Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells.
Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells.
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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。
DOI：
10.1021/bi00475a024
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Castellino,FJ
中科院分区：
文献类型：
--
作者：
Davidson,DJ;Fraser,MJ;Castellino,FJ
印第安纳州圣母大学化学和生物化学系，1989年12月6日收到；1990年2月2日收到修订稿摘要：比较了人血浆纤溶酶原Asn289连接的寡糖结构和在鳞翅目昆虫细胞中表达的重组人纤溶酶原，这些细胞被含有人纤溶酶原全长的重组杆状病毒感染后。利用阴离子交换液相色谱对糖肽酶F从蛋白质中切割出的低聚糖单元进行作图，与标准低聚糖结构的洗脱位置进行比较，并结合单糖组成分析，我们发现人血浆蛋白只含有双唾液-双触角复合型碳水化合物和无唾液双触角复合型碳水化合物，证实了本实验室早先发表的工作。昆虫细胞表达的重组人纤溶酶原的糖基化模式表现出相当大的微观异质性，包括可识别的高甘露糖碳水化合物(Man9GlcNAc2)和截短的高甘露糖低聚糖(Man5GlcNAc2、Man4GlcNAc2和Man3GlcNAc2)。最重要的是，大约40%的寡糖群体由复杂的碳水化合物(双唾液-双天线)组成，在结构上与人类血浆蛋白相同。这是首次直接鉴定昆虫细胞中产生的蛋白质中的复杂碳水化合物，并表明可以将高甘露糖碳水化合物修剪和加工成复合型低聚糖。我们的数据表明，在这些细胞中存在正常和替代的途径来掺入和修剪高甘露糖低聚糖，甘露糖苷酶以及半乳糖基、己糖氨基和唾液酸基转移酶存在于这些细胞中，并且/或者可以被诱导。从这些观察中，我们得出结论，氨基酸序列和/或蛋白质构象特性可以控制寡糖加工事件。
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 Received December 6, 1989; Revised Manuscript Received February 2, 1990 abstract: A comparison has been made betweenthe Asn289-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed inlepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteinsby glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, we find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate (Man9GlcNAc2) and truncated high-mannose oligosaccharide (Man5GlcNAc2, Man4GlcNAc2, and Man3GlcNAc2). Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This is the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. Our data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexos-aminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, we conclude that aminoacid sequences and/or protein conformationalproperties can control oligosaccharide processing events.