Glycoengineering of plant expression systems is a prerequisite for the production of biopharmaceuticals that are compatible with animal-derived glycoproteins. Large amounts of high-mannose glycans such as Man₇GlcNAc₂, Man₈GlcNAc₂, and Man₉GlcNAc₂ (Man7/8/9), which can be favorably modified by chemical conjugation of mannose-6-phosphate, are desirable for lysosomal enzyme targeting. This study proposed a rice cell-based glycoengineering strategy using two different mannosidase inhibitors, kifunensine (KIF) and swainsonine (SWA), to increase Man7/8/9 glycoforms of recombinant human acid α-glucosidase (rhGAA), which is a therapeutic enzyme for Pompe disease. Response surface methodology was used to investigate the effects of the mannosidase inhibitors and to evaluate the synergistic effect of glycoengineering on rhGAA. Both inhibitors suppressed formation of plant-specific complex and paucimannose type N-glycans. SWA increased hybrid type glycans while KIF significantly increased Man7/8/9. Interestingly, the combination of KIF and SWA more effectively enhanced synthesis of Man7/8/9, especially Man9, than KIF alone. These changes show that SWA in combination with KIF more efficiently inhibited ER α-mannosidase II, resulting in a synergistic effect on synthesis of Man7/8/9. In conclusion, combined KIF and SWA treatment in rice cell culture media can be an effective method for the production of rhGAA displaying dominantly Man7/8/9 glycoforms without genetic manipulation of glycosylation.

中文翻译：

---

使用甘露糖苷酶抑制剂的N-糖基改造，以增加转基因水稻细胞培养物中酸性α-葡萄糖苷酶上的高甘露糖聚糖。

植物表达系统的糖工程是生产与动物衍生糖蛋白相容的生物药物的前提。大量的高甘露糖聚糖，例如Man ₇ GlcNAc ₂，Man ₈ GlcNAc ₂和Man ₉ GlcNAc ₂（Man7 / 8/9），其可以通过对6-磷酸甘露糖进行化学缀合而被有利地修饰，对于溶酶体酶靶向而言是合乎需要的。这项研究提出了一种基于水稻细胞的糖工程策略，该方法使用两种不同的甘露糖苷酶抑制剂（kifunensine（KIF）和swainsonine（SWA））来增加重组人酸性α-葡萄糖苷酶（rhGAA）的​​Man7 / 8/9糖型。庞贝病。响应面方法用于研究甘露糖苷酶抑制剂的作用，并评估糖工程对rhGAA的协同作用。两种抑制剂均抑制了植物特异性复合物和甘露糖型N-聚糖的形成。SWA增加杂合型聚糖，而KIF显着增加Man7 / 8/9。有趣的是，KIF和SWA的组合可更有效地增强Man7 / 8/9的合成，特别是Man9，而不是仅靠KIF。这些变化表明，SWA与KIF结合可以更有效地抑制ERα-甘露糖苷酶II，从而对Man7 / 8/9的合成产生协同作用。总之，在水稻细胞培养基中结合KIF和SWA处理可能是生产展示主要显示Man7 / 8/9糖型而无需进行糖基化遗传操作的rhGAA的有效方法。