NOTCH1(N1)是一种跨膜受体,与相对细胞上的膜束缚配体相互作用,介导许多细胞命运决定所必需的直接细胞-细胞相互作用。蛋白O-岩藻糖基转移酶1(POFUT1)将O-岩藻糖添加到NOTCH1胞外域中的表皮生长因子(EGF)样重复序列,这是贩运和信号激活所必需的。我们以前表明POFUT1S162L导致患者POFUT1活性丧失90%和整体发育缺陷;然而,POFUT1导致这些症状的机制尚不清楚.与对照组相比,POFUT1S162L患者成纤维细胞在细胞表面上具有等量的N1,但显示DLL1配体结合的60%减少和JAG1配体结合的70%减少。为了确定POFUT1S162L患者成纤维细胞中N1上O-岩藻糖的减少是否是这些影响的原因,我们免疫纯化了对照和患者成纤维细胞的内源性N1,并使用质谱糖蛋白质组学方法分析了O-岩藻糖基化.N1EGF8至EGF12包含配体结合域,EGF8和EGF12上的O-岩藻糖与配体物理相互作用以增强亲和力。来自POFUT1S162L患者成纤维细胞的N1的糖蛋白质组学显示在所有分析位点的WT岩藻糖基化水平,除了在EGF9处大幅降低和在EGF12处完全不存在O-岩藻糖。由于已知EGF12上的O-岩藻糖的损失对N1活性有显著影响,这可以解释在POFUT1S162L患者中观察到的症状.
NOTCH1 is a transmembrane receptor interacting with membrane-tethered ligands on opposing cells that mediate the direct cell-cell interaction necessary for many cell fate decisions. Protein O-fucosyltransferase 1 (POFUT1) adds O-
fucose to Epidermal Growth Factor (EGF)-like repeats in the NOTCH1 extracellular domain, which is required for trafficking and signaling activation. We previously showed that POFUT1 S162L caused a 90% loss of POFUT1 activity and global developmental defects in a patient; however, the mechanism by which POFUT1 contributes to these symptoms is still unclear. Compared to controls, POFUT1 S162L patient fibroblast cells had an equivalent amount of NOTCH1 on the cell surface but showed a 60% reduction of DLL1 ligand binding and a 70% reduction in JAG1 ligand binding. To determine if the reduction of O-
fucose on NOTCH1 in POFUT1 S162L patient fibroblasts was the cause of these effects, we immunopurified endogenous NOTCH1 from control and patient fibroblasts and analyzed O-fucosylation using mass spectral glycoproteomics methods. NOTCH1 EGF8 to EGF12 comprise the ligand binding domain, and O-
fucose on EGF8 and EGF12 physically interact with ligands to enhance affinity. Glycoproteomics of NOTCH1 from POFUT1 S162L patient fibroblasts showed WT fucosylation levels at all sites analyzed except for a large decrease at EGF9 and the complete absence of O-
fucose at EGF12. Since the loss of O-
fucose on EGF12 is known to have significant effects on NOTCH1 activity, this may explain the symptoms observed in the POFUT1 S162L patient.