Substrate specificity of the glycosyl donor for oligosaccharyl transferase

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 e ndoplasmic reticulum. (ER). The glycosyl acceptor requires an Asn-Xaa-Thr/S er sequon, where Xaa can be any natural amino acid except proline, for N-li nked glycosylation to occur. To address the substrate specificity of the gl ycosyl donor, three unnatural dolichol-linked disaccharide analogues (Dol-P P-GlcNTFA-GlcNAc 1c, Dol-PP-2DFGlc-GlcNAc Id, and Dol-PP-GlcNAc-Glc le) wer e synthesized and evaluated as substrates or inhibitors for OT from yeast. The synthetic analogue Dol-PP-GlcNAc-Glc le, with substitution in the dista l sugar, was found to be a substrate (K-mapp = 26 muM for OT. On the other hand, the analogues Dol-PP-GlcNTFA-GlcNAc le (Ki = 154 muM) and Dol-PP-2DFG lc-GlcNAc 1d (K-i = 25 muM), with variations in the proximal sugar, were in hibitors for OT. The dolichol-linked monosaccharide Dol-PP-GlcNAc 3 was fou nd to be the minimum unit for glycosylation to occur.