Elsevier

Methods in Enzymology

Volume 417, 2006, Pages 127-136
Methods in Enzymology

Roles of O‐Fucose Glycans in Notch Signaling Revealed by Mutant Mice

https://doi.org/10.1016/S0076-6879(06)17010-XGet rights and content

Abstract

Notch receptor signaling is important for many developmental processes in the metazoa. Insights into how Notch receptor signaling is regulated have been obtained from the characterization of mutants of model organisms in which Notch signaling is perturbed. Here we describe the effects of mutations that alter the glycosylation of Notch receptors and Notch ligands in the mouse. The extracellular domain of Notch receptors and Notch ligands carries N‐glycans and O‐glycans, including O‐fucose and O‐glucose glycans. Mutations in several genes that inhibit the synthesis of O‐fucose glycans, and one that also affects the maturation of N‐glycans, cause Notch signaling defects and disrupt development.

Section snippets

Overview

Notch receptors belong to a family of single transmembrane glycoproteins containing 29–36 EGF repeats in their extracellular domain (ECD). Notch receptor signaling is critical for cell fate determination, cell growth control, and development in metazoans. In mammals, there are four Notch receptors (Notch1–Notch4) and five Notch ligands (Jagged1, Jagged2, Delta1, Delta3, and Delta4). Each Notch receptor is synthesized in the endoplasmic reticulum (ER) as a single polypeptide and later cleaved in

Glycosylation of Notch Receptors and Notch Ligands

The extracellular domain of Notch receptors is glycosylated with N‐glycans (Johansen et al., 1989) and O‐glycans, including O‐fucose and O‐glucose glycans (Moloney et al., 2000b). O‐glucosylation occurs at Ser or Thr in the consensus sequence C1XXPS/TC2 between the first and second Cys residue in Notch EGF repeats. A trisaccharide may be present at O‐glucose sites. Although the structure of the trisaccharide is unknown, it is predicted to contain xylose in the structure Xylβ1,3Xylβ1,3Glc (Fig. 1

Pofut1 Is an Essential Component of the Canonical Notch Signaling Pathway

O‐Fucose on Notch receptors was first shown to play a role in Notch signaling in Lec13 Chinese hamster ovary (CHO) cells that make very low amounts of GDP‐fucose (Moloney 2000a, Chen 2001). Jagged1‐induced Notch signaling in a co‐culture reporter assay is markedly reduced in Lec13 cells. These cells transfer little fucose to glycoproteins but can be corrected by exogenous fucose and thus have normal function of fucosyltransferases including Pofut1. The fact that Lec13 cells exhibit little Notch

Fringe Is a Modulator of Notch Signaling

Fringe is a β1,3N‐acetylglucosaminyltransferase (β1,3GlcNAcT) that transfers GlcNAc to O‐fucose on EGF repeats of Notch receptors (Haines and Irvine, 2003). In mammals, there are three Fringe homologs, Lunatic, Manic, and Radical Fringe (Lfng, Mfng, and Rfng). They function in the Golgi, but all of them are also secreted.

Lfng is expressed in many cell types during embryonic stages, but its dynamic expression in mouse somites indicated a functional role during segmentation (Johnston et al., 1997

β4GalT‐1 Is a Novel Regulator of Notch Signaling

The elongation of the GlcNAcβ3Fuc disaccharide on Notch EGF repeats by β4GalT‐1 was found to be required for Lfng and Mfng inhibition of Jagged1‐induced Notch signaling in a co‐culture assay (Chen et al., 2001). Neither the Lec20 CHO mutant that lacks β4GalT‐1 and β4GalT‐6 nor the Lec8 CHO mutant that cannot transport UDP‐Gal into the Golgi exhibits Lfng or Mfng modulation of Notch signaling. When corrected with a cDNA encoding β4GalT‐1, Lec20 cells are rescued for Fng effects. To investigate

Mutations in Other Glycosyltransferase Genes

There are now many strains of mice that have a null mutation in a glycosyltransferases gene that affects the synthesis of N‐glycans or mucin O‐glycans or O‐Mannose glycans (Lowe and Marth, 2003). Although Notch receptors and their ligands carry N‐glycans, none of the mutant mice defective in N‐glycan synthesis with the exception of the mice lacking β4GalT‐1 described previously have been found to exhibit an overt phenotype consistent with defective Notch signaling. However, they may well have a

Acknowledgments

This work was supported by NCI grant RO1 95022 to PS.

References (48)

  • R. Rampal et al.

    Highly conserved O‐fucose sites have distinct effects on Notch1 function

    J. Biol. Chem.

    (2005)
  • L. Shao et al.

    Fringe modifies O‐fucose on mouse Notch1 at epidermal growth factor‐like repeats within the ligand‐binding site and the Abruptex region

    J. Biol. Chem.

    (2003)
  • K. Shimizu et al.

    Manic fringe and lunatic fringe modify different sites of the Notch2 extracellular region, resulting in different signaling modulation

    J. Biol. Chem.

    (2001)
  • D.B. Sparrow et al.

    Mutation of the Lunatic Fringe gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype

    Am. J. Hum. Genet.

    (2006)
  • N. Zhang et al.

    A mutation in the Lunatic fringe gene suppresses the effects of a Jagged2 mutation on inner hair cell development in the cochlea

    Curr. Biol.

    (2000)
  • M. Asano et al.

    Growth retardation and early death of beta‐1,4‐galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells

    EMBO J.

    (1997)
  • D.J. Becker et al.

    Strain‐specific modification of lethality in fucose‐deficient mice

    Mamm. Genome.

    (2003)
  • Y. Bessho et al.

    Periodic repression by the bHLH factor Hes7 is an essential mechanism for the somite segmentation clock

    Genes Dev.

    (2003)
  • J. Chen et al.

    Negative feedback loop formed by Lunatic fringe and Hes7 controls their oscillatory expression during somitogenesis

    Genesis

    (2005)
  • J. Chen et al.

    Fringe modulation of Jagged1‐induced Notch signaling requires the action of beta4galactosyltransferase‐1

    Proc. Natl. Acad. Sci. USA

    (2001)
  • R. Cordes et al.

    Specification of vertebral identity is coupled to Notch signalling and the segmentation clock

    Development

    (2004)
  • Y.A. Evrard et al.

    lunatic fringe is an essential mediator of somite segmentation and patterning

    Nature

    (1998)
  • K.L. Hahn et al.

    Lunatic fringe null female mice are infertile due to defects in meiotic maturation

    Development

    (2005)
  • K.L. Hahn et al.

    A loss of lunatic fringe is associated with female infertility

    Development

    (2006)
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