N-acetyl-beta-glucosaminidase accounts for differences in glycosylation of influenza virus hemagglutinin expressed in insect cells from a baculovirus vector.
The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (Sf9) cells and in Estigmene acrea cells by using a baculovirus vector. Structural analysis revealed that the endo-H-resistant N-glycans of HA from Sf9 cells were predominantly trimannosyl core oligosaccharides, whereas in E. acrea cells most of these cores were elongated by at least one terminal N-acetylglucosamine residue. To understand the difference in carbohydrate structures, enzymes involved in N-glycan processing have been analyzed. The results revealed that the different glycosylation patterns observed are due to an N-acetyl-beta-glucosaminidase activity that was found in Sf9 cells but not in E. acrea cells. This enzyme specifically used the GlcNAcMan(3)GlcNAc(2) oligosaccharide as a substrate. When N-acetyl-beta-glucosaminidase or alpha-mannosidase II was inhibited by specific inhibitors, the amount of terminal N-acetylglucosamine in hemagglutinin from Sf9 cells was significantly enhanced. These results demonstrate that N glycosylation in both cell lines follows the classical pathway up to the stage of GlcNAcMan(3)GlcNAc(2) oligosaccharide side chains. Whereas these structures are the end product in E. acrea cells, they are degraded in Sf9 cells to Man(3)GlcNAc(2) cores by N-acetyl-beta-glucosaminidase.
Wagner
R
R
Institut für Virologie, Philipps-Universität, Marburg, Germany.
Geyer
H
H
Geyer
R
R
Klenk
H D
HD
D000118:Acetylglucosaminidase / Q000502:physiology*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002240:Carbohydrate Sequence; D005822:Genetic Vectors; D006031:Glycosylation; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000737:chemistry* / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D018411:Spodoptera; D013379:Substrate Specificity
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D011994:Recombinant Proteins; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase