Glycosylation of ovalbumin in a heterologous cell: analysis of oligosaccharide chains of the cloned glycoprotein in mouse L cells.
In an effort to understand the factors that influence protein glycosylation, we are studying the expression of the chicken ovalbumin gene in a heterologous cell. Ovalbumin synthesized in mouse L-353 cells is glycosylated, as judged by incorporation of [3H]mannose and susceptibility to endo-beta-N-acetylglucosaminidases. Sequential digestion of ovalbumin synthesized in L cells with trypsin and chymotrypsin yields material migrating as one peak on HPLC coincident with similarly treated material from chicken ovalbumin, suggesting that the protein synthesized in L cells is glycosylated at the correct site. As in the case of chicken ovalbumin, the protein synthesized in L cells contains large amounts of hybrid oligosaccharides. Approximately 50% of the [3H]mannose incorporated into ovalbumin secreted by L cells is found in such hybrid structures. These results suggest that it is the polypeptide chain of ovalbumin that is responsible for proper glycosylation and subsequent processing of a substantial fraction of the oligosaccharide chains to hybrid structures. However, differences do exist between ovalbumin synthesized in L cells and the chicken glycoprotein. The hybrid oligosaccharides of ovalbumin secreted by L cells are completely sialylated and do not contain a bisecting GlcNAc residue, distinguishing them from hybrid chains in chicken ovalbumin. In addition to high-mannose and hybrid oligosaccharide chains, ovalbumin synthesized in L cells contains oligosaccharides of the complex type. To date, this type of sugar chain has not been observed in chicken ovalbumin. These differences in fine structure, between the oligosaccharides derived from ovalbumin secreted by L cells and those known to be present in the chicken egg glycoprotein, suggest that the cell type also plays a role in oligosaccharide processing.
Sheares
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D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000032:analysis; D006023:Glycoproteins / Q000378:metabolism*; D007739:L Cells; D051379:Mice; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis; D010047:Ovalbumin / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; D010047:Ovalbumin