Hamilton Stephen R, Bobrowicz Piotr, Bobrowicz Beata, Davidson Robert C, Li Huijuan, Mitchell Teresa, Nett Juergen H, Rausch Sebastian, Stadheim Terrance A, Wischnewski Harry, Wildt Stefan, Gerngross Tillman U
Thayer School of Engineering and the Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA.
Science. 2003 Aug 29;301(5637):1244-6. doi: 10.1126/science.1088166.
We report the humanization of the glycosylation pathway in the yeast Pichia pastoris to secrete a human glycoprotein with uniform complex N-glycosylation. The process involved eliminating endogenous yeast glycosylation pathways, while properly localizing five active eukaryotic proteins, including mannosidases I and II, N-acetylglucosaminyl transferases I and II, and uridine 5'-diphosphate (UDP)-N-acetylglucosamine transporter. Targeted localization of the enzymes enabled the generation of a synthetic in vivo glycosylation pathway, which produced the complex human N-glycan N-acetylglucosamine2-mannose3-N-acetylglucosamine2 (GlcNAc2Man3GlcNAc2). The ability to generate human glycoproteins with homogeneous N-glycan structures in a fungal host is a step toward producing therapeutic glycoproteins and could become a tool for elucidating the structure-function relation of glycoproteins.
我们报道了在毕赤酵母中糖基化途径的人源化，以分泌具有均匀复杂N-糖基化的人糖蛋白。该过程涉及消除内源性酵母糖基化途径，同时正确定位五种活性真核蛋白，包括甘露糖苷酶I和II、N-乙酰葡糖胺转移酶I和II以及尿苷5'-二磷酸（UDP）-N-乙酰葡糖胺转运蛋白。这些酶的靶向定位使得能够生成一条合成的体内糖基化途径，该途径产生复杂的人N-聚糖N-乙酰葡糖胺2-甘露糖3-N-乙酰葡糖胺2（GlcNAc2Man3GlcNAc2）。在真菌宿主中生成具有均匀N-聚糖结构的人糖蛋白的能力是朝着生产治疗性糖蛋白迈出的一步，并且可能成为阐明糖蛋白结构-功能关系的一种工具。
