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

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 C 1 XSXPC 2 . 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, nontraditional 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: C 1 XSX(A/P)C 2 . 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.The Notch family of single-pass transmembrane receptors is essential for early metazoan development, activating the expression of many genes involved in cell differentiation and tissue morphogenesis (1-3). Defects in Notch signaling have been implicated in a number of human diseases, including several forms of cancer, vascular defects such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (4 -6), multiple sclerosis (6), and a number of developmental syndromes (4, 7-10). The canonical Notch signaling pathway is initiated by the interaction of Notch with its ligand on an apposed cell. Upon ligand binding, Notch undergoes presenilin-1-dependent proteolysis, releasing a soluble Notch intracellular domain, which enters the cell nucleus to interact directly with the transcription factors from the CSL family and regulate Notch target genes (2). There are four members of the Notch family in vertebrates (Notch1 to Notch4) interacting with several classes of Notch ligands: ligands of the DSL family (Delta-like 1, 3, and 4 and Jagged 1 and 2) (1) and newly characterized ligands without the DSL domain, such as DNER and MAGP-1 and -2 (11-13).The Notch proteins consist of a large extracellular domain (ECD), 5 a transmembrane region, and a large intracellular domain (1, 2, 14). The majority of the ECD consists of tandem epidermal growth factor-like (EGF) repeats (36 EGF repeats are ...

Fringe-mediated extension of O -linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands

“…It is interesting that, whereas in our study prokaryotically expressed hN1 [11][12][13] containing either the T466A or the T466S substitutions both show reduced binding to Jagged1, in previous coculture assays, full-length murine Notch1 containing the T466A substitution showed no activity in the presence of cells expressing DLL1, whereas the T466S mutant was both O-fucosylated and behaved similarly to WT protein (38,44). In addition, T466 from mouse Notch1 is modified at high stoichiometry with O-fucose in cells (16), suggesting that O-fucose is present on this site in vivo. In light of these studies, our data highlight the importance of the presence of O-fucose glycans in this region in increasing the affinity of the interaction between Notch and its ligands and provide an explanation for the observation that the Notch receptor needs to be O-fucosylated in order for optimal signaling to occur (28,30).…”

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch

“…Analysis of Glycopeptides by Nano-LC-Electrospray Ionization-MS-Purified Notch protein was reduced, alkylated, and subjected to in-gel digestions with either trypsin, chymotrypsin, or V8 protease and analyzed by nano-liquid chromatography/tandem mass spectrometry using an Agilent 6340 ion trap mass spectrometer with a nano-HPLC CHIP cube interface autosampler as reported previously (28,44). Constant neutral loss searches as well as generation of extracted ion chromatograms and multiple-reaction monitoring chromatograms were performed as reported previously using the Data Analysis software from Agilent (44,61).…”

“…Whether this O-fucose trisaccharide occurs on Notch remains to be resolved. Notch EGF repeats are O-glucosylated by protein O-glucosyltransferase 1 (Rumi in flies, POGLUT1 in mammals) at a serine within a consensus sequence between the first and second conserved cysteine (C 1 XSX(A/P)C 2 ) (28,29). O-Glucosylation is also essential for Notch function in flies because loss of rumi in flies results in temperature-sensitive Notch-null phenotypes (29,30).…”

“…This disaccharide can be further extended to a trisaccharide by the addition of a terminal xylose, generating Xyl␣1-3Xyl␣1-3Glc␤-O-Ser, by xyloside ␣3-xylosyltransferase (XXYLT1) (35). We have previously observed the O-glucose trisaccharide as the predominant O-glucose glycoform on NOTCH1 expressed in mammalian cells, whereas O-glucose has been observed in mono-, di-, and trisaccharide forms on Drosophila Notch, depending on the EGF repeat examined (28,33).…”

Galactose Differentially Modulates Lunatic and Manic Fringe Effects on Delta1-induced NOTCH Signaling

“…Attempts to identify such differences using radiolabeled GlcNAc and NOTCH1 ECD fragments were difficult to interpret. Future efforts will focus on a mass spectrometry strategy that looks very promising (39).…”