REGULATION & BIOSYNTHESIS OF VITAMIN K-DEPENDENT FACTORS

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The overall aim of this project is to achieve an enhanced understanding of the elements involved in transcription and post-translational processing of the vitamin K-dependent clotting factors. Using human Factor X as a paradigm for study, experiments proposed here will (1) determine cis and trans regulatory elements required for the expression of Factor X. Using footprinting assays, gel shift, and mutagenesis followed by transfection assays with reporter genes, the location of functional transcription factor (TF) binding sites within the F.X promoter will be determined. Binding sites for several liver-specific TF's (HNF-3 and C/EBP) will be sought and co-transfection assays used to determine the effects of these TF's on the F.X promoter. These findings will be used to explore two other phenomena, the pathophysiology of hemophilia B Leyden, and the pattern of clotting factor expression in HepG2 cells. (2) We shall explore the function of the signal sequences in these proteins. The signal sequences of secreted proteins function to target these proteins to the endoplasmic reticulum. We have described a Factor X variant (Factor X Santo Domingo) in which a mutation in the signal sequence results in severe Factor X deficiency. We propose to carry out further experiments to determine the intracellular fate of this mutant protein. In the course of characterizing this variant, we have established for the first time the signal peptidase (SP) cleavage site in F.X. We propose to determine the SP cleavage sites for Factors VII and II as well. This will, as a consequence, define the start sites of the propeptides, which are required for the posttranslational carboxylation of the vitamin K-dependent proteins. (3) We shall determine the role of specific Gla residues in Factor X through the study of naturally occurring and recombinant Factor X variants with mutations in the Gla domain. Using site directed mutagenesis, Gla(16) to Asp and Gla(20) to Asp variants will be constructed. These, along with a naturally occurring Gla(26) to Asp variant, will be characterized by physicochemical means and by coagulation assays, to define the roles of specific residues in maintaining conformation and effecting phospholipid binding.