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Case Reports
. 2013 Oct 1;587(19):3249-53.
doi: 10.1016/j.febslet.2013.08.019. Epub 2013 Aug 27.

Replacement of the Y450 (c234) phenyl ring in the carboxyl-terminal region of coagulation factor IX causes pleiotropic effects on secretion and enzyme activity

Alessio Branchini  1 , Matteo Campioni, Maria Gabriella Mazzucconi, Francesca Biondo, Rosella Mari, Maria Patrizia Bicocchi, Francesco Bernardi, Mirko Pinotti
Affiliations
Case Reports

Replacement of the Y450 (c234) phenyl ring in the carboxyl-terminal region of coagulation factor IX causes pleiotropic effects on secretion and enzyme activity

Alessio Branchini et al. FEBS Lett. .

Abstract

The interplay between impaired protein biosynthesis and/or function caused by missense mutations, particularly in relation to specific protein regions, has been poorly investigated. As model we chose the severe p.Y450C mutation in the carboxyl-terminal region of coagulation factor IX (FIX) and, by expression of a panel of recombinant variants, demonstrated the key role of the tyrosine phenyl group for both FIX secretion and coagulant activity. Comparison among highly homologous coagulation serine proteases indicate that additive or compensatory pleiotropic effects on secretion and function by carboxyl-terminal mutations produce life-threatening or mild phenotypes in the presence of similarly reduced protein amounts.

Keywords: Carboxyl-terminal region; Coagulation factor IX; Dysfunctional enzyme; Gene expression; Impaired secretion; Missense mutations.

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Figures

Fig. 1
Fig. 1
Recombinant FIX variants and sequence alignment of highly homologous coagulation factors. Schematic representation of the FIX structure with the natural (in bold) and artificial amino acid substitutions reported on the top. LC, light chain; HC, heavy chain. The box reports the sequence alignment of the carboxyl-terminal region of FIX, FVII, PC and FX and the numbers indicate the protein residues including the pre-propeptide sequence. The arrow indicates the position (c234, chymotrypsin numbering) of the FIX mutation under study.
Fig. 2
Fig. 2
Expression levels of the recombinant FIX variants (A) Protein (white bars) and coagulant activity (grey bars) levels of secreted rFIX variants. Results are expressed as the percentage of rFIX-wt, and are reported as mean ± standard deviation from three independent experiments. Inset. Western Blotting analysis in non-reducing conditions of rFIX variants in cell lysates. MW, molecular weight marker. (B) Secreted levels of the rFIX-450C (■), rFIX-450S (□), rFIX-Y450F (○) and rFIX-wt (●) variants in time-course experiments. Conditioned media were collected at 6, 18, 24, 30 and 48 h after transfection. Protein levels are expressed as the log10 of concentration.
Fig. 3
Fig. 3
Relationship between secreted protein levels and specific activity of FIX, FVII and PC carboxyl-terminal variants. The specific activity was calculated as the ratio between activity and protein levels. The wild-type specific activity (referred as 1.00) is indicated by the dotted line. (A) FIX variants are: rFIX-wt (●), rFIX-450C (■), rFIX-450S (□), rFIX-450F (▾) and rFIX-450H variant (▴) ; °, rFIX-450P variant (not detectable, [11]). The rFIX-450C and rFIX-450S are superposed. The chemical structure of the side chains at position 450 is displayed aside. (B) FVII variants are: rFVII-466X (□), rFIX-465X (○) rFVII-464X (♢),rFVII-463X () rFVII-462A (○), rFVII-462 W (△), rFVII-462Q (∇). ∗, gain-of-function R462X variant . PC variants are: rPC-459X (■), rPC-456X (▾), rPC-453X (▴) and rPC-452X (●). †, further deletions of the PC carboxyl-terminus resulted in undetectable secreted PC protein levels.
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