De novo synthesis of type 1 lacto-series glycolipids in human colonic adenocarcinoma cells: efficient synthesis of the Le(a) antigen and absence of brefeldin A-induced inhibition of its synthesis in Colo 205 cells.
De novo biosynthesis of lacto-series type 1 chain glycolipids was studied by [3H]serine metabolic labeling of ceramide in Colo 205 and SW403 cells. Highly efficient synthesis of III4FucLc4 (Le(a)) from lactosylceramide was found to be characteristic of Colo 205 cells. Although de novo synthesized, 3H-labeled Le(a) was found to accumulate after a 4-h labeling period, a 3H-labeled pool of Le(a) precursors was not detected until after 8 to 12 h. This is due presumably to hydrolysis of the initial Le(a) product. Labeling of neutral glycolipids from SW403 cells instead indicated a stepwise elongation of oligosaccharide chains providing labeled pools of type 1 and 2 chain precursors followed by their fucosylated derivatives. Analysis of neutral glycolipids isolated from Golgi membranes fractionated by sucrose density gradient centrifugation confirmed the efficient synthesis of type 1 antigens in early Golgi fractions of Colo 205 cells along with the results from SW403 cells observed using whole cells. De novo glycolipid synthesis in the presence of brefeldin A indicated a disruption of the efficient Le(a) synthesis in Colo 205 cells with the appearance of biosynthetic intermediates of Le(a) synthesis. However, Le(a) remained the de novo product accumulated in these as well as SW403 cells. The results suggest efficient trafficking of glycolipid precursors between successive glycosyltransferases coupled with the relative distribution of beta 1-->3- vs beta 1-->4galactosyltransferase in intracellular membranes are properties associated with preferential synthesis of type 1 chain antigens as occurs in Colo 205 cells.
Pacific Northwest Research Foundation, Seattle, Washington 98122.
E H
EH
Holmes
T G
TG
Greene
D000230:Adenocarcinoma / Q000378:metabolism*; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000096:biosynthesis*; D020126:Brefeldin A; D018395:CA-19-9 Antigen; D002240:Carbohydrate Sequence; D003110:Colonic Neoplasms / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology; D005732:Gangliosides / Q000096:biosynthesis*; D006017:Glycolipids / Q000096:biosynthesis*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D014407:Tumor Cells, Cultured
D015295:Antigens, Tumor-Associated, Carbohydrate; D018395:CA-19-9 Antigen; D003517:Cyclopentanes; D005732:Gangliosides; D006017:Glycolipids; D020126:Brefeldin A; C064564:sialyl Le(a) ganglioside