EFFECTS OF OVEREXPRESSION OF BETA-1,4-GALACTOSYLTRANSFERASE ON GLYCOPROTEIN-BIOSYNTHESIS IN F9 EMBRYONAL CARCINOMA-CELLS

Beta,1,4-galactosyltransferase (GalTase) plays a central role in the b iosynthesis of N-acetyllactosamine-containing oligosaccharides. Howeve r, despite this seemingly important function, little is known about ho w changes in the levels of GalTase affect oligosaccharide biosynthesis . We have examined the effects of overexpressing GalTase on the glycos ylation of endogenous glycoproteins in F9 mouse embryonal carcinoma ce lls. Cells transfected with either the short form of the GalTase cDNA (encoding a protein of 386 amino acids) or the long form of the GalTas e cDNA (encoding a protein of 399 amino acids) had a 3-fold increase i n total GalTase activity, relative to control F9 cells. Analysis of pr onase-digested glycopeptides obtained from control and transfected cel ls after metabolic labelling with [6-H-3]galactose revealed no signifi cant qualitative or quantitative differences, as assessed by Bio-Gel P -6 gel filtration chromatography and Tomato lectin affinity chromatogr aphy. Furthermore, SDS-PAGE analysis of immunoprecipitated [H-3]galact ose-labelled lysosomal-associated membrane protein-1 (LAMP-1) glycopro tein showed no difference in amounts or mobility. Pronase digestion an d subsequent analysis of the gel-fractionated LAMP-1 glycoproteins als o indicated no differences between the various cell lines. The inabili ty of elevated GalTase activity to affect glycosylation was not due to limiting levels of GalTase substrates, since an excess of substrates was detectable in lysed cells using either endogenous or exogenous Gal Tase and UDP-[H-3]galactose. Finally, the subcellular distribution of GalTase, as assessed by sucrose gradient fractionation, was similar be tween all cell types, thus suggesting that GalTase was appropriately c ompartmentalized in the transfected cells. More importantly, GalTase s pecific activities in the Golgi membranes of the transfected cells wer e 3-4 times greater than in control cells. These results show that sel ectively increasing GalTase activity does not alter glycoprotein biosy nthesis in F9 cells.