Fig. 5: MGAT1-mediated glycosylation of CD73 orchestrates its dimerization and translocation to the membrane.

a The CD73 protein levels and molecular sizes in MDA-MB468 and MDA-MB231 cells with MGAT1 OE/KD were determined by immunoblotting. The samples derived from the same experiment but different gels for CD73 and MGAT1, and β-ACTIN were processed in parallel. b Immunoblotting of CD73 in MGAT1 OE/KD MDA-MB468 lysates treated with Endo H/PNGase F. c Flow cytometry analysis of membrane CD73 in MGAT1 OE/KD MDA-MB468 cells. d The membrane distribution of CD73 (green) was visualized with stimulated emission depletion microscopy imaging in MDA-MB468 cells with MGAT1 OE/KD with membrane dye (red). e, f CD73 dimerization in MGAT1 OE/KD MDA-MB468 cells was assessed by glutaraldehyde cross-linking (e) and semi-native gel (f) immunoblotting. g Structural analysis of CD73 dimerization in inactive (PDB: 4H2G) and active (PDB: 6TVX) states, highlighting N-glycosylation sites (cyan) and C-terminal dimerization interface (magenta). h The scheme of investigating CD73 dimerization with a split-GFP assay. The GFP 1-10 or GFP 11 × 7 was attached to the N-terminus of WT CD73, CD73-4NQ, or CD73Δ480-537 and co-transfected into cells to investigate their dimerization abilities. i The split-GFP signal generated from dimerized WT CD73, CD73-4NQ, or CD73Δ480-537 was visualized by confocal microscopy in HEK-293T cells. j Flow cytometry of membrane CD73 + cells in MGAT1 OE/KD MDA-MB231 and MDA-MB468 with or without VAMP3 KD. k Schematic model showing how MGAT1-mediated glycosylation of CD73 orchestrates its dimerization and further translocation to cell membranes. Failure of CD73 glycosylation impedes CD73 dimerization and membrane translocation. Created in BioRender. Zhu, Y. (2025) https://BioRender.com/i34w446. l Spearman’s rank correlation analysis shows the MGAT1 protein expression is highly positively correlated with the THBS1 signaling pathways. m Adenosine levels in MGAT1 OE/KD MDA-MB468 and MDA-MB231 cells treated with THBS1. Data (mean ± SEM), images, western blot, and flow cytometry are representative of at least three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (j) or two-tailed unpaired t test (m). Source data are provided as a Source Data file.