Figure 1.

MALDI-TOF-MS analysis of Drosophila Notch EGF20 expressed in HEK293T cells. (A) The schematic diagram of EGF domains modified with the most extended forms of O-glycans. In HEK293T cells, O-Fuc modified by POFUT1 remains as a monosaccharide due to lack of Fringe activity responsible for elongation of O-Fuc. O-Glc modified by POGLUT1 can be elongated into a trisaccharide by the action of two distinct xylosyltransferases. Atypical O-Glc modification was found on Ser residue (light blue) of EGF11. Also shown are EOGT-catalyzed O-GlcNAc on Ser/Thr residue (red) and beta-hydroxylation (dark blue). (B) The amino acid sequence of wild-type Drosophila EGF20 (dEGF20^WT) and its variants. Theoretical molecular weight (Mw) of dEGF20^WT is 7603.16; dEGF20^{ΔGlcΔFucΔGlcNAc}, 7555.16; dEGF20^{ΔFucΔGlcNAc}, 7571.16, dEGF20^{ΔGlcΔGlcNAc}, 7557.13; dEGF20^ΔGlcΔFuc, 7585.19. Modification sites for O-Glc (blue), O-Fuc (green), and O-GlcNAc (red) were highlighted. (C) Wild type dEGF20 and its variants were transiently expressed in HEK293T cells, isolated with anti-His antibody, and analyzed by MALDI-TOF-MS in a positive linear mode. Molecular mass of major peaks in each spectrum are shown. Peak-to-peak mass increments corresponding to glycosylation are indicated by double-headed arrows. Where indicated, EOGT is overexpressed in HEK293T cells. Gray circle, glucose; gray star, xylose; yellow circle, galactose; blue square, GlcNAc; purple diamond, NeuAc; gray triangle, fucose.