PDF(Pubmed)
Abstract：
Cell-free, chemoenzymatic platforms are emerging technologies towards generating glycoconjugates with defined and homogeneous glycoforms. Recombinant oligosaccharyltransferases can be applied to glycosylate \"empty,\" i.e., aglycosyalted, peptides and proteins. While bacterial oligosaccharlytransferases have been extensively investigated, only recently a recombinant eukaryotic single-subunit oligosaccharyltransferase has been successfully used to in vitro N-glycosylate peptides. However, its applicability towards synthesizing full-length glycoproteins and utilizing glycans beyond mannose-type glycans for the transfer have not be determined. Here, we show for the first time the synthesis of hybrid- and complex-type glycans using synthetic lipid carriers as substrates for in vitro N-glycosylation reactions. For this purpose, transmembrane-deleted human β-1,2 N-acetylglucosamintransferase I and II (MGAT1ΔTM and MGAT2ΔTM) and β-1,4-galactosyltransferase (GalTΔTM) have been expressed in Escherichia coli and used to extend an existing multi-enzyme cascade. Both hybrid and agalactosylated complex structures were transferred to the N-glycosylation consensus sequence of peptides (10 amino acids: G-S-D-A-N-Y-T-Y-T-Q) by the recombinant oligosaccharyltransferase STT3A from Trypanosoma brucei.
摘要：
无细胞,化学酶平台是产生具有确定和均质糖型的糖缀合物的新兴技术。重组寡糖转移酶可以应用于糖基化\“空，\"即，aglycosylted,肽和蛋白质。虽然细菌寡糖转移酶已被广泛研究，直到最近，重组真核单亚基寡糖基转移酶已成功用于体外N-糖基化肽。然而,尚未确定其在合成全长糖蛋白和利用甘露糖型聚糖以外的聚糖进行转移方面的适用性。这里,我们首次展示了使用合成脂质载体作为体外N-糖基化反应底物的杂交型和复合型聚糖的合成。为此,跨膜缺失的人β-1,2N-乙酰葡糖胺转移酶I和II（MGAT1ΔTM和MGAT2ΔTM）和β-1,4-半乳糖基转移酶（GalTΔTM）已在大肠杆菌中表达，并用于扩展现有的多酶级联。杂合和半乳糖化的复合结构均通过来自布鲁氏锥虫的重组寡糖转移酶STT3A转移到肽的N-糖基化共有序列（10个氨基酸：G-S-D-A-N-Y-T-Y-T-Q）。
