Abstract
Post-translational processing of Factor IX includes glycosylation, cleavage of the signal peptide and propeptide, vitamin K-dependent carboxylation of specific glutamic acid residues to form gamma-carboxyglutamic acid, and beta-hydroxylation of aspartic acid at residue 64 to form beta-hydroxyaspartic acid. The human Factor IX cDNA coding sequence was modified in the propeptide region (residue -18 to -1) using oligonucleotide-directed site-specific mutagenesis, and the altered Factor IX cDNA was expressed in Chinese hamster ovary cells. The effects of the mutations on proteolytic processing, gamma-carboxylation, and beta-hydroxylation were assessed by direct structural analysis. After purification, the molecular weight of each of the recombinant Factor IX species and its NH2-terminal amino acid sequence were shown to be identical to those of plasma Factor IX. gamma-Carboxyglutamic acid and beta-hydroxyaspartic acid analyses revealed that recombinant wild-type Factor IX contained 9.2 gamma-carboxyglutamic acid and 0.3 beta-hydroxyaspartic acid residues/molecule compared with 11.4 gamma-carboxyglutamic acid and 0.39 beta-hydroxyaspartic acid residues in plasma Factor IX. When the 18-residue propeptide was deleted or when the cells were grown in the presence of sodium warfarin, secreted Factor IX contained no detectable gamma-carboxyglutamic acid but 0.36 and 0.40 residues of beta-hydroxyaspartic acid, respectively. Point mutations leading to substitution of alanine for phenylalanine at residue -16 or glutamic acid for alanine at residue -10 contained 0.2 and 1.7 gamma-carboxyglutamic acid residues, respectively, and 0.2 residues of beta-hydroxyaspartic acid. These data confirm that the propeptide mutations made do not interfere with proteolytic processing and that the Factor IX propeptide contains a recognition site that designates the adjacent glutamic acid-rich domain for gamma-carboxylation. In contrast, beta-hydroxylation of aspartic acid 64 is an independent process which does not require vitamin K and is mediated through a hydroxylation recognition site in the mature Factor IX, not in the propeptide.
Highlights
From the Centerfor Hemostasis and Thrombosis Research, Division of Hematology-Oncology, Department of Medicine, New England Medical Center and Tufts UniversitySchool of Medicine, Boston, Massachusetts02111
Plasma Factor IX. y-Carboxyglutamic acid and 8-hy- (8).&Hydroxyaspartic acid is formed post-translationally by droxyaspartic acid analyses revealedthat recombinant the enzymatic hydroxylation of a single aspartic acid residue wild-type Factor IX contained 9.2 y-carboxyglutamic in Factor IX
When the 18-residue propeptide was deleted or when the cells were grown in the presence of sodium warfarin, secreted Factor IX contained no detectable ycarboxyglutamic acid but 0.36 and 0.40 residues of Bhydroxyaspartic acid, respectively
Summary
Mutagenesis and Expression of Factor IX cDNA-Construction of the Factor IX expression plasmid (pMT2-IX/wt) has been described (13). Bilized on Sepharose and used for the immunoaffinity purification of the recombinant Factor IX species secreted in the Protein Analysis--SDS'-gel electrophoresis was performed on 10- cell culture supernatants or plasma Factor IX, respectively. These results confirm our prior results in which these mutant Factor IX forms in cell culture supernatants were analyzed using Western blotting techniques (13) These data indicate the apparent homogeneity of the protein preparations and the apparent absence of a leader sequence (e.g. sensitivity of the system was about 10pmol for each derivatized signal peptide, propeptide) associatwedith the secreted Factor amino acid. Therewere no secondary acid and aspartic acid in a given sample and compared to the ratio sequences observed that corresponded to the NH2 terminus obtained for a plasma Factor IX sample hydrolyzed and derivatized of the signal peptide or the propeptide These results indiat thesame time, assuming 1mol of Factor IX to contain 0.39 mol of p-hydroxyaspartic acid.
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