the relationship of secretion and activity of recombinant factor ix with n-glycosylation

Background: human coagulation factor ix (hfix) is a glycoprotein with two n-glycosylation sites at the activation peptide. since the activation peptide is removed in mature hfix, the exact role of n-glycosylation is unclear. to investigate the role of n-glycosylation in the secretion and activity of hfix, we inhibited n-glycosylation by tunicamycin in the stable human embryonic kidney (hek)-coagulation factor ix (fix) cells. materials and methods: after the treatment of stable fix-expressing hek cells in the presence or absence of tunicamycin, the expression and activity of the recombinant fix (rfix) were determined in culture medium and cell lysate with enzyme-linked immunosorbent assay and clotting test, respectively. results: based on the data analysis, total concentrations of fix in stable hek-fix was the same in the media with and without tunicamycin. but throughout the post-induction period, the intracellular and secreted levels of fix in tunicamycin-treated hek-fix cells increased and decreased, respectively, compared with those of control hek-fix cells, though the results were not significant. these results indicate that disrupting the synthetic process may slightly reduce the fix levels secreted in hek-fix cells. conclusion: although glycosylation plays a vital role in the folding and secretion of the proteins, it does not affect the secretion of fix. besides, the n-glycosylation of the produced fix failed to play a significant role in its activity.