Data_Sheet_1_Efficient N-Glycosylation of the Heavy Chain Tailpiece Promotes the Formation of Plant-Produced Dimeric IgA.PDF

Production of monomeric IgA in mammalian cells and plant expression systems such as Nicotiana benthamiana is well-established and can be achieved by co-expression of the corresponding light and heavy chains. In contrast, the assembly of dimeric IgA requires the additional expression of the joining chain and remains challenging especially in plant-based systems. Here, we examined factors affecting the assembly and expression of HER2 binding dimeric IgA1 and IgA2m(2) variants transiently produced in N. benthamiana. While co-expression of the joining chain resulted in efficient formation of dimeric IgAs in HEK293F cells, a mixture of monomeric, dimeric and tetrameric variants was detected in plants. Mass-spectrometric analysis of site-specific glycosylation revealed that the N-glycan profile differed between monomeric and dimeric IgAs in the plant expression system. Co-expression of a single-subunit oligosaccharyltransferase from the protozoan Leishmania major in N. benthamiana increased the N-glycosylation occupancy at the C-terminal heavy chain tailpiece and changed the ratio of monomeric to dimeric IgAs. Our data demonstrate that N-glycosylation engineering is a suitable strategy to promote the formation of dimeric IgA variants in plants.

在哺乳动物细胞与本氏烟草（Nicotiana benthamiana）等植物表达系统中，单体免疫球蛋白A（monomeric IgA）的生产技术已十分成熟，仅需共表达对应的轻重链即可实现。与之相对，二聚体免疫球蛋白A（dimeric IgA）的组装需要额外表达连接链（joining chain），且该过程仍存在诸多挑战，在植物表达系统中尤为突出。本研究针对本氏烟草中瞬时表达的、可结合人类表皮生长因子受体2（HER2）的二聚体IgA1及IgA2m(2)变体，考察了影响其组装与表达的各类因素。尽管连接链的共表达可使HEK293F细胞高效组装出二聚体IgA，但在植物样本中检测到的却是单体、二聚体与四聚体变体的混合产物。对位点特异性糖基化的质谱分析结果显示，植物表达系统内单体与二聚体IgA的N-聚糖（N-glycan）谱存在显著差异。在本氏烟草中共表达源自利什曼原虫（Leishmania major）的单亚基寡糖转移酶（single-subunit oligosaccharyltransferase），可提升重链C末端尾肽处的N-糖基化占据率，并改变单体与二聚体IgA的比例。本研究数据证实，N-糖基化工程（N-glycosylation engineering）是推动植物系统中二聚体IgA变体组装的适宜策略。