Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells

Abstract

Mammalian Notch receptors require modification by fucose on epidermal growth factor-like (EGF) repeats of their extracellular domain to respond optimally to signal induction by canonical Notch ligands. Inactivation of the Golgi GDP-fucose transporter Slc35c1 in mouse or human does not cause marked defects in Notch signaling during development, and shows milder fucosylation defects than those observed in mice unable to synthesize GDP-fucose, indicating the existence of another mechanism for GDP-fucose transport into the secretory pathway. We show here that fibroblasts from mice or humans lacking Slc35c1 exhibit robust Notch signaling in co-culture signaling assays. A potential candidate for a second GDP-fucose transporter is the related gene Slc35c2. Overexpression of Slc35c2 reduces expression of the fucosylated epitopes Lewis X and sialylated Lewis X in CHO cells, indicating competition with Slc35c1. The fucosylation of a Notch1 EGF repeat fragment that occurs in the endoplasmic reticulum was increased in CHO transfectants overexpressing Slc35c2. In CHO cells with low levels of Slc35c2, both Delta1-and Jagged1-induced Notch signaling were reduced, and the fucosylation of a Notch1 fragment was also decreased. Immunofluorescence microscopy of rat intestinal epithelial cells and HeLa cells, and analysis of rat liver membrane fractions showed that Slc35c2 is primarily colocalized with markers of the cis-Golgi network and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The combined results suggest that Slc35c2 is either a GDP-fucose transporter that competes with Slc35c1 for GDP-fucose, or a factor that otherwise enhances the fucosylation of Notch and is required for optimal Notch signaling in mammalian cells.

Negative feedback regulation of Wnt signaling via N-linked fucosylation in zebrafish

“…We reasoned that over-expressing a transporter might provide a more robust means to perturb fucosylation. Based on the high degree of similarity between zebrafish and mouse Slc35c1, the fully characterized mouse Myc-tagged Slc35c1 (Lu et al, 2010) might similarly promote N-linked fucosylation in zebrafish embryos, as previously reported in cell culture (Lu et al, 2010). To test this notion we expressed mouse slc35c1-myc ( mSlc35c1 ) mRNA in zebrafish and detected the Slc35c1-Myc protein produced using an anti-Myc antibody immuno-blot (IB).…”

“…In previous work, knocking out an individual GDP-Fuc transporter only caused mild phenotypes in flies and in mice, likely due to functional redundancy between transporters (Geisler et al, 2012; Hellbusch et al, 2007; Ishikawa et al, 2010; Lu et al, 2010). We reasoned that over-expressing a transporter might provide a more robust means to perturb fucosylation.…”

“…However, the nature of such regulation remains unknown. In this study, we found that expression of slc35c1, a rate-limiting step regulating fucosylation (Lu et al, 2010; Moriwaki et al, 2007), fluctuates dramatically during development. This suggests that besides functioning as a “housekeeping gene”, slc35c1 may also play some regulatory role through N-linked fucosylation in specific developmental processes.…”

“…GDP-Fuc availability in cellular compartments is a limiting factor for fucosylation (Lu et al, 2010; Moriwaki et al, 2007). GDP-Fuc transporters play a key role in directing GDP-Fuc to cellular compartments, and thus limit the GDP-Fuc available for protein or glycan modification (Ma et al, 2006).…”

“…As links between GDP-Fuc production and usage, GDP-Fuc transporters are critical regulators of the fucosylation level (Lu et al, 2010; Ma et al, 2006; Moriwaki et al, 2007). In vertebrates, Slc35c1 is the primary transporter for GDP-Fuc into the Golgi apparatus, where Futs modify glycosylated substrates, primarily N-linked glycans (Hellbusch et al, 2007; Ma et al, 2006) (Fig.…”