Wang Qiong, Betenbaugh Michael J
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, U.S.A.
Emerg Top Life Sci. 2018 Oct 26;2(3):433-442. doi: 10.1042/ETLS20180056.
As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.
作为一种复杂且常见的翻译后修饰，N-糖基化会影响重组糖蛋白的生物活性和功效。例如，α1,6-岩藻糖基化显著影响抗体依赖的细胞毒性，而α2,6-唾液酸化对于抗体的抗炎活性至关重要。末端唾液酸化对糖蛋白的循环半衰期很重要。中国仓鼠卵巢（CHO）细胞是目前主要的重组蛋白生产平台，在本综述中，总结了CHO糖基化的特点。此外，还描述了过去几十年来深入研究的用于调整CHO细胞中糖蛋白岩藻糖基化和唾液酸化的最新及当前代谢工程策略。一种减少α1,6-岩藻糖基化的方法是通过敲低和敲除方法抑制岩藻糖基转移酶（FUT8）的表达。另一种调节岩藻糖基化的方法是通过抑制岩藻糖基化生物合成途径中的多个基因或使用化学抑制剂。为了调节抗体可结晶片段的唾液酸化，单独或与氨基酸突变一起表达唾液酸转移酶和半乳糖转移酶可以影响抗体糖型，并进一步影响抗体效应功能。抑制唾液酸酶表达和化学补充也是提高重组糖蛋白唾液酸化水平的有效且互补的方法。对CHO细胞或蛋白质序列进行工程改造以控制糖型以产生更均一的聚糖是一个新兴的研究课题。为了调节糖基化代谢途径，详细介绍了多个糖基因敲除和敲入的相互作用以及多种方法的组合，包括基因操作、蛋白质工程和化学补充，以在重组糖蛋白上实现特定的聚糖谱，从而获得卓越的生物学功能和有效性。
