Structures of the asparagine-289-linked oligosaccharides assembled on recombinant human plasminogen expressed in a Mamestra brassicae cell line (IZD-MBO503).
Structures of the asparagine-289-linked oligosaccharides assembled on recombinant human plasminogen expressed in a Mamestra brassicae cell line (IZD-MBO503).
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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。
DOI：
10.1021/bi00241a008
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发表时间：
1991
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;Castellino,FJ
圣母大学化学与生物化学系，圣母大学，印第安纳州 46556 收稿日期：1990 年 12 月 12 日；修订稿于 1991 年 4 月 17 日收到摘要：在本报告中，我们强化并扩展了之前的一项研究 [Davidson, DJ, Fraser, M. J., & Castellino, FJ (1990) Biochemistry 29, 5584-5590]，其中我们首次证明鳞翅目昆虫 (Spodoptera frugiperda) 细胞 (IPLB-SF-21 AE）能够在人类蛋白质（纤溶酶原）上组装 N 连接复合寡糖，其 cDNA 已通过杆状病毒载体的重组 DNA 技术插入到这些细胞中。为了研究鳞翅目昆虫细胞是否存在产生复杂寡糖的更普遍的能力，并鉴定此类昆虫细胞能够组装的寡糖类型的化学性质，我们用含有[R561E]人纤溶酶原（HPg）cDNA的重组（r）杆状病毒感染Mamestra芸苔（IZD-MBO503）细胞48小时，并表征了糖肽酶F的性质(GF)释放了这些细胞表达的r-HPg的Asn289上包含的N-连接寡糖。我们发现，大约 63% 的 N-连接寡糖属于复合型，其中双唾液酸双触角 (28%)、脱唾液酸双触角 (7%)、岩藻糖基化双唾液酸双触角 (25%) 和岩藻糖基化脱唾液酸双触角 (3%) 寡糖代表了主要的复合型碳水化合物种类。其余寡糖属于高甘露糖类型，其中（甘露糖）9（7V-乙酰氨基葡萄糖）2（22%）、（甘露糖）5（N-乙酰氨基葡萄糖）2（13%）和（甘露糖）3（7V-乙酰氨基葡萄糖）2（2%）代表观察到的主要寡糖。对另一种鳞翅目昆虫细胞系 Manduca sexta (CM-1) 中 r-HPg 表达的研究也清楚地表明，纯化的蛋白质上存在 (a2, 6) 连接的唾液酸，这表明昆虫细胞在 r-HPg 上组装复合型寡糖的能力本质上是普遍的。这些研究表明，尽管观察到内源性昆虫细胞蛋白显然不含N-连接复合寡糖，但组装此类结构所需的糖基转移酶基因存在于这些细胞中，并且能够在适当的条件下被利用。由此产生的寡糖加工的改变似乎是鳞翅目昆虫细胞的一个广泛特性，在这种情况下，这是通过用含有 HPg cDNA 的重组杆状病毒感染来实现的。该系统可以作为阐明一些控制蛋白质糖基化的调节因素的通用探针。
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 Received December 12, 1990; Revised Manuscript Received April 17, 1991 abstract: In this report, we have fortified and extended a previous investigation [Davidson, DJ, Fraser, M. J., & Castellino, FJ (1990) Biochemistry 29, 5584-5590] in which we demonstrated for the first time that lepidopteran insect (Spodoptera frugiperda) cells (IPLB-SF-21 AE) were capable of assembling N-linked complex oligosaccharide on a human protein (plasminogen), the cDNA of which had been inserted into these cells via recombinant DNA technology with a baculovirus vector. In order to investigate whether a more general capability of lepidopteran insect cells to produce complex oligosaccharide existed, and to identify the chemical nature of the types of oligosaccharides that such insect cells were able to assemble, we have infectedMamestra brassicae (IZD-MBO503) cells for 48 h with a recombinant (r) baculovirus containing the [R561E] human plasminogen (HPg) cDNA and characterized the nature of the glycopeptidase F (GF) released N-linked oligosaccharides contained on Asn289 of the r-HPg expressed by these cells. We found that approximately 63% of the total N-linked oligosaccharides were of the complex type, with bisialo-biantennary (28%), asialo-biantennary (7%), fucosylated bisaialo-biantennary (25%), and fucosylated asialo-biantennary (3%) oligosaccharides representing the major complex-type carbohydrate species. The remainder of the oligosaccharides were of the high-mannose type, with (mannose) 9 (7V-acetyIglucosamine) 2 (22%),(mannose) 5 (iV-acetylglucosamine) 2 (13%), and (mannose) 3 (7V-acetylglucosamine) 2 (2%) representing the major oligosaccharides observed. Investigations with r-HPg expression in another lepidopteran insect cell line, Manduca sexta (CM-1), also clearly demonstrated that (a2, 6)-linked sialic acid was present on the purified protein, suggesting that the ability of insect cells to assemble complex-type oligosaccharide on r-HPg is general in nature. These studies demonstrate that, despite the observations that endogenous insect cell proteins apparently do not contain N-linked complex oligosaccharide, the glycosyltransferase genes required for assembly of such structures are present in these cells and are capable of being utilized under proper conditions. The resulting alteration of oligosaccharide processing appears to be a broad property of lepidopteran insect cells, which is effected in this case by infection with a recombinant baculovirus containing the cDNA for HPg. This system may serve as a general probe for elucidation of some of the regulatory factors governing protein glycosylation.