Effect of altered oligosaccharide structure on the cell surface number, distribution and turnover of the high molecular weight acidic glycoproteins of CHO cells.
The influence of altered carbohydrate structure on the surface number, distribution and turnover of plasma membrane glycoproteins has been studied in Chinese hamster ovary (CHO) cells by comparing three lines that are resistant to the cytotoxic effects of wheat germ agglutinin (WGA) with parental CHO cells. The glycoproteins investigated were members of a group of high molecular weight acidic glycoproteins (HMWAG). On parental cells these represent the major surface components that become labelled by lactoperoxidase-catalysed iodination. They are the only plasma membrane glycoproteins that bind WGA. The mutant lines also possess iodinatable surface polypeptides of high molecular weight, but these were less acidic and electrophoretically less diffuse than those from parental cells. These polypeptides in general did not bind [125I]WGA when two-dimensional polyacrylamide gels were overlaid with iodinated lectin. Mutant cells treated with fluorescein-conjugated WGA showed low surface fluorescence. However, the nuclear envelope and a small region in the perinuclear zone fluoresced strongly. Together, these results confirm that the surface glycoproteins of mutant cells had altered carbohydrate structure. Mouse antiserum prepared against the HMWAG, however, bound equally effectively to the mutant lines as to the parental lines. Indirect immunofluorescence experiments showed that the HMWAG had a fairly uniform distribution over the surface, and that internalization induced by second antibody occurred at a similar rate and in a similar manner in all lines, including the mutants. Electron microscopic observations using immunoperoxidase procedures confirmed the similarities in glycoprotein distribution on mutant and parental cells. Two mouse monoclonal antibodies raised against the HMWAG also revealed no difference in the number or topography of surface glycoproteins. Finally, the half-lives of several HMWAG in a parental and a mutant line (15B) maintained on low-serum medium were compared by means of a 125I/131I double-label technique. Half-lives of HMWAG from the former averaged 12 h and from the latter 11 h. It is concluded that the lack of complex termini on oligosaccharides of this particular group of CHO plasma membrane glycoproteins has no effect on their number, distribution or turnover.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D037102:Lectins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D009154:Mutation; D009844:Oligosaccharides; D010053:Ovary / Q000166:cytology; D011485:Protein Binding; D014909:Wheat Germ Agglutinins
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D037102:Lectins; D008565:Membrane Proteins; D009844:Oligosaccharides; D014909:Wheat Germ Agglutinins