would like to thank Dr. Kelly Ten Hagen for giving us an opportunity to write this manuscript, and Drs. Bernadette C. Holdener, Hamed Jafar-Nejad and Haltiwanger lab members for helpful comments. Primary work was supported by NIH grant GM061126 (to RSH) and the research grant from Mizutani Foundation for Glycoscience (to HT).

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