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

Linchao Lu,Xinghua Hou,Shaolin Shi,Christian Körner,Pamela Stanley

doi:10.1074/jbc.m110.126003

Linchao Lu, Xinghua Hou + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.126003

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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.

Highlights

Modified with O-fucose attached to Ser or Thr by protein O-fucosyltransferase-1 (Pofut1)4 in mammals and OFUT1 in Drosophila [3]
We previously reported that overexpression of Slc35c2 decreases expression of fucosylated epitopes termed Lewis X and sialyl-Lewis X in LEC11B Chinese hamster ovary (CHO) cells [32]
In this article we show that overexpressed Slc35c2 increases, whereas knockdown reduces, O-fucosylation of Notch1 epidermal growth factor-like (EGF) repeats, and that optimal Notch signaling requires Slc35c2 in mammalian cells

Summary

The abbreviations used are

Protein O-fucosyltransferase-1; PE, phycoerythrin; SSEA-1, stage-specific embryonic antigen-1; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; GSI, ␥-secretase inhibitor; Fut6B, ␣1,3-fucosyltransferse 6B; Endo H, endoglycosidase H; IEC, intestinal epithelial cell; PDI, protein disulfide isomerase; ManII, ␣-mannosidase II; LADII, leukocyte adhesion deficiency type II; ER, endoplasmic reticulum; MEF, mouse embryonic fibroblast; PNGase, peptide:N-glycosidase; PSA, Pisum sativum agglutinin. Sequence comparisons revealed a gene that clusters with SLC35C1 termed SLC35C2 in human [31], to be the most likely candidate for an alternative GDP-fucose transporter. We previously reported that overexpression of Slc35c2 decreases expression of fucosylated epitopes termed Lewis X and sialyl-Lewis X in LEC11B CHO cells [32]. An alternative GDP-fucose transporter recently identified in Drosophila as important for Notch signaling is the homologue of human SLC35B4 [33]. We investigated the effects of overexpression and reduced expression of mouse and CHO Slc35c2 on the fucosylation and activation of Notch signaling in mammalian cells. In this article we show that overexpressed Slc35c2 increases, whereas knockdown reduces, O-fucosylation of Notch EGF repeats, and that optimal Notch signaling requires Slc35c2 in mammalian cells

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