O-GLYCOSYLATION AND CELLULAR-DIFFERENTIATION IN A SUBPOPULATION OF MUCIN-SECRETING HT-29 CELL-LINE

Malignant transformation of epithelial cells is associated with abnorm al glycosylation of mucins, The aim of this work was to evaluate the c hanges in the O-glycosylation processes during differentiation of tumo r cells by performing in vitro reactions using crude microsomal prepar ations obtained from a subpopulation of HT-29 cells capable of differe ntiating into mucin-secreting cells (HT-29 MTX cells), The reactions o f O-glycosylation were carried out at different times of culture: befo re confluence (Day 5), when cells are still undifferentiated, and afte r confluence (Day 21), when cells display a mucin-secreting phenotype, As acceptor for the UDP-N-acetylgalactosamine:polypeptide N-acetylgal actosaminyltransferase (GalNAc transferase), the peptide motif TTSAPTT S (tandem repeat deduced from MUC5AC human gastric gene, expressed in HT-29 MTX cells) was used, A higher rate of enzyme activity was observ ed in preconfluent cells, and analysis by capillary electrophoresis an d electrospray mass spectrometry showed a different, pattern of galact osaminylation in pre-and postconfluent cells. Core 1 UDP-galactose:N-a cetyl-alpha-galactosaminyl-R 3-beta-galactosyltransferase (3-beta-gala ctosyltransferase) activity also decreased with the differentiation, w hereas CMP-neuraminic acid:galactose-beta-1, 3-N-acetyl-alpha-galactos aminyl-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 acti vities were already at high rates; (ii) the mucins purified from postc onfluent cells showed a high content of sialic acid in an alpha-2,3-li nkage 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 cel lular differentiation (C) 1997 Academic Press.