The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engineering of amino acid -4

Abstract

Homologous “propeptide” regiona are present in a family of vitamin κ-dependent mammalian proteins, including clotting factors II, VII, IX, X, protein C, protein S and bone “gla” proteins. To test the hypothesis that the propeptide is a signal for the correct γ-carboxylation of the adjacent γ-carboxy region, we have mutated amino acid -4 of human factor IX from an arginim to a glutamine residue, by M13-directed site-specific mutagenesis of a cDNA clone. After expression of mutant factor IX in dog kidney cells, we find that it is secreted into the medium in a precursor form containing the propeptide, and is inefficiently γ-carboxylated compared to the control, wild-type, recombinant factor IX. This result supports the hypothesis that the propeptide region is required for efficient γ-carboxylation of factor IX in dog kidney cells. Furthermore, it comfirms previous results that arginine at amino acid -4 is required for correct propeptide processing.