GLYCOSYLATION AND SECRETION OF HUMAN-TISSUE PLASMINOGEN-ACTIVATOR IN RECOMBINANT BACULOVIRUS-INFECTED INSECT CELLS
GLYCOSYLATION AND SECRETION OF HUMAN-TISSUE PLASMINOGEN-ACTIVATOR IN RECOMBINANT BACULOVIRUS-INFECTED INSECT CELLS
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DOI：
10.1128/mcb.9.1.214
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发表时间：
1989-01-01
影响因子：
5.3
通讯作者：
SUMMERS, MD
中科院分区：
文献类型：
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作者：
JARVIS, DL;SUMMERS, MD
从鳞翅目昆虫果翅夜蛾(Sf9)建立的细胞系通常被用来作为重组杆状病毒载体表达外源蛋白的宿主。我们以人组织型纤溶酶原激活物(t-PA)为模型，研究了这些细胞中蛋白质糖基化和分泌的途径。在Sf9细胞中表达的t-PA既是N糖基化的，又是分泌的。至少胞外t-PA中N-连接的寡糖的一个子集对内切-β-N-乙酰-D-氨基葡萄糖苷酶H具有抗性，该酶能去除未成熟的高甘露糖型低聚糖。这驳斥了以前研究得出的普遍结论，即Sf9细胞不能将未成熟的N-连接低聚糖加工成内切-β-N-乙酰-D-氨基葡萄糖苷酶H抗性的形式。即使脉冲标记时间很短，在Sf9细胞中也没有检测到非糖基化的t-PA前体。这表明t-PA的哺乳动物信号序列在Sf9细胞中被有效识别，并且它可以介导发生共翻译N糖基化的粗面内质网膜上的快速转位。然而，t-PA的分泌相当缓慢，半衰期约为1.6h，因此，在t-PA多肽的转位和N糖基化之后，分泌的一个限速步骤(S)发生。用衣霉素处理Sf9细胞，但不用寡糖加工抑制剂处理，可以阻止t-PA在细胞外培养上清液中的出现。这表明，在杆状病毒感染的Sf9细胞中，t-PA的分泌直接或间接需要N糖基化本身，而不是N连接的寡糖的加工。最后，分泌的相对效率随着感染时间的延长而急剧下降，这表明Sf9宿主细胞的分泌途径在杆状病毒感染的后期受到了损害。
Cell lines established from the lepidopteran insect Spodoptera frugiperda (fall armyworm; Sf9) are used routinely as hosts for the expression of foreign proteins by recombinant baculovirus vectors. We have examined the pathway of protein glycosylation and secretion in these cells, using human tissue plasminogen activator (t-PA) as a model. t-PA expressed in Sf9 cells was both N glycosylated and secreted. At least a subset of the N-linked oligosaccharides in extracellular t-PA was resistant to endo-.beta.-N-acetyl-D-glucosaminidase H, which removes immature, high-mannose-type oligosaccharides. This refutes the general conclusion from previous studies that Sf9 cells cannot process immature N-linked oligosaccharides to an endo-.beta.-N-acetyl-D-glucosaminidase H-resistant form. A nonglycosylated t-PA precursor was not detected in Sf9 cells, even with very short pulse-labeling times. This suggests that the mammalian signal sequence of t-PA is efficiently recognized in Sf9 cells and that it can mediate rapid translocation across the membrane of the rough endoplasmic reticulum, where cotranslational N glycosylation takes place. However, t-PA was secreted rather slowly, with a half-time of about 1.6 h. Thus, a rate-limiting step(s) in secretion occurs subsequent to translocation and N glycosylation of the t-PA polypeptide. Treatment of Sf9 cells with tunicamycin, but not with inhibitors of oligosaccharide processing, prevented the appearance of t-PA in the extracellular medium. This suggests that N glycosylation per se, but not processing of the N-linked oligosaccharides, is required directly or indirectly in baculovirus-infected Sf9 cells for the secretion of t-PA. Finally, the relative efficiency of secretion decreased dramatically with time of infection, suggesting that the Sf9 host cell secretory pathway is compromised during the later stages of baculovirus infection.