The Propeptides of the Vitamin K-dependent Proteins Possess Different Affinities for the Vitamin K-dependent Carboxylase

Abstract

The vitamin K-dependent ␥-glutamyl carboxylase catalyzes the modification of specific glutamates in a number of proteins required for blood coagulation and associated with bone and calcium homeostasis. All known vitamin K-dependent proteins possess a conserved eighteen-amino acid propeptide sequence that is the primary binding site for the carboxylase. We compared the relative affinities of synthetic propeptides of nine human vitamin K-dependent proteins by determining the inhibition constants (K i ) toward a factor IX propeptide/ ␥-carboxyglutamic acid domain substrate. The K i values for six of the propeptides (factor X, matrix Gla protein, factor VII, factor IX, PRGP1, and protein S) were between 2-35 nM, with the factor X propeptide having the tightest affinity. In contrast, the inhibition constants for the propeptides of prothrombin and protein C are ϳ100-fold weaker than the factor X propeptide. The propeptide of bone Gla protein demonstrates severely impaired carboxylase binding with an inhibition constant of at least 200,000-fold weaker than the factor X propeptide. This study demonstrates that the affinities of the propeptides of the vitamin K-dependent proteins vary over a considerable range; this may have important physiological consequences in the levels of vitamin Kdependent proteins and the biochemical mechanism by which these substrates are modified by the carboxylase.The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamates to ␥-carboxyglutamate (Gla) 1 in a number of proteins. Most vitamin K-dependent proteins are involved in the hemostatic process (prothrombin, factors VII, IX, and X, and proteins C, S, and Z), whereas two others (bone Gla protein and matrix Gla protein) are associated with bone (1-4). Two new putative vitamin K-dependent proteins of unassigned function, proline-rich Gla proteins (PRGP1 and PRGP2), were identified by sequence homology searches and are believed to be membrane proteins (5).A conserved eighteen-amino acid sequence essential for substrate recognition is found in all vitamin K-dependent proteins and was first identified by Pan and Price (6) based on sequence comparisons of the blood and bone vitamin K-dependent proteins. The conserved region is present as a propeptide sequence amino-terminal to the highly conserved Gla domains of the vitamin K-dependent blood proteins and is proteolytically removed to form the mature protein. With bone Gla protein, this sequence is also present as a propeptide amino-terminal to the mature form of the protein, whereas with matrix Gla protein the vitamin K-dependent propeptide-like sequence is part of the mature form of the protein (7). Confirmation of the importance of the propeptide sequence in carboxylation is demonstrated by experiments where deletion of the propeptide abrogates carboxylation of factor IX or protein C expressed in cell culture (8, 9). In addition, mutagenesis studies have identified a number of highly conserved amino acids (e.g. Phe Ϫ16, Ala Ϫ10, Leu Ϫ6) as well as les...

Identification of the N-Linked Glycosylation Sites of Vitamin K-Dependent Carboxylase and Effect of Glycosylation on Carboxylase Function

“…Wild-type human GGCX cDNA with a HPC4 tag (EDQVDPRLIDGK) at the carboxyl terminus (29) was engineered into the baculovirus expression vector pFastBac1 by BamH I and EcoR I sites. The resulting plasmid, pFastBac1-GGCX-HPC4, was transformed into E. coli.…”

“…Expression of GGCX was performed by infection of 2 × 10 6 /mL Sf9 cells with the recombinant virus at a multiplicity of infection of 1. Cells were collected after 48 h of infection, and the expressed GGCX was purified by affinity chromatography using anti-HPC4 antibody-coupled Sepharose resin as previously described (29).…”

Transmembrane Domain Interactions and Residue Proline 378 Are Essential for Proper Structure, Especially Disulfide Bond Formation, in the Human Vitamin K-Dependent γ-Glutamyl Carboxylase

“…In these experiments we used 25 mM MOPS (pH 6.8) in buffer A instead of 25 mM Tris-HCl (pH 7.4), since NEM reacts more specifically with cysteines at the lower pH. Excess unreacted NEM was removed by washing the microsome pellet twice with 60 mL of ice-cold buffer A. Solubilization of the microsomal pellet and the subsequent purification of carboxylase using the C-terminal peptide antibody affinity resin were performed as described (26).…”

“…Carboxylation activity was determined by the incorporation of 14 CO 2 into the pentapeptide substrate FLEEL in the presence of propeptide as described (26). The concentration of active carboxylase was determined from the fraction of protein binding to the fluorescein-labeled consensus propeptide by fluorescence anisotropy as described previously (16).…”

The Elusive Role of the Potential Factor X Cation-binding Exosite-1 in Substrate and Inhibitor Interactions

“…First, to facilitate purification of the expressed material, a sequence coding for the 9E10 epitope at the C-terminal end of the FX coding region was replaced with the sequence DQVDPRLIDGK, recognized by monoclonal antibody HPC-4 (Roche Applied Science, Meylan, France). Second, to improve ␥-carboxylation, the sequence coding for the propeptide of FX was replaced in a three-step mutagenesis procedure with the homologous sequence in human prothrombin (20,21). Following these modifications, the vector was used as a template to prepare vectors for variants E36Q/E37Q/E39Q, E36K/E37K/E39K, E74Q/E76Q/E77Q, and E74K/E76K/E77K of FX (Table I).…”