Wenzel Lisa, Hoffmann Marcus, Rapp Erdmann, Rexer Thomas F T, Reichl Udo
Department of Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.
glyXera GmbH, Magdeburg, Germany.
Front Mol Biosci. 2023 Sep 12;10:1266431. doi: 10.3389/fmolb.2023.1266431. eCollection 2023.
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 -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 -glycosylation reactions. For this purpose, transmembrane-deleted human β-1,2 -acetylglucosamintransferase I and II (MGAT1ΔTM and MGAT2ΔTM) and β-1,4-galactosyltransferase (GalTΔTM) have been expressed in and used to extend an existing multi-enzyme cascade. Both hybrid and agalactosylated complex structures were transferred to the -glycosylation consensus sequence of peptides (10 amino acids: G-S-D-A-N-Y-T-Y-T-Q) by the recombinant oligosaccharyltransferase STT3A from .
无细胞化学酶平台是用于生成具有明确且均一糖型的糖缀合物的新兴技术。重组寡糖基转移酶可用于对“空的”，即无糖基化的肽和蛋白质进行糖基化修饰。虽然细菌寡糖基转移酶已得到广泛研究，但直到最近，一种重组真核单亚基寡糖基转移酶才成功用于对肽进行N-糖基化修饰。然而，其在合成全长糖蛋白以及利用除甘露糖型聚糖以外的聚糖进行转移方面的适用性尚未确定。在此，我们首次展示了使用合成脂质载体作为N-糖基化反应底物合成杂合型和复合型聚糖。为此，跨膜缺失的人β-1,2-N-乙酰葡糖胺转移酶I和II（MGAT1ΔTM和MGAT2ΔTM）以及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反应。杂合型和无半乳糖基化的复合型结构均通过来自酿酒酵母的重组寡糖基转移酶STT3A转移至肽的N-糖基化共有序列（10个氨基酸：G-S-D-A-N-Y-T-Y-T-Q）。
