title: Purification and characterization of the MUC1 mucin-type glycoprotein, epitectin, from human urine: structures of the major oligosaccharide alditols.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Penn State University College of Medicine, Hershey 17033, USA. vbhavana@bcmic.hmc.psu.edu
authors.forename: V P
authors.initials: VP
authors.lastname: Bhavanandan
authors.forename: Q
authors.initials: Q
authors.lastname: Zhu
authors.forename: K
authors.initials: K
authors.lastname: Yamakami
authors.forename: N A
authors.initials: NA
authors.lastname: Dilulio
authors.forename: S
authors.initials: S
authors.lastname: Nair
authors.forename: C
authors.initials: C
authors.lastname: Capon
authors.forename: J
authors.initials: J
authors.lastname: Lemoine
authors.forename: B
authors.initials: B
authors.lastname: Fournet
mesh_terms: D000596:Amino Acids / Q000032:analysis; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry / Q000652:urine*; D002240:Carbohydrate Sequence; D002499:Centrifugation, Density Gradient; D002845:Chromatography / Q000379:methods; D006001:Glycoconjugates / Q000032:analysis; D006801:Humans; D007822:Laryngeal Neoplasms / Q000276:immunology; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009005:Monosaccharides / Q000032:analysis; D018396:Mucin-1 / Q000737:chemistry; D014407:Tumor Cells, Cultured
chemical_list: D000596:Amino Acids; D015295:Antigens, Tumor-Associated, Carbohydrate; D006001:Glycoconjugates; D009005:Monosaccharides; D018396:Mucin-1