Notch receptors initiate a signaling pathway essential for development in all metazoans. Defects in the pathway cause a number of congenital birth defects and cancers. Notch can function as either an oncogene or tumor suppressor in cancers, so therapies that can increase or decrease Notch activity are needed. Notch is regulated at numerous levels, but glycosylation of the Notch extracellular domain (ECD) has emerged as a major regulator that can increase or decrease Notch activity depending on context. The Notch ECD contains up to 36 tandem Epidermal Growth Factor-like (EGF) repeats, many of which contain consensus sequences for O-linked fucose. O-Fucose is added to EGF repeats by Protein O-fucosyltransferase 1 (POFUT1) and is essential for Notch function in all contexts examined, while extension of O-fucose by the Fringe family of ?3-N- acetylglucosaminyltransferases is modulatory. Fringe regulation of Notch has become a paradigm for regulation of a signaling receptor by altering its glycosylation status. Fringe modifications typically enhance Notch1 (N1) activation by the Delta-like family of ligands (DLL1, DLL4), but inhibit activation by Jagged ligands (J1, J2). In the past grant cycle we made significant progress on the molecular mechanisms by which Fringe modifications differentiate between ligands by identifying which EGF repeats of N1 are modified by each Fringe enzyme, and determining which of those EGF repeats are responsible for the modulatory effect. Our results demonstrate that Fringes ?mark? the Notch ECD, with some marks activating DLL1-N1 activation, and distinct marks inhibiting J1-N1 activation. In addition, we have recently shown that fucose analogs (fucose with modifications to carbon 6) inhibit Notch activation in a ligand-specific manner, providing proof of concept that we can use small molecules to alter Notch glycosylation and regulate its function. Our results have led to the overall hypothesis for this application: O-glycans at specific sites on Notch regulate its activity by directly modulating initial Notch-ligand binding, or by modulating events subsequent to ligand binding but prior to proteolytic receptor activation. We will test this hypothesis in three aims.
Aim 1 seeks to address how Fringe modifications inhibit J1-N1 activation. Our published work shows that Fringes enhance binding of DLL1 to N1, providing a molecular explanation for enhanced DLL1-N1 activation, but we also showed that Fringes enhance J1 binding to N1. Thus, Fringe modification must inhibit J1-N1 activation by affecting a step subsequent to ligand binding but prior to proteolytic receptor activation. The experiments in Aim 1 will examine several possible mechanisms for this effect.
Aim 2 examines the basis for the striking site-specific elongation of O- fucose on Notch EGF repeats by the Fringes. This knowledge will allow us to eliminate Fringe modification at specific sites without affecting addition of O-fucose. Finally, Aim 3 examines how the fucose analogs affect Notch activity, whether we can generate more potent analogs with distinct activities, and whether we can use the analogs to inhibit growth of cancer cells dependent on Notch activity for division.

Public Health Relevance

The Notch signaling pathway is essential for development, and defects in the pathway result in a number of human diseases including birth defects (e.g. congenital heart defects) as well as cancers. Since hyperactivation of Notch causes cancer in some contexts (e.g. T-cell acute lymphoblastic leukemia), while reduction of Notch activity causes cancer in others (e.g. squamous cell carcinomas), methods to increase or decrease Notch activity could lead to novel therapies. Here we analyze how glycosylation of the Notch receptor can either increase or decrease Notch activity and examine small molecules that alter glycosylation and inhibit Notch activity.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Intercellular Interactions Study Section (ICI)
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Bond, Michelle Rueffer
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University of Georgia
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Takeuchi, Hideyuki; Schneider, Michael; Williamson, Daniel B et al. (2018) Two novel protein O-glucosyltransferases that modify sites distinct from POGLUT1 and affect Notch trafficking and signaling. Proc Natl Acad Sci U S A 115:E8395-E8402
Takeuchi, Hideyuki; Wong, Derek; Schneider, Michael et al. (2018) Variant in human POFUT1 reduces enzymatic activity and likely causes a recessive microcephaly, global developmental delay with cardiac and vascular features. Glycobiology 28:276-283
Schneider, Michael; Kumar, Vivek; Nordstrøm, Lars Ulrik et al. (2018) Inhibition of Delta-induced Notch signaling using fucose analogs. Nat Chem Biol 14:65-71
Weh, Eric; Takeuchi, Hideyuki; Muheisen, Sanaa et al. (2017) Functional characterization of zebrafish orthologs of the human Beta 3-Glucosyltransferase B3GLCT gene mutated in Peters Plus Syndrome. PLoS One 12:e0184903
Hubmacher, Dirk; Schneider, Michael; Berardinelli, Steven J et al. (2017) Unusual life cycle and impact on microfibril assembly of ADAMTS17, a secreted metalloprotease mutated in genetic eye disease. Sci Rep 7:41871
Luca, Vincent C; Kim, Byoung Choul; Ge, Chenghao et al. (2017) Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity. Science 355:1320-1324
Takeuchi, Hideyuki; Yu, Hongjun; Hao, Huilin et al. (2017) O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking. J Biol Chem 292:15964-15973
Sheikh, M Osman; Halmo, Stephanie M; Patel, Sneha et al. (2017) Rapid screening of sugar-nucleotide donor specificities of putative glycosyltransferases. Glycobiology 27:206-212
Kakuda, Shinako; Haltiwanger, Robert S (2017) Deciphering the Fringe-Mediated Notch Code: Identification of Activating and Inhibiting Sites Allowing Discrimination between Ligands. Dev Cell 40:193-201
Schneider, Michael; Al-Shareffi, Esam; Haltiwanger, Robert S (2017) Biological functions of fucose in mammals. Glycobiology 27:601-618

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