Expression of human interferon omega 1 in Sf9 cells. No evidence for complex-type N-linked glycosylation or sialylation.
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.
Voss
T
T
Ernst-Boehringer Institut für Arzneimittelforschung, Department of Protein Chemistry, Bender & Co., Vienna, Austria.
Ergülen
E
E
Ahorn
H
H
Kubelka
V
V
Sugiyama
K
K
Maurer-Fogy
I
I
Glössl
J
J
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007370:Interferon Type I / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009036:Moths; D019158:N-Acetylneuraminic Acid; D011487:Protein Conformation; D012794:Sialic Acids / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D002241:Carbohydrates; D017931:DNA Primers; D007370:Interferon Type I; D012794:Sialic Acids; C063743:interferon omega 1; D019158:N-Acetylneuraminic Acid