Regulation of Notch signaling by glycosylation

Abstract

Notch receptors are approximately 300 kDa cell surface glycoproteins whose activation by Notch ligands regulates cell fate decisions in the metazoa. The extracellular domain of Notch receptors has many epidermal growth factor like repeats that are glycosylated with O-fucose and O-glucose glycans as well as N-glycans. Disruption of O-fucose glycan synthesis leads to severe Notch signaling defects in Drosophila and mammals. Removal or addition of O-fucose glycan consensus sites on Notch receptors also leads to Notch signaling defects. Ligand binding and ligand-induced Notch signaling assays have provided insights into how changes in the O-fucose glycans of Notch receptors alter Notch signaling.

Fig. 1. Canonical Notch signal transduction pathway

Mammalian Notch1 is given as an example of Notch receptors in mammals and Drosophila. Notch receptors are cleaved by furin in the Golgi compartment to form heterodimers that span the plasma membrane once. The EGF repeats of Notch1 are colored according to the O-fucose, O-glucose and complex N-glycans attached as described in Fig. 2. Notch ligands also carry EGF repeats but to date no functional effects have been observed in the absence of O-fucose glycans. Ligands require their N-terminal DSL (Delta/Serrate/Lag-2) domain to bind to Notch receptors. Ligand binding is followed by endocytosis of NECD into the ligand cell, cleavage by an ADAM protease at a juxtamembrane site followed by cleavage by a complex containing γ-secretase in the transmembrane region (leaving Val 1744 N-terminal in the case of Notch1). Released NICD goes to the nucleus to complex with the CSL transcriptional repressor bound to the consensus GCTGATAG in promoter regions of genes. The co-activator MAML is recruited and target genes are activated. Well characterized Notch target genes include the Hes and Hey transcriptional repressors.

Fig. 2. The Glycans of Mouse Notch1

Notch1 receptor EGF repeats have consensus sites for O-fucose (C²X_4-5S/TC³) [27] and O-glucose (C¹XSXPC²) glycans [23] while N-glycans are added at certain AsnXSer/Thr consensus sequons. Structural analyses have been performed on Notch1 synthesized as ECD fragments in mammalian cells. The largest O-fucose glycan observed to date is sialic acidα(2,3)Galβ(1,4)GlcNAcβ(1,3)Fuc-O and the largest O-glucose glycan is Xylα(1,3)Xylα(1,3)O-Glc-O. O-glycans initiated by O-GalNAc on Ser/Thr may also be present but have not been reported.