The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engineering of amino acid -4.
Homologous "propeptide" regions are present in a family of vitamin K-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 gamma-carboxylation of the adjacent gamma-carboxy region, we have mutated amino acid -4 of human factor IX from an arginine 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 gamma-carboxylated compared to the control, wild-type, recombinant factor IX. This result supports the hypothesis that the propeptide region is required for efficient gamma-carboxylation of factor IX in dog kidney cells. Furthermore, it confirms previous results that arginine at amino acid -4 is required for correct propeptide processing.
Galeffi
P
P
Sir William Dunn School of Pathology, University of Oxford, UK.
Brownlee
G G
GG
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D019735:Carbon-Carbon Ligases; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005164:Factor IX / Q000032:analysis / Q000235:genetics*; D005973:Glutamine; D006801:Humans; D008025:Ligases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals / Q000378:metabolism*
D021382:Protein Sorting Signals; D005973:Glutamine; D005164:Factor IX; D001120:Arginine; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase