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Glycobiology vol 3 no 2 pp. 155-163, 1993
© 1993

research-article

Effects of overexpression of ß1, 4-galactosyltransferase on glycoprotein biosynthesis in F9 embryonal carcinoma cells

Adel Youakim and Barry D. Shur

Department of Biochemistry and Molecular Biology, Box 117, The University of Texas M.D.Anderson Cancer Center, 1515 Holcombe Blvd Houston, TX 77030, USA

Received on October 24, 1992; accepted on December 9, 1992

ß1,4-Galactosyltransferase (GalTase) plays a central role in the biosynthesis of N-acetyllactosamine-containing oligo-saccharides. 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 chroma-tography. Furthermore, SDS-PAGE analysis of immuno-precipitated [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. More importantly, GalTase specific activities in the Golgi membranes of the transfected cells were 3–4 times greater than in control cells. These results show that selectively increasing GalTase activity does not alter glycoprotein biosynthesis in F9 cells.

F9 cells galactosyltransferase glycoprotein biosynthesis


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H. Hathaway and B. Shur
Mammary gland morphogenesis is inhibited in transgenic mice that overexpress cell surface beta1,4-galactosyltransferase
Development, January 9, 1996; 122(9): 2859 - 2872.
[Abstract] [PDF]


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