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Glycosyltransferase Changes upon Differentiation of CaCo-2 Human Colonic Adenocarcinoma Cells¹

Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 [I. B.]; and McGill Cancer Centre, McGill University, Montreal, Quebec, Canada H3G 1Y6 [P. A. R., A. H.]

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 ß-galactosidase activities and extremely high pyrophosphatase activities in differentiated cells were effectively inhibited by 5 mM -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 (Galß4GlcNAcß3- repeating units), blood group i UDP-GlcNAc:Galß-R ß3-GlcNAc transferase and UDP-Gal:GlcNAc ß4-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:Galß3GalNAc-R [GlcNAc to N-acetylgalactosamine (GalNAc)] ß6-GlcNAc transferase activity. In contrast, O-glycan core 1 UDP-Gal:GalNAc-R ß3-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:GlcNAcß3Galß-R (Glc-NAc to Gal) ß6-GlcNAc transferase, O-glycan core 3 UDP-Glc-NAc:GalNAc-R ß3GlcNAc transferase and O-glycan core 4 UDP-GlcNAc:GlcNAcß3GalNAc-R (GlcNAc to GalNAc) ß6-GlcNAc transferase.

¹ This research was supported by a grant from the Canadian Cystic Fibrosis Foundation to Harry Schachter (Hospital for Sick Children, Toronto), and by grants from the Cancer Research Society and the Canadian Foundation for Ileitis and Colitis to A. H. and the Medical Research Council of Canada to I. B.

² To whom requests for reprints should be addressed, at the Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.

Received 12/10/90. Accepted 4/ 4/91.

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