Abstract
Fringe plays a key role in the specification of boundaries during development by modulating the ability of Notch ligands to activate Notch receptors. Fringe is a fucose-specific beta1,3-N-acetylglucosaminyltransferase that modifies O-fucose moieties on the epidermal growth factor-like (EGF) repeats of Notch. To investigate how the change in sugar structure caused by Fringe modulates Notch activity, we have analyzed the sites of O-fucose and Fringe modification on mouse Notch1. The extracellular domain of Notch1 has 36 tandem EGF repeats, many of which are predicted to be modified with O-fucose. We recently proposed a broadened consensus sequence for O-fucose, C(2)X(3-5)(S/T)C(3) (where C(2) and C(3) represent the second and third conserved cysteines), significantly expanding the potential number of modification sites on Notch. Here we demonstrate that sites predicted using this broader consensus sequence are modified with O-fucose on mouse Notch1, and we present evidence suggesting that the consensus can be further refined to C(2)X(4-5)(S/T)C(3). In particular, we demonstrate that EGF 12, a portion of the ligand-binding site, is modified with O-fucose and that this site is evolutionarily conserved. We also show that endogenous Fringe proteins in Chinese hamster ovary cells (Lunatic fringe and Radical fringe) as well as exogenous Manic fringe modify O-fucose on many but not all EGF repeats of mouse Notch1. These findings suggest that the Fringes show a preference for O-fucose on some EGF repeats relative to others. This specificity appears to be encoded within the amino acid sequence of the individual EGF repeats. Interestingly, our results reveal that Manic fringe modifies O-fucose both at the ligand-binding site (EGF 12) and in the Abruptex region. These findings provide insight into potential mechanisms by which Fringe action on Notch receptors may influence both the affinity of Notch-ligand binding and cell-autonomous inhibition of Notch signaling by ligand.
Highlights
The Notch signaling pathway plays an essential role in multiple stages of development in metazoans [1]
We show that endogenous Fringe proteins in Chinese hamster ovary cells (Lunatic fringe and Radical fringe) as well as exogenous Manic fringe modify O-fucose on many but not all epidermal growth factor-like (EGF) repeats of mouse Notch1
We demonstrated that the Notch1 protein from Chinese hamster ovary cells is modified with both O-fucose and O-glucose, suggesting that these consensus sites can be used to accurately predict whether a protein will bear the modifications [8]
Summary
Tion sites have not been mapped on either Notch or its ligands. Recent studies have demonstrated that O-fucose modifications play an essential role in Notch function. To better understand the mechanism of how the O-fucose structures modulate Notch function, we have begun to map sites of both O-fucose and Fringe modification on mouse Notch. We show that the broadened Ofucose consensus sequence (C2X3–5(S/T)C3) [10] can be used to accurately predict sites of O-fucosylation, and we observed that Fringe modifies O-fucose on EGF repeats that have important biological roles, including ligand binding and cell-autonomous inhibition by ligand. These findings suggest specific mechanisms for how the change in O-fucose glycan structure can modulate Notch function
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