GLYCOFORM HETEROGENEITY OF PORCINE INTERFERON-GAMMA EXPRESSED IN SF9 CELLS

Porcine interferon-gamma (SfPoIFN-gamma) was expressed with high effic iency in Spodoptera frugiperda (Sf9) cells by means of the baculovirus expression system. Up to 10(5) U/ml of antivirally active SfPoIFN-gam ma could be tracked down in the culture medium at 64 h postinfection. Three proteins (17, 19, and 21 kDa), which under nondenaturing conditi ons primarily exist as mutual-dimeric combinations, were purified by i mmunoaffinity chromatography. Carbohydrate labeling and kinetic deglyc osylation studies suggested that the 19- and 21-kDa proteins are N-gly cosylated variants of a single 17-kDa protein carrying no N-linked sug ars, in which one respectively two N-glycosylation sequons are occupie d by glycans of 2 kDa. Both the quantitative recovery of SfPoIFN-gamma from a Con A column at 0.2 M methyt-alpha-mannopyranoside and the res ults of lectin blots, revealing strong affinity of the 19- and 21-kDa species for Galanthus nivalis agglutinin, support the presence of N-gl ycosidically linked high mannose-type chains in the carbohydrate moiet y 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 agglutinin s specifically recognize sialic acid linked alpha(2-6) and alpha(2-3), respectively, to penultimate galactose residues. Their affinity for t he larger glycoforms of PoIFN-gamma suggests that the biosynthetic pat hways in Sf9 cells are able to modify oligomannose structures to compl ex or hybrid glycans.