Novel glycan modification found to regulate notch signaling

A research group including Professor Hideyuki Takeuchi and Assistant Professor Yohei Tsukamoto of the Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka; Professor Tetsuya Okajima of the Department of Molecular Biochemistry, Nagoya University Graduate School of Medicine; and Associate Professor Hiroyuki Hosokawa and Assistant Professor Yuichi Kama of the Division of Host Defense Mechanism, Basic Medical Sciences, Tokai University School of Medicine, together with collaborators from three other institutions, has discovered a previously unknown glycan modification on Notch receptors, which play essential roles in cell development and differentiation. The researchers found that the novel glycan modification suppresses Notch1 signaling. The findings were published in PNAS.

The Notch signaling pathway plays a crucial role in the development and differentiation of multicellular organisms. Notch signaling is initiated when ligand proteins on the surface of one cell bind to the large receptor protein NOTCH on the surface of a neighboring cell. This interaction ultimately alters gene expression, transmitting signals within the cell. Dysfunction of this receptor is associated with numerous congenital disorders and cancers.

Proper Notch receptor function depends on O-glycosylation, in which sugars such as glucose and fucose are attached to the receptor's extracellular domain. However, many aspects of how these modifications regulate Notch receptor activity remain unclear.

Using advanced analytical techniques, including liquid chromatography-mass spectrometry (LC-MS), the researchers identified a previously unknown glycan modification on Notch receptors and clarified its function. Specifically, they discovered a novel structure, S-Glc-Gal-Neu5Ac, among O-glucose (Glc) glycans attached to serine residues within epidermal growth factor-like (EGF) repeats. Although this structure was already known as a glycan modification found on glycolipids, this is the first report of its occurrence on glycoproteins.

Mass spectrometry analysis revealed that the novel glycan modification is found exclusively on the tenth EGF repeat (EGF10) among the 36 EGF repeats present in the extracellular domain of NOTCH1. The researchers further showed that O-glucose on EGF10 can be extended not only by the previously known addition of xylose but also by a newly identified galactose extension.

By analyzing glycans derived from glycosyltransferase-deficient cells, the team identified the glycosyltransferases responsible for their biosynthesis. They found that B4GALT1 mediates the addition of galactose to O-glucose, while ST3GAL4 catalyzes the addition of Neu5Ac to galactose-extended O-glucose. These enzymes differ from those responsible for synthesizing the same Glc-Gal-Neu5Ac structure on glycolipids, demonstrating that glycoproteins and glycolipids can use distinct biosynthetic pathways despite carrying similar glycan structures.

The researchers also showed that B4GALT1-dependent galactose extension of O-glucose occurs in HEK293T cells, which do not express α-lactalbumin, a protein previously thought to be essential for this process. They further identified alanine at position 396 (A396) as a key determinant of galactose extension. Substituting this residue with the aromatic amino acids tyrosine (A396Y) or phenylalanine (A396F) completely abolished galactose extension of the O-glucose glycan.

The researchers also investigated how the novel glycan modification affects Notch signaling. To examine its function, they generated NOTCH proteins carrying different glycan structures using A396 mutants, glycosyltransferase-deficient cells, and glycosyltransferase-overexpressing cells.

Experiments using recombinant NOTCH1 proteins showed that xylose extension enhanced ligand binding, whereas galactose extension suppressed it. The team then measured Notch signaling by stimulating cells expressing full-length recombinant NOTCH1 proteins with recombinant ligands. In A396Y and A396F mutants, which lack galactose extension, signaling activity was enhanced compared with wild-type NOTCH1.

Finally, the researchers overexpressed either wild-type NOTCH1 or the A396Y and A396F mutant forms in mouse lymphoid progenitor cells and induced differentiation by co-culturing the cells with ligand-expressing OP9 cells. These lymphoid progenitor cells (Cas9-LPs) lack the EBF1 gene required for B-cell differentiation and therefore differentiate exclusively into T cells in a Notch signaling-dependent manner.

Compared with cells expressing wild-type NOTCH1, progenitor cells expressing the galactose-deficient mutants showed markedly enhanced differentiation into T cells.

Taken together, the results show that galactose extension suppresses binding between NOTCH1 and its ligands, whereas xylose extension enhances it. As a consequence, the absence of galactose extension leads to enhanced Notch signaling. When this occurs in lymphoid progenitor cells, the strengthened NOTCH1 signaling appears to promote differentiation into T cells.

The study demonstrates that a difference in a single amino acid can dramatically alter glycan structure and, in turn, regulate the Notch signaling pathway, which is essential for multicellular organisms. Beyond expanding our understanding of the diversity of glycan modifications, the findings provide new insight into the mechanisms that regulate Notch receptors.

The results may also contribute to future drug discovery efforts aimed at controlling Notch signaling through glycan modifications.

Journal Information
Publication: PNAS
Title: Differential O-glucose elongation on a specific EGF repeat within the canonical ligand-binding domain regulates DLL1/4-NOTCH1 signaling
DOI: 10.1073/pnas.2504827122