Substrate recognition by oligosaccharyl transferase. Inhibition of co-translational glycosylation by acceptor peptides.

Abstract

In a microsome system rendered competent in protein translation by the addition of rabbit reticulocyte lysate, co-translational insertion and glycosylation of N-linked glycoproteins is observed when the appropriate mRNA is supplied. We have utilized this system to examine the ability of acceptor tripeptides of the type Asn-X-Thr/Ser to inhibit co-translational glycosylation. Using endogenous oligosaccharide-lipid as the carbohydrate donor, dog pancreas microsomes efficiently glycosylated N alpha-[3H]Ac-Asn-Leu-Thr-NHCH3 (apparent Km = 100 microM). Glycopeptide formation was essentially complete within 20 min. In the presence of mRNA from vesicular stomatitis virus or chicken ovalbumin, a similar tripeptide, N alpha-Ac-Asn-Leu-Thr-NH2, inhibited co-translational glycosylation. Translocation of the nascent chains was not affected. Thus, in the absence of peptide, all translated G protein was glycosylated and found within the microsomes, whereas in the presence of the peptide a mixture of glycosylated and nonglycosylated G protein was sequestered. Inhibition of nascent chain glycosylation was competitive and not merely the result of oligosaccharide lipid depletion, because preincubation of the microsomes with the peptide followed by its removal did not affect subsequent glycosylation of ovalbumin or G protein. Six derivatives of Asn-Leu-Thr-NH2, three of which were acceptors and three of which were not, were tested for their ability to inhibit co-translational glycosylation. The three acceptor peptides, N alpha-Ac-Asn-Leu-Thr-NH2, N alpha-Oc-Asn-Leu-Thr-NH2, and N alpha-Bz-Asn-Leu-Thr-NH2, effectively inhibited nascent chain glycosylation. In contrast, the three nonacceptors, N alpha-Ac-Gln-Leu-Thr-NH2, N alpha-Ac-Asn(N beta-Me)-Leu-Thr-NH2, and Asn-Leu-Thr-NH2, had no effect. Taken together, these data indicate that the inhibition of co-translational glycosylation by a peptide is dependent on its ability to compete for the active site of the oligosaccharyl transferase.