N-Glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans
N-Glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans
复制标题
DOI：
10.1093/glycob/cwr009
复制
发表时间：
2011-06-01
期刊：
影响因子：
4.3
通讯作者：
Steinkellner, Herta
中科院分区：
文献类型：
--
作者：
Castilho, Alexandra;Gattinger, Pia;Steinkellner, Herta
糖工程越来越被认为是一种强大的工具，以产生重组糖蛋白与定制的N-糖基化模式。在这里，我们证明了植物糖基化途径的调制对人类型的二分和分支复杂的N-聚糖的形成。缺乏植物特异性N-糖基化的糖工程烟草（即β 1，2-木糖和核心α 1，3-岩藻糖）用于瞬时表达人促红细胞生成素（hEPO）和人转铁蛋白（hTF）以及人β 1，4-甘露糖基-β 1，4-N-乙酰葡糖胺转移酶（GnTIII）、α 1，3-甘露糖基-β 1，4-N-乙酰葡糖胺转移酶（GnTIV）和α 1，6-甘露糖基-β 1，6-N-乙酰葡糖胺转移酶（GnTV）。将hEPO表达为与IgG-Fc结构域的融合物（EPO-Fc），并通过蛋白A亲和层析纯化。重组hTF从渗透的植物叶片的细胞内液中分离。hEPO和hTF的基于质谱的N-聚糖分析揭示了二等分（GnGnbi）和三触角以及四触角复合N-聚糖（Gn[GnGn]、[GnGn]Gn和[GnGn][GnGn]）的定量形成。GnTIII与GnTIV和GnTV的共表达导致了平分的四触角复合物N-聚糖的有效产生。我们的研究结果表明，在很大的均匀性与人型N-糖基化的重组蛋白的产生。本文所述的策略提供了一种用于在重组糖蛋白上产生具有确定结构的拟南芥型N-连接聚糖的稳健且直接的方法，其可以推进糖蛋白研究并加速基于蛋白质的治疗剂的开发。
Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. beta 1,2-xylose and core alpha 1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human beta 1,4-mannosyl-beta 1,4-N-acetylglucosaminyltransferase (GnTIII), alpha 1,3-mannosyl-beta 1,4-N-acetylglucosaminyltransferase (GnTIV) and alpha 1,6-mannosyl-beta 1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.