Tai V W, Imperiali B
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
J Org Chem. 2001 Sep 21;66(19):6217-28. doi: 10.1021/jo0100345.
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.
寡糖基转移酶（OT）催化多萜醇连接的十四糖（Dol-PP-GlcNAc(2)Man(9)Glc(3)，1a）在内质网（ER）腔内新生多肽的天冬酰胺侧链上进行共翻译转移。糖基受体需要一个Asn-Xaa-Thr/Ser序列（其中Xaa可以是除脯氨酸外的任何天然氨基酸）才能发生N-连接糖基化。为了研究糖基供体的底物特异性，合成了三种非天然的多萜醇连接的二糖类似物（Dol-PP-GlcNTFA-GlcNAc 1c、Dol-PP-2DFGlc-GlcNAc 1d和Dol-PP-GlcNAc-Glc 1e），并将其作为酵母OT的底物或抑制剂进行评估。发现远端糖有取代的合成类似物Dol-PP-GlcNAc-Glc 1e是OT的底物（K(m)(app)() = 26 microM）。另一方面，近端糖有变化的类似物Dol-PP-GlcNTFA-GlcNAc 1c（K(i) = 154 microM）和Dol-PP-2DFGlc-GlcNAc 1d（K(i) = 252 microM）是OT的抑制剂。发现多萜醇连接的单糖Dol-PP-GlcNAc 3是发生糖基化的最小单位。
