Primary structural requirements for N- and O-glycosylation of yeast mannoproteins

Abstract

Primary structural requirements both for N- and O-glycosylation have been studied using a series of synthetic peptides and a membrane fraction from Saccharomyces cerevisiae. N-Glycosylation: the tripeptide sequence Asn-Xaa-Thr/Ser was found to be necessary for the transfer of saccharide units from oligosaccharide-lipid to asparagine. Substitution of asparagine by aspartic acid or glutamine, or replacement of threonine by valine in the hexapeptide Tyr- Asn -Leu- Thr -Ser-Val prevents its glycosylation. Also, a proline residue in the position of Xaa makes the peptide unable to function as an acceptor. Transfer onto asparagine occurs only efficiently if both the α-amino group of asparagine and the α-carboxyl moiety of the hydroxy amino acid are blocked. Yield of glycosylation improves with increasing peptide chain length. With regard to the glycosyl donor dolichyl diphosphate-bound GlcNAc 2Man 9Glc 3 is the preferred substrate. Non-glucosylated glycolipid Dol- PP-GlcNAc 2Man 9 is a poor donor, whereas smaller precursors Dol- PP-GlcNAc 2 and Dol- PP-GlcNAc 2Man 1 allow reasonable transfer. O-Glycosylation: no marker sequence can be derived for the formation of an O-glycosidic linkage via Dol- P-Man. Introduction of a proline residue in vicinity to the hydroxy amino acid leads to a significant improvement of glycosyl transfer. It is postulated that accessibility of potential O-glycosylation sites rather than a specific sequence may be a prerequisite for O-glycosylation.