Recombinant MUC1 Probe Authentically Reflects Cell-specific O-Glycosylation Profiles of Endogenous Breast Cancer Mucin

Knowledge about the O-linked glycan chains of tumor-associated MUC1 is primarily based on enzymatic and immunochemical evidence. To obtain structural information and to overcome limitations by the scarcity of endogenous mucin, we expressed a recombinant glycosylation probe corresponding to six MUC1 tandem repeats in four breast cancer cell lines. Comparative analyses of theO-glycan profiles were performed after hydrazinolysis and normal phase chromatography of 2-aminobenzamide-labeled glycans. Except for a general reduction in the O-glycan chain lengths and a high density glycosylation, no common structural pattern was revealed. T47D fusion protein exhibits an almost complete shift from core 2 to core 1 expression with a preponderance of sialylated glycans. By contrast, MCF-7, MDA-MB231, and ZR75–1 cells glycosylate the MUC1 repeat peptide preferentially with core 2-based glycans terminating mostly with α3-linked sialic acid (MDA-MB231, ZR75–1) or α2/3-linked fucose (MCF-7). Endogenous MUC1 from T47D and MCF-7 cell supernatants revealed almost identicalO-glycosylation profiles compared with the respective recombinant probes, indicating that the fusion proteins reflected the authentic O-glycan profiles of the cells. The structural patterns in the majority of cells under study are in conflict with biosynthetic models of MUC1 O-glycosylation in breast cancer, which claim that the truncation of normal core 2-based polylactosamine structures to short sialylated core 1-based glycans is due to the reduced activity of core 2-forming β6-N-acetylglucosaminyltransferases and/or to overexpression of competitive α3- sialyltransferase.