Factors affecting glycosylation site occupancy of Asn-184 of tissue-type plasminogen activator produced in Chinese hamster ovary cells

The presence or absence of an oligosaccharide (glycosylation site occupancy) can be important for the physical and biological properties of a glycoprotein. For example, tissue-type plasminogen activator (t-PA) with three sites occupied is more soluble but less active in vitro than t-PA with two sites occupied. Cultured cells produce both fully occupied and partially occupied t-PA (known as variable glycosylation site occupancy). The mechanism underlying variable glycosylation site occupancy is unknown.;Factors in the cell culture environment could potentially affect the percentage of fully glycosylated molecules. In this study, factors that influence the rate at which a protein is synthesized (translation elongation rate) were shown to affect site occupancy of t-PA produced in Chinese hamster ovary cells. Several factors known to slow down the elongation rate increased site occupancy, presumably by allowing more time for the enzyme-catalyzed transfer of the oligosaccharide to the glycosylation site on the protein. The protein synthesis inhibitors cycloheximide and fusidic acid, as well as the amino acid analog histidinol, increased site occupancy from approximately 40% (control) to a maximum of 80%, 58% and 78%, respectively. The methylxanthines theophylline and caffeine, shown to increase elongation rate in vitro, decreased site occupancy to 33% and 34%, respectively. A change in an environmental parameter (lowering temperature) increased site occupancy, presumably by decreasing elongation rate. All of these results are consistent with the hypothesis that elongation rate affects site occupancy.;Factors that potentially act through mechanisms other than elongation rate were investigated for their effect in this system. Several factors that affect other variably-occupied glycoproteins had little effect in this system. Thus, the culture conditions which can affect glycosylation site occupancy of variably-occupied sites appear to be protein-dependent. Factors contributing to the mechanism(s) underlying variable glycosylation site occupancy may therefore be different for t-PA than for other glycoproteins. Whatever the mechanism, the variably-occupied glycosylation site on t-PA is not normally available for a sufficient length of time to be fully glycosylated, and increasing the time through decreasing elongation rate increases site occupancy.