Immunoaffinity purification of protein C by using conformation-specific monoclonal antibodies to protein C-calcium ion complex

We isolated protein C from a barium citrate-adsorbed fresh plasma and human factor IX concentrate by immunoaffinity chromatography on a column of Sepharose coupled with monoclonal antibodies to protein C. The antibodies used were conformation-specific monoclonal antibodies to the calcium-induced structure of protein C. Protein C was bound to antibodies coupled with Sepharose in the presence of calcium ions and was eluted with EDTA. This immunopurification resulted in a 13,000-fold purification of the fully functional zymogen from plasma. The immunoaffinity-isolated protein C was found to have higher amounts of single-chain protein C than conventionally isolated protein C when analyzed by sodium dodecyl sulfate-polyacrylamide gels under reduced conditions. The factor IX concentrate was applied to this Ca2+-dependent antibody JTC-3-immobilized Sepharose in the presence of 5 mM CaCl2, and protein C with its gamma-carboxyglutamic acid (Gla) domain intact was firstly bound to this column and then eluted by metal chelation with EDTA. When flow-through fractions were applied again in the presence of Ca2+ to this column, modified protein C which had lost its N-terminal 42-residue peptide was weakly bound to this column. It was eluted in the absence of Ca2+. However, only a low percentage of modified protein C was detectable by an enzyme-linked immunosorbent assay using Ca2+-dependent monoclonal antibody JTC-3 and peroxidase-labeled immunopurified polyclonal antibody. These results indicate that factor IX concentrate has both Gla-domain-intact and Gla-domainless protein C. Moreover, it suggests that Ca2+-dependent monoclonal antibody JTC-3 may recognize the coupled conformational change of protein C induced by the combined effect of Ca2+ binding to the Gla domain and to other parts of protein C.     

Conformation-specific monoclonal antibodies to the calcium-induced structure of protein C

Monoclonal antibodies to various domains of human protein C were characterized, and the cross-reactivity of these antibodies with other vitamin K-dependent proteins was explored. Three antibodies, JTC-1, -2, and -3 reacted with protein C only in the presence of Ca2+ and were shown to bind to the light chain of protein C. It is suggested that these antibodies recognize a gamma-carboxyglutamic acid domain-related conformational change induced by metal ions, evidenced by the fact that half-maximal binding was observed at calcium concentration of 0.5, 0.6, and 0.7 mM, respectively, by the fact that these antibodies, even in the presence of Ca2+, do not react with gamma-carboxyglutamic acid domainless protein C, and by the fact that Zn2+ and Tb3+ support binding in essentially the same way. Each cell line was stabilized by recloning five times. In addition each antibody had a single isoelectric point and was of the IgG1 kappa class. The interaction of antibodies JTC-1, -2; and -3 with protein C-Ca2+ was characterized by a single class of binding sites with Kd of 3.98 X 10(-9) M, 4.01 X 10(-9) M, and 6.76 X 10(-9) M, respectively. However, antibodies JTC-1, -2, and -3 bound to prothrombin-Ca2+ with Kd of 7.81 X 10(-9) M, 2.0 X 10(-7) M, and higher than 1.0 X 10(-5) M, respectively. In addition they had weak affinity for factor X in the presence of Ca2+. The results indicate that the antibodies JTC-1, -2, and -3 are conformation-specific monoclonal antibodies directed against an at least partially common metal ion-induced three-dimensional structure in protein C, prothrombin, and factor X.     

Calcium binding to the epidermal growth factor homology region of bovine protein C

A high affinity calcium binding site that is independent of the gamma-carboxyglutamic acid-rich amino-terminal region, has been demonstrated in bovine protein C, as well as in the other vitamin K-dependent proteins (except prothrombin) involved in blood coagulation. gamma-Carboxyglutamic acid-independent calcium binding in protein C is required for its rapid activation by the thrombin-thrombomodulin complex. We have now isolated a Ca2+-binding fragment from a tryptic digest of bovine protein C. The isolated fragment contains the two domains that are homologous to the epidermal growth factor precursor from the light chain of protein C, and a small disulfide bound peptide derived from the heavy chain. The isolated fragment bound 1 mol of Ca2+/mol of protein with a dissociation constant (Kd) of approximately 1 x 10(-4) M. This is similar to the Kd previously determined for binding of a single Ca2+ ion to protein C lacking the gamma-carboxyglutamic acid region. Immunochemical evidence indicated that Ca2+ binding induced a conformational change both in protein C lacking the gamma-carboxyglutamic acid region and in the isolated fragment.     

The interaction of a Ca2+-dependent monoclonal antibody with the protein C activation peptide region. Evidence for obligatory Ca2+ binding to both antigen and antibody

Protein C undergoes Ca2+-induced conformational changes required for activation by the thrombin-thrombomodulin complex. A Ca2+-dependent monoclonal antibody (HPC4) that blocks protein C activation was used to study conformational changes near the activation site in protein C. The half-maximal Ca2+ dependence was similar for protein C and gamma-carboxy-glutamic acid-domainless protein C for binding to HPC4 (205 +/- 23 and 110 +/- 29 microM Ca2+, respectively), activation rates (214 +/- 22 and 210 +/- 37 microM), and intrinsic fluorescence of gamma-carboxyglutamic acid-domainless protein C (176 +/- 34 microM). Protein C heavy chain binding to HPC4 was half-maximal at 36 microM Ca2+, although neither the heavy chain nor HPC4 separately bound Ca2+ with high affinity. The epitope was lost when the activation peptide was released. A synthetic peptide, P (6-17), which spans the activation site, exhibited Ca2+-dependent binding to HPC4 (half-maximal binding = 6 microM Ca2+). Thus, each decrease in antigen structure resulted in a reduced Ca2+ requirement for binding to HPC4. Tb3+ and Ca2+ binding studies demonstrated a Ca2+-binding site in HPC4 required for high affinity antigen binding. These studies provide the first direct evidence for a Ca2+-induced conformational change in the activation region of a vitamin K-dependent zymogen. Furthermore, Ca2+ binding to HPC4 is required for antigen binding. The multiple roles of Ca2+ described may be useful in interpretation of other metal-dependent antibody/antigen interactions.     

Localization of the metal-induced conformational transition of bovine prothrombin

The calcium-stabilized antigenic determinants on bovine prothrombin were localized to the NH2-terminal 1-42 residues using conformation-specific antibodies. Polyclonal antibodies to the bovine prothrombin-Ca(II) complex were raised in rabbits, and purified antibody subpopulations were isolated by sequential immunoabsorption and affinity chromatography. Anti-prothrombin-Ca(II) antibodies, characterized by their absolute specificity for the prothrombin-metal complex (Tai, M. M., Furie, B. C., and Furie, B. (1980) J. Biol. Chem. 255, 2790-2795), bound to prothrombin, fragment 1, reduced and carboxymethylated fragment 1, and CNBr fragment (1-72) in solution. However, these antibodies do not bind significantly to the gamma-carboxyglutamic acid-rich fragment (1-39), CNBr fragment (73-156), or prethrombin 1. To obviate the complex analysis of possible reasons for the lack of antibody binding to small peptides in solution, conformation-specific antibodies directed against defined regions of the whole prothrombin molecule were isolated. The influence of calcium ions on the binding of these site-specific antibody subpopulations to 125I-labeled prothrombin fragment 1 was evaluated. Anti-(1-39)N, anti-(1-42)N, anti-(1-72)N, and anti-(reduced and carboxymethylated fragment 1)N showed enhanced binding to prothrombin fragment 1 in the presence of Ca(II), indicating the presence of calcium-stabilized antigenic determinants within each of these regions on fragment 1. In contrast, calcium ions had no effect on the interaction of anti-des-(1-42)prothrombin, anti-prethrombin 1, anti-(43-72)N, and anti-(73-156)N antibodies with prothrombin fragment 1. These results indicate that the metal-induced conformational transition, monitored immunochemically, is localized to the NH2-terminal, gamma-carboxyglutamic acid-rich region of prothrombin between residues 1-42.     

The high affinity calcium-binding site involved in protein C activation is outside the first epidermal growth factor homology domain.

Binding Ca2+ to a high affinity site in protein C and 4-carboxyglutamic acid (Gla)-domainless protein C results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. It has been hypothesized that this high affinity Ca(2+)-binding site is located in the NH2-terminal epidermal growth factor (EGF) homology region of protein C. We have expressed in human 293 cells a deletion mutant of protein C (E2-PD) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of protein C. Ca2+ inhibits activation of E2-PD or Gla-domainless protein C by thrombin with half-maximal inhibition occurring at Ca2+ concentrations of 103 +/- 11 and 70 +/- 7 microM, respectively, but is required for both E2-PD and Gla-domainless protein C activation by the thrombin-thrombomodulin complex with half-maximal acceleration occurring at Ca2+ concentrations of 87 +/- 8 and 89 +/- 8 microM, respectively. Both E2-PD and Gla-domainless protein C exhibit a reversible, Ca(2+)- but not Mg(2+)-dependent decrease (6 +/- 1%) in fluorescence emission intensity with Kd = 38 +/- 3 microM Ca2+. We conclude that the high affinity Ca(2+)-binding site important for the activation of protein C is located outside of the NH2-terminal EGF homology region and that the metal-binding site in the NH2-terminal EGF homology region may not be a high affinity site in intact protein C.     

Calcium binding of bovine protein S. Effect of thrombin cleavage and removal of the gamma-carboxyglutamic acid-containing region

Thrombin cleaves protein S at arginine residues 52 and 70 resulting in loss of cofactor activity and reduced Ca2+ ion binding. After thrombin cleavage the NH2-terminal region containing gamma-carboxyglutamic acid (Gla) is linked to the large COOH-terminal fragment by a disulfide bond. Measurements of the rate of disulfide bond reduction by thioredoxin in intact protein S showed that the disulfide bonds are largely inaccessible to thioredoxin in the presence of Ca2+ ions, whereas in the presence of EDTA apparently all of the disulfide bonds are rapidly reduced. Probing the reactivity of the disulfide bonds in thrombin-modified proteins indicated that the thrombin cleavage induces a conformational change in the protein. After thrombin cleavage of protein S, the domain containing gamma-carboxyglutamic acid could be removed by selective reduction with thioredoxin followed by alkylation of the sulfhydryl groups. Ca2+ ion binding was compared in intact protein S, thrombin-modified protein S, and Gla domainless protein S. The intact protein S bound several Ca2+ ions, and the binding was not saturable. Thrombin-modified protein S, whether intact or with the Gla domain removed by selective reduction, bound two to three Ca2+ ions with a KD of 15-20 microM. The Gla domain in thrombin-modified protein S thus does not contribute significantly to the high affinity Ca2+ ion binding. Thrombin cleavage of protein S may be of physiological importance in the regulation of blood coagulation.     

Monoclonal antibodies against human abnormal (des-gamma-carboxy)prothrombin specific for the calcium-free conformer of prothrombin

A monoclonal antibody JO1 X 1 was prepared against human abnormal prothrombin using the hybridoma technique. The clone secreting this antibody was selected on the basis of the ability of this antibody to bind to abnormal prothrombin, but not to prothrombin, in the presence of calcium ions. The antibodies were purified by affinity chromatography in EDTA on columns of prothrombin-Sepharose. Bound antibodies were eluted with 15 mM CaCl2. The kinetics of dissociation of antibody from the antibody-prothrombin complex with the addition of calcium ions fit a first-order kinetic model. Increasing CaCl2 concentration increased the rate of antibody-prothrombin dissociation. Ca(II) and Mn(II) inhibited antibody-prothrombin interaction; half-maximal binding was observed at 0.9 and 4 mM, respectively. Mg(II) had little effect on antibody-antigen interaction. The JO1 X 1 antibody bound fragment 1, fragment (1-39), abnormal prothrombin, and prothrombin equivalently in the presence of EDTA, but did not bind to des(1-44)prothrombin in the presence of EDTA or prothrombin in the presence of CaCl2. These results indicate that the monoclonal antibody JO1 X 1 is conformation specific for the calcium-free conformer of prothrombin and directed against an antigenic determinant near the NH2 terminus of prothrombin expressed in the 1-39 region of the protein. This analysis provides confirmation of the presence of a metal-free conformer of prothrombin.     

Characterization of monoclonal antibodies against human protein C specific for the calcium ion-induced conformation or for the activation peptide region

Three monoclonal antibodies have been produced that are specific for the activation peptide region in human protein C. These antibodies inhibited the activation of protein C by thrombin and by the thrombin-thrombomodulin complex. A fourth monoclonal antibody specifically recognized the Ca2+-stabilized conformation in protein C. This antibody bound both intact protein C and protein C from which the gamma-carboxyglutamic acid-containing region had been removed by limited proteolysis. These results indicate that this antibody recognizes the conformation in protein C stabilized by Ca2+ bound to the single binding site that is independent of gamma-carboxyglutamic acid.     

The interaction of thrombomodulin with Ca2+.

Thrombomodulin (TM) is a cofactor for protein C activation by thrombin and each residue of a consensus Ca2+ site in the sixth epidermal growth factor domain (EGF6) is essential for this cofactor activity [Nagashima, M., Lundh, E., Leonard, J.C., Morser, J. & Parkinson, J.F. (1993) J. Biol. Chem. 268, 2888-2892]. Three soluble analogs of the extracellular domain of TM, solulin (Glu4-Pro490), TME1-6 (Cys227-Cys462) and TMEi4-6 (Val345-Cys462) were prepared for equilibrium dialysis experiments by exhaustive dialysis against Ca2+-depleted buffer. However, all three analogs still contained one tightly bound Ca2+ (Kd approximately 2 microm), which could only be removed by EDTA. Epitope mapping with Ca2+-dependent monoclonal antibodies to EGF6 provided further localization of this tight Ca2+ site. Equilibrium dialysis of the soluble TM analogs in [45Ca2+] between 10 and 200 microm revealed a second Ca2+ site (Kd = 30 +/- 10 microm) in both solulin and TME1-6, but not in TMEi4-6. Ca2+ binding to this second site was unaffected by bound thrombin and we attribute it to the consensus Ca2+ site in EGF3. A 75-fold decrease in the binding affinity of thrombin to TM was observed with immobilized solulin treated with EDTA to remove the high affinity Ca2+ by measuring kassoc and kdiss rates in a BIAcoretrade mark instrument. Ca2+-dependent conformational transitions detected by CD spectroscopy in the far UV indicate a more ordered structure upon Ca2+ binding. Bound Ca2+ stabilized soluble TM against protease digestion at a trypsin-like protease-sensitive site between Arg456 and His457 in EGF6 compared with protease treatment in EDTA. Finally, TM containing EGF domains 4-6, but lacking the interdomain loop between EGF3 and 4 (TME4-6), has an identical Ca2+ dependence for the activation of protein C as found for TMEi4-6, indicating this interdomain loop is not involved in Ca2+ binding.     

The binding of calcium to a salivary phosphoprotein, protein C, and comparison with calcium binding to protein A, a related salivary phosphoprotein.

The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.     

Calcium-binding properties of cardiac and skeletal troponin C as determined by circular dichroism and ultraviolet difference spectroscopy.

Calcium titration of the conformational change in cardiac and skeletal troponin C (TN-C) was followed by circular dichroism (CD) at pH values in the range from 5.2 to 7.4. Computer analysis was used to resolve the contributions from the different classes of Ca2+ -binding sites. At pH 6.94 in skeletal TN-C, apparent affinity constants for calcium of 1.8 x 10(7) and 4.5 x 10(5) M-1 were determined for the two classes of binding sites. The more sophisticated computer analysis of the data has revealed a substantial CD contribution from the low-affinity sites (approximately 30% of the high affinity contribution at pH 6.94) and suggests that skeletal TN-C with Ca2+ bound at the low-affinity sites is in a different conformation from that when just the high-affinity sites are occupied, in agreement with a recent nuclear magnetic resonance (NMR) study on this system (Seaman, K. B., Hartshorne, D. J. & Bothener-By, A. A. (1977) Biochemistry 16,4039-4046). With the cardiac protein at pH 7.07, an apparent affinity constant for calcium of 2.0 x 10(7) M-1 was calculated while no low-affinity site at this pH was detected by CD. On the other hand, at lower pH values, such as 6.05, a CD contribution from the cardiac low-affinity Ca2+ -binding site is detected with an apparent binding constant of 3.7 +/- 0.7 x 10(4) M-1. At the lower pH values, protonation of a class of carboxyl groups in each protein which possesses a high pKa (6.2-6.3) elicits the conformational change at the high-affinity sites with a corresponding decrease in the overall magnitude of the Ca2+ -evoked changes. The expression of a conformational change upon Ca2+ binding at the level of the low-affinity sites is enchanced by protonation of a class of carboxyls with a pKa of 6.3 in cardiac TN-C and 6.7-6.8 with the skeletal homologue. In both cases, this contribution is reduced upon protonation of carboxyls with pKa less than or equal to 5.5. It was also observed that the low-affinity sites of skeletal TN-C have a much larger role to play in the total conformational change than the low-affinity sites of cardiac TN-C, a finding probably related to the inability of site 1 in the cardiac protein to bind calcium. In the cardiac protein, the Ca2+ -induced tyrosine difference-spectrum maximum is reduced from deltaepsilonM,287nm =330M-1.cm-1 to 20M-1.cm-1 by protonation of a class of groups with a pKa of 6.4, presumably the same carboxyl groups as those invoved in the CD conformational contribution from the high-affinity binding sites. No such effect was observed for the skeletal protein where deltaepsilonM,287nm was constant at 110M-1 .cm-1 over the pH range studied. The dramatic alterations in the tyrosine environment of cardiac TN-C with pH are attributed to either or both of the tyrosines located in the two high-affinity Ca2+ -binding sites (sites 3 and 4)...     

Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes

Monoclonal antibodies (mAbs) raised against reduced and alkylated thrombospondin (TSP) were screened for the ability to react with Ca2+-replete TSP versus EDTA-treated TSP. Two mAbs designated A6.1 and D4.6 were found to react much more strongly with TSP after EDTA treatment. The dissociation constants for these mAbs were measured in 5 mM EDTA and found to be 6 X 10(-10) M for A6.1 and 7 X 10(-9) M for D4.6. Binding to A6.1 was undetectable in the presence of 1 mM Ca2+ while binding of D4.6 occurred with about 100-fold lower affinity. The Ca2+ concentration dependence of A6.1 binding was broad with a midpoint near 50 microM free Ca2+ while that of D4.6 showed a sharp transition below 0.1 microM. Upon dialysis of EDTA-treated TSP into Ca2+ containing buffer, the binding of the mAbs was prevented or decreased, indicating reversibility of the conformational transition induced by the initial removal of Ca2+ . Mg2+ can compete with the Ca2+ binding sites involved in mAb binding, but TSP dialyzed from Ca2+ into Mg2+ binds the two mAbs as well as EDTA-treated TSP, indicating that Mg2+ cannot maintain the Ca2+-replete structure of TSP. The proteolytic fragments of TSP with which the two mAbs react were determined by probing Western blots of digests of TSP with the mAbs. A6.1 reacts with the 70-kDa fragment generated by chymotrypsin in EDTA which contains the interchain disulfide bonds of TSP and the binding site(s) for type V collagen (Mumby, S. M., Raugi, G. J., and Bornstein, P. (1984) J. Cell Biol. 98, 646-652). D4.6 reacts with fragments of 140 and 120 kDa found in digests of Ca2+-replete TSP which are absent from digests in EDTA. Electron microscopy of rotary shadowed, carbon-coated replicas of TSP mAb complexes confirms the Ca2+ sensitivity of mAb binding and has been used to localize the epitopes for both mAbs on the three-dimensional structure of TSP.     

Calcium-dependent interaction between the epidermal growth factor precursor-like region of human protein C and a monoclonal antibody

Protein C, like the other vitamin K-dependent plasma proteins that participate in blood coagulation, except prothrombin, has at least one high affinity calcium-binding site that is independent of gamma-carboxyglutamic acid. Calcium binding to this site is required for activation of protein C by the thrombin-thrombomodulin complex. In an attempt to localize this calcium-binding site, we subjected protein C to limited tryptic digestion. A monoclonal antibody that recognizes a calcium-dependent epitope both in intact protein C, in gamma-carboxyglutamic acid-domainless protein C, and in activated protein C, was used to isolate a fragment from the tryptic digest. The fragment was derived from the light chain of protein C and consisted of the two domains that are homologous to the epidermal growth factor precursor. Half-maximal binding of the intact protein and of the isolated fragment by the antibody occurred at 100-200 microM Ca2+. The results suggest the presence of a Ca2+-binding site in the epidermal growth factor homology region of protein C.     

Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation

Changes in the affinity of the heavy subunit of blood coagulation factor Va (Vh) for prothrombin are thought to be important in regulating the rate of thrombin production. Using analytical ultracentrifugation, we have measured the affinity of bovine Vh for prothrombin and for the prethrombin 1 fragment of prothrombin at 23.3 degrees C, pH 7.65, in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 0.1 mM benzamidine, and either 2 mM Ca2+ or 2 mM ethylenediaminetetraacetate (EDTA). Under these conditions a 1:1 complex of Vh with prothrombin is formed that is governed by a dissociation constant (Kd) of 10 microM, regardless of whether the buffer contains Ca2+ or EDTA. An identical Kd is observed when prethrombin 1 is substituted for prothrombin. This indicates that the fragment 1 portion of prothrombin, containing the gamma-carboxyglutamic acid residues, does not influence the association. Substitution of human prethrombin 1 for the bovine molecule also results in a 1:1 Vh-prethrombin 1 complex governed by a slightly weaker Kd (27 microM). Discrete proteolysis of bovine Vh by the anticoagulant activated protein C converts the Vh to a form with little or no affinity for prethrombin 1 (Kd greater than 1 mM), without detectable change in the mass of the Vh.     

Structural elucidation of critical residues involved in binding of human monoclonal antibodies to hepatitis C virus E2 envelope glycoprotein

Human monoclonal antibodies derived from B cells of HCV-infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acid residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein.     

Binding of Ca 2+ to normal and dicoumarol-induced prothrombin

The Ca²⁺ binding properties of normal bovine prothrombin have been studied and compared with those of an abnormal bovine prothrombin induced by dicoumarol. The normal prothrombin binds up to 10–12 Ca²⁺ per mole of protein. The three first Ca²⁺ were bound to sites which exhibited positive cooperativity. A Ca²⁺ dependent conformational change was demonstrated during the binding of the first three Ca²⁺. In contrast with normal prothrombin, the dicoumarol-induced prothrombin had only one high affinity binding site. No ligand-induced conformational change was detected in this prothrombin.     

A conserved epitope on several human vitamin K-dependent proteins. Location of the antigenic site and influence of metal ions on antibody binding

A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 X 10(-8) to 1 X 10(-6) M. Other vitamin K-dependent proteins including Factor IX and protein S did not inhibit or inhibited only at the highest concentration binding of radiolabeled protein C to the immobilized antibody. Chemical treatment of prothrombin with a variety of agents including denaturation by sodium dodecyl sulfate, reduction with mercaptoethanol followed by carboxymethylation with iodoacetic acid, citraconylation of lysine residues, removal of metal ion with EDTA, or heat decarboxylation did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. Chymotrypsin digestion of prothrombin and isolation on QAE-Sephadex of the peptide representing amino-terminal residues 1-44 of prothrombin further localized the antigenic site recognized by the monoclonal antibody to the highly conserved gamma-carboxyglutamic acid-containing domain. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Antibody H-11 bound specifically to synthetic peptides corresponding to residues 1-12 of Factor VII and 1-22 of protein C. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. The glutamic acid residues in this peptide segment are the first 2 gamma-carboxyglutamic acid residues near the amino-terminal end in the native proteins. Increasing concentrations of Ca2+, Mg2+, or Mn2+ partially inhibited binding of 125I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monoclonal antibodies against type II rat brain protein kinase C

Monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C were used for the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II protein kinase C in a dose-dependent manner but did neither to the type I nor III isozyme. Immunoblot analysis of the tryptic fragments from protein kinase C revealed that all three antibodies recognized the 27-38-kDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-kDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained 70-80% of the kinase activity which was dependent on Ca2+ and phosphatidylserine and further activated by diacylglycerol or tumor-promoting phorbol ester. With antibody 8/1, the kinetic parameters with respect to Km for ATP and histone and K alpha for phosphatidylserine and phorbol 12,13-dibutyrate were not significantly influenced. However, the antibody causes variable effects on the K alpha for Ca2+ under different assay conditions. When determined in the presence of phosphatidylserine, the K alpha for Ca2+ was reduced by an order of magnitude (37 +/- 8 to 2.0 +/- 1.8 microM); in the presence of phosphatidylserine and phorbol 12,13-dibutyrate, the K alpha for Ca2+ was not significantly altered; and in the presence of phosphatidylserine and dioleoylglycerol, the kinase became an apparently Ca2+-independent enzyme. The effects of antibody 8/1 on the kinetic parameters of the enzyme for phorbol ester binding were different from those for kinase activity. This antibody causes a 20-30% reduction in phorbol ester binding and a 2-fold increase (1.9 +/- 0.2 to 3.9 +/- 0.3 micrograms/ml) in the concentration of phosphatidylserine required for half-maximal binding, but is without significant influence on those parameters for Ca2+ and phorbol 12,13-dibutyrate. The differential effects of antibody 8/1 on kinase activity and phorbol ester binding with respect to the kinetic parameter of phosphatidylserine suggest that the roles of this phospholipid in supporting phorbol ester binding and kinase activation are different. In the presence of the antibody, the autophosphorylations of the phospholipid/phorbol ester-binding domain and the kinase domain were reduced; the reduction was more pronounced for the former than for the latter. These results suggest that the epitope for antibody 8/1 is localized within the phospholipid/phorbol ester-binding domain at the region adjacent to the kinase domain so that the autophosphorylations of both domains are affected.     

Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain.

Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca2+, respectively. This study investigates the mechanism of Ca2+-induced binding of PKC betaII to anionic membranes mediated by the C2 domain. Stopped-flow fluorescence spectroscopy reveals that Ca2+-induced binding of the isolated C2 domain to anionic vesicles proceeds via at least two steps: (1) rapid binding of two or more Ca2+ ions to the free domain with relatively low affinity and (2) diffusion-controlled association of the Ca2+-occupied domain with vesicles. Ca2+ increases the affinity of the C2 domain for anionic membranes by both decreasing the dissociation rate constant (k(off)) and increasing the association rate constant (k(on)) for membrane binding. For binding to vesicles containing 40 mol % anionic lipid in the presence of 200 microM Ca2+, k(off) and k(on) are 8.9 s(-1) and 1.2 x 10(10) M(-1) x s(-1), respectively. The k(off) value increases to 150 s(-1) when free Ca2+ levels are rapidly reduced, decreasing the average lifetime of the membrane-bound C2 domain (tau = k(off)(-1)) from 110 ms in the presence of Ca2+ to 6.7 ms when Ca2+ is rapidly removed. Experiments addressing the role of electrostatic interactions reveal that they stabilize either the initial C2 domain-membrane encounter complex or the high-affinity membrane-bound complex. Specifically, lowering the phosphatidylserine mole fraction or including MgCl2 in the binding reaction decreases the affinity of the C2 domain for anionic vesicles by both reducing k(on) and increasing k(off) measured in the presence of 200 microM Ca2+. These species do not affect the k(off) value when Ca2+ is rapidly removed. Studies with PKC betaII reveal that Ca2+-induced binding to membranes by the full-length protein proceeds minimally via two kinetically resolvable steps: (1) a rapid bimolecular association of the enzyme with vesicles near the diffusion-controlled limit and, most likely, (2) subsequent conformational changes of the membrane-bound enzyme. As is the case for the C2 domain, k(off) for full-length PKC betaII increases when Ca2+ is rapidly removed, reducing tau from 11 s in the presence of Ca2+ to 48 ms in its absence. Thus, both the C2 domain and the slow conformational change prolong the lifetime of the PKC betaII-membrane ternary complex in the presence of Ca2+, with rapid membrane release triggered by removal of Ca2+. These results provide a molecular basis for cofactor regulation of PKC whereby the C2 domain searches three-dimensional space at the diffusion-controlled limit to target PKC to relatively common anionic phospholipids, whereupon a two-dimensional search is initiated by the C1 domain for the more rare, membrane-partitioned DG.