Figure 1: PD-L1 is glycosylated in cancer cells.
From: Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity
(a) Expression of PD-L1 protein in primary breast cancer patient samples. Western blot analysis of PD-L1 in representative breast cancer patient samples. (b) Western blot analysis of PD-L1 in four breast cancer, four melanoma and three lung and three colon cancer cells. (c) Western blot analysis of PD-L1 expression in shCTRL, two independent shPD-L1 stable clones and reconstitution of PD-L1 restored expression in the shPD-L1#5 clone of BT549 cells (left). Western blot analysis of PD-L1 expression in CRISPR/Cas9-mediated PD-L1 knockout BT549 cells (right panel). (d) Glycosylation pattern of PD-L1 protein in BT549 and MDA-MB-231 cells. Cell lysates were treated with PNGase F and analysed by western blot analysis. Black circle, glycosylated PD-L1; arrowhead, non-glycosylated PD-L1. (e) Glycosylation pattern of PD-L1-GFP, HA-PD-L1 and PD-L1-Flag proteins. Cell lysates were treated with PNGase F and Endo H and analysed by western blot analysis. (f) Schematic diagram of PD-L1 protein. Full-length PD-L1 was separated into extracellular domain (ECD) and intracellular domain (ICD). SP, signal peptide; TM, transmembrane domain. Four putative NXT motifs in the ECD domain are labelled in red. The numbers indicate amino-acid positions. (g) LC-MS/MS-based identification of N-glycopeptides. LC-MS/MS-based identification of N-glycopeptides corresponding to one of the four N-glycosylation sites, N35. The LC-MS profiles (top) are shown as spectra averaged over a period of elution time (as labelled in figures) when a representative subset of glycoforms were detected. For each N-glycosylation site, one representive HCD MS2 spectrum (bottom) is shown to exemplify its identification based on detection of y1 ion (tryptic peptide backbone carrying the GlcNAc attached to the N-glycosylated Asn), along with the b and y ions defining its peptide sequence. The cartoon symbols used for the glycans (see inset) conform to the standard representation recommended by the Consortium for Functional Glycomics. (h) Western blot analysis of the protein expression pattern of PD-L1 WT and its NQ mutants. Non-glycosylated form in lane 14 indicates PD-L1 WT with overnight treatment with TM. Black circle, glycosylated PD-L1; arrowhead, non-glycosylated PD-L1.
