Analysis of endogenous NOTCH1 fromPOFUT1 S162Lpatient fibroblasts reveals the importance of theO-fucose modification on EGF12 in human development

Abstract

NOTCH1 (N1) 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. ProteinO-fucosyltransferase 1 (POFUT1) addsO-fucose to Epidermal Growth Factor (EGF)-like repeats in the NOTCH1 extracellular domain, which is required for trafficking and signaling activation. We previously showed thatPOFUT1 S162Lcaused 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 S162Lpatient fibroblast cells had an equivalent amount of N1 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 ofO-fucose on N1 inPOFUT1 S162Lpatient fibroblasts was the cause of these effects, we immunopurified endogenous N1 from control and patient fibroblasts and analyzedO-fucosylation using mass spectral glycoproteomics methods. N1 EGF8 to EGF12 comprise the ligand binding domain, andO-fucose on EGF8 and EGF12 physically interact with ligands to enhance affinity. Glycoproteomics of N1 fromPOFUT1 S162Lpatient fibroblasts showed WT fucosylation levels at all sites analyzed except for a large decrease at EGF9 and the complete absence ofO-fucose at EGF12. Since the loss ofO-fucose on EGF12 is known to have significant effects on N1 activity, this may explain the symptoms observed in thePOFUT1 S162Lpatient.