No abstract

The MUC1 glycoprotein, epitectin, a component of the human bladder epithelium, was purified from human urine. Sedimentation equilibrium analysis and gel filtration using polysaccharide or protein standards revealed a polydisperse preparation with molecular weights ranging from about 0.9 to 1.3 x 10(6). This suggests that in the native state epitectin exists as aggregates of three or four monomer units of 350-400 kDa. Epitectin was found to have significant affinity to hexyl-, octyl- or phenyl agarose indicating that hydrophobic interactions and possibly carbohydrate-carbohydrate interactions may be responsible for the self-association. Chemical and enzymic deglycosylation of [125I]-labeled urine epitectin and metabolically labeled H.Ep.2 epitectin resulted in extremely polydisperse products. The buoyant densities of epitectin purified from urine and H.Ep.2 cells were found to be 1.39-1.40 g ml(-1), suggesting that the total carbohydrate content of these preparations is not significantly different. The O-linked saccharides of epitectin were fractionated by HPLC and analyzed by permethylation and FAB-MS. The neutral saccharides from both sources contain three common structures, namely Gal1 --> 3GalNAc, GlcNAc1 --> 6 (Gal1 --> 3) GalNAc and Gal1 --> 4GlcNAc --> 6 (Gal1 --> 3)GalNAc. The sialic acid of urine epitectin consisted entirely of N-acetylneuraminic acid. The two sources of epitectin, in vitro labeled on sialic acid, were found to have the same sialyl oligosaccharides but in different proportions. Metabolic labeling and N-glycanase susceptibility experiments firmly established the presence of N-linked saccharides in epitectin as minor components. The remarkable similarities in the total carbohydrate content, the carbohydrate composition and structures of saccharides between epitectin from urine, a non-malignant source, and H.Ep.2 cells is surprising in view of the prevailing view that MUC1 glycoproteins of cancer cells are underglycosylated compared to those produced by non-malignant cells.

The treatment of H.Ep.2 cells with 4 mM aryl-N-acetyl-alpha-galactosaminide, an inhibitor of the elongation of O-linked saccharides of glycoproteins, resulted in a 70-80% decrease in the incorporation of [3H]glucosamine into epitectin (a MUC 1 mucin-type glycoprotein). Gel electrophoresis of the partially glycosylated epitectin immunoprecipitated from the extracts of treated cells showed two bands of apparent mol. wts 420 and 460 kDa on SDS-PAGE, compared to bands of 350 and 390 kDa for the native epitectin. Analysis of the saccharides released by beta-elimination from the partially glycosylated epitectin showed the presence of substantial levels of beta 1-->3 galactosylated and sialylated saccharides. The data indicate that the inhibition of elongation of Ser/Thr-linked N-acetyl-galactosamine in epitectin by aryl-N-acetyl-alpha-galactosaminides is only partial.