Notch signaling is essential for proper development of multicellular organisms. In humans, dysregulation of Notch signaling has been implicated in several cancers and other diseases such as CADASIL (Cerebral Autosomal-Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy) and Alagille syndrome. Glycosylation of the Notch receptor is integral for the regulation of Notch signaling and activation. So far, three major forms of O-glycosylation are known to occur at predicted consensus sequences within the epidermal growth factor-like (EGF) repeats of the extracellular domain of Notch: O-glucosylation, O-fucosylation, and O-GlcNAcylation. Glycosyltransferases Protein O-glucosyltransferase 1 (POGLUT1), POFUT1, and EOGT are responsible for these modifications, respectively. A recent co-crystal between a portion of Notch1 and Delta-like ligand 4 (DLL4) revealed an additional hexose modification on EGF11 at a novel site that interacts with DLL4. EGF11 has previously been implicated in the binding interface of Notch and its ligands, potentially linking this novel modification to the regulation of Notch signaling. We have identified two homologs of POGLUT1, POGLUT1 and POGLUT2, and through radioactive enzyme activity assays have shown that these enzymes add glucose to this novel site on EGF11. To further confirm these two enzymes are responsible for this modification, single and double knockout HEK293 cells were generated using CRISPR/Cas9 gene editing. Mass spectral analysis of mouse Notch1 EGF extracellular domain expressed in these cells confirmed loss of the glucose modification on EGF11 when both POGLUT2 and POGLUT3 were knocked out. We performed rescue experiments with either POGLUT2 or POGLUT3, and confirmed the restoration of the glucose modification on EGF11 of mouse Notch1. The site of modification appears to be conserved on Notch3 and Notch4, but not Notch2. Elimination of the novel O-glucose site on EGF11 by mutagenesis reduces Notch1 activity, suggesting that POGLUT2 and POGLUT3 are novel modulators of Notch activity. These results lay the groundwork for future studies on these two enzymes in order to enhance our understanding of Notch O-glycosylation and how it regulates Notch signaling, as well as identifying additional proteins these novel enzymes may be modifying. This work was supported by NIH grant GM061126.
