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 biosynthesis of N-acetyllactosamine-containing oligosaccharides. However, despite this seemingly important function, little is known about how changes in the levels of GalTase affect oligosaccharide biosynthesis. We have examined the effects of overexpressing GalTase on the glycosylation of endogenous glycoproteins in F9 mouse embryonal carcinoma cells. Cells transfected with either the short form of the GalTase cDNA (encoding a protein of 386 amino acids) or the long form of the GalTase cDNA (encoding a protein of 399 amino acids) had a 3-fold increase in total GalTase activity, relative to control F9 cells. Analysis of pronase-digested glycopeptides obtained from control and transfected cells after metabolic labelling with [6-3H]galactose revealed no significant qualitative or quantitative differences, as assessed by Bio-Gel P-6 gel filtration chromatography and Tomato lectin affinity chromatography. Furthermore, SDS-PAGE analysis of immunoprecipitated [3H]galactose-labelled lysosomal-associated membrane protein-1 (LAMP-1) glycoprotein showed no difference in amounts or mobility. Pronase digestion and subsequent analysis of the gel-fractionated LAMP-1 glycoproteins also indicated no differences between the various cell lines. The inability 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 GalTase and UDP-[3H]galactose. Finally, the subcellular distribution of GalTase, as assessed by sucrose gradient fractionation, was similar between all cell types, thus suggesting that GalTase was appropriately compartmentalized in the transfected cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Youakim
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Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Shur
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D015703:Antigens, CD; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004247:DNA / Q000235:genetics; D054278:Embryonal Carcinoma Stem Cells; D005690:Galactose / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis*; D006031:Glycosylation; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism; D009237:N-Acetyllactosamine Synthase / Q000235:genetics*; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D011402:Pronase / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
D015703:Antigens, CD; D006023:Glycoproteins; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D014531:Uridine Diphosphate Galactose; D004247:DNA; D009237:N-Acetyllactosamine Synthase; D011402:Pronase; D005690:Galactose