Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.
Agroscience Research Laboratories, Hokusan Co., Ltd., 27-4, Kitanosato, Kitahiroshima, Hokkaido 061-1111, Japan.
J Biosci Bioeng. 2014 Oct;118(4):448-54. doi: 10.1016/j.jbiosc.2014.04.005. Epub 2014 May 3.
Production of pharmaceutical glycoproteins, such as therapeutic antibodies and cytokines, in plants has many advantages in safety and reduced costs. However, plant-made glycoproteins have N-glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a)) epitope, Galβ(1-3)[Fucα(1-4)]GlcNAc. Because it is likely that these sugar residues and glycan structures are immunogenic, many attempts have been made to delete them. Previously, we reported the simultaneous deletion of the plant-specific core α-1,3-fucose and α-1,4-fucose residues in Le(a) epitopes by repressing the GDP-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants (rGMD plants, renamed to ΔGMD plants) (Matsuo and Matsumura, Plant Biotechnol. J., 9, 264-281, 2011). In the present study, we generated a core β-1,2-xylose residue-repressed transgenic N. benthamiana plant by co-suppression of β-1,2-xylosyltransferase (ΔXylT plant). By crossing ΔGMD and ΔXylT plants, we successfully generated plants in which plant-specific sugar residues were repressed (ΔGMDΔXylT plants). The proportion of N-glycans with deleted plant-specific sugar residues found in total soluble protein from ΔGMDΔXylT plants increased by 82.41%. Recombinant mouse granulocyte/macrophage-colony stimulating factor (mGM-CSF) and human monoclonal immunoglobulin G (hIgG) harboring N-glycans with deleted plant-specific sugar residues were successfully produced in ΔGMDΔXylT plants. Simultaneous repression of the GMD and XylT genes in N. benthamiana is thus very useful for deleting plant-specific sugar residues.
生产药用糖蛋白，如治疗性抗体和细胞因子，在植物中有许多优势，在安全性和降低成本。然而，植物制造的糖蛋白具有植物特异性糖残基（核心β-1,2-木糖和α-1,3-岩藻糖）和 Lewis a（Le(a)）表位，Galβ(1-3)[Fucα(1-4)]GlcNAc。由于这些糖残基和聚糖结构可能具有免疫原性，因此已经进行了许多尝试来删除它们。以前，我们通过抑制与 GDP-L-岩藻糖生物合成相关的 GDP-D-甘露糖 4,6-脱水酶（GMD）基因，在 Nicotiana benthamiana 植物中同时删除了 Le(a)表位中的植物特异性核心α-1,3-岩藻糖和α-1,4-岩藻糖残基（rGMD 植物，重命名为ΔGMD 植物）（Matsuo 和 Matsumura，植物生物技术杂志，9,264-281,2011）。在本研究中，我们通过共抑制β-1,2-木糖基转移酶（ΔXylT 植物）生成了核心β-1,2-木糖残基抑制的转基因 N. benthamiana 植物。通过杂交ΔGMD 和ΔXylT 植物，我们成功生成了植物特异性糖基被抑制的植物（ΔGMDΔXylT 植物）。从ΔGMDΔXylT 植物的总可溶性蛋白中发现的具有删除的植物特异性糖残基的 N-糖链的比例增加了 82.41%。成功在ΔGMDΔXylT 植物中生产了具有删除的植物特异性糖残基的重组小鼠粒细胞/巨噬细胞集落刺激因子（mGM-CSF）和人单克隆免疫球蛋白 G（hIgG）。因此，在 N. benthamiana 中同时抑制 GMD 和 XylT 基因对于删除植物特异性糖基非常有用。
