ALTERATION OF PROPTIDE SEQUENCE IMPAIRS BIOLOGICAL ACTIVITY OF HUMAN FACTOR VII

Abstract

We have investigated the effect of altering the leader sequence of human factor VII on its biological activity. Factor VII is a vitamin K-dependent blood coagulation protein whose activity depends on the presence of gamma-carboxyglutamic acid (gla) residues in its amino terminal region. Since factor VII and other vitamin K-dependent proteins exhibit structural homology in the propeptide, it has been suggested that the propeptide is involved in gamma-carboxylation. Recently, two factor IX patients were identified with point mutations which prevented the processing of the propeptide and generated a factor IX with greatly reduced biological activity (Diuguid et al., PNAS 83; 5803; Bentley et al., Cell 45: 343). To examine this question using recombinant DNA technology, we altered the sequence of the factor VII propeptide by in vitro mutagenesis of the factor VII cDNA and then expressed the altered genes in baby hamster kidney (BHK) cells. For the 60 and 38 aa leader forms of factor VII, the arg (R) at -1 was changed to ser (S), yielding the sequence HRRRS before the +1 ala. In addition, for the 60 aa leader form, a ser was inserted after the arg at -1, resulting in the sequence HRRRRS before the +1 ala. As determined by ELISA, the mutant proteins were synthesized and secreted by BHK cells at levels comparable to the wild-type forms of factor VII. Analysis by radioimmune precipitation and SDS-PAGE indicated that substitution of arg by ser at -1 prohibits processing of the factor VII propeptide, whereas, insertion of a ser after the four arg's does not. However, all three proteins have reduced biological activity by approximately 5-fold when compared to the wild-type forms with the one-stage clotting assay. All three proteins are also quantitatively precipitated by Ba citrate, indicating they are at least partially gamma-carboxylated. These results suggest that the correct sequence of the propeptide, not just cleavage of the propeptide, is necessary for generating a biologically active molecule. The effect of these sequence alterations on gamma-carboxylation will be evaluated further by analysis of the amino acid sequence and composition of the mutant proteins.