From the Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China.
the College of Basic Medical Sciences, Dalian Medical University, Dalian 116044, Liaoning, China, and.
J Biol Chem. 2018 Apr 13;293(15):5572-5584. doi: 10.1074/jbc.M117.813030. Epub 2018 Feb 23.
Recombinant therapeutic proteins are becoming very important pharmaceutical agents for treating intractable diseases. Most biopharmaceutical proteins are produced in mammalian cells because this ensures correct folding and glycosylation for protein stability and function. However, protein production in mammalian cells has several drawbacks, including heterogeneity of glycans attached to the produced protein. In this study, we established cell lines with high-mannose-type -linked, low-complexity glycans. We first knocked out two genes encoding Golgi mannosidases ( and ) in HEK293 cells. Single knockout (KO) cells did not exhibit changes in -glycan structures, whereas double KO cells displayed increased high-mannose-type and decreased complex-type glycans. In our effort to eliminate the remaining complex-type glycans, we found that knocking out a gene encoding the endoplasmic reticulum mannosidase I () in the double KO cells reduced most of the complex-type glycans. In triple KO (, , and ) cells, Man9GlcNAc2 and Man8GlcNAc2 were the major -glycan structures. Therefore, we expressed two lysosomal enzymes, α-galactosidase-A and lysosomal acid lipase, in the triple KO cells and found that the glycans on these enzymes were sensitive to endoglycosidase H treatment. The -glycan structures on recombinant proteins expressed in triple KO cells were simplified and changed from complex types to high-mannose types at the protein level. Our results indicate that the triple KO HEK293 cells are suitable for producing recombinant proteins, including lysosomal enzymes with high-mannose-type -glycans.
重组治疗蛋白正成为治疗难治性疾病的重要药物。大多数生物制药蛋白是在哺乳动物细胞中产生的，因为这确保了蛋白质的正确折叠和糖基化，以保持其稳定性和功能。然而，哺乳动物细胞中的蛋白质生产有几个缺点，包括与产生的蛋白质连接的聚糖的异质性。在这项研究中，我们建立了具有高甘露糖型-连接、低复杂度聚糖的细胞系。我们首先敲除了 HEK293 细胞中编码高尔基体甘露糖苷酶（和）的两个基因。单敲除（KO）细胞没有表现出 -糖链结构的变化，而双 KO 细胞显示出增加的高甘露糖型和减少的复杂型聚糖。在我们努力消除剩余的复杂型聚糖的过程中，我们发现敲除双 KO 细胞中编码内质网甘露糖苷酶 I（）的基因可以减少大多数复杂型聚糖。在三 KO（、和）细胞中，Man9GlcNAc2 和 Man8GlcNAc2 是主要的 -糖链结构。因此，我们在三 KO 细胞中表达了两种溶酶体酶，α-半乳糖苷酶-A 和溶酶体酸性脂肪酶，并发现这些酶上的聚糖对内切糖苷酶 H 处理敏感。在三 KO 细胞中表达的重组蛋白的 -糖链结构在蛋白质水平上被简化，并从复杂型转变为高甘露糖型。我们的结果表明，三 KO HEK293 细胞适合生产包括具有高甘露糖型 -聚糖的溶酶体酶在内的重组蛋白。
