Notch receptors are cell surface glycoproteins that transduce signals to control cell fate and cell division in metazoans. Constitutive or disregulated signaling leads to developmental defects and cancer. The ligands that bind to Notch and stimulate signal transduction are Delta and Serrate/Jagged. We have shown in collaboration with others that Drosophila and mammalian fringe proteins have a beta3N-acetylglucosaminyltransferase activity and act directly on Notch to add GIcNAc to O-fucose on Notch EGF repeats, thereby modulating ligand-induced Notch signaling. In Drosophila and mammalian cells, fringe acts cell autonomously to inhibit the Notch response to Serrate(Jagged) while potentiating the Notch response to Delta. We have used a co-culture reporter assay to show that manic or lunatic fringe in Notch expressing CHO cells inhibit the response to Jagged1. We have used a panel of CHO glycosylation mutants to show that inhibition of Notch signaling by fringe does not require complex or hybrid N-glycans or sialic acid but does require the action of fringe on O-fucose attached to Notch EGF repeats, and the subsequent action of beta4GalT- 1. Thus O. fucosyltransferase and beta4GalT-1 are novel modulators of Notch signaling. Using CHO glycosylation mutants, co-culture Notch signaling and soluble ligand binding assays, and mouse mutants, we propose 1 )To identify the different 0-fucose glycans required for Jagged- and Delta-induced signaling by Notch receptors 1, 2, 3 and 4 and for the modulation of Notch signaling by mammalian fringes (manic, lunatic and radical); 2) To determine the biochemical mechanism by which fringe action perturbs Notch signaling; and 3) To identify in vivo consequences to Notch signaling in the mouse of inactivating the genes encoding new modulators of Notch signaling.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA095022-04
Application #
6878134
Study Section
Pathobiochemistry Study Section (PBC)
Program Officer
Woodhouse, Elizabeth
Project Start
2002-04-01
Project End
2007-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
4
Fiscal Year
2005
Total Cost
$408,358
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
Varshney, Shweta; Stanley, Pamela (2017) Notch Ligand Binding Assay Using Flow Cytometry. Bio Protoc 7:
Song, Yinghui; Kumar, Vivek; Wei, Hua-Xing et al. (2016) Lunatic, Manic, and Radical Fringe Each Promote T and B Cell Development. J Immunol 196:232-43
Wang, Weihuan; Yu, Shuiliang; Zimmerman, Grant et al. (2015) Notch Receptor-Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention. Stem Cells 33:2280-93
Tashima, Yuko; Stanley, Pamela (2014) Antibodies that detect O-linked ?-D-N-acetylglucosamine on the extracellular domain of cell surface glycoproteins. J Biol Chem 289:11132-42
Yao, David; Huang, Yuanshuai; Huang, Xiaoran et al. (2011) Protein O-fucosyltransferase 1 (Pofut1) regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions. Blood 117:5652-62
Visan, Ioana; Yuan, Julie S; Liu, Ying et al. (2010) Lunatic fringe enhances competition for delta-like Notch ligands but does not overcome defective pre-TCR signaling during thymocyte beta-selection in vivo. J Immunol 185:4609-17
Guilmeau, Sandra; Flandez, Marta; Bancroft, Laura et al. (2008) Intestinal deletion of Pofut1 in the mouse inactivates notch signaling and causes enterocolitis. Gastroenterology 135:849-60, 860.e1-6
Stahl, Mark; Uemura, Kazuhide; Ge, Changhui et al. (2008) Roles of Pofut1 and O-fucose in mammalian Notch signaling. J Biol Chem 283:13638-51
Stanley, Pamela (2007) Regulation of Notch signaling by glycosylation. Curr Opin Struct Biol 17:530-5
Shi, Shaolin; Ge, Changhui; Luo, Yi et al. (2007) The threonine that carries fucose, but not fucose, is required for Cripto to facilitate Nodal signaling. J Biol Chem 282:20133-41

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