Abstract
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 the O-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 alpha 3-linked sialic acid (MDA-MB231, ZR75-1) or alpha 2/3-linked fucose (MCF-7). Endogenous MUC1 from T47D and MCF-7 cell supernatants revealed almost identical O-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 beta 6-N-acetylglucosaminyltransferases and/or to overexpression of competitive alpha 3- sialyltransferase.
Highlights
Knowledge about the O-linked glycan chains of tumorassociated MUC1 is primarily based on enzymatic and immunochemical evidence
MFP6 is a clone with six tandem repeats and was chosen as a glycosylation probe (Fig. 1A), since we noticed that fusion proteins with smaller repeat numbers were expressed poorly in the breast cancer cell lines used in this study, and substantially larger ones were expected to be difficult to chromatograph in reversed-phase HPLC
The utility of the approach using an artificial protein to probe in vivo O-glycosylation in tumor cell lines was validated by comparison with the endogenous mucin and the demonstration that the natural, cell-specific profiles of O-linked glycans are authentically reflected on the fusion protein
Summary
Knowledge about the O-linked glycan chains of tumorassociated MUC1 is primarily based on enzymatic and immunochemical evidence. The bulk of glycosylation, which can make up between 50 and 80% of the total mass, is O-linked to numerous threonine and serine residues in the mucin domain This domain comprises a variable number of tandemly repeated 20-amino acid sequences, each containing five potential O-glycosylation sites [10, 11]. Tumor-associated glycoforms, which had been isolated from T47D [17] and BT 20 [18] breast carcinoma cell line supernatants or from cell lysates [19], were demonstrated to contain truncated precursor structures like core-GalNAc or the core 1 disaccharide Gal(1–3)GalNAc as well as its mono- and disialylated derivatives
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