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J. Biol. Chem., Vol. 278, Issue 43, 42340-42345, October 24, 2003
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Modulation of Notch-Ligand Binding by Protein O-Fucosyltransferase 1 and Fringe*
From the Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854
Notch receptors are glycoproteins that mediate a wide range of developmental processes. Notch is modified in its epidermal growth factor-like domains by the addition of fucose to serine or threonine residues. O-Fucosylation is mediated by protein O-fucosyltransferase 1, and down-regulation of this enzyme by RNA interference or mutation of the Ofut1 gene in Drosophila or by mutation of the Pofut1 gene in mouse prevents Notch signaling. To investigate the molecular basis for the requirement for O-linked fucose on Notch, we assayed the ability of tagged, soluble forms of the Notch extracellular domain to bind to its ligands, Delta and Serrate. Down-regulation of OFUT1 by RNA interference in Notch-secreting cells inhibits both Delta-Notch and Serrate-Notch binding, demonstrating a requirement for O-linked fucose for efficient binding of Notch to its ligands. Conversely, overexpression of OFUT1 in cultured cells increases Serrate-Notch binding but inhibits Delta-Notch binding. These effects of OFUT1 are consistent with the consequences of OFUT1 overexpression on Notch signaling in vivo. Intriguingly, they are also opposite to, and are suppressed by, expression of the glycosyltransferase Fringe, which specifically modifies O-linked fucose. Thus, Notch-ligand interactions are dependent upon both the presence and the type of O-fucose glycans.
Received for publication, August 6, 2003
* This work was supported by the Howard Hughes Medical Institute and by National Institutes of Health Grant R01-GM54594. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A long term fellow of the Human Frontier Science Program (Grant LT00582).
To whom correspondence should be addressed. Tel.: 732-445-2332; Fax: 732-445-7431; E-mail: irvine{at}waksman.rutgers.edu.
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