PERMANENT EXPOSURE OF MUCIN-SECRETING HT-29 CELLS TO BENZYL-N-ACETYL-ALPHA-D-GALACTOSAMINIDE INDUCES ABNORMAL O-GLYCOSYLATION OF MUCINS ANDINHIBITS CONSTITUTIVE AND STIMULATED MUC5AC SECRETION

Previous work has shown that treatment of HT-29 methotrexate (MTX) cel ls with benzyl-N-acetyl-alpha-D-galactosaminide results in profound ch anges in mucin oligosaccharide chains. To analyse in depth the effect of this drug, we first determined the structure of mucin oligosacchari de chains synthesized by HT-29 MTX cells and the changes induced by pe rmanent drug exposure. Mucins from untreated cells contained nine mono sialylated structures (core types 1, 2, 3 and 4) and four disialylated structures (types 1, 2 and 4). Core 1 structures predominated, in par ticular NeuAc alpha 2-3Gal beta 1-3GalNAc-ol. Exposure of HT-29 MTX ce lls 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 (Gal beta 1-3GalNAc alpha-O-Ser/ Thr) and Tn (GalNAc alpha-O-Ser/Thr) antigenicity. A 3-fold increase i n both Gal beta 1-3GalNAc alpha 2,3-sialyltransferase activity and mRN A expression was detected. At the ultrastructural level, T-antigen was not detectable in mucin droplets in control cells, but was strongly e xpressed 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 secretagogu e-induced MUC5AC secretion was reduced and no mucus layer was detected . In conclusion, benzyl-N-acetyl-alpha-D-galactosaminide induces abnor mal O-glycosylation and altered regulation of MUC5AC secretion.