Manic Fringe and Lunatic Fringe Modify Different Sites of the Notch2 Extracellular Region, Resulting in Different Signaling Modulation

Abstract

Three mammalian fringe proteins are implicated in controlling Notch activation by Delta/Serrate/Lag2 ligands during tissue boundary formation. It was proved recently that they are glycosyltransferases that initiate elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats in the extracellular domain of Notch molecules. Here we demonstrate the existence of functional diversity among the mammalian fringe proteins. Although both manic fringe (mFng) and lunatic fringe (lFng) decreased the binding of Jagged1 to Notch2 and not that of Delta1, the decrease by mFng was greater in degree than that by lFng. We also found that both fringe proteins reduced Jagged1-triggered Notch2 signaling, whereas neither affected Delta1-triggered Notch2 signaling. However, the decrease in Jagged1-triggered Notch2 signaling by mFng was again greater than that by lFng. Furthermore, we observed that each fringe protein acted on a different site of the extracellular region of Notch2. Taking these findings together, we propose that the difference in modulatory function of multiple fringe proteins may result from the distinct amino acid sequence specificity targeted by these glycosyltransferases.The Notch family of proteins consists of transmembrane receptors that play a critical role in cell fate choices and the formation of compartment border, preventing distinct cell populations from intermixing, during the development of both vertebrates and invertebrates (1-4). In higher vertebrates, four Notch genes, Notch1 through Notch4, have been identified (5-10). An important structural characteristic of these is the common presence of multiple repeats of epidermal growth factor (EGF 1 )-like sequence in the extracellular domain. Ligands for the Notch receptors are designated Delta/Serrate/Lag2 (DSL) proteins (or DSL ligands), five of which have been identified thus far in mammals (Jagged1/Serrate1 (11-13), Jagged2/Serrate2 (14 -16), Delta1 (17), , and Delta4 (19)).Drosophila and mammalian fringe proteins modulate the formation of compartment border in the developing embryo through affecting Notch activation by the ligands (20 -25). Drosophila fringe inhibits a group of cells from responding to the ligand Serrate and potentiates them to respond to another ligand, Delta (20). In higher vertebrates, three fringe proteins, manic fringe (mFng), lunatic fringe (lFng), and radical fringe (rFng) have been identified (22,26). The mammalian fringe proteins modulate Notch signaling when expressed in Drosophila (26), and lFng null mouse phenotypes are similar to those described for mice deficient in components of the Notch signaling pathway (27,28). A weak sequence similarity shared by Drosophila fringe and a class of bacterial glycosyltransferases predicted that fringe proteins might be a glycosyltransferase (29). In a recent work, it was proven experimentally that Drosophila and mammalian fringe proteins have a fucose-specific ␤1,3-N-acetylglucosaminyltransferase activity that catalyzes the elongation of O-linked fucose...

Protein O -fucosyltransferase 1 is an essential component of Notch signaling pathways

“…Notch1 has Ͼ20 EGF repeats that contain a consensus sequence for O-fucosylation and most may be modified by Fringe (7). It is known that the action of Fringe in transferring GlcNAc to EGF-O-fucose causes changes in soluble ligand binding to Notch receptors (5,(13)(14)(15). We have shown that Lec13 mutant cells with low fucose in glycoproteins exhibit reduced Notch signaling for both Jagged1 and Delta1 ligands (refs.…”

“…It is found on urokinase-type plasminogen activator (uPA) (8), on Notch receptors and their ligands (6,9), on Cripto (10,11), and on other proteins, including blood clotting factors. O-Fucose is required for signaling by uPA through the uPA receptor (12), for Fringe modulation of Notch receptor signaling in Drosophila (4,5) and in coculture assays (4,(13)(14)(15)(16), and for Cripto to mediate Nodal signaling (10,11).…”

Regulation of Notch signaling by glycosylation

“…In general, Lunatic Fringe (Lfng) and Manic Fringe (Mfng) inhibit Jagged1-induced Notch signaling and increase Delta1-induced Notch signaling. However, differences between the effects of Lfng and Mfng on Notch2 signaling have been observed [18], and Radical Fringe (Rfng) overexpression in NIH3T3 cells enhances both Delta1-and Jagged1-induced Notch signaling [19]. Lfng, Mfng and Rfng differ in in vitro specific activity [20], and their activities may not be equivalent in different cell types.…”

Fringe-mediated extension of O -linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands

“…Many of the O-fucose monosaccharides on Notch can be extended with GlcNAc by a single Fringe enzyme in Drosophila and by any of three Fringe enzymes-Lunatic fringe (Lfng), Manic fringe (Mfng), or Radical fringe (Rfng) in mammalian organisms (24), each of which affects Notch activity in slightly different ways (17,(25)(26)(27). The simplest case appears in flies, where Fringe modification has a positive effect on Delta-mediated signaling and a negative effect on Serrate-mediated signaling.…”