Lopez M, Coddeville B, Langridge J, Plancke Y, Sautière P, Chaabihi H, Chirat F, Harduin-Lepers A, Cerutti M, Verbert A, Delannoy P
Laboratoire de Chimie Biologique, Unité Mixte de Recherche du CNRS n 111, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Glycobiology. 1997 Jul;7(5):635-51. doi: 10.1093/glycob/7.5.635.
The development of therapeutic glycoprotein production using the baculovirus expression system depends on the ability of insect cell lines to reproduce site specific mammalian-like N-glycans. A combination of 1H-NMR and mass spectrometry techniques (MALD-MS, ES-MS, and CID-MS-MS) allowed us to elucidate the N-linked oligosaccharides microheterogeneity on three different N-glycosylation sites, Asn233, Asn476, and Asn545, of a baculovirus-expressed recombinant bovine lactoferrin produced in Mamestra brassicae. Two families of N-glycan structures have been found: first, oligomannosidic glycans (Man[9-5]GlcNAc2) and secondly, short truncated partially fucosylated glycans (Man(3-2)[Fuc(0-1)]GlcNAc2). These results indicate that Mamestra brassicae cell line is not able to synthesize complex N-glycans, even if an alpha1,6-linked fucose residue is frequently present on the asparagine-bound N-acetylglucosamine residue of short truncated structures. Nevertheless, we have shown that Mamestra brassicae ensures the same N-glycosylation pattern as found on natural bovine lactoferrin showing the same distribution between complex and high-mannose type glycans on the different glycosylation sites. Sites which are naturally occupied by high-mannose glycans (Asn233 and Asn545) are substituted essentially by the same type of N-glycans in the recombinant counterpart, and the site Asn476,which carries sialylated complex type chains in the natural glycoprotein, is substituted by short, truncated, partially fucosylated chains in Mamestra brassicae-expressed bovine lactoferrin. These various results lead us to the conclusion that bovine lactoferrin is an interesting model to determine the potential of glycosylation of the baculovirus/insect cell expression systems.
利用杆状病毒表达系统生产治疗性糖蛋白，其发展取决于昆虫细胞系产生位点特异性哺乳动物样N-聚糖的能力。1H-NMR和质谱技术（MALD-MS、ES-MS和CID-MS-MS）相结合，使我们能够阐明在甘蓝夜蛾中产生的杆状病毒表达的重组牛乳铁蛋白的三个不同N-糖基化位点（Asn233、Asn476和Asn545）上N-连接寡糖的微观异质性。已发现两类N-聚糖结构：第一类是寡甘露糖型聚糖（Man[9-5]GlcNAc2），第二类是短截短的部分岩藻糖基化聚糖（Man(3-2)[Fuc(0-1)]GlcNAc2）。这些结果表明，甘蓝夜蛾细胞系不能合成复杂的N-聚糖，即使在短截短结构的天冬酰胺结合的N-乙酰葡糖胺残基上经常存在α1,6-连接的岩藻糖残基。然而，我们已经表明，甘蓝夜蛾确保了与天然牛乳铁蛋白相同的N-糖基化模式，在不同糖基化位点上复杂型和高甘露糖型聚糖之间显示出相同的分布。天然被高甘露糖聚糖占据的位点（Asn233和Asn545）在重组对应物中基本上被相同类型的N-聚糖取代，而天然糖蛋白中携带唾液酸化复杂型链的位点Asn476在甘蓝夜蛾表达的牛乳铁蛋白中被短的、截短的、部分岩藻糖基化的链取代。这些不同的结果使我们得出结论，牛乳铁蛋白是确定杆状病毒/昆虫细胞表达系统糖基化潜力的一个有趣模型。
