Massive in vitro synthesis of tagged oligosaccharides in 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside treated HT-29 cells.

Abstract

Permanent exposure of differentiated HT-29 cells to the sugar analogue, 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha- O -bn) leads to marked effects upon the phenotypic properties of mucin-secreting or enterocyte-like HT-29 cells: an inhibition in the secretion of mucins, a blockade in the apical targeting of membrane brush border glycoproteins and a swelling of cells with intracellular accumulation of numerous vesicles. Folch extraction and partition of treated enterocyte-like HT-29 cells revealed a very important accumulation of orcinol and/or resorcinol reactive material in the upper phase (usually containing gangliosides), as compared with untreated HT-29 cells and with treated and untreated Caco-2 cells. Structural analysis indicated the accumulation of a series of GalNAcalpha- O -bn derived oligosaccharides, most of them with the common core Galbeta1-3GalNAcalpha- O -bn. These oligosaccharides contained residues of GlcNAc, Gal, Neu5Ac, or Fuc. In particular, the tagged sialyl-Lewis(x)was identified, as well as more complex sialylated derivatives, including the sialyl-Lewis(x)substituted by an additional Neu5Ac residue. The benzylated oligosaccharides were not significantly detected in the culture medium except for Galbeta1-3GalNAcalpha- O -bn. Upon reversion of the treatment, these derivatives dis-appeared from the cells within few days, however were not recovered as such in the culture medium. Intracellular degradation occurred with desialylation and defucosylation as the first steps. The spectacular accumulation of benzylated oligosaccharides in HT-29 cell, permanently exposed to GalNAcalpha- O -bn very likely plays an important role in the alterations of cellular processes previously described in this cell line. The HT-29 cell culture system also appears to be an efficient source of several tagged oligosaccharides.