All post-translational modifications except propeptide cleavage are required for optimal secretion of coagulation factor VII.
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.
Bolt
Gert
G
Mammalian Cell Technology, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark. bolt@novonordisk.com
Steenstrup
Thomas D
TD
Kristensen
Claus
C
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005167:Factor VII / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D009154:Mutation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D014813:Vitamin K Deficiency; D014859:Warfarin / Q000494:pharmacology
D014859:Warfarin; D005167:Factor VII