INTRACELLULAR EVALUATION OF ER TARGETING ELUCIDATES A MILD FORM OF INHERITED COAGULATION DEFICIENCY
Abstract number: O-M-021
Rizzotto1 L., Pinotti1 M., Pinton2 P., Rizzuto2 R., Bernardi1 F.
11Biochemistry and Molecular Biology 22Department of Experimental and Diagnostic Medicine, Section of General Pathology, Telethon Center fo, University of Ferrara, Ferrara, Italy
How-to-cite Rizzotto L, Pinotti M, Pinton P, Rizzuto R, Bernardi F. INTRACELLULAR EVALUATION OF ER TARGETING ELUCIDATES A MILD FORM OF INHERITED COAGULATION DEFICIENCY. J Thromb Haemost 2007; 5 Supplement 2: O-M-021
Abstract
Introduction: Missense mutations reduce protein levels through several mechanisms. Among them altered targeting to endoplasmic reticulum (ER), and its relationship with patient phenotypes, are poorly known. We studied the mutations associated with mild coagulation factor VII (FVII) deficiency and occurring at crucial positions of the prepropeptide, the L-48P (hydrophobic core), L-42P (position –3 preceding the signal-peptidase cleavage site) and V-39I (signal-peptidase cleavage site).
Methods: We expressed in BHK cells the variants of i) FVII to evaluate secreted and intracellular protein levels, and ii) chimaeric FVII-GFP to study ER targeting in living cells. mRNA splicing was studied by RT-PCR upon expression of FVII minigenes.
Results: Expression levels of the –48PFVII (9%) and -42PFVII (2%) in media were decreased. Markedly reduced protein levels were found in cell organelles for -48PFVII (10.54.9 ng/ml; WtFVII, 13043.4 ng/ml) and -42PFVII (~5 ng/ml), to suggest impaired ER targeting.
In the FVII-GFP fluorescent model, the WtFVII-GFP showed a bright fluorescence that was maintained after plasma membrane permeabilization with digitonin, thus confirming correct ER targeting. Fluorescence of the –48PFVII-GFP and –42PFVII-GFP declined upon digitonin treatment, to demonstrate their mislocalization in the cytosol. Noticeably, a residual fluorescence of –48PFVII-GFP (10%) and –42PFVII-GFP (20%) in organelles was observed.
The V-39I did not affect levels of secreted FVII or ER targeting. Studies at the mRNA level also failed to show a detrimental effect for this mutation, occurring at the donor splice site of intron 1A. These findings, at least in our system, suggest caution in conferring a causative role to mutations in the absence of experimental insights.
Conclusions: The study with the native and chimaeric proteins, extendable to other coagulation proteases, indicated that the L-48P and L-42P mutations were associated to residual expression of FVII, which explained the mild form of FVII deficiency in patients.
