Intracellular evaluation of ER targeting elucidates a mild form of inherited coagulation deficiency

Missense mutations reduce protein levels through several molecular mechanisms. Among them, altered targeting to endoplasmic reticulum (ER) and its relationship with clinical phenotypes in patients have been poorly investigated. To address this point, we studied the prepeptide mutations (L-48P, L-42P) associated with mild deficiency of factor VII (FVII), the serine-protease triggering blood coagulation. Mutations were introduced into the native FVII to evaluate secreted and intracellular protein levels, and into a chimeric FVII-GFP to study ER targeting in living cells. In conditioned medium from stably or transiently transfected cells, expression levels of the -48PFVII (9% and 55%, respectively) and particularly those of the -42PFVII (2% and 12%) were decreased compared with those of WtFVII, indicating the causative nature of mutations. Markedly reduced protein levels were observed in cell organelles for -48PFVII (10.5 +/- 4.9 ng/mL; Wt-FVII, 130 +/- 43.4 ng/mL) and -42PFVII (approximately 5 ng/mL), thus suggesting impaired ER targeting. Fluorescence of the -48PFVII-GFP and -42PFVII-GFP was diffuse, covered the nucleus, and declined upon plasma membrane permeabilization with digitonin, which demonstrated mislocalization of variants in the cytosol. Noticeably, the residual fluorescence of -48PFVII-GFP (10%) and -42PFVII-GFP (20%) in organelles was fairly compatible with FVII levels in patients' plasma. The studies with the native and chimeric proteins indicated that both prepeptide mutations were associated with residual expression of normal FVII, which explained the mild form of FVII deficiency in patients. This approach, extendable to other coagulation serine proteases, clearly contributed to elucidate the relationship of genotype with plasma and clinical phenotype