Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells.
Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells.
复制标题
重组IFN-γ在中国仓鼠卵巢和昆虫细胞中转运和糖基化的限制。
DOI：
--
复制
发表时间：
1999
影响因子：
3.8
通讯作者：
D. C. James
中科院分区：
文献类型：
--
作者：
A. Hooker;Nicola H. Green;A. J. Baines;A. T. Bull;N. Jenkins;P. G. Strange;P. G. Strange;D. C. James
在这项研究中，我们比较了哺乳动物和昆虫细胞中重组糖蛋白的胞内转运和加工。通过基质辅助激光解吸质谱法对重组人IFN-γ的N-糖基化的详细分析表明，由中国仓鼠卵巢和杆状病毒感染的昆虫Sf 9细胞分泌的蛋白质分别与复杂的唾液酸化或截短的三甘露糖基核心聚糖相关。然而，在这两种情况下，细胞内蛋白质主要与高甘露糖型寡糖（Man-6至Man-9）相关，表明内质网至顺式高尔基体转运是两种表达系统中的主要限速步骤。在CHO细胞中，尽管存在与分泌产物相同的唾液酸化IFN-γ糖型的较小细胞内亚群（因此与晚期高尔基体隔室或分泌囊泡相关），但没有其他中间体是明显的。因此，在高尔基体中的顺行运输过程不限制分泌。在Sf 9昆虫细胞中，除了核心岩藻糖基化和去甘露糖基化（两者都是糖基化位点特异性的）之外，没有直接证据表明ER后聚糖加工事件。为了研究核苷酸-糖可用性对细胞特异性糖基化的影响，通过阴离子交换HPLC定量测定哺乳动物和昆虫细胞中核苷酸-糖底物的细胞含量。在两种宿主细胞类型中，UDP-己糖和UDP-N-乙酰己糖胺相对于其他底物的丰度更高。然而，与CHO细胞不同，未感染或杆状病毒感染的Sf 9细胞中不存在唾液酸转移酶活性和CMP-NeuAc底物。对于其他昆虫细胞宿主Sf 21和Ea 4获得了类似的数据。我们的结论是，虽然在哺乳动物和昆虫细胞中的重组蛋白的细胞内运输和分泌的限制是相似的，在Sf昆虫细胞中的N-聚糖加工是有限的，并且在这些昆虫细胞系中的N-聚糖加工的遗传修饰将受到限制的底物的末端半乳糖基化。
In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.
登录
查看更多内容
鳞翅目昆虫（草地贪夜蛾）细胞表达人纤溶酶原 cDNA 时的低聚糖加工。
影响因子：
2.9
作者：
Davidson,DJ;Fraser,MJ;Castellino,FJ
通讯作者：
Castellino,FJ
天冬酰胺-289 连接寡糖在 Mamestra 芸苔细胞系 (IZD-MBO503) 中表达的重组人纤溶酶原上组装的结构。
影响因子：
2.9
作者：
Davidson,DJ;Castellino,FJ
通讯作者：
Castellino,FJ
来自鳞翅目昆虫细胞系 Sf9 的 α1,2-甘露糖苷酶 cDNA 的分离和表征。
影响因子：
4.3
作者：
Kawar,Z;Herscovics,A;Jarvis,DL
通讯作者：
Jarvis,DL
来自杆状病毒感染的鳞翅目昆虫细胞 (IPLB-SF21AE) 的高尔基衍生 (α 1,2)-甘露糖苷酶-I 的纯化和特性，对甘露糖 6-N-乙酰葡糖胺 2 具有优先活性。
影响因子：
2.9
作者：
Ren,J;Bretthauer,RK;Castellino,FJ
通讯作者：
Castellino,FJ
大鼠肝脏完整高尔基体制剂中寡糖的生物合成。
DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Hayes,BK;Freeze,HH;Varki,A
通讯作者：
Varki,A