Glycans that regulate Notch signaling in the intestine

Abstract

Intestinal homeostasis is key to the maintenance of good health. The small intestine plays important roles in absorption, digestion, hormonal and immune functions. Crypt base columnar (CBC) stem cells residing at the bottom of crypts are nurtured by Paneth cells, and together create the stem cell niche, the foundation of intestinal homeostasis. CBC stem cells replicate to replenish their number, or differentiate into a variety of epithelial cells with specialized functions. Notch signaling is a cell-cell signaling pathway that regulates both the proliferation and differentiation of CBC stem cells. NOTCH1 and NOTCH2 stimulated by canonical Notch ligands DLL1 and DLL4 mediate Notch signaling in the intestine that, in concert with other signaling pathways including the WNT and BMP pathways, determines cell fates. Importantly, interactions between Notch receptors and canonical Notch ligands are regulated by O-glycans linked to Ser/Thr in epidermal growth factor-like (EGF) repeats of the Notch receptor extracellular domain (NECD). The O-glycans attached to NECD are key regulators of the strength of Notch signaling. Imbalances in Notch signaling result in altered cell fate decisions and may lead to cancer in the intestine. In this review, we summarize the impacts of mutations in Notch pathway members on intestinal development and homeostasis, with a focus on the glycosyltransferases that transfer O-glycans to EGF repeats of NOTCH1, NOTCH2, DLL1 and DLL4.

Keywords: Notch signalling pathway; O-glycans; glycosyltransferases; intestinal development; mouse models.

Figure 1.. Notch signaling in intestinal epithelium.

Lgr5+ CBC stem cells expressing NOTCH1 and NOTCH2 are present at the bottom of crypts interspersed with Paneth cells. Paneth cells express Notch ligands DDL1, DLL4 and JAG1 which stimulate Notch signaling in CBC stem cells. O-glycans attached to EGF repeats on the ECD of Notch receptors regulate receptor and ligand binding. Notch ligands also have EGF repeats modified by O-glycans, and bind Notch receptors via their N-terminal Delta-Serrate-LAG-2 (DSL) domain. POFUT1 initiates formation of O-fucose glycans by adding O-fucose to the consensus site present in numerous EGF repeats of NECD. O-fucose is extended by the addition of GlcNAc by LFNG or RFNG. LFNG is expressed in CBC stem cells, at increased levels in progenitors of the TA zone, and by goblet cells, enteroendocrine (EE) and Tuft cells. RFNG is expressed by Paneth cells. Interactions between Notch receptor O-glycans and Notch ligands determine the strength of Notch signaling, which regulates the replication and differentiation of CBC stem cells. Secretory lineage progenitors expressing DLL1 and LFNG induce lateral inhibition of enterocyte progenitors in the TA zone to control the balance of cell fate decisions and differentiated cells of the villus. The figure was created in part with Biorender.com.

Figure 2.. Roles of O-glycans in Notch signaling in small intestine.

Mouse NOTCH1 ECD consists of 36 EGF repeats which are modified by various O-glycans signified by the symbol for the initiating sugar. Each O-glycan is initiated at a consensus site in relevant EGF repeats by a specific glycosyltransferase as shown. The EGF repeats shown to be modified by a particular O-glycan are based on recent reviews for O-fucose and O-glucose (60) glycans, and for O-GlcNAc glycans (39). Further extension of the initial sugar by other glycosyltransferases leads to the formation of an O-glycan. The diagram summarizes the consequences of deleting different glycosyltransferase genes in the intestine (X, gene deleted; red arrow, decrease; green arrow, increase). Proposed or ongoing experiments investigating intestinal consequences in compound mutant mice are also presented.