| Abstract
| - Oligosaccharyl transferase (OT) catalyzes the co-translational transfer of a dolichol-linked tetradecasaccharide (Dol-PP-GlcNAc2Man9Glc3, 1a) to an asparagine side chain of a nascent polypeptideinside 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 glycosylationto occur. To address the substrate specificity of the glycosyl donor, three unnatural dolichol-linkeddisaccharide 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. Thesynthetic analogue Dol-PP-GlcNAc-Glc 1e, with substitution in the distal sugar, was found to bea substrate (Kmapp = 26 μM) for OT. On the other hand, the analogues Dol-PP-GlcNTFA-GlcNAc1c (Ki = 154 μM) and Dol-PP-2DFGlc-GlcNAc 1d (Ki = 252 μM), with variations in the proximalsugar, were inhibitors for OT. The dolichol-linked monosaccharide Dol-PP-GlcNAc 3 was found tobe the minimum unit for glycosylation to occur.
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