PURIFICATION AND CHARACTERIZATION OF THE MUC1 MUCIN-TYPE GLYCOPROTEIN, EPITECTIN, FROM HUMAN URINE - STRUCTURES OF THE MAJOR OLIGOSACCHARIDE-ALDITOLS

The MUC1 glycoprotein, epitectin, a component of the human bladder epi thelium, was purified from human urine. Sedimentation equilibrium anal ysis and gel filtration using polysaccharide or protein standards reve aled a polydisperse preparation with molecular weights ranging from ab out 0.9 to 1.3x10(6). This suggests that in the native state epitectin exists as aggregates of three or four monomer units of 350-400 kDa. E pitectin was found to have significant affinity to hexyl-, octyl-or ph enyl agarose indicating that hydrophobic interactions and possibly car bohydrate-carbohydrate interactions may be responsible for the self-as sociation. Chemical and enzymic deglycosylation of [I-125]-labeled uri ne epitectin and metabolically labeled H.Ep.2 epitectin resulted in ex tremely polydisperse products. The buoyant densities of epitectin puri fied 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 i s not significantly different. The O-linked saccharides of epitectin w ere fractionated by HPLC and analyzed by permethylation and FAB-MS. Th e neutral saccharides from both sources contain three common structure s, namely Gal1-->3GalNAc, GlcNAc1-->6 (Gal1-->3) GalNAc and Gal1-->4 G lcNAc-->6 (Gal1-->3)GalNAc. The sialic acid of urine epitectin consist ed entirely of N-acetylneuraminic acid. The two sources of epitectin, in vitro labeled on sialic acid, were found to have the same sialyl ol igosaccharides but in different proportions. Metabolic labeling and N- glycanase susceptibility experiments firmly established the presence o f N-linked saccharides in epitectin as minor components. The remarkabl e similarities in the total carbohydrate content, the carbohydrate com position and structures of saccharides between epitectin from urine, a non-malignant source, and H.Ep.2 cells is surprising in view of the p revailing view that MUC1 glycoproteins of cancer cells are underglycos ylated compared to those produced by non-malignant cells.