Abstract
Factor VII (FVII) is a key serine protease in blood coagulation. N-glycosylation in FVII has been shown to be critical for protein secretion. To date, however, the underlying biochemical mechanism remains unclear. Recently, we found that N-glycans in the transmembrane serine protease corin are critical for calnexin-assisted protein folding and extracellular expression. In this study, we tested the hypothesis that N-glycans in the FVII protease domain mediate calnexin-assisted protein folding and that naturally occurring F7 mutations abolishing N-glycosylation impair FVII secretion. We expressed human FVII wild-type (WT) and mutant proteins lacking one or both N-glycosylation sites in HEK293 and HepG2 cells in the presence or absence of a glucosidase inhibitor. FVII expression, secretion and binding to endoplasmic reticulum chaperones were examined by immune staining, co-immunoprecipitation, Western blotting, and ELISA. We found that N-glycosylation at N360 in the protease domain, but not N183 in the pro-peptide domain, of human FVII is required for protein secretion. Elimination of N-glycosylation at N360 impaired calnexin-assisted FVII folding and secretion. Similar results were observed in WT FVII when N-glycan-calnexin interaction was blocked by glucosidase inhibition. Naturally occurring F7 mutations abolishing N-glycosylation at N360 reduced FVII secretion in HEK293 and HepG2 cells. These results indicate that N-glycans in the FVII protease domain mediate calnexin-assisted protein folding and subsequent extracellular expression. Naturally occurring F7 mutations abolishing N-glycosylation in FVII may impair this mechanism, thereby reducing FVII levels in patients.