The effect of protein synthesis inhibitors on the glycosylation site occupancy of recombinant human prolactin

Abstract

The relationship between synthesis and N-linked glycosylation siteoccupancy of recombinant human prolactin produced from C127 cellswas studied with the aid of a battery of protein synthesis inhibitors.Non-lethal concentrations of sodium fluoride, gougerotin, puromycin,anisomycin, and emetine did not alter site occupancy, but low concentrations(< 10 micrograms ml-1) of cycloheximide increased the fraction ofsecreted prolactin bearing oligosaccharide from 20% to 80% of thetotal. Cycloheximide is an inhibitor of the elongation step of proteinsynthesis. The observed increase in glycosylation site occupancyupon addition of cycloheximide is consistent with the current opinionthat the initial glycosylation event occurs cotranslationally duringa limited time period. Cycloheximide may extend this time periodby reducing elongation rate. However, the absence of any effect fromtreatment with other inhibitors of elongation suggests that cycloheximideis unique in its behavior on this system.