Notch receptors play an essential role in numerous stages of development, and deregulation of Notch signaling leads to a variety of human pathologies, including cancers, vascular disorders, and developmental disorders. The Notch extracellular domain (ECD) contains up to 36 tandem epidermal growth factor-like (EGF) repeats, many of which are modified by two unusual forms of glycosylation: O-fucose and O-glucose. O-Fucose modifications are essential for Notch function. Genetic ablation of the enzyme that adds O-fucose to EGF repeats causes severe Notch-like phenotypes in either mice or flies, and elongation of O-fucose by the Fringe family of ss3-N-acetylglucosaminyltransferases modulates Notch signaling. Fringe functions by altering the interaction between Notch and its ligands: Delta or Serrate/Jagged. Interestingly, Fringe modification increases Notch-Delta interactions while inhibiting Notch-Serrate/Jagged interactions. We have recently shown that O-glucose modifications are also essential for Notch function. Mutations in the gene encoding the enzyme responsible for addition of O-glucose to EGF repeats (protein O-glucosyltransferase, or Rumi), cause a temperature sensitive Notch-like phenotype in flies. Although both O-fucose and O-glucose modifications are required for Notch function, the molecular details for how they mediate their effects are not known. Our hypothesis is that the O-fucose and O-glucose glycans affect Notch function by affecting the conformation of the Notch ECD. This hypothesis is based on exciting recent structural studies suggesting that the Notch ECD has regions of flexibility that are evolutionarily conserved. In the first aim we will analyze how Fringe modification alters Notch-ligand binding. Using quantitative mass spectral methods, we will map Fringe modification sites, and correlate these modifications with changes in Notch-ligand binding. These studies will tell us which regions of the Notch ECD are involved in regulating Notch-ligand binding. We will examine the possibility that some regions of the Notch ECD inhibit ligand binding, and we will test whether Fringe modulates this inhibitory activity. Finally, we will examine whether Fringe induces changes in the shape of the Notch ECD using protease sensitivity, analytical ultracentrifugation, and an innovative cryoEM method. In the second aim, we will identify and characterize mammalian homologue of Rumi, as well as the xylosyltransferases that elongate O-glucose with xyloses.
The final aim i s designed to test whether elimination of Rumi (or the xylosyltransferases identified in Aim 2) in mammalian cells (using siRNA strategies) causes a loss of Notch activity as was seen in flies. In addition, we will examine whether loss of O-glucose causes conformational changes in the Notch ECD using the same methodologies described in Aim 1 for the effects of Fringe on Notch. These experiments will provide molecular details for how these unusual carbohydrate modifications alter Notch function. Public Health Relevance: Defects in the Notch signaling pathway lead to a variety of human pathologies, including several types of cancer, vascular disorders, and developmental disorders. Notch is regulated at numerous levels, including by glycosylation (modification of proteins by sugars). Our studies are aimed at understanding how glycosylation affects Notch activity so that we can take advantage of its ability to regulate Notch to design potential therapies for Notch-related diseases.

Public Health Relevance

to human health: Defects in the Notch signaling pathway lead to a variety of human pathologies, including several types of cancer, vascular disorders, and developmental disorders. Notch is regulated at numerous levels, including by glycosylation (modification of proteins by sugars). Our studies are aimed at understanding how glycosylation affects Notch activity so that we can take advantage of its ability to regulate Notch to design potential therapies for Notch-related diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061126-12
Application #
8197516
Study Section
Intercellular Interactions (ICI)
Program Officer
Marino, Pamela
Project Start
2001-01-01
Project End
2013-09-15
Budget Start
2011-12-01
Budget End
2013-09-15
Support Year
12
Fiscal Year
2012
Total Cost
$626,355
Indirect Cost
$171,492
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Vasudevan, Deepika; Takeuchi, Hideyuki; Johar, Sumreet Singh et al. (2015) Peters plus syndrome mutations disrupt a noncanonical ER quality-control mechanism. Curr Biol 25:286-95
Vasudevan, Deepika; Haltiwanger, Robert S (2014) Novel roles for O-linked glycans in protein folding. Glycoconj J 31:417-26
Takeuchi, Hideyuki; Haltiwanger, Robert S (2014) Significance of glycosylation in Notch signaling. Biochem Biophys Res Commun 453:235-42
Taylor, Paul; Takeuchi, Hideyuki; Sheppard, Devon et al. (2014) Fringe-mediated extension of O-linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands. Proc Natl Acad Sci U S A 111:7290-5
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Al-Shareffi, Esam; Chaubard, Jean-Luc; Leonhard-Melief, Christina et al. (2013) 6-alkynyl fucose is a bioorthogonal analog for O-fucosylation of epidermal growth factor-like repeats and thrombospondin type-1 repeats by protein O-fucosyltransferases 1 and 2. Glycobiology 23:188-98
Takeuchi, Hideyuki; Haltiwanger, Robert S (2013) Enzymatic analysis of the protein O-glycosyltransferase, Rumi, acting toward epidermal growth factor-like (EGF) repeats. Methods Mol Biol 1022:119-28
Lee, Tom V; Sethi, Maya K; Leonardi, Jessica et al. (2013) Negative regulation of notch signaling by xylose. PLoS Genet 9:e1003547

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