Inhibition of N-linked glycosylation.

Treatment of cells with inhibitors of the enzymes that synthesize N-linked oligosaccharide chains results in production of glycoproteins containing missing or altered chains. This approach is useful for examining potential functional role(s) of this class of oligosaccharides on specific proteins or intact cells. This unit describes the use of inhibitors to prevent N-linked glycosylation of proteins in cultured cells. First, the optimal concentration of inhibitor for the experiment (i.e., highest nontoxic concentration) is determined by monitoring [(35)S]methionine incorporation as a measure of protein biosynthesis. The ability of the inhibitor to hinder oligosaccharide processing is then determined by analyzing cells labeled with [(3)H]mannose using TCA precipitation or endo H digestion. Further suggestions are given on how to use methods for identifying a specific glycoprotein (if available) to measure the effect of the inhibitor on its N-linked oligosaccharide chains. A Support Protocol details a method for concentrating proteins by acetone precipitation.