title: Protein engineering of the propeptide of human factor IX.
abstract: Vitamin-K-dependent plasma proteins contain a highly conserved propeptide sequence located between the classical hydrophobic leader sequence and the N-terminus of the mature protein. This acts as a recognition sequence for the vitamin-K-dependent carboxylase which catalyses the conversion of specific glutamate residues to gamma-carboxyglutamate (Gla) residues in the adjacent Gla domain. Protein engineering of the 18 residue propeptide from human factor IX has highlighted the importance of residues -16Phe and -10Ala with respect to carboxylase recognition. In addition, studies of haemophilia B patients have shown that C-terminal propeptide residues -4Arg and -1Arg are required for proteolysis of the propeptide from the mature protein. To extend these previous studies we have introduced two novel mutations into the propeptide of human factor IX at positions -17(Val----Asp) and -6(Leu----AsP), and studied the effect of these changes on gamma-carboxylation and proteolytic processing. Both mutations reduce the expression of a calcium-dependent epitope in the Gla domain; however, only -6Leu----Asp shows reduced binding to barium sulphate. In addition, this latter mutation prevents proteolytic processing of the propeptide. These data support the current hypothesis that the propeptide contains two recognition elements: one for carboxylase recognition located towards the N-terminus, and one for propeptidase recognition located near the C-terminus.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, UK.
authors.forename: P A
authors.initials: PA
authors.lastname: Handford
authors.forename: P R
authors.initials: PR
authors.lastname: Winship
authors.forename: G G
authors.initials: GG
authors.lastname: Brownlee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D004285:Dogs; D005164:Factor IX / Q000235:genetics*; D002836:Hemophilia B; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
chemical_list: D000911:Antibodies, Monoclonal; D011498:Protein Precursors; D011994:Recombinant Proteins; C049838:factor IX Cambridge; D005164:Factor IX