ER quality control of Crumbs2 and Notch proteins by GXYLT1-dependent xylosyl elongation of O-glucose glycans.

Protein O-glucosyltransferase (POGLUT1/Rumi) transfers an O-linked glucose (O-Glc) monosaccharide from UDP-Glc to a serine residue in epidermal growth factor-like (EGF) repeats with a specific consensus sequence. In mammals, GXYLT1 and GXYLT2 transfer xylose to this O-Glc monosaccharide. Previous studies showed that genetic deletion of Poglut1 in mice leads to embryonic lethality with defects in Crumbs2 (CRB2) trafficking and NOTCH1 signaling during development. Both CRB2 and Notch receptors have multiple EGF repeats with the O-Glc modification site. However, the roles of xylosyl elongation of O-Glc in protein trafficking and mammalian embryonic development are unknown. Here, we demonstrated that xylosyl elongation of O-Glc occurs in the ER and comprehensively analyzed O-Glc glycosylation on CRB2, NOTCH1, and NOTCH2 expressed in HEK293T cells by mass spectrometry. We found that most EGF repeats containing the consensus sequence were modified with O-Glc glycans, while the degree of xylosyl elongation varied among different EGF repeats, suggesting EGF repeat-specific regulation. GXYLT1 knockout cells showed reduced or lost xylosyl elongation, but GXYLT2 knockout cells did not. Cell-based assays demonstrated decreased secretion of substrate proteins in GXYLT1 knockout cells, which was rescued by wild-type GXYLT1. Analysis of Gxylt1 and Gxylt2 knockout mice revealed that Gxylt1 deletion, but not Gxylt2 deletion, caused embryonic lethality. Altogether, our data suggest that GXYLT1 plays a major role in xylosyl elongation of O-Glc glycans and thereby contributes to the ER quality control and trafficking of CRB2, NOTCH1, and NOTCH2 in HEK293T cells while GXYLT1 and GXYLT2 have distinct biological functions during mouse embryonic development.