Roles of Pofut1 and O-Fucose in Mammalian Notch Signaling

Mark Stahl,Kazuhide Uemura,Changhui Ge,Shaolin Shi,Yuko Tashima,Pamela Stanley

doi:10.1074/jbc.m802027200

Abstract

Mammalian Notch receptors contain 29-36 epidermal growth factor (EGF)-like repeats that may be modified by protein O-fucosyltransferase 1 (Pofut1), an essential component of the canonical Notch signaling pathway. The Drosophila orthologue Ofut1 is proposed to function as both a chaperone required for stable cell surface expression of Notch and a protein O-fucosyltransferase. Here we investigate these dual roles of Pofut1 in relation to endogenous Notch receptors of Chinese hamster ovary and murine embryonic stem (ES) cells. We show that fucosylation-deficient Lec13 Chinese hamster ovary cells have wild type levels of Pofut1 and cell surface Notch receptors. Nevertheless, they have reduced binding of Notch ligands and low levels of Delta1- and Jagged1-induced Notch signaling. Exogenous fucose but not galactose rescues both ligand binding and Notch signaling. Murine ES cells lacking Pofut1 also have wild type levels of cell surface Notch receptors. However, Pofut1-/- ES cells do not bind Notch ligands or exhibit Notch signaling. Although overexpression of fucosyltransferase-defective Pofut1 R245A in Pofut1-/- cells partially rescues ligand binding and Notch signaling, this effect is not specific. The same rescue is achieved by an unrelated, inactive, endoplasmic reticulum glucosidase. Therefore, mammalian Notch receptors require Pofut1 for the generation of optimally functional Notch receptors, but, in contrast to Drosophila, Pofut1 is not required for stable cell surface expression of Notch. Importantly, we also show that, under certain circumstances, mammalian Notch receptors are capable of signaling in the absence of Pofut1 and O-fucose.

Highlights

Numerous modulators of the canonical Notch signaling pathway have been identified, most of which act intracellularly
We investigate the relative roles of protein O-fucosyltransferase 1 (Pofut1) and O-fucose in mediating the cell surface expression of mammalian Notch receptors as well as their abilities to bind Notch ligands and to transduce a Notch signal
We show that in murine embryonic stem (ES) cells or Chinese hamster ovary (CHO) cells, Pofut1 is not required for stable cell surface expression of mammalian Notch receptors

Summary

EXPERIMENTAL PROCEDURES

Cells and Cell Culture—CHO cells were cultured in ␣-modified minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum (Gemini, West Sacramento, CA) unless otherwise indicated. Cells (5 ϫ 105) were incubated with soluble Notch ligands (Delta1-Fc or Jagged1-Fc) or anti-NECD antibodies in 0.2 ml of ligand binding buffer for 30 – 60 min at room temperature or at 4 °C with gentle rotation. To examine binding of anti-Notch antibodies to fixed and permeabilized ES cells, washed cells (1 ϫ 106) were fixed with Fix & Perm Reagent A (Fixation Medium; Caltag Laboratories, Burlingame, CA) for 15 min at room temperature, centrifuged, resuspended in 1 ml of binding buffer, and divided into two aliquots. Cells were incubated with PE-labeled secondary antibody in binding buffer for 15 min at room temperature and in Fix & Perm Reagent A (Fixation Medium) according to the manufacturer’s instructions (Caltag Laboratories, Burlingame, CA). L, soluble Jagged1-Fc (0.5 ␮g/ml) binding to Lec cells expressing Lfng (solid line) or control vector (dashed line)

RESULTS

Overexpression of wild type

Average Notch signaling ratio