Permanent exposure of mucin-secreting HT-29 cells to benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation of mucins and inhibits constitutive and stimulated MUC5AC secretion.
Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-alpha-D-galactosaminide results in profound changes in mucin oligosaccharide chains. To analyse in depth the effect of this drug, we first determined the structure of mucin oligosaccharide chains synthesized by HT-29 MTX cells and the changes induced by permanent drug exposure. Mucins from untreated cells contained nine monosialylated structures (core types 1, 2, 3 and 4) and four disialylated structures (types 1, 2 and 4). Core 1 structures predominated, in particular NeuAcalpha2-3Galbeta1-3GalNAc-ol. Exposure of HT-29 MTX cells to benzyl-N-acetyl-alpha-D-galactosaminide from days 2-21 resulted in a decrease in intracellular mucins and both their sialic acid and galactose content, and an increased T (Galbeta1-3GalNAcalpha-O-Ser/Thr) and Tn (GalNAcalpha-O-Ser/Thr) antigenicity. A 3-fold increase in both Galbeta1-3GalNAc alpha2, 3-sialyltransferase activity and mRNA expression was detected. At the ultrastructural level, T-antigen was not detectable in mucin droplets in control cells, but was strongly expressed in intracytoplasmic vesicles in treated cells. In these cells, MUC1 and MUC3 transcripts were up-regulated, whereas MUC2, MUC5B and MUC5AC were down-regulated. Furthermore, constitutive and secretagogue-induced MUC5AC secretion was reduced and no mucus layer was detected. In conclusion, benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation and altered regulation of MUC5AC secretion.
Unité de Recherches sur la Biologie et la Physiopathologie des Cellules Mucipares, INSERM U377, Place de Verdun, 59045 Lille cedex, France.
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Hennebicq-Reig
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T
Lesuffleur
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Capon
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De Bolos
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Kim
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Moreau
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Richet
B
B
Hémon
M A
MA
Recchi
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Maës
J P
JP
Aubert
F X
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Real
A
A
Zweibaum
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Delannoy
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Degand
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Huet
D000116:Acetylgalactosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000230:Adenocarcinoma; D001593:Benzyl Compounds / Q000494:pharmacology*; D002240:Carbohydrate Sequence; D003110:Colonic Neoplasms; D005690:Galactose / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D055271:Mucin 5AC; D009077:Mucins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012794:Sialic Acids / Q000378:metabolism; D014407:Tumor Cells, Cultured
D001593:Benzyl Compounds; C524678:MUC5AC protein, human; D055271:Mucin 5AC; D009077:Mucins; D009844:Oligosaccharides; D012794:Sialic Acids; C080739:benzyl-alpha-N-acetylgalactosamine; D016695:Glycosyltransferases; D000116:Acetylgalactosamine; D005690:Galactose