ENZYMATIC N-GLYCOSYLATION AND O-GLYCOSYLATION OF SYNTHETIC PEPTIDE ACCEPTORS BY DOLICHOL-LINKED SUGAR-DERIVATIVES IN YEAST

Using synthetic peptides we have investigated structural requirements for enzymatic N and O‐glycosylation via dolichol‐linked sugar derivatives in membranes from Saccharomyces cerevisiae. Dolichyl diphosphate chitobiose, Dol‐PP‐(GlcNAc)2, was used as a glycosyl donor for the formation of the N‐glycosidic linkage to asparagine. This reaction simulates glycosyl transfer in vitro from lipid‐linked oligosaccharides. The structural requirement of the carbohydrate acceptor for the transfer of chitobiose is the tripeptide sequence Asn‐X‐Ser/Thr. Moreover, the rate of glycosylation is affected by the chain length of peptides. Dinitrophenylation and dansylation of peptides reveal that other criteria are also of importance for glycosyl transfer in vitro. In contrast to the asparagine sequon, a marker sequence for the formation of the O‐glycosidic linkage via Dol‐P‐Man cannot be deduced. However, glycosyl transfer requires at least a minimum chain length of a tripeptide. With increasing chain length acceptor properties become significantly better. Presumably accessibility rather than recognition of a specific sequence might be the key for O‐glycosylation. The mannose unit was found to be transferred from Dol‐P‐β‐d‐mannose with inversion of its configuration to form α‐d‐mannosyl peptide. In addition, newly formed mannosyl peptide could be used as an acceptor for chain elongation via GDP‐Man giving rise to mannobiosyl peptide, a reaction that occurs in the glycosylation process of endogenous membrane‐bound acceptor. Thus synthetic peptides may be useful tools, not only to study structural requirements for glycosylation, but also to study dolichol‐mediated reactions independent of endogenous substrate. Copyright © 1979, Wiley Blackwell. All rights reserved