All post-translational modifications except propeptide cleavage are required for optimal secretion of coagulation factor VII

Gert Bolt; Thomas D. Steenstrup; Claus Kristensen

doi:10.1160/TH07-05-0332

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2007; 98(05): 988-997
DOI: 10.1160/TH07-05-0332

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

All post-translational modifications except propeptide cleavage are required for optimal secretion of coagulation factor VII

Authors

Gert Bolt

¹Mammalian Cell Technology, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark
Thomas D. Steenstrup

¹Mammalian Cell Technology, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark
Claus Kristensen

¹Mammalian Cell Technology, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark

Abstract

Summary

Human coagulation factor VII (FVII) has two N-glycosylation sites (N145 and N322) and two O-glycosylation sites (S52 and S60). In transiently transfected COS-7 cells, all combinations of N- and O-glycosylation knock-out mutations reduced the release of FVII to the medium. Pulse-chase analysis of CHO-K1 cell lines expressing recombinant FVII demonstrated that virtually all wild-type FVII synthesized was secreted from the cells, whereas both N- and O- glycosylation knock-out mutations induced partial intracellular degradation of the synthesized FVII. Likewise, two thirds of the FVII synthesized in vitamin K-depleted and warfarin-treated CHO cells was degraded intracellularly, demonstrating the importance of gamma-carboxylation for the secretion of FVII. The furin inhibitor decanoyl-R-V-K-R-chloromethylketone inhibited propeptide cleavage, but FVII with propeptide appeared to be secreted equally well as FVII without propeptide. Propeptide cleavage was not inhibited by vitamin K depletion and warfarin treatment, suggesting that for FVII, correct gamma-carboxylation is not required for optimal processing of the propeptide. In conclusion, all post-translational modifications of FVII except propeptide cleavage were important for complete secretion of the synthesized FVII and to avoid intracellular degradation. Thus, the extensive post-translational modification of FVII seems critical for the intracellular stability of the protein and is required for keeping the protein in the secretory pathway.

Keywords

Factor VII - protein processing - post-translational - glycosylation - gamma-carboxylation

Full Text

References

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