title: Substrate specificity of the glycosyl donor for oligosaccharyl transferase.
abstract: Oligosaccharyl transferase (OT) catalyzes the co-translational transfer of a dolichol-linked tetradecasaccharide (Dol-PP-GlcNAc(2)Man(9)Glc(3), 1a) to an asparagine side chain of a nascent polypeptide inside the lumen of the endoplasmic reticulum (ER). The glycosyl acceptor requires an Asn-Xaa-Thr/Ser sequon, where Xaa can be any natural amino acid except proline, for N-linked glycosylation to occur. To address the substrate specificity of the glycosyl donor, three unnatural dolichol-linked disaccharide analogues (Dol-PP-GlcNTFA-GlcNAc 1c, Dol-PP-2DFGlc-GlcNAc 1d, and Dol-PP-GlcNAc-Glc 1e) were synthesized and evaluated as substrates or inhibitors for OT from yeast. The synthetic analogue Dol-PP-GlcNAc-Glc 1e, with substitution in the distal sugar, was found to be a substrate (K(m)(app)() = 26 microM) for OT. On the other hand, the analogues Dol-PP-GlcNTFA-GlcNAc 1c (K(i) = 154 microM) and Dol-PP-2DFGlc-GlcNAc 1d (K(i) = 252 microM), with variations in the proximal sugar, were inhibitors for OT. The dolichol-linked monosaccharide Dol-PP-GlcNAc 3 was found to be the minimum unit for glycosylation to occur.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: V W
authors.initials: VW
authors.lastname: Tai
authors.forename: B
authors.initials: B
authors.lastname: Imperiali
mesh_terms: D004186:Disaccharidases / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology*; D004286:Dolichols / Q000378:metabolism*; D006020:Glycopeptides / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases; D007700:Kinetics; D008565:Membrane Proteins; D009842:Oligopeptides / Q000378:metabolism; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D014166:Transferases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D015003:Yeasts / Q000378:metabolism
chemical_list: D004286:Dolichols; D006020:Glycopeptides; D008565:Membrane Proteins; D009842:Oligopeptides; D011103:Polyisoprenyl Phosphate Monosaccharides; C018439:N-acetylglucosaminylpyrophosphoryldolichol; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D004186:Disaccharidases