title: The carboxyl-terminal region is NOT essential for secreted and functional levels of coagulation factor X.
abstract: BACKGROUND
  The homologous coagulation factor X (FX), VII (FVII), IX (FIX) and protein C (PC) display striking differences in the carboxyl-terminus, with that of FX being the most extended. This region is essential for FVII, FIX and PC secretion.
  
  
  OBJECTIVE
  To provide experimental evidence for the role of the FX carboxyl-terminus.
  
  
  METHODS
  Recombinant FX (rFX) variants were expressed in multiple eukaryotic cell systems. Protein and activity levels were evaluated by ELISA, coagulant and amidolytic assays.
  
  
  CONCLUSIONS
  Expression of a panel of progressively truncated rFX variants in HEK293 cells revealed that the deletion of up to 21 residues in the carboxyl-terminus did not significantly affect secreted protein levels, as confirmed in HepG2 and BHK21 cells. In contrast, chimeric rFX-FVII variants with swapped terminal residues showed severely reduced levels. The truncated rFX variants revealed normal amidolytic activity, suggesting an intact active site. Intriguingly, these variants, which included that resembling the activated FXβ form once cleaved, also displayed remarkable or normal pro-coagulant capacity in PT- and aPTT-based assays. This supports the hypothesis that subjects with nonsense mutations in the FX carboxyl-terminus, so far never identified, would be asymptomatic.
  
  
  CONCLUSIONS
  For the first time we demonstrate that the FX carboxyl-terminal region downstream of residue K467 is not essential for secretion and provides a modest contribution to pro-coagulant properties. These findings, which might suggest an involvement of the carboxyl-terminal region in the divergence of the homologous FX, FVII, FIX and PC, help to interpret the mutational pattern of FX deficiency.
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.|LTTA Centre, University of Ferrara, Ferrara, Italy.
authors.forename: A
authors.identifier: http://orcid.org/0000-0002-6113-2694
authors.initials: A
authors.lastname: Branchini
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.|LTTA Centre, University of Ferrara, Ferrara, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Baroni
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Burini
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Puzzo
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Nicolosi
authors.affiliation: Centre for Haemostasis and Thrombosis, Haematology Section, Department of Medical Sciences, University of Ferrara, Ferrara, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Mari
authors.affiliation: Centre for Haemostasis and Thrombosis, Haematology Section, Department of Medical Sciences, University of Ferrara, Ferrara, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Gemmati
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.|LTTA Centre, University of Ferrara, Ferrara, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Bernardi
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.|LTTA Centre, University of Ferrara, Ferrara, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Pinotti
mesh_terms: D000818:Animals; D001777:Blood Coagulation; D006224:Cricetinae; D005170:Factor X / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010314:Partial Thromboplastin Time; D017434:Protein Structure, Tertiary; D011517:Prothrombin Time; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D005170:Factor X
keywords: blood coagulation factors; factor X; mutagenesis; recombinant proteins; secretion; sequence deletion