O-glycosylation and cellular differentiation in a subpopulation of mucin-secreting HT-29 cell line

Malignant transformation of epithelial cells is associated with abnormal glycosylation of mucins, The aim of this work was to evaluate the changes in the O-glycosylation processes during differentiation of tumor cells by performing in vitro reactions using crude microsomal preparations obtained from a subpopulation of HT-29 cells capable of differentiating into mucin-secreting cells (HT-29 MTX cells), The reactions of O-glycosylation were carried out at different times of culture: before confluence (Day 5), when cells are still undifferentiated, and after confluence (Day 21), when cells display a mucin-secreting phenotype, As acceptor for the UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc transferase), the peptide motif TTSAPTTS (tandem repeat deduced from MUC5AC human gastric gene, expressed in HT-29 MTX cells) was used, A higher rate of enzyme activity was observed in preconfluent cells, and analysis by capillary electrophoresis and electrospray mass spectrometry showed a different, pattern of galactosaminylation in pre-and postconfluent cells. Core 1 UDP-galactose:N-acetyl-alpha-galactosaminyl-R 3-beta-galactosyltransferase (3-beta-galactosyltransferase) activity also decreased with the differentiation, whereas CMP-neuraminic acid:galactose-beta-1, 3-N-acetyl-alpha-galactosaminyl-R 3-alpha-sialyltransferase activity increased, In comparison, the evolving process of mucin biosynthesis was tested by the analysis of purified mucins of HT-29 MTX cells, in amino acid and carbohydrate composition, and immunoreactivity assays using several antibodies and lectins. The results suggested that (i) no mucins were detected at Day 5, while the GalNAc transferase and 3-beta-galactosyltransferase activities were already at high rates; (ii) the mucins purified from postconfluent cells showed a high content of sialic acid in an alpha-2,3-linkage to galactose residues; and (iii) cellular differentiation seemed to be accompanied by more regulated processes of glycosylation. This study of the O-glycosylation in HT-29 MTX cells is thus an interesting approach to analyzing the regulation of mucin biosynthesis during cellular differentiation (C) 1997 Academic Press.