XXYLT1 inhibits NOTCH1 activation in Jurkat cells while promoting the cell proliferation

ABSTRACT: Glycosylation, one of the major forms of post-translational modification, regulates the protein functions in many contexts. Epidermal growth factor-like repeats are known to undergo domain-specific O-glycosylation such as O-glucosylation, O-fucosylation, and O-GlcNAc’ylation. O-Glucose glycans are attached to the specific serine residues by the action of POGLUT1 and can be elongated with two xylose residues by GXYLT1 or GXYLT2 and XXYLT1 in mammals. The very intriguing fact of xylosyl elongation of O-glucose as a negative regulator was recently reported. However, the stoichiometries of O-glucose on Drosophila Notch and mammalian Notch appear to be distinct, so that the roles of xylosylation in mammalian Notch signaling remain elusive. Here, we aimed to clarify the contribution of terminal xylosylation mediated by XXYLT1 to NOTCH1 signaling in Jurkat cells, a T-cell acute lymphoblastic leukemia cell line, which shows the cell cell-autonomous NOTCH1 activation due to the juxtamembrane expansion mutation. Mass spectrometric analysis on NOTCH1 fragments overexpressed in Jurkat cells demonstrates that the O-glucose site on NOTCH1 EGF10 is modified with various elongating patterns of O-glucose in Jurkat cells, which are similar to what was observed in HEK293T cells. Genetic deletion of XXYLT1 in Jurkat cells leads to the enhanced activation of NOTCH1, suggesting that XXYLT1 inhibits NOTCH1 activation in Jurkat cells, while the cell surface expression of NOTCH1 is not altered. Unexpectedly, the cell proliferation of Jurkat cells is impaired in XXYLT1 KO cells, which is accompanied by the downregulation of MYC, one of the known target genes of Notch signaling. Our results for the first time revealed that mammalian Notch activation is fine-tuned by xylosylation in Jurkat cells as proposed in Drosophila, highlighting the potential Notch agonism by the inhibition of xylosylation. In addition, our additional findings on the cell proliferation underscore the notion that there are possibly other substrates than NOTCH1 which XXYLT1 modifies, thereby and regulatinges their functions in Jurkat cells.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hideyuki Takeuchi 

PROVIDER: PXD056048 | JPOST Repository | Tue Oct 13 00:00:00 BST 2026

REPOSITORIES: jPOST