Glycoform heterogeneity of porcine interferon-gamma expressed in Sf9 cells.
Porcine interferon-gamma (SfPoIFN-gamma) was expressed with high efficiency in Spodoptera frugiperda (Sf9) cells by means of the baculovirus expression system. Up to 10(5) U/ml of antivirally active SfPoIFN-gamma could be tracked down in the culture medium at 64 h postinfection. Three proteins (17, 19, and 21 kDa), which under nondenaturing conditions primarily exist as mutual-dimeric combinations, were purified by immunoaffinity chromatography. Carbohydrate labeling and kinetic deglycosylation studies suggested that the 19- and 21-kDa proteins are N-glycosylated variants of a single 17-kDa protein carrying no N-linked sugars, in which one respectively two N-glycosylation sequons are occupied by glycans of 2 kDa. Both the quantitative recovery of SfPoIFN-gamma from a Con A column at 0.2 M methyl-alpha-mannopyranoside and the results of lectin blots, revealing strong affinity of the 19- and 21-kDa species for Galanthus nivalis agglutinin, support the presence of N-glycosidically linked high mannose-type chains in the carbohydrate moiety of SfPoIFN-gamma. Intriguingly, both 19- and 21-kDa glycoforms, but not their sialidase-treated derivatives, showed clear reactivity with the Sambucus nigra and Maackia amurensis agglutinins. These agglutinins specifically recognize sialic acid linked alpha(2-6) and alpha(2-3), respectively, to penultimate galactose residues. Their affinity for the larger glycoforms of PoIFN-gamma suggests that the biosynthetic pathways in Sf9 cells are able to modify oligomannose structures to complex or hybrid glycans.
Vandenbroeck
K
K
Rega Institute for Medical Research, Leuven, Belgium.
Willems
L
L
Billiau
A
A
Opdenakker
G
G
Huybrechts
R
R
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003470:Culture Media; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D027801:Galanthus; D005822:Genetic Vectors; D006031:Glycosylation; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008715:Methionine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins; D018411:Spodoptera; D013462:Sulfur Radioisotopes; D013552:Swine; D013997:Time Factors; D014162:Transfection
D003470:Culture Media; D017931:DNA Primers; D011134:Polysaccharides; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D007371:Interferon-gamma; D008715:Methionine