In vivo N-glycosylation and fate of Asn-X-Ser/Thr tripeptides.

Abstract

The minimum primary structural requirement for a tripeptide to serve as a substrate for oligosaccharyl transferase is the sequence -Asn-X-Ser/Thr-. In the present study the activities of three structurally different tripeptides containing acceptor sequences for oligosaccharyl transferase were compared in three systems: Xenopus oocytes, in which they were introduced into the cytoplasm by microinjection, cultured mammalian cells, and isolated rat liver microsomes. In the last two systems, the peptides were added exogenously to the culture or to the incubation medium, respectively. On the basis of lectin column and paper chromatographic analysis it was established that the microinjected acceptor tripeptides were glycosylated in Xenopus oocytes. However, lectin column analysis and retention of sensitivity to endoglycosidase H revealed that none of the three glycopeptides was processed to complex oligosaccharide chains and none was subsequently secreted. Rather, over a 24-h period the glycopeptides were degraded. Chloroquine was found to block this degradation process, but even under these conditions, the glycopeptides were not secreted into the medium. In the isolated microsomes the glycosylation of the acceptor tripeptides was time-dependent and the tripeptide with an iodotyrosine residue in the X position was found to be a poor substrate. When added to cultured mammalian cells, all three of the tripeptides were taken up, glycosylated, and subsequently secreted. These results are discussed in the context of the wide differences in glycosylation of the three peptides and their lack of secretion after glycosylation in Xenopus oocytes.