Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium.
Mass Spectrometry Laboratory-MolSys Research Unit, ULiege, Liège, Belgium.
Plant Biotechnol J. 2023 Sep;21(9):1773-1784. doi: 10.1111/pbi.14074. Epub 2023 Jun 2.
Production of recombinant pharmaceutical glycoproteins has been carried out in multiple expression systems. However, N-glycosylation, which increases heterogeneity and raises safety concerns due to the presence of non-human residues, is usually not controlled. The presence and composition of N-glycans are also susceptible to affect protein stability, function and immunogenicity. To tackle these issues, we are developing glycoengineered Nicotiana tabacum Bright Yellow-2 (BY-2) cell lines through knock out and ectopic expression of genes involved in the N-glycosylation pathway. Here, we report on the generation of BY-2 cell lines producing deglycosylated proteins. To this end, endoglycosidase T was co-expressed with an immunoglobulin G or glycoprotein B of human cytomegalovirus in BY-2 cell lines producing only high mannose N-glycans. Endoglycosidase T cleaves high mannose N-glycans to generate single, asparagine-linked, N-acetylglucosamine residues. The N-glycosylation profile of the secreted antibody was determined by mass spectrometry analysis. More than 90% of the N-glycans at the conserved Asn297 site were deglycosylated. Likewise, extensive deglycosylation of glycoprotein B, which possesses 18 N-glycosylation sites, was observed. N-glycan composition of gB glycovariants was assessed by in vitro enzymatic mobility shift assay and proven to be consistent with the expected glycoforms. Comparison of IgG glycovariants by differential scanning fluorimetry revealed a significant impact of the N-glycosylation pattern on the thermal stability. Production of deglycosylated pharmaceutical proteins in BY-2 cells expands the set of glycoengineered BY-2 cell lines.
已在多种表达系统中进行了重组药物糖蛋白的生产。然而，由于存在非人类残基，N-糖基化通常不受控制，从而增加了异质性并引起了安全性问题。N-聚糖的存在和组成也容易影响蛋白质的稳定性、功能和免疫原性。为了解决这些问题，我们通过敲除和异位表达参与 N-糖基化途径的基因，正在开发糖基工程化的烟草 Nicotiana tabacum Bright Yellow-2（BY-2）细胞系。在这里，我们报告了产生去糖基化蛋白的 BY-2 细胞系的生成。为此，内切糖苷酶 T 与仅产生高甘露糖 N-聚糖的 BY-2 细胞系中产生的免疫球蛋白 G 或人巨细胞病毒糖蛋白 B 共表达。内切糖苷酶 T 将高甘露糖 N-聚糖切割成单个天冬酰胺连接的 N-乙酰葡萄糖胺残基。通过质谱分析确定分泌抗体的 N-糖基化谱。保守的 Asn297 位点上超过 90%的 N-聚糖被去糖基化。同样，观察到具有 18 个 N-糖基化位点的糖蛋白 B 的广泛去糖基化。通过体外酶促迁移率变动测定评估 gB 糖型的 N-聚糖组成，并证明与预期的糖型一致。通过差示扫描荧光法比较 IgG 糖型，发现 N-糖基化模式对热稳定性有重大影响。BY-2 细胞中去糖基化药物蛋白的生产扩展了糖基工程化 BY-2 细胞系的范围。
