XXYLT1 inhibits NOTCH1 activation in Jurkat cells while promoting cell proliferation

Glycosylation, a key post-translational modification, regulates protein function in many contexts. Epidermal growth factor-like repeats undergo domain-specific O-glycosylation such as O-glucosylation, O-fucosylation, and O-GlcNAc'ylation. O-Glucose glycans are attached to specific serine residues by the action of protein O-glucosyltransferase 1 (POGLUT1) and can be elongated with two xylose residues by glucoside α1-3xylosyltransferase 1 (GXYLT1) or glucoside α1-3xylosyltransferase 2 (GXYLT2) and xyloside α1-3xylosyltransferase 1 (XXYLT1) in mammals. The xylosyl elongation of O-glucose as a negative regulator of Notch in Drosophila has recently been reported, but its role in mammalian Notch signaling remains elusive. Here, we investigated the impact of terminal xylosylation by XXYLT1 on NOTCH1 signaling in Jurkat cells, a T-cell acute lymphoblastic leukemia cell line with cell-autonomous NOTCH1 activation due to the juxtamembrane expansion mutation. Mass spectrometry analysis of NOTCH1 fragments overexpressed in Jurkat cells demonstrated that the O-glucose site on NOTCH1 EGF10 was modified with various elongating patterns of O-glucose. Genetic deletion of XXYLT1 in Jurkat cells led to enhanced activation of NOTCH1, suggesting that XXYLT1 inhibited NOTCH1 activation in Jurkat cells, whereas the cell surface expression of NOTCH1 was not altered. Unexpectedly, the proliferation of Jurkat cells was impaired in XXYLT1 knockout cells, with accompanying MYC downregulation, a Notch target gene. Our results revealed for the first time that mammalian Notch activation is fine-tuned by xylosylation in Jurkat cells, thus highlighting the potential of Notch agonism by the inhibition of xylosylation. Additionally, our findings regarding cell proliferation underscore the notion that there are possibly substrates other than NOTCH1 that XXYLT1 modifies, thereby regulating their functions in Jurkat cells.