Roles of Notch Glycoslation in Signaling

Abstract

Notch receptors are single pass transmembrane glycoproteins with an extracellular domain (ECD) comprised of many epidermal growth factor-like (EGF) repeats. Specific Ser and Thr residues in numerous EGF repeats have an attached O-glycan initiated by fucose, glucose, or N-acetylglucosamine (GlcNAc), and elongated with additional sugars. Notch receptors signal following binding of Notch ligands to the Notch receptor ECD, leading to release of the intracellular domain and activation of many Notch target genes. Notch signaling regulates cell fate decisions in many organs during development and differentiation. Dysregulation of Notch signaling leads to developmental disorders and to cancer. Lunatic (LFNG), Manic (MFNG) and Radical (RFNG) Fringe are GlcNAc transferases that extend Notch EGF repeats which already carry a fucose. EOGT is also a GlcNAc-transferase that transfers O-GlcNAc directly to EGF repeats. We are investigating roles for EOGT and each Fringe in T and B cell development using mice lacking various combinations of the Eogt, Lfng, Mfng and Rfng genes. We are also identifying individual roles for Fng genes using a co-culture assay of bone marrow hemopoietic stem cells (HSC) from compound mutant mice stimulated by OP9 stromal cells expressing one of the four Notch ligands, DLL1, DLL4, JAG1 or JAG2. The combined results show that the O-fucose glycans on NOTCH1 are key regulators of the strength of NOTCH1 signaling which, in turn, determines differentiation of HSC to T cell progenitors.