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Purification and characterization of the MUC1 mucin-type glycoprotein, epitectin, from human urine: structures of the major oligosaccharide alditols

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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 1.3×106. 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 001contain three common structures, namely Gal1→3GalNAc, GlcNAc1→6 (Gal1→3) GalNAc and Gal1→4 GlcNAc→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.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Penn State University College of Medicine, Hershey Pennsylvania, 17033, USA

    V.P Bhavanandan, Qin Zhu, Kazuo Yamakami, Nancy A DiIulio & Sukumaran Nair

  2. Laboratoire de ChimieBiologique, U.M.R. n° 111 du C.N.R.S., Universite des Sciences et Technologies de Lille, 59655, Villeneuve de’Ascq Cedex, France

    Calliope Capon, Jerome Lemoine & Bernard Fournet

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  1. V.P Bhavanandan
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  2. Qin Zhu
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  3. Kazuo Yamakami
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  4. Nancy A DiIulio
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  5. Sukumaran Nair
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  7. Jerome Lemoine
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Bhavanandan, V., Zhu, Q., Yamakami, K. et al. Purification and characterization of the MUC1 mucin-type glycoprotein, epitectin, from human urine: structures of the major oligosaccharide alditols. Glycoconj J 15, 37–49 (1998). https://doi.org/10.1023/A:1006987315827

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