title: Glycosyltransferase changes upon differentiation of CaCo-2 human colonic adenocarcinoma cells.
abstract: The spontaneous differentiation of CaCo-2 human colonic adenocarcinoma cells to enterocytes in culture is associated with a decrease in polylactosaminoglycans, particularly those attached to the lysosomal membrane glycoprotein h-lamp-1 (Youakim et al., Cancer Res., 49:6889-6895, 1989). To elucidate the biosynthetic mechanisms leading to these alterations we have compared glycosyltransferase activities that are involved in the synthesis of polylactosaminoglycans and of the N- and O-glycan structures that provide the framework for the attachment of these chains. Glycosyltransferase activities in cell homogenates obtained from undifferentiated and differentiated CaCo-2 cells were assayed by high pressure liquid chromatography separation of enzyme products. The beta-galactosidase activities and extremely high pyrophosphatase activities in differentiated cells were effectively inhibited by 5 mM gamma-galactonolactone and 10 mM AMP, respectively. CaCo-2 cells contain most of the enzymes that are involved in N-glycan branching [N-acetylglucosamine (GlcNAc) transferases I to V] with the exception of GlcNAc transferase VI. The levels of GlcNAc transferase I activities were comparable in undifferentiated and differentiated cells, but GlcNAc transferase II to V activities were significantly increased upon differentiation. The enzyme activities that are directly involved in the synthesis of linear polylactosaminoglycans (Gal beta 4GlcNAc beta 3- repeating units), blood group i UDP-GlcNAc:Gal beta-R beta 3-GlcNAc transferase and UDP-Gal:GlcNAc beta 4-Gal transferase, were found at similar levels in undifferentiated and differentiated CaCo-2 cells. Since GlcNAc transferase III activity is known to inhibit further branching and galactosylation, these results suggest that its increased activity in differentiated CaCo-2 cells may be partly responsible for the decreased synthesis of fucosylated polylactosaminoglycans. Differentiated cells showed a 2-fold increase in O-glycan core 2 UDP-GlcNAc:Gal beta 3GalNAc alpha-R [GlcNAc to N-acetylgalactosamine (GalNAc)] beta 6-GlcNAc transferase activity. In contrast, O-glycan core 1 UDP-Gal:GalNAc alpha-R beta 3-Gal transferase activity was found decreased. Several enzymes that are found in homogenates from normal human colonic tissue are absent or barely detectable in CaCo-2 cells. These include blood group I UDP-GlcNAc:GlcNAc beta 3Gal beta-R (GlcNAc to Gal) beta 6-GlcNAc transferase, O-glycan core 3 UDP-GlcNAc:GalNAc alpha-R beta 3 GlcNAc transferase and O-glycan core 4 UDP-GlcNAc:GlcNAc beta 3GalNAc-R (GlcNAc to GalNAc) beta 6-GlcNAc transferase.
authors.affiliation: Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: I
authors.initials: I
authors.lastname: Brockhausen
authors.forename: P A
authors.initials: PA
authors.lastname: Romero
authors.forename: A
authors.initials: A
authors.lastname: Herscovics
mesh_terms: D000230:Adenocarcinoma; D000606:Amino Sugars / Q000096:biosynthesis*; D002240:Carbohydrate Sequence; D002454:Cell Differentiation; D002460:Cell Line; D003110:Colonic Neoplasms; D006602:Hexosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000096:biosynthesis*; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D000606:Amino Sugars; D011134:Polysaccharides; C039448:lactosaminoglycan; D006602:Hexosyltransferases; D001616:beta-Galactosidase