Glycosylation of ovalbumin in a heterologous cell: analysis of oligosaccharide chains of the cloned glycoprotein in mouse L cells.

Abstract

In 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.Based on extensive studies, the enzymatic steps involved in the biosynthesis of oligosaccharide chains of asparaginelinked glycoproteins have been elucidated (1). Initially, the oligosaccharide chains are synthesized on a lipid-linked carrier; subsequently, they are cotranslationally transferred as a unit to the appropriate asparagine residues of the nascent polypeptide chain. The asparagine to be glycosylated must be part of a specific tripeptide acceptor sequence, Asn-XaaSer/Thr, this sequence being necessary but not sufficient for glycosylation. After transfer to protein, the oligosaccharide chain is processed, yielding either simple high-mannose chains or, after more extensive processing, complex-type sugar chains. In a few instances, as in the case of ovalbumin, hybrid structures are found containing features common to both high-mannose and complex oligosaccharides. Although the basic steps of the glycosylation pathway for asparagine-linked glycoproteins are known, the factors governing glycosylation and processing remain ambiguous. Because we are interested in determining the influence of the protein structure and cell type on proper-site glycosylation and oligosaccharide chain processing, we have begun to study the expression of cloned glycoproteins in heterologous cells. In this report, we examine the glycosylation of a model glycoprotein, chicken ovalbumin, in a mouse L-cell/herpes simplex virus type 1 (HSV-1) system (2).Mature chicken ovalbumin has two potential glycosylation sites, Asn-293 and Asn-312, of which only the first site is glycosylated in vivo. Chicken ovalbumin is heterogeneous with respect to its oligosaccharide chains, and at least nine basic structures have been characterized (3-6). The oligosaccharides fall into two major categories, high-mannose and hybrid. Although the exact distribution of high-mannose and hybrid chains is uncertain, the sum total of hybrid oligosaccharide chains is believed to be approximately equal to that of the high-mannose oligosaccharides (6). As (2) have shown that these cells synthesize an ovalbumin precursor that is processed and secreted into the extracellular medium after superinfection of the cells with HSV-1. These cells contain a plasmid (pRB353) with ovalbumin genomic DNA located downstream from and in the same transcriptional orientation as the a-regulated promoter of a protein 4 from HSV-1.Cells were maintained at 34°C in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and hypoxanthine (0.11 mM), aminopterin (0.45 ,uM), and thymidine (20.6 ,M). Radiolabeled ovalbumin was prepared essentially as described (2). HSV-1 ts502A305 carries a temperature-sensitive mutation such th...

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