Atherocalcin, a gamma-carboxyglutamic acid containing protein from atherosclerotic plaque.

The specialized calcium binding amino acid, γ-carboxyglutamic acid (Gla) is quantitated in developing atherosclerotic plaque relative to progression of the disease, and a Gla-containing protein isolated from calcified atherosclerotic plaque is partially characterized. Low levels of Gla are found in fatty streak and fibrous plaque lesions, and a marked increase in Gla content occurs in calcified plaque. A unique Gla-containing protein is purified from 0.5M EDTA (pH 8.0) extracts of calcified plaque, named atherocalcin. The protein containing 19 Gla residues/1000 amino acids is 80,000 molecular weight, with a pI of 4.16 – 4.3 and is uniquely different from other known Gla-containing proteins. The implications of this work for the further understanding of the pathogenesis and therapy of atherosclerosis are discussed.     

A single gamma-carboxyglutamic acid residue in a novel cysteine-rich secretory protein without propeptide

Gamma-glutamyl carboxylase catalyzes the modification of specific glutamyl residues to gamma-carboxyglutamyl (Gla) residues in precursor proteins that possess the appropriate gamma-carboxylation recognition signal within the propeptide region. We describe the immunopurification and first biochemical characterization of an invertebrate high molecular weight Gla-containing protein with homologues in mammals. The protein, named GlaCrisp, was isolated from the venom of the marine cone snail Conus marmoreus. GlaCrisp gave intense signals in Western blot experiments employing the Gla-specific antibody M3B, and the presence of Gla was chemically confirmed by amino acid analysis after alkaline hydrolysis. Characterization of a full-length cDNA clone encoding GlaCrisp deduced a precursor containing an N-terminal signal peptide but, unlike other Gla-containing proteins, no apparent propeptide. The predicted mature protein of 265 amino acid residues showed considerable sequence similarity to the widely distributed cysteine-rich secretory protein family and closest similarity (65% identity) to the recently described substrate-specific protease Tex31. In addition, two cDNA clones encoding the precursors of two isoforms of GlaCrisp were identified. The predicted precursor isoforms differed at three amino acid positions (-6, 9, and 25). Analysis by Edman degradation and nanoelectrospray ionization mass spectrometry, before and after methyl esterfication, identified a Gla residue at amino acid position 9 in GlaCrisp. This is the first example of a Gla-containing protein without an obvious gamma-carboxylation recognition site. The results define a new class of Gla proteins and support the notion that gamma-carboxylation of glutamyl residues is phylogenetically older than blood coagulation and the vertebrate lineage.     

Rapid assay for gamma-carboxyglutamic acid in urine and bone by precolumn derivatization and reversed-phase liquid chromatography

A reversed-phase high-performance liquid chromatographic assay for the analysis of gamma-carboxyglutamic acid (Gla) in urine and bone protein hydrolyzates is described. The method employs precolumn derivatization with o-phthalaldehyde and mercaptoethanol. Gla was quantified by reference to an internal standard (beta-carboxyaspartic acid). The "within-run" coefficient of variation of the assay for Gla in urine was between 2.1 and 3.4%, and that for bone protein hydrolyzates was 3.2%. The "between-run" coefficient of variation ranged from 4.1 to 5.5%. There was good agreement between the measurement of urinary Gla by high-performance liquid chromatography and amino acid analyzer. Free Gla could not be detected in serum.     

gamma-Carboxyglutamic acid content of hepatocellular carcinoma-associated des-gamma-carboxy prothrombin

Serum des-gamma-carboxy prothrombin (DCP) is a useful marker for the diagnosis of hepatocellular carcinoma (HCC), but the exact mechanism of its synthesis and its structural properties in liver diseases are unknown. DCP is measured by the monoclonal antibody MU-3. The purpose of this study was to examine the epitope of MU-3 and to characterize the differences in DCP between HCC and benign liver diseases. The epitope of MU-3 was examined by ELISA using prothrombin Gla domain polypeptides and was determined to be amino acid residues 17-27 of the prothrombin Gla domain, which has four gamma-carboxyglutamic acid residues (Gla) at positions 19, 20, 25 and 26. Peptides having a glutamic acid residue (Glu) at these positions reacted strongly to MU-3 but lost reactivity when Glu 19 or 20 was changed to Gla. In the order of gamma-carboxylation, MU-3 reacted strongly to DCP containing 0-1 Gla, weakly to 2-4 Gla and not at all to DCP containing more than five Gla. After adsorbing normal prothrombin with barium carbonate, DCP reaction to MU-3 was measured by determining the amount of DCP that was adsorbed by MU-3-coated beads. The proportion of DCP reacting to MU-3 in HCC was 41.0-76.8%, whereas in patients with benign liver diseases, only 0-42.1% reacted to MU-3. These results indicate that DCP variants preferentially synthesized in HCC have less than four Gla, which are restricted to positions 16, 25, 26 and 29, whereas DCP variants in benign liver diseases have more than five Gla.     

Direct identification of gamma-carboxyglutamic acid in the sequencing of vitamin K-dependent proteins.

We report the first direct method for the identification of the vitamin K-dependent Ca2+ binding amino acid, gamma-carboxyglutamic acid (Gla), in the sequencing of proteins. The carboxyl groups on the protein are first converted to methyl esters with methanolic HCl, a procedure that reduces the polarity of the resulting ATZ derivative of dimethyl-Gla and so greatly improves its extraction from the polybrene-treated glass fiber filter. After conversion to the PTH derivative in methanolic HCl, the resulting dimethyl ester of PTH Gla can be identified directly by a simple modification of the standard HPLC program for the separation of PTH derivatives. This methylation procedure can be used to identify Gla residues in proteins bound to PVDF membranes, as we demonstrate for matrix Gla protein and prothrombin, and to evaluate directly the degree of partial gamma-carboxylation at given glutamic acid residues, as we demonstrate for the 50% gamma-carboxylation of residue 17 in human bone Gla protein.     

Discovery of bone gamma-carboxyglutamic acid protein in mineralized scales. The abundance and structure of Lepomis macrochirus bone gamma-carboxyglutamic acid protein.

The mineralized scale of the freshwater sunfish Lepomis macrochirus (bluegill) contains a Gla protein. The protein was identified in extracts of scale by a new colorimetric assay for Gla-containing proteins. The protein was purified by gel filtration chromatography followed by reversed phase high performance liquid chromatography (HPLC). Several tests establish the identity of scale Gla protein and bone Gla protein (BGP). First, the proteins exhibit identical mobilities on electrophoresis and by reversed phase HPLC. Second, they have identical amino-terminal amino acid sequences. Finally, identical peptides are generated by proteolytic digestion. The 45-residue amino acid sequence of the bone Gla protein from L. macrochirus has a high sequence homology with swordfish, as well as homology to mammalian bone Gla protein. The BGP of bluegill shares with swordfish BGP a truncated NH2 terminus and an extended COOH terminus. These features may be unique to fish, as they have not been observed in terrestrial vertebrates. The bluegill BGP is the first vitamin K-dependent protein to contain a non-gamma-carboxylated residue to the NH2-terminal side of all of its Gla residues. In all other vitamin K-dependent proteins, Gla always appears to the NH2-terminal side of the first Glu. The implications of this result are discussed. The bluegill rib bone is curiously enriched in BGP, as are other mineralized tissues of this species. One hypothesis is that this may be due to the acellular nature of the bone in this species. The abundance of BGP in the bones of this fish may provide clues to the unknown function of this bone protein.     

An improved method for the determination of γ-carboxyglutamic acid in proteins,bone, and urine

A method of high-performance liquid chromatographic separation of the fluorescence derivative of γ-carboxyglutamic acid (Gla) is presented. Alkaline hydrolysates of protein samples were reacted with o-phthalaldehyde in the presence of ethanethiol for 2 min, and the fluorescence derivative of γ-carboxyglutamic acid was resolved from the other amino acids by a short column packed with silica-based anion exchanger under isocratic conditions. By this method, as low as 200 fmol of γ-carboxyglutamic acid can be quantitatively analyzed within 10 min. The method presented here shortened the analysis time for Gla and was at least 10 times more sensitive than the method we described previously (Anal. Biochem.117, 259–265, 1981). The application of this method to the formic acid-soluble or insoluble γ-carboxyglutamic acid-containing proteins in chicken bone and the concomitant increase of γ-carboxyglutamic acid content in chicken bone with age are reported.     

Matrix Gla protein, a new gamma-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone

A new protein has been isolated from CaCl2/urea extracts of demineralized bovine bone matrix. This protein has five to six residues of the vitamin K-dependent amino acid, gamma-carboxyglutamic acid (Gla), and we have accordingly designated it matrix Gla protein. Matrix Gla protein is a 15,000 dalton protein whose amino acid composition includes a single disulfide bond. The absence of 4-hydroxyproline in matrix Gla protein demonstrates that it is not a precursor to bone Gla protein, 5,800 dalton protein which has a residue of 4-hydroxyproline at position 9 in its sequence. Matrix Gla protein also does not cross-react with antibodies raised against bone Gla protein.     

Rapid assay for γ-carboxyglutamic acid in urine and bone by precolumn derivatization and reversed-phase liquid chromatography

A reversed-phase high-performance liquid chromatographic assay for the analysis of γ-carboxyglutamic acid (Gla) in urine and bone protein hydrolyzates is described. The method employs precolumn derivatization with o-phthalaldehyde and mercaptoethanol. Gla was quantified by reference to an internal standard (β-carboxyaspartic acid). The “within-run” coefficient of variation of the assay for Gla in urine was between 2.1 and 3.4%, and that for bone protein hydrolyzates was 3.2%. The “between-run” coefficient of variation ranged from 4.1 to 5.5%. There was good agreement between the measurement of urinary Gla by high-performance liquid chromatography and amino acid analyzer. Free Gla could not be detected in serum.     

A single amino acid replacement results in the Ca2+-induced self-assembly of a helical conantokin-based peptide

Conantokins are short (17-27 amino acid residues), gamma-carboxyglutamate (Gla)-rich peptide components of the venoms of marine snails of the genus Conus. They display high apo and/or Ca(2+)-induced helicity and act as potent and selective inhibitors of the N-methyl-d-aspartate receptor (NMDAR). We have previously established that one of the conantokins, conantokin-G (con-G), self-associates in the presence of Ca(2+) with high specificity for antiparallel chain orientation [Dai, Q., Prorok, M., and Castellino, F. J. (2004) J. Mol. Biol. 336, 731-744]. The dimerization appears to be driven by interhelical Ca(2+) coordination between the following residue pairings: Gla(3)-Gla(14)('), Gla(7)-Gla(10)('), Gla(10)-Gla(7)('), and Gla(14)-Gla(3)('). A second member of the conantokin family, conantokin-T (con-T), shares sequence identity with con-G at 8 of 21 amino acids, including 4 Gla residues. These similarities notwithstanding, several primary and secondary structural differences exist between con-T and con-G. Particularly notable is that con-T contains a Lys, rather than a Gla, at position 7. Moreover, unlike con-G, con-T does not undergo Ca(2+)-triggered self-assembly. In the present study, sedimentation equilibrium ultracentrifugation is employed to demonstrate that a single amino acid replacement analogue of con-T, con-T[K7gamma], assumes a dimeric superstructure in the presence of Ca(2+) at pH values consistent with the ionization of Gla carboxylate groups. Furthermore, HPLC-monitored thiol-disulfide folding and rearrangement assays with Cys-containing con-T variants suggest that the relative chain alignment preference in the noncovalent complex is antiparallel. Our results suggest that interchain Ca(2+) coordination in con-T[K7gamma] is incumbent upon an "i, i + 4, i +7, i +11" arrangement of Gla residues, as occurs in native con-G.     

The amino acid sequence of Emu osteocalcin: gas phase sequencing of Gla-containing proteins

Osteocalcin (OC), the major gamma carboxyglutamic acid (Gla)-containing protein of vertebrate bone, has been isolated from bones of the emu (Dromaius novaehollandae) and the primary structure determined by a combination of gas phase N-terminal sequencing of the intact molecule and a proteolytic fragment, and carboxypeptidase Y C-terminal sequencing. Gla residues were located by counting tritium radioactivity in fractions from the N-terminal sequencing of the tritiated/thermally decarboxylated molecule. Emu OC consists of 48 amino acid residues containing 3 Gla residues, and a single disulphide bond. The C-terminal 29 residues are identical to those of the human and sheep OC sequences. Alignment of the N-terminal sequence against those of other OCs reveals greater sequence homology with chicken OC than with mammalian OCs.     

Synthesis and proof of structure of the new amino acid in prothrombin

The first synthesis of the new amino acid γ-carboxyglutamic acid (Gla) found in prothrombin is reported. The structure previously proposed is thus confirmed. Part of the proof of identity with the natural product is based upon an unusual chemical modification occurring during acetylation in methanol.     

Amino acid determinants for NMDA receptor inhibition by conantokin-T

Several derivatives of conantokin-T (con-T), a naturally occurring, gamma-carboxyglutamate (Gla)-containing peptide with NMDA receptor (NMDAR) antagonist properties, were synthesized and evaluated for their ability to displace [(3)H]MK-801 from adult rat forebrain membranes. Analyses of progressive C-terminal truncation analogs of the parent 21-mer revealed gradual losses in activity with decreased chain length. In this series, con-T[1-8] was identified as the shortest variant capable of manifesting inhibitory activity (< 1% of the parent peptide). Ala substitution studies of individual residues identified Gly1, Gla3, Met8 and Leu12 as important for activity, while Glu2, Gla4 and Tyr5 were shown to be essential in this regard. The effect of side-chain length and charge in the N-terminal region was probed by single amino acid replacements. No correlation was observed between potencies and circular dichroism-derived helical contents of the con-T derivatives. Further elaboration of structure-function relationships in con-T was effected through the design and synthesis of helically constrained and destabilized analogs. The results of the current study were compared with those of a previous investigation on con-G, a related conantokin. Substantial differences in activity requirements were noted between the peptides, particularly in the C-terminal regions. Chimeras of con-T and con-G were generated and revealed virtually no interchangeability of residues between these two peptides. Finally, single amino acid substitutions that resulted in analogs with enhanced inhibitory properties were combined to yield superior conantokin-based NMDAR inhibitors.     

Distribution of gamma-carboxyglutamic acid residues in partially carboxylated human prothrombins

The role of gamma-carboxyglutamic acid in prothrombin has been examined using partially carboxylated variant prothrombins isolated from a person with a hereditary defect in vitamin K-dependent carboxylation. These species differ in gamma-carboxyglutamic acid content, distribution, and function, as monitored by metal binding properties, conformational transitions, phospholipid binding, and calcium-dependent coagulant activity (Borowski, M., Furie, B. C., Goldsmith, G. H., and Furie, B. (1985) J. Biol. Chem. 260, 9258-9264). The distribution of gamma-carboxyglutamic acids in the variant prothrombin species was determined by specific tritium incorporation into gamma-carboxyglutamic acid residues, thermal decarboxylation, and automated Edman degradation. gamma-Carboxyglutamic acid residues in the partially carboxylated prothrombins were identified by the assay of tritium in the resultant glutamic acid residues in the acarboxyprothrombins. The results indicate that variant prothrombins 1-3 are nearly homogeneous populations of partially carboxylated prothrombins. The ability of prothrombin to undergo a metal-induced conformational change and to bind to phospholipid vesicles correlated closely to the presence of a gamma-carboxyglutamic acid at residue 16. This residue is likely involved in the formation of a critical high affinity metal-binding site, possibly formed by Gla 16 and Gla 25 and/or Gla 26. A second high affinity metal-binding site, present in all of the variant prothrombin species, is defined, as an upper limit, by Gla 6, Gla 14, Gla 19, and Gla 20. This region is likely responsible for the interaction of certain of the conformation-specific antibodies to the metal-stabilized conformer of prothrombin.     

Novel gamma-carboxyglutamic acid-containing peptides from the venom of Conus textile

The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.     

Calcium binding to a human factor IXa derivative lacking gamma-carboxyglutamic acid: evidence for two high-affinity sites that do not involve beta-hydroxyaspartic acid

A derivative of human blood clotting factor IXa beta lacking gamma-carboxyglutamic acid (Gla) residues was prepared by limited proteolysis with chymotrypsin, and subsequently examined for its ability to bind calcium ions. By amino acid analysis, Gla-domainless human factor IXa beta contained 0.3-0.4 moles of beta-hydroxyaspartic acid per mole of protein. Equilibrium dialysis experiments demonstrated that Gla-domainless human factor IXa beta retained two high-affinity calcium binding sites (Kd=52 microM), a finding essentially identical to that observed for Gla-domainless bovine factor IX that contains 0.8-0.9 moles of beta-hydroxyaspartic acid per mole of protein. These data strongly suggest that the beta-hydroxyaspartic acid residue in these proteins does not participate in their high affinity calcium sites.     

Simplified procedure for the analysis of 3- and 4-hydroxyproline

A column chromatographic analysis of 3-hydroxyproline (3-Hyp), 4-hydroxyproline (4-Hyp), and γ-carboxyglutamic acid (Gla) is described. The analyses of urine and plasma were performed with a JLC-6AH amino acid analyzer. A 0.15 M sodium citrate buffer, pH 2.1, was used for elution. Urinary Gla, 3-Hyp, and 4-Hyp were among the seventeen peaks eluted before asparti acid. Hyp, Gla, glutamine, and asparagine in plasma were separated by elution with 0.2 M sodium citrate buffer, pH 3.25, containing 10% methanol. This single-column procedure achieves the sequential separation and quantitation of Gla, 3-Hyp, and 4-Hyp in urine as well as plasma, and is applicable to the diagnosis of collager, metabolism disorders.     

Proton, calcium, and magnesium binding by peptides containing gamma-carboxyglutamic acid.

gamma-Carboxyglutamic acid (Gla) is believed to bind Ca [II] ions and Mg [II] ions in prothrombin and other coagulation proteins. Binding constants for H+, Ca [II] ions, and Mg [II] ions to Gla-containing peptides are determined using pH and ion selective electrode titrations. The binding constants for peptides containing a single Gla residue are similar to the constants for malonic acid. Peptides containing two Gla residues in sequence (di-Gla peptides) bind Ca [II] ions and Mg [II] ions more strongly. KMgL for the di-Gla peptides is similar to the site-binding constant for Ca [II] ions in denatured BF1. These di-Gla peptides may be useful analogs for metal binding by the disordered Gla domain in BF1.     

γ-Carboxyglutamic acid content of hepatocellular carcinoma-associated des-γ-carboxy prothrombin

Serum des-γ-carboxy prothrombin (DCP) is a useful marker for the diagnosis of hepatocellular carcinoma (HCC), but the exact mechanism of its synthesis and its structural properties in liver diseases are unknown. DCP is measured by the monoclonal antibody MU-3. The purpose of this study was to examine the epitope of MU-3 and to characterize the differences in DCP between HCC and benign liver diseases. The epitope of MU-3 was examined by ELISA using prothrombin Gla domain polypeptides and was determined to be amino acid residues 17–27 of the prothrombin Gla domain, which has four γ-carboxyglutamic acid residues (Gla) at positions 19, 20, 25 and 26. Peptides having a glutamic acid residue (Glu) at these positions reacted strongly to MU-3 but lost reactivity when Glu 19 or 20 was changed to Gla. In the order of γ-carboxylation, MU-3 reacted strongly to DCP containing 0–1 Gla, weakly to 2–4 Gla and not at all to DCP containing more than five Gla. After adsorbing normal prothrombin with barium carbonate, DCP reaction to MU-3 was measured by determining the amount of DCP that was adsorbed by MU-3-coated beads. The proportion of DCP reacting to MU-3 in HCC was 41.0–76.8%, whereas in patients with benign liver diseases, only 0–42.1% reacted to MU-3. These results indicate that DCP variants preferentially synthesized in HCC have less than four Gla, which are restricted to positions 16, 25, 26 and 29, whereas DCP variants in benign liver diseases have more than five Gla.     

Identification and purification of vitamin K-dependent proteins and peptides with monoclonal antibodies specific for gamma -carboxyglutamyl (Gla) residues

Novel monoclonal antibodies that specifically recognize gamma-carboxyglutamyl (Gla) residues in proteins and peptides have been produced. As demonstrated by Western blot and time-resolved immunofluorescence assays the antibodies are pan-specific for most or all of the Gla-containing proteins tested (factors VII, IX, and X, prothrombin, protein C, protein S, growth arrest-specific protein 6, bone Gla protein, conantokin G from a cone snail, and factor Xa-like proteins from snake venom). Only the Gla-containing light chain of the two-chain proteins was bound. Decarboxylation destroyed the epitope(s) on prothrombin fragment 1, and Ca(2+) strongly inhibited binding to prothrombin. In Western blot, immunofluorescence, and surface plasmon resonance assays the antibodies bound peptides conjugated to bovine serum albumin that contained either a single Gla or a tandem pair of Gla residues. Binding was maintained when the sequence surrounding the Gla residue(s) was altered. Replacement of Gla with glutamic acid resulted in a complete loss of the epitope. The utility of the antibodies was demonstrated in immunochemical methods for detecting Gla-containing proteins and in the immunopurification of a factor Xa-like protein from tiger snake venom. The amino acid sequences of the Gla domain and portions of the heavy chain of the snake protein were determined.