Enzymatic amplification involving glycosyltransferases forms the basis for the increased size of asparagine-linked glycans at the surface of NIH 3T3 cells expressing the N-ras proto-oncogene

• Published in: The Journal of biological chemistry 1991-11, Vol.266 (32), p.21674-21680
• Main Authors: EASTON, E. W, BOLSCHER, J. G. M, VAN DEN EIJNDEN, D. H
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Animals; Asparagine; Biological and medical sciences; Carbohydrate Conformation; Carbohydrate Sequence; Cell physiology; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Fundamental and applied biological sciences. Psychology; Genes, ras; Glycopeptides - isolation & purification; Hexosyltransferases - metabolism; Mammary Tumor Virus, Mouse - genetics; Membrane Glycoproteins - biosynthesis; Membrane Glycoproteins - genetics; Mice; Molecular and cellular biology; Molecular Sequence Data; Oligosaccharides - biosynthesis; Protein Processing, Post-Translational; Repetitive Sequences, Nucleic Acid; Substrate Specificity; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)54689-5

Expression of ras oncogenes in NIH 3T3 fibroblasts results in the acquisition by these cells of an invasive potential concomitant with the appearance of cell surface asparagine-linked complex-type glycan structures of a higher average molecular weight (Bolscher, J.G. M., van der Bijl, M. M. W., Neefjes, J. J., Hall, A., Smets, L.A., and Ploegh, H.L. (1988) EMBO J. 7, 3361-3368). We have investigated the enzymatic basis for the altered glycosylation by assessing the activities of all major Golgi glycosyltransferases involved in the synthesis of these structures. Use was made of a stable transfectant cell line (T15) containing the N-ras-protooncogene under the control of a glucocorticoid-inducible mouse mammary tumor virus promoter. Upon induction of the ras gene with dexamethasone: 1) the levels of N-acetylglucosaminyltransferase I and II were essentially unaltered, indicating an unaffected potential to synthesize complex-type glycans; 2) the activities of the branching N-acetylglucosaminyltransferase III and V were elevated 2- to 2.5-fold suggesting the formation of increased amounts of bisected glycans and of structures carrying a Gal beta 1---GlcNAc beta 1---6Man-branch; 3) the levels of the elongating beta 4-galactosyltransferase and beta 3-N-acetylglucosaminyl-transferase were increased 5- to 7-fold indicating a strongly enhanced capacity to synthesize polylactosaminoglycan chains; 4) the level of the major chain-terminating enzyme, alpha 3-galactosyltransferase, was slightly decreased (0.7-fold), whereas those of the alpha 3- and alpha 6-sialyltransferases were slightly elevated (1.3- and 2-fold, respectively), suggesting a shift from termination by alpha-galactosyl residues to termination by sialic acid moieties. Studies on the acceptor specificities of the different glycosyltransferases indicate that these changes occur in a coordinated manner in which the effects of altered glycosyltransferase expression levels amplify each other. Analysis of the size of cell surface complex-type glycopeptides before and after digestion with neuraminidase and endo-beta-galactosidase suggested an increased sialic acid density, an increase in the number and/or length of polylactosaminoglycan chains, and an increased branching of the glycans upon N-ras induction. The enzymatic results explain these structural changes and allow us to define the alterations in glycosylation pathways associated with ras expression.