Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK.
Faculty for Science and Engineering, Biosciences, Åbo Akademi University, Turku, Finland.
Sci Signal. 2022 Oct 11;15(755):eabo3507. doi: 10.1126/scisignal.abo3507.
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.
跨膜配体 Jagged/Serrate 和 Delta 家族的规范成员具有细胞外的氨基末端 C2 结构域，该结构域与磷脂结合，是 Notch 受体最佳激活所必需的。在人类 JAGGED1（JAG1）中，导致 C2 结构域中氨基酸取代的体细胞突变已在肿瘤中被鉴定出来。我们在报告细胞测定中发现，影响 N-糖基化位点的突变会降低配体激活 Notch 的能力。该 N-糖基化位点位于 C2 结构域中，在 Jagged/Serrate 家族中保守，但在 Delta 家族中缺失。JAG1 氨基末端的位点特异性聚糖分析表明，该位点被复合型或高甘露糖 N-聚糖占据是在报告细胞测定中完全激活 Notch 所必需的。与 Notch 结合缺陷的 JAG1 变体类似，通过糖基化位点的突变或内切糖苷酶处理去除 N-聚糖，会降低受体激活。N-聚糖变体在 Notch 信号依赖性血管平滑肌细胞分化测定中也降低了受体激活。C2 N-聚糖的缺失将 JAG1 与脂质体的结合降低到与整个 C2 结构域缺失相似的程度。分子动力学模拟表明，N-聚糖的存在限制了 JAG1 相对于膜的取向，从而促进了 Notch 结合。这些数据与 N-聚糖在促进脂质结合构象中的关键作用一致，该构象对于 Jagged 在细胞膜上的定向以实现 Notch 的完全激活是必需的。
