student research day - Case Western Reserve University School of ...

David Yao
The Regulation of O-linked fucose on Notch Signaling and Notch-
Ligand Interactions
David Yao, Bronislawa Petryniak, Jeongsup Shim, John Lowe
Department of Pathology
Case Western Reserve University School of Medicine
Notch receptors play an essential role in cellular differentiation, proliferation and apoptosis, thus controlling the
development of multiple biological systems. In vertebrates, Notch is a family of transmembrane proteins that includes
Notch1, Notch2, Notch3 and Notch4. These molecules interact with both Delta-like and Jagged ligands. The extracellular
domain of Notch1 is composed of multiple tandem EGF-like repeat motifs. Each contains six highly conserved cysteine
residues as well as serine/threonine residues that may be decorated with O-linked fucose. These O-linked fucose moieties
can be further elongated by Fringe glycosyltransferases, which append N-acetylglucosamine molecules to fucose through
a ß-1,3-linkage. Although O-fucosylation of Notch receptors is clearly evident, the physiologic function of O-linked fucose
in the activation of Notch signaling and Notch-ligand binding are still unclear. In this report, we first demonstrated that
Fringe elongation of O-linked fucose on Notch1 enhances the binding affinity between Notch1 and Delta-like ligands
including DLL1 and DLL4. Furthermore, this modification up-regulates DLL1- and DLL4-mediated Notch signaling. On the
contrary, the Fringe modification does not appear to regulate the Notch1-Jagged1 (J1) interaction and thus J1-mediated
Notch signaling. To further address the function of O-linked fucose, we studied the induction of Notch signaling in the
presence of Lunatic Fringe after an individual O-linked fucose site was abolished. Our results suggest that multiple Olinked
fucose sites on Notch1 differentially participate in DLL1- and DLL4-mediated Notch activation. Mutations at both
EGF9 and EGF12 reduced Notch activity significantly. We further examined the regulation of individual O-fucose sites in
Notch-ligand binding. By comparing to the wild-type control, the affinity of soluble DLL1, DLL4, and Jagged-2 ligands to
Notch1 expressing cells was reduced in the presence of individual EGF9 and EGF12 mutations. These findings indicate
that O-fucose sites play an essential role in Notch-ligand binding and hence the activation of Notch signaling.
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