Fig. 1
O-Glycome repertoire of 5637 and T24 bladder cancer cell lines showing a predominance of sialylated T antigens (sialyl-T and di-sialyl-T, herein generally termed ST antigens). A) Typical cellular O-Glycome Reporter/Amplification nanoLC-ESI-MS spectra for 5637 and T24 cells. Both mass spectra present mono and di-sialylated T antigens at m/z 933.5 and 1294.7 as dominant glycan species. Less abundant ions include T antigen (m/z 572.3), fucosylated T antigen (m/z 746.4) and sialylated and/or fucosylated core 2 glycans (m/z 1178.6, 1382.7, 1556.8, and 1743.9). Traces of the sialyl-Tn (STn) antigen could also be observed at m/z 729.4. B) MS/MS spectrum for the sialyl-T antigen at m/z 933.5. The MS/MS showed a major ion corresponding to the loss of the benzyl tag (Bn) and typical ions derived from glycosidic linkage cleavages (m/z 336.2, 354.3, 376.2, 450.3, 558.1, 580.5), corresponding to sugars diagnostic ions of sialyl-T structure. C) Relative quantification of major O-glycans contributing to the O-glycome for 5637 and T24 cells showing similar expression patterns. The graph shows that all cell lines present similar O-glycomes, emphasizing mono- and di-sialylated T antigens as the most abundant glycans. Notably, T24 cells appear enriched for di-sialylated T antigens whereas 5637 present a higher percentage of mono-sialylated species. D-F) Immunofluorescence microscopy and flow cytometry analysis of 5637 and T24 reveals very low levels of T antigens and an overexpression of sialylated species at the cell membrane. Panel D did not reveal T antigens but shows a significant accumulation of ST antigens at the cell surface. The histogram in panel E displays the expression of T antigen in bladder cancer cells before and after sialidase treatment for evaluation of ST antigens expression, highlighting the striking overexpression of ST antigens compared to its neutral form in these cell lines. Graph F compares T antigen levels after de-sialylation in the two cell lines, suggesting the higher expression in 5637 cells