Voss T, Ergülen E, Ahorn H, Kubelka V, Sugiyama K, Maurer-Fogy I, Glössl J
Ernst-Boehringer Institut für Arzneimittelforschung, Department of Protein Chemistry, Bender & Co., Vienna, Austria.
Eur J Biochem. 1993 Nov 1;217(3):913-9. doi: 10.1111/j.1432-1033.1993.tb18321.x.
Human interferon omega 1 (IFN-omega 1) was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system. Half of the protein purified by immunoaffinity chromatography was shown to be N-glycosylated at the same site as the natural IFN-omega 1. The degree of glycosylation was independent of the expression rate. While natural IFN-omega 1 was shown to carry complex-type oligosaccharides [Adolf, G. R., Maurer-Fogy, I., Kalsner, I. & Cantell, K. (1990) J. Biol. Chem. 265, 9290-9295], the insect cell produced protein which was demonstrated by lectin blot, mass spectroscopy and HPLC analysis to contain only the core oligosaccharide. Two different structures, (Man)2(GlcNAc)2[Fuc] and (Man)3(GlcNAc)2[Fuc] were identified. The fucosylation was identified to be (alpha 1-6)-linked to the core saccharide. Sialic acid residues were clearly absent. IFN-omega 1 expressed in S. frugiperda cells was shown to be partially truncated at the C-terminus by nine residues; its antiviral activity when glycosylated was significantly lower than the activity of IFN-omega 1 produced by Sendai-virus-stimulated leukocytes. Circular dichroism and fluorescence spectroscopy did not reveal any structural differences between glycosylated and nonglycosylated IFN-omega 1. This implies the importance of a complex-type glycosylation for the maximal biological activity of human IFN-omega 1.
利用杆状病毒表达系统在草地贪夜蛾Sf9昆虫细胞中表达了人ω-1干扰素（IFN-ω1）。经免疫亲和层析纯化的蛋白质中有一半在与天然IFN-ω1相同的位点发生了N-糖基化。糖基化程度与表达率无关。虽然天然IFN-ω1被证明带有复合型寡糖[阿道夫，G.R.，毛雷尔-福吉，I.，卡尔纳，I. & 坎特尔，K.（1990年）《生物化学杂志》265卷，9290 - 9295页]，但通过凝集素印迹、质谱分析和高效液相色谱分析表明，昆虫细胞产生的蛋白质仅含有核心寡糖。鉴定出了两种不同的结构，即（甘露糖）2（N-乙酰葡糖胺）2[岩藻糖]和（甘露糖）3（N-乙酰葡糖胺）2[岩藻糖]。岩藻糖基化被鉴定为与核心糖以（α1 - 6）键相连。明显不存在唾液酸残基。在草地贪夜蛾细胞中表达的IFN-ω1在C末端被部分截短了9个残基；其糖基化后的抗病毒活性显著低于仙台病毒刺激的白细胞产生的IFN-ω1的活性。圆二色性和荧光光谱分析未揭示糖基化和非糖基化的IFN-ω1之间存在任何结构差异。这意味着复合型糖基化对于人IFN-ω1的最大生物学活性很重要。
