Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)-deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR-Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVNLec1. We found that Sf-RVN and Sf-RVNLec1 cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVNLec1 cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVNLec1 cells were Endo H-cleavable Man5GlcNAc2 structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.

中文翻译：

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一种新的昆虫细胞系，用于生产具有可切割 N-聚糖的重组糖蛋白。

糖蛋白难以结晶，因为它们具有由多个单糖组成的异质聚糖，它们的 O-糖苷键具有相当大的旋转自由度。研究 N-糖蛋白的晶体学家通常通过使用 β1,2-N-乙酰氨基葡萄糖转移酶 I (MGAT1) 缺陷型哺乳动物细胞系来规避这个问题，该细胞系可产生具有未成熟 N-聚糖的重组糖蛋白。这些聚糖支持蛋白质折叠和质量控制，但可以使用内切-β-N-乙酰氨基葡萄糖苷酶 H (Endo H) 去除。许多晶体学家还使用杆状病毒-昆虫细胞系统 (BICS) 为他们的工作生产重组蛋白，但无法使用 MGAT1 缺陷型昆虫细胞系来促进该系统中的糖蛋白结晶。因此，我们使用 BICS 特异性 CRISPR-Cas9 载体编辑弹状病毒阴性草地贪夜蛾细胞系 (Sf-RVN) 的 Mgat1 基因，并分离出具有多个 Mgat1 缺失的亚克隆，我们将其命名为 Sf-RVNLec1。我们发现 Sf-RVN 和 Sf-RVNLec1 细胞具有相同的生长特性，并且同样可以作为杆状病毒介导的重组糖蛋白生产的宿主。N-聚糖分析表明，从 Sf-RVNLec1 细胞中分离的总内源性糖蛋白部分仅具有未成熟和高甘露糖型 N-聚糖。最后，N-聚糖分析和内切糖苷酶分析表明，由 Sf-RVNLec1 细胞产生的三种重组糖蛋白上的绝大多数 N-聚糖是 Endo H 可切割的 Man5GlcNAc2 结构。因此，该研究产生了一种新的 BICS 昆虫细胞系，可用于生产具有 Endo H 可切割 N-聚糖的重组糖蛋白。这将使研究人员能够将 BICS 的高生产率与对重组糖蛋白去糖基化的能力相结合，这将有助于通过 X 射线晶体学确定糖蛋白结构的努力。