Background The homologous coagulation factor X (FX), VII (FVII), IX (FIX) and protein C (PC) display striking differences in the carboxyl-terminus, which varies both in length and amino acid composition. This region represents a key determinant for the efficient secretion of FVII, FIX and PC, as demonstrated by nonsense, missense as well as frameshifted variants, either natural or artificial. So far, very little is known about FX, which is charactrized by the most extended carboxyl-terminal region. Our aim was to provide experimental evidence on the role of FX carboxyl-terminus on protein secretion and function by a deletion scanning approach. Methods A panel of recombinant FX (rFX) variants was created by site-directed mutagenesis and expressed in multiple eukaryotic cell systems. Protein and activity levels were evaluated by ELISA, coagulant and amidolytic assays. Results An initial deletion scanning stemming from analysis of FX, FVII, FIX and PC primary sequences identified key residues in FX carboxyl-terminus mimicking the length of FVII (rFX-482X, FVII-like), PC (rFX-480X, PC-like) and FIX (rFX-470X, FIX-like), which is the shortest family member. In addition, five truncated proteins with intermediate length (rFX-484X, rFX-479X, rFX-478X, rFX-474X, and rFX-472X) were explored, together with truncated FX variants stemming from the sequential removal of single amino acids (rFX-470X, rFX-469X, and rFX-468X). Expression of this panel of progressively truncated (484X-468X) 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. However, a further one amino acid deletion from position 467 (rFX-467X) led to remarkable reduced secreted levels. These results were confirmed by expression studies 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, the transiently-expressed truncated variants, which included that resembling the activated FXbeta form once cleaved (rFX-470X), displayed remarkable or normal pro-coagulant capacity in PT- and aPTT-based assays, thus confirming the marginal impact of the carboxyl-terminus on functional properties. These observations are consistent with the virtual absence of patients with nonsense mutations affecting the carboxylterminus of FX as compared with those described in FVII, FIX and PC, mostly associated with deficiency. 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 467 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.

The carboxyl-terminal region is not essential for secreted and functional levels of coagulation factor X

BRANCHINI, Alessio;BARONI, Marcello;MARI, Rosella;GEMMATI, Donato;BERNARDI, Francesco;PINOTTI, Mirko
2016

Abstract

Background The homologous coagulation factor X (FX), VII (FVII), IX (FIX) and protein C (PC) display striking differences in the carboxyl-terminus, which varies both in length and amino acid composition. This region represents a key determinant for the efficient secretion of FVII, FIX and PC, as demonstrated by nonsense, missense as well as frameshifted variants, either natural or artificial. So far, very little is known about FX, which is charactrized by the most extended carboxyl-terminal region. Our aim was to provide experimental evidence on the role of FX carboxyl-terminus on protein secretion and function by a deletion scanning approach. Methods A panel of recombinant FX (rFX) variants was created by site-directed mutagenesis and expressed in multiple eukaryotic cell systems. Protein and activity levels were evaluated by ELISA, coagulant and amidolytic assays. Results An initial deletion scanning stemming from analysis of FX, FVII, FIX and PC primary sequences identified key residues in FX carboxyl-terminus mimicking the length of FVII (rFX-482X, FVII-like), PC (rFX-480X, PC-like) and FIX (rFX-470X, FIX-like), which is the shortest family member. In addition, five truncated proteins with intermediate length (rFX-484X, rFX-479X, rFX-478X, rFX-474X, and rFX-472X) were explored, together with truncated FX variants stemming from the sequential removal of single amino acids (rFX-470X, rFX-469X, and rFX-468X). Expression of this panel of progressively truncated (484X-468X) 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. However, a further one amino acid deletion from position 467 (rFX-467X) led to remarkable reduced secreted levels. These results were confirmed by expression studies 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, the transiently-expressed truncated variants, which included that resembling the activated FXbeta form once cleaved (rFX-470X), displayed remarkable or normal pro-coagulant capacity in PT- and aPTT-based assays, thus confirming the marginal impact of the carboxyl-terminus on functional properties. These observations are consistent with the virtual absence of patients with nonsense mutations affecting the carboxylterminus of FX as compared with those described in FVII, FIX and PC, mostly associated with deficiency. 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 467 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.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2364282
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