An N-glycan on the C2 domain of JAGGED1 is important for Notch activation

Abstract

The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand’s ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling–dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation.

The Role of C2 Domains in Two Different Phosphatases: PTEN and SHIP2

“…Although SHIP2 and PTEN are both phosphatases and contain a catalytic Ptase domain and an adjacent C2 domain, the role of their C2 domains appears to be different. The C2 domain of PTEN participates significantly in membrane binding and is important for the positioning of the active site in the Ptase domain towards the lipid substrate-a role also observed for other C2 domains [37]. In contrast, the C2 domain in SHIP2 bound relatively weakly to anionic phospholipid membranes, and the Ptase domain was able to gain active conformation in the absence of the C2 domain.…”

The Role of C2 Domains in Two Different Phosphatases: PTEN and SHIP2

“…Although SHIP2 and PTEN are both phosphatases and contain a catalytic Ptase domain and an adjacent C2 domain, the role of their C2 domains appear to be different. The C2 domain of PTEN participates significantly in membrane binding, and is important for positioning of the active site in the Ptase domain towards the lipid substrate; a role also observed for other C2 domains [37]. In contrast, the C2 domain in SHIP2 bound relatively weakly to anionic phospholipid membranes and the Ptase domain was able to gain the active conformation in the absence of the C2 domain.…”

Biomedical engineering approaches for the delivery of JAGGED1 as a potential tissue regenerative therapy

“…At the N-terminal of the protein is a phospholipid-binding C2 domain that contributes to NOTCH activation by providing both cell membrane and NOTCH interaction sites. A study done by Meng et al (2022) found that N-glycosylation of this C2 domain is required for efficient phospholipid binding and NOTCH activation by JAG1. This is because the N-glycan promotes the required orientation of JAG1 for NOTCH signaling.…”