Abstract：
Microalgae are considered as attractive expression systems for the production of biologics. As photosynthetic unicellular organisms, they do not require costly and complex media for growing and are able to secrete proteins and perform protein glycosylation. Some biologics have been successfully produced in the green microalgae Chlamydomonas reinhardtii. However, post-translational modifications like glycosylation of these Chlamydomonas-made biologics have poorly been investigated so far. Therefore, in this study, we report on the first structural investigation of glycans linked to human erythropoietin (hEPO) expressed in a wild-type C. reinhardtii strain and mutants impaired in key Golgi glycosyltransferases. The glycoproteomic analysis of recombinant hEPO (rhEPO) expressed in the wild-type strain demonstrated that the three N-glycosylation sites are 100% glycosylated with mature N-glycans containing four to five mannose residues and carrying core xylose, core fucose and O-methyl groups. Moreover, expression in C. reinhardtii insertional mutants defective in xylosyltransferases A and B and fucosyltransferase resulted in drastic decreases of core xylosylation and core fucosylation of glycans N-linked to the rhEPOs, thus demonstrating that this strategy offers perspectives for humanizing the N-glycosylation of the Chlamydomonas-made biologics.
摘要：
微藻被认为是生产生物制品的有吸引力的表达系统。作为光合单细胞生物，它们不需要昂贵和复杂的培养基来生长，并且能够分泌蛋白质和进行蛋白质糖基化。已在绿色微藻衣藻中成功生产了一些生物制剂。然而,到目前为止，对这些衣藻制成的生物制品的翻译后修饰如糖基化的研究还很少。因此,在这项研究中，我们报道了与人类促红细胞生成素（hEPO）相关的聚糖在野生型莱茵衣原体菌株和关键高尔基体糖基转移酶受损的突变体中表达的首次结构研究。在野生型菌株中表达的重组hEPO(rhEPO)的糖蛋白质组分析表明,三个N-糖基化位点100%被含有4至5个甘露糖残基并携带核心木糖的成熟N-聚糖糖基化,核心岩藻糖和O-甲基。此外，在木糖基转移酶A和B和岩藻糖基转移酶缺陷的C.reinhardtii插入突变体中的表达导致与rhEPO连接的聚糖的核心木糖糖基化和核心岩藻糖基化的急剧下降，因此证明了该策略为使衣藻属生物制品的N-糖基化人源化提供了前景。
