Fig. 7

Sialylated HOMER3 glycoforms can be found at the cell-surface of bladder cancer cells and display significant inter-patient structural variability. A) HOMER3 at the cell membrane co-localizes in the same tumour area with sialylated Tn and T antigens in bladder cancer. According to immunohistochemistry, HOMER3 was diffusely expressed in wide tumour areas, both at the cytoplasm and cell membranes. These areas co-localized with very high STn and/or ST expressions areas. B) Immunofluorescence microscopy highlighting the co-localization (in white) of HOMER3 (violet) with sialylated Tn and T antigens (green) at the cell membrane of bladder cancer cells. HOMER3 was detected in both cytoplasm and cell membrane of a high number of cancer cells in invasive tumours. The tumours were also screened for the Tn and T antigens and their sialylated counterparts STn and ST. Tumours were negative for neutral glycans but showed high sialospecies, in accordance with immunohistochemistry analysis in panel A. C) MS/MS HOMER3 glycopeptide with cancer-associated glycans isolated from an invasive bladder tumour. Briefly, invasive tumours showing no Tn and T antigens were elected for this analysis. Glycoproteins were extracted from these tumours and digested with neuraminidase to expose Tn and T antigens derived from their sialylated counterparts. VVA and PNA lectins were then used to pulldown glycoproteins carrying these glycans for downstream analysis by nanoLC-MS/MS. Peptide and glycopeptide fragmentations as well as typical glycan oxonium ions at m/z 204.09 (GalNAc) and 366.14 (GalNAc-Gal) were used for glycoproteins and glycosites annotations. D) HOMER3 glycosites mapping by nanoLC-EThcD-MS/MS demonstrated significant inter-patients’ variability. HOMER3 was isolated from five tumour samples of different patients by lectin affinity and characterized in relation to their glycosylation pattern by nanoLC-EThcD-MS/MS. This analysis identified 16 glycosites in the extracellular region of HOMER3 with little homology between different patients. Notably, the identification of glycopeptides carrying the Tn and T antigens strongly supports previous sialylation, since the neutral glycans were not detected in the original tumours