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• W2047550920 abstract "Site-directed mutagenesis of the 40 N-terminal residues (γ-carboxyglutamic acid domain) of blood clotting factor VII was carried out to identify sites that improve membrane affinity. Improvements and degree of change included P10Q (2-fold), K32E (13-fold), and insertion of Tyr at position 4 (2-fold). Two other beneficial changes, D33F (2-fold) and A34E (1.5-fold), may exert their impact via influence of K32E. The modification D33E (5.2-fold) also resulted in substantial improvement. The combined mutant with highest affinity, (Y4)P10Q/K32E/D33F/A34E, showed 150–296-fold enhancement over wild-type factor VIIa, depending on the assay used. Undercarboxylation of Glu residues at positions 33 and 34 may result in an underestimate of the true contributions of γ-carboxyglutamic acid at these positions. Except for the Tyr4 mutant, all other beneficial mutations were located on the same surface of the protein, suggesting a possible membrane contact region. An initial screening assay was developed that provided faithful evaluation of mutants in crude mixtures. Overall, the results suggest features of membrane binding by vitamin K-dependent proteins and provide reagents that may prove useful for research and therapy. Site-directed mutagenesis of the 40 N-terminal residues (γ-carboxyglutamic acid domain) of blood clotting factor VII was carried out to identify sites that improve membrane affinity. Improvements and degree of change included P10Q (2-fold), K32E (13-fold), and insertion of Tyr at position 4 (2-fold). Two other beneficial changes, D33F (2-fold) and A34E (1.5-fold), may exert their impact via influence of K32E. The modification D33E (5.2-fold) also resulted in substantial improvement. The combined mutant with highest affinity, (Y4)P10Q/K32E/D33F/A34E, showed 150–296-fold enhancement over wild-type factor VIIa, depending on the assay used. Undercarboxylation of Glu residues at positions 33 and 34 may result in an underestimate of the true contributions of γ-carboxyglutamic acid at these positions. Except for the Tyr4 mutant, all other beneficial mutations were located on the same surface of the protein, suggesting a possible membrane contact region. An initial screening assay was developed that provided faithful evaluation of mutants in crude mixtures. Overall, the results suggest features of membrane binding by vitamin K-dependent proteins and provide reagents that may prove useful for research and therapy. γ-carboxyglutamic acid factor VII wild-type active site-blocked wild-type factor VIIa tissue factor 5-dimethylaminonaphthalene-1-sulfonyl phosphatidylserine phosphatidylcholine matrix-assisted laser desorption ionization time-of-flight Interaction between the vitamin K-dependent plasma proteins and a membrane surface is essential for hemostasis (1Furie B. Furie B.C. Cell. 1998; 53: 505-518Google Scholar,2Kalafatis M. Swords N.A. Rand M.D. Mann K.G. Biochim. Biophys. Acta. 1994; 1227: 113-129Google Scholar). This interaction is mediated through contact of the γ-carboxyglutamic acid (Gla)1 domains of these proteins with membranes containing acidic phospholipids (3Nelsestuen G.L. Shah A.M. Harvey S.B. Vitam. Horm. 2000; 58: 355-389Google Scholar, 4Neuenschwander P.F. Morrissey J.H. J. Biol. Chem. 1994; 269: 8007-8013Google Scholar). The Gla domain consists of ∼40 N-terminal residues of which 9–12 glutamic acid residues are post-translationally modified to Gla (5Magnusson S. Sottrup-Jensen L. Peterson T.E. Morris H.R. Dell A. FEBS Lett. 1974; 44: 189-193Google Scholar, 6Nelsestuen G.L. Zytokovicz T.H. Howard J.B. J. Biol. Chem. 1974; 249: 6347-6350Google Scholar, 7Stenflo J. J. Biol. Chem. 1974; 249: 5527-5535Google Scholar). In the presence of calcium, the Gla domain adopts structure and binds to membranes by a mechanism that is not fully understood. The Gla domains of the different vitamin K-dependent plasma proteins show striking sequence homology, yet have quite different affinities for phospholipid membranes (8McDonald J.F. Shah A.M. Schwalbe R.A. Kisiel W. Dahlback B. Nelsestuen G.L. Biochemistry. 1997; 36: 5120-5127Google Scholar). If the Gla domain constitutes the membrane contact region, individual amino acid residues within this domain should contribute to these different affinities. This suggestion has been supported by mutations of the Gla domain that affect membrane affinity. For example, the P10H mutant of bovine protein C shows an ∼10-fold enhancement in membrane affinity, whereas the H10P mutant of human protein C shows an ∼3-fold decline in membrane affinity (9Shen L. Shah A.M. Dahlback B. Nelsestuen G.L. Biochemistry. 1997; 36: 16025-16031Google Scholar). Although a major difference among the vitamin K-dependent proteins is a Gla residue versusanother amino acid at position 32, the Q32E mutation of protein C has no impact on membrane affinity (10Zhang L. Jhingan A. Castellino F.J. Blood. 1992; 80: 942-952Google Scholar, 11Shen L. Shah A.M. Dahlback B. Nelsestuen G.L. J. Biol. Chem. 1998; 273: 31086-31091Google Scholar). Similarly, replacement of Glu32 in factor X has little impact on protein function (12Larson P.J. Camire R.M. Wong D. Fasano N.C. Monroe D.M. Tracy P. High K.A. Biochemistry. 1998; 37: 5029-5038Google Scholar), and Glu32 of human prothrombin was described as only moderately important (13Ratcliffe J.V. Furie B. Furie B.C. J. Biol. Chem. 1993; 268: 24339-24345Google Scholar), having little impact on eitherK m or V max of the prothrombinase reaction. However, a double mutant of human protein C, S11G/Q32E, shows an ∼10-fold enhancement in membrane affinity (11Shen L. Shah A.M. Dahlback B. Nelsestuen G.L. J. Biol. Chem. 1998; 273: 31086-31091Google Scholar), and a double mutant of factor VII, P10Q/K32E, has a 25-fold enhancement in membrane affinity (14Shah A.M. Kisiel W. Foster D.C. Nelsestuen G.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4229-4234Google Scholar). Under appropriate conditions, these proteins show similar improvement in function (11Shen L. Shah A.M. Dahlback B. Nelsestuen G.L. J. Biol. Chem. 1998; 273: 31086-31091Google Scholar, 14Shah A.M. Kisiel W. Foster D.C. Nelsestuen G.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4229-4234Google Scholar, 15Nelsestuen G.L. Stone M. Martinez M.B. Harvey S.B. Foster D. Kisiel W. J. Biol. Chem. 2001; 276: 39825-39831Google Scholar, 16Henderson N. Key N.S. Christie B. Kisiel W. Foster D. Nelsestuen G.L. Thromb. Haemostasis. 2002; 88: 98-103Google Scholar). Although previous studies showed that proteins with enhanced membrane affinity can be created, the contribution of individual amino acid residues was not determined, and possible improvement by changes in other sites was not shown. This study used human factor VII (FVII) as a model Gla domain to investigate these questions and presents a simple functional assay that allows evaluation of minute quantities of impure protein as an initial screen of mutant protein activity. Single-site mutants as well as those with the highest affinity were purified and characterized. The results indicate that nearly all functional improvements arose from membrane binding affinity. The largest enhancements in membrane affinity resulted from mutation in the region of residue 32, a position that is located quite far from the N-terminal end of the protein, where membrane contact is generally suggested (39Freedman S.J. Blostein M.D. Baleja J.D. Jacobs M. Furie B.C. Furie B. J. Biol. Chem. 1996; 271: 16227-16236Google Scholar). The mutant with highest function, (Y4)P10Q/K32E/D33F/A34E, showed 150–296-fold improvement over wild-type FVII. Overall, the proteins described in this study contribute to a better understanding of the membrane contact site and the binding forces involved while providing novel reagents to probe coagulation reaction mechanisms that may be of use in therapy. Cloning and mutagenesis were performed by ATG Inc. (Eden Prairie, MN) following standard procedures (17Cormack B. Ausubel F.M. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1991: 8.5.7-8.5.9Google Scholar). Human FVII cDNA was cloned from a human liver cDNA library and then subcloned into the vector pRc-CMV. Mutagenesis was verified by sequencing of the entire Gla domain of all variant FVII proteins, including untranslated pre- and propeptide segments. Proteins were expressed in fetal human kidney 293 cells that were stably transfected using the agent LipofectAMINETM2000 (Invitrogen) following the manufacturer's instructions. Following previously outlined procedures (9Shen L. Shah A.M. Dahlback B. Nelsestuen G.L. Biochemistry. 1997; 36: 16025-16031Google Scholar), Geneticin-resistant colonies were selected, and high producing clones were grown to confluence in three layered flasks (Nunc) containing Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1.0 mmnonessential amino acids, 50 units/ml penicillin, 50 μg/ml streptomycin, 10 μg/ml vitamin K1, and 100–200 μg/ml Geneticin. Confluent cells were rinsed with and then cultured in serum-free Dulbecco's modified Eagle's medium containing 1.0 mm nonessential amino acids, 10 μg/ml vitamin K1, and 0.5 mm benzamidine hydrochloride. EDTA (pH 7.4) and benzamidine hydrochloride were added to conditioned medium intended for purification to concentrations of 5.0 and 2.0 mm, respectively. The conditioned medium was vacuum-filtered twice through Whatman double-filter paper (qualitative No. 1) and then through a 0.22-μm polyether sulfone membrane (Corning Inc.). If not immediately purified, the filtered medium was stored at −70 °C. The filtered medium was diluted 1:1 with distilled and deionized water containing 4.0 mm EDTA (pH 7.4) and 2.0 mmbenzamidine hydrochloride and then applied to a High Q Macro-Prep anion-exchange column (Bio-Rad). The column was equilibrated prior to loading and washed extensively with Tris buffer (50 mm Tris, 100 mm NaCl, and 0.02% (w/w) NaN3 (pH 7.4)) containing 2.0 mm EDTA and 2.0 mm benzamidine hydrochloride. The column was eluted isocratically with the same buffer containing 400 mm NaCl and no EDTA. Eluted fractions containing FVII activity were pooled and diluted 1:1 with Tris buffer containing 30 mm CaCl2 and 2 mm benzamidine hydrochloride. The pooled and diluted fractions were subjected to immunoaffinity chromatography using a calcium-dependent anti-human FVII monoclonal antibody (CaFVII22, provided by Dr. Walter Kisiel) coupled to Affi-Prep®Hz support (Bio-Rad). The column was washed with Tris buffer containing calcium and then eluted with Tris buffer containing 15 mm EDTA and 2.0 mm benzamidine hydrochloride. Fractions containing FVII activity were pooled and subjected to a final ion-exchange chromatography step using a Mono Q HR5/5 anion-exchange column (Amersham Biosciences). The column was equilibrated and washed extensively with Tris buffer. Proteins were eluted with a linear gradient of 100–500 mm NaCl over 30 min (flow rate of 1.0 ml/min). Eluted protein was concentrated by centrifugation filtration (Millipore Ultrafree,M r 10,000 cutoff) and then stored at −70 °C. SDS-PAGE analysis indicated that the proteins were highly pure and contained only zymogen FVII and FVIIa in both nonreducing and reducing gels. The percentage of FVIIa ranged from 40 to 95%. Prior to assay for enzyme activity, the proteins were fully activated using a commercial source of tissue factor (TF; Innovin, Dade Behring Corp.). A commercial preparation of FVIIa (NovoSeven, Nordisk) was used as the standard for all measurements. Protein concentrations were determined by the Bradford assay (40Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar). The amidolytic activity for the chromogenic substrate S-2288 (Chromogenix) was also used as a standard of comparison. Plasma clotting assays, described previously (15Nelsestuen G.L. Stone M. Martinez M.B. Harvey S.B. Foster D. Kisiel W. J. Biol. Chem. 2001; 276: 39825-39831Google Scholar), were used to determine FVIIa concentration. To ensure that FVIIa was the limiting component, the assay was conducted in the presence of excess TF (Innovin). TF concentrations in the Innovin preparation were determined from the concentration of NovoSeven needed to generate maximum activity. The TF concentration in the preparation used in this study was 2.7 nm. FVII was activated by mixing either crude or purified protein with 20 μl of Innovin solution, followed by incubation at 37 °C until activation was complete. (Typically, 1.0 μl of solution resulted in a final concentration of ∼0.3 nm FVII.) Activation of FVII was monitored by adding 2.65 μl of the activation mixture to 112.5 μl of prewarmed standard Tris buffer (0.05 m) containing 100 mm NaCl, 1.0 mg/ml bovine serum albumin, and 6.67 mmCaCl2. To initiate coagulation, 37.5 μl of prewarmed FVII-deficient plasma (Sigma) was added, and clotting time was determined by the hand tilt-test method. Full activation occurred within 1 h, and concentrations of FVIIa were determined by comparison with the NovoSeven standard. The results for the different assay comparisons gave the same protein concentration within the estimated error of the assays (±20%). Active site-blocked wild-type FVIIa (WT-FVIIai) was produced as previously described (15Nelsestuen G.L. Stone M. Martinez M.B. Harvey S.B. Foster D. Kisiel W. J. Biol. Chem. 2001; 276: 39825-39831Google Scholar) using the active-site inhibitor dansyl-glutamylglycylarginyl chloromethyl ketone (Dr. Walter Kisiel) and NovoSeven as the source of WT-FVIIa. WT-FVIIai concentrations were determined using the Bradford assay (40Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar). Highly pure phospholipids in organic solvent (bovine brain phosphatidylserine (PS) and egg phosphatidylcholine (PC), Sigma) were mixed at proper ratios and thoroughly dried, first under a stream of argon gas and then under vacuum for 14 h. Dried phospholipids were solubilized in Tris buffer and sonicated on ice for 3-s bursts every 10 s for 3 min (Heat Systems-Ultrasonics W-385). This was repeated four times. The sonicated vesicles were applied to a Sepharose 4B size-exclusion column (Amersham Biosciences). Eluted fractions containing vesicles with average diameters of 32–38 nm (LSA2 photon correlation spectrometer, Langley Ford Co.) were pooled for use in membrane binding studies. The concentrations of phospholipid were determined by phosphorus analysis (18Chen P.S. Toribara T.Y. Warner H. Anal. Chem. 1956; 28: 1756-1758Google Scholar) using a phospholipid/phosphorus weight ratio of 25:1. Protein-membrane binding was determined by relative light scattering at 90° using methods previously described (19Nelsestuen G.L. Lim T.L. Biochemistry. 1977; 16: 4164-4170Google Scholar). Briefly, for a constant particle concentration and for particles that are small compared with the wavelength of light, the ratio of the light scattering intensity of a protein·vesicle complex (I 2) to that of the vesicles alone (I 1) is related to the ratio of the molecular weight of the protein·vesicle complex (M 2) to that of the vesicles alone (M 1) by the relationship in Equation 1, I2/I1=(M2/M1)2(∂n/∂c)3)2Equation 1 where ∂n/∂c is the change in refractive index as a function of concentration of the light scattering species and was estimated as described (19Nelsestuen G.L. Lim T.L. Biochemistry. 1977; 16: 4164-4170Google Scholar). Light scattering contributions of the buffer and protein were subtracted to obtainI 1 and I 2. Values ofM 2/M 1 were estimated at various amounts of added protein and were plotted versus the protein (P)/phospholipid (PL) ratio (w/w). Dissociation constants (K D) for protein-membrane binding were estimated from the relationship in Equation 2.KD=[P]free[P⋅PLmax−P⋅PL]/[P⋅PL]Equation 2 The amount of free protein ([P]free) was estimated from the known weight concentrations of phospholipid and protein in the solution and the difference betweenM 2/M 1 and the theoretical value of M 2/M 1 if all of the added protein were bound. Weight concentrations of bound and free FVII were converted to molar concentration with a M rof 50,000 for all FVII variants. The concentration of P·PL was estimated from the known concentration of phospholipid and the ratioM 2/M 1. P·PLmax was calculated by assuming saturation binding at a ratio of 1:1 protein/phospholipid in the complex (M 2/M 1(max) = 2.0) (19Nelsestuen G.L. Lim T.L. Biochemistry. 1977; 16: 4164-4170Google Scholar). MALDI-TOF mass spectrometry was used to confirm the identity of the variant FVII proteins tested and to assess the extent of carboxylation. Purified proteins were incubated at 37 °C for 30 min in the presence of either chymotrypsin or trypsin at a ratio of 500:1 (w/w) FVII protein/protease. Reaction solutions were dried by vacuum centrifugation and solubilized in a 5:95 acetonitrile/water solution containing 0.1% trifluoroacetic acid. The solutions were desalted using reverse-phase chromatography (ZipTip, Millipore Corp.), mixed 1:1 with a saturated matrix solution (5-methoxysalicylic acid in 50:50 ethanol/water solution), spotted on the spectrometer target, allowed to dry, and then subjected to MALDI-TOF mass spectrometry (BiflexTM III, Bruker). Minimum laser power was used to obtain spectra. Moderate increases in power above this minimum did not result in changes in the distribution of the various carboxylated species observed. The percentage of each carboxylation species was determined by measurement of peak areas using integration software provide by the spectrometer manufacturer. The relative activities of FVIIa variants were determined by a method outlined previously (15Nelsestuen G.L. Stone M. Martinez M.B. Harvey S.B. Foster D. Kisiel W. J. Biol. Chem. 2001; 276: 39825-39831Google Scholar). Full activation of FVII was first ensured by incubation with TF (18 pm; Innovin) in 50 μl of Tris buffer containing 5.0 mm CaCl2 and 1.0 mg/ml bovine serum albumin. The mutant FVII proteins with higher membrane affinity (K32E, P10Q/K32E, P10Q/D33E, and (Y4)P10Q/K32E/D33F/A34E) were added to a final protein concentration of 3.0 nm. WT-FVIIa, P10Q, and K32E were added to a final concentration of 10 nm. Full activation of the FVIIa preparation was achieved after incubation for 1 h. A range of WT-FVIIai concentrations was added, and the mixture was allowed to equilibrate for another 2 h at 37 °C. Factor X (Enzyme Research Laboratories) was added to a concentration of 200 nm to initiate the reaction. After 10 min, the reaction was stopped by addition of excess EDTA (15 mm). The concentration of factor Xa was measured as activity for chromogenic substrate (0.32 mm S-2222, Chromogenix) by monitoring the absorbance change at 405 nm in a Beckman DU-70 UV-visible spectrophotometer. The amount of FVIIa that was bound to tissue factor (FVIIa·TF) was estimated from the activity observed relative to that of a standard with no WT-FVIIai. The concentration of WT-FVIIai bound to TF (WT-FVIIai·TF) was estimated from the fraction activity that was lost. Results are presented in a Hill-type plot represented by Equation 3 (15Nelsestuen G.L. Stone M. Martinez M.B. Harvey S.B. Foster D. Kisiel W. J. Biol. Chem. 2001; 276: 39825-39831Google Scholar), log([WT-FVIIai⋅TF]/[FVIIa⋅TF]) =log([WT-FVIIai]/[FVIIa])+log KD(VIIa)/KD(VIIai)Equation 3 where K D(VIIai) is the dissociation constant for the WT-FVIIai·TF complex andK D(VIIa) is the dissociation constant for the FVIIa·TF complex. Comparison of a plot of log([WT-FVIIai·TF]/[FVIIa·TF]) versuslog[WT-FVIIai] for two FVIIa variants at an identical and constant FVIIa concentration will allow estimation of their relative affinities for TF. Equation 3 represents free protein concentrations. Therefore, conditions were selected to ensure that the total protein concentration was in large excess over TF, so total protein was approximately equal to free protein. Pure FVII preparations or conditioned medium containing FVII were added to Innovin (0.1 ml) in an amount to generate ∼0.3 nm FVII. This concentration of pure FVII provided a final coagulation time of 25 s for all samples. Because all FVII variants have the same clotting time in the absence of inhibitor (see pure protein analysis below and Ref. 15Nelsestuen G.L. Stone M. Martinez M.B. Harvey S.B. Foster D. Kisiel W. J. Biol. Chem. 2001; 276: 39825-39831Google Scholar), use of a constant clotting time allowed the determination of the FVII concentration in an unpurified sample. It was important that TF was maintained in excess over FVII. Activation of FVII was allowed to proceed to completion (60 min at 37 °C). Activated FVII solution (containing 2.5 μl of Innovin) plus varying amounts of WT-FVIIai were mixed with Tris buffer containing 6.67 mm CaCl2 and 1.0 mg/ml bovine serum albumin to create 112.5-μl aliquots, which were incubated for 1 h at 37 °C to achieve equilibrium binding between TF, WT-FVIIai, and FVIIa. Coagulation was initiated by addition of 37.5 μl of prewarmed FVII-deficient plasma. Clotting times (CT) were determined, and results were evaluated by a plot of log(CT/CTo)versus log[WT-FVIIai], where CTo is the clotting time without inhibitor. Relative function of the different FVIIa variants was estimated by offset of the plots for the two proteins. The identities of purified recombinant FVII proteins were verified by mass spectrometry of Gla domains released from the intact proteins by limited protease digestion. The Gla domains consisted of either 1–40 (containing all Gla residues) or 1–32 (less one carboxylation site at residue 35) N-terminal residues. Use of the methoxysalicylic acid matrix and the lowest practical laser power resulted in a low level of undercarboxylated peptide species (Fig.1). In most cases, the fully carboxylated peptide (theoretical m/z of 5235 for the +1 charge state of K32E) was the most abundant peak (Fig. 1 A). However, in-source decarboxylation of Gla residues occurred during MALDI-TOF analysis and reached quantitative levels when the sinapinic acid matrix was used (20Martinez M.B. Harvey S.B. Higgins L. Krick T. Shen T. Kisiel W. Foster D. Brown T. Evans Jr., T.C. Shah A.M. Nelsestuen G.L. Proceedings of the 49th Conference on Mass Spectrometry and Allied Topics, Chicago, May 27–31, 2001. American Society for Mass Spectrometry, Santa Fe, NM2001Google Scholar). Undercarboxylation was detected by peaks separated by 44 mass units and a small peak corresponding to the fully decarboxylated peptide (m/z 4751) (Fig. 1 A). The quantitative distribution among the different species was very consistent for replicate samples as well as for many plasma-derived versusrecombinant proteins (20Martinez M.B. Harvey S.B. Higgins L. Krick T. Shen T. Kisiel W. Foster D. Brown T. Evans Jr., T.C. Shah A.M. Nelsestuen G.L. Proceedings of the 49th Conference on Mass Spectrometry and Allied Topics, Chicago, May 27–31, 2001. American Society for Mass Spectrometry, Santa Fe, NM2001Google Scholar). This consistency suggested that comparison of quantitative MALDI-TOF data could be used to detect relative differences in the carboxylation states of various proteins. For example, repeated measurement of different preparations of plasma-derived bovine factor X gave 77 ± 4% signal intensity in the fully carboxylated peptide. This level of fully carboxylated peptide corresponded to 97–98% carboxylation of all Glu residues. Similar results were obtained for bovine prothrombin and human protein C. Consequently, the somewhat lower yield of the fully carboxylated peptide of recombinant WT-FVII (46%) suggested undercarboxylation of the parent protein (Fig. 1 B). In fact, undercarboxylation at position 35 of recombinant WT-FVIIa has been previously observed (21Jurlander B. Thim L. Klausen N.K. Persson E. Kjalke M. Rexen P. Jorgensen T.B. Ostergaard P.B. Erhardtsen E. Bjorn S.E. Semin. Thromb. Hemost. 2001; 27: 373-383Google Scholar,22Thim L. Bjoern S. Christensen M. Nicolaisen E.M. Lund-Hansen T. Pedersen A.H. Hedner U. Biochemistry. 1988; 27: 7785-7793Google Scholar). That position 35 of recombinant WT-FVII was the major site of undercarboxylation was also suggested by MALDI-TOF analysis of peptide 1–32, which gave a high yield of the fully carboxylated state (70%) (Fig. 1 B). Undercarboxylation at position 35 of FVII (and a corresponding residue in factor IX (23Gillis S. Furie B.C. Furie B. Patel H. Huberty M.C. Switzer M. Foster B.W. Scoble H.A. Bond M.D. Protein Sci. 1997; 6: 185-196Google Scholar)) had no detected impact on the activity of the mature proteins (21Jurlander B. Thim L. Klausen N.K. Persson E. Kjalke M. Rexen P. Jorgensen T.B. Ostergaard P.B. Erhardtsen E. Bjorn S.E. Semin. Thromb. Hemost. 2001; 27: 373-383Google Scholar, 22Thim L. Bjoern S. Christensen M. Nicolaisen E.M. Lund-Hansen T. Pedersen A.H. Hedner U. Biochemistry. 1988; 27: 7785-7793Google Scholar). The total carboxylation levels of WT-FVII and P10Q/K32E, determined by this procedure, were 9.3 (of 10 theoretical) and 10.4 (of 11 theoretical) residues per Gla domain, respectively. These estimates were nearly identical to the values of 9.6 ± 0.9 and 10.7 ± 0.8 obtained by amino acid analysis after base hydrolysis (14Shah A.M. Kisiel W. Foster D.C. Nelsestuen G.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4229-4234Google Scholar). Comparative analysis by MALDI-TOF was used to estimate the carboxylation states for the FVII mutants produced in this study. Five of the seven proteins showed >93% of theoretical Gla levels (Fig.1 B, last column), suggesting a carboxylation state of the parent protein similar to that of commercial FVIIa. Two mutants showed substantially lower levels of carboxylation, 8.9 of 11 theoretical residues for P10Q/D33E and 10.9 of 12 theoretical residues for (Y4)P10Q/K32E/D33F/A34E. The latter mutants contained additional Glu residues beyond position 32. Given that undercarboxylation occurs at position 35 of recombinant WT-FVIIa, it was possible that the additional Glu residues at positions 33 and 34 were undercarboxylated as well. If correct, the functional impact of Gla residues at positions 33 and 34, detected in the following studies, may underestimate the true impact of Gla at these positions. When small unilamellar vesicles of 25:75 PS/PC were used to measure protein binding, the variant proteins displayed increasing membrane affinity in the order WT-FVII < P10Q < K32E (Fig.2 A). Mutants with higher affinity (K32E) bound at the theoretical limit (Fig. 2 A), so equilibrium binding constants could not be estimated. Because binding affinity is dependent on PS content of the membrane, use of a lower PS content (10:90 PS/PC) (Fig. 2 B) provided an equilibrium of bound and free protein for most mutants. Affinity increased in the order WT-FVII < P10Q < K32E < P10Q/D33E < P10Q/K32E. Dissociation constants estimated from these results are reported in Table I. TheK D value obtained for P10Q/K32E (0.16 ± 0.08 μm) compared well with the value of 0.22 μmfor small unilamellar vesicles of 10:90 PS/PC reported by Shah et al. (14Shah A.M. Kisiel W. Foster D.C. Nelsestuen G.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4229-4234Google Scholar). Even lower PS content was needed to estimate the binding constant for the highest affinity mutant (5:95 PS/PC) (Fig.2 C). Estimated K D values indicated a 3-fold enhancement in membrane affinity for the (Y4)P10Q/K32E/D33F/A34E mutant over the P10Q/K32E variant.Table IImpact of mutagenesis on FVII activity and membrane affinityFactor X activation (relative function)aWT-FVIIai concentration at 50% inhibition of 10 nm (lightface) and 3.0 nm (boldface) factor VIIa. Values in parentheses indicate the -fold change relative to WT-FVIIa.Clotting assaybInhibitor concentration required to increase clotting time by 60% (log(CT/CTo = 0.2. Fold changes in mutant activity relative to WT-FVIIa are in parentheses and are based on a 43-fold improvement of P10Q/K32E (first column)., cValues of the average of the K D values determined from each titration point. Values in parentheses indicate the -fold change relative to WT-FVIIa.K DcΔΔG dValues determined from the difference inK D for WT-FVII and variants.Purified proteinConditioned Medium10% PS5% PSnmnmμmkcal/molWT-FVII0.8 (1.0)ND (1.0)ND5.8 ± 0.6 (1.0)ND0.0P10Q2.4 (3.1)NDND3.2 ± 0.3 (1.8)ND−0.52K32E10/3.4 (13)0.17 (23)0.250.69 ± 0.1 (8.4)ND−1.28P10Q/K32E10.4 (43)0.31 (43)0.42 (43)0.16 ± 0.08 (36)1.5 ± 0.3(36)−2.06P10Q/D33E3.8 (16)0.15 (21)0.190.48 ± 0.08 (12)ND−1.52(Y4)P10Q/K32E/D33F/A34E35 (146)1.2 (166)2.9 (296)ND0.6 ± 0.2(95)−2.8 to −3.5a WT-FVIIai concentration at 50% inhibition of 10 nm (lightface) and 3.0 nm (boldface) factor VIIa. Values in parentheses indicate the -fold change relative to WT-FVIIa.b Inhibitor concentration required to increase clotting time by 60% (log(CT/CTo = 0.2. Fold changes in mutant activity relative to WT-FVIIa are in parentheses and are based on a 43-fold improvement of P10Q/K32E (first column).c Values of the average of the K D values determined from each titration point. Values in parentheses indicate the -fold change relative to WT-FVIIa.d Values determined from the difference inK D for WT-FVII and variants. Open table in a new tab Functional evaluation of the FVIIa mutants was carried out in a purified system that detected factor X activation. Experiments were performed under equilibrium competition conditions in which the FVIIa variants must compete with inhibitor, WT-FVIIai, for TF (described in Ref. 15Nelsestuen G.L. Stone M. Martinez M.B. Harvey S.B. Foster D. Kisiel W. J. Biol. Chem. 2001; 276: 39825-39831Google Scholar). To allow comparison of results for different proteins, the FVIIa species and WT-FVIIai concentrations were maintained in great excess over TF, so the total FVIIa and FVIIai concentration approximated the respective free concentration. The ability of the lowest affinity FVIIa variants (10 nm) to displ" @default.
• W2047550920 created "2016-06-24" @default.
• W2047550920 creator A5006868361 @default.
• W2047550920 creator A5022250856 @default.
• W2047550920 creator A5023957817 @default.
• W2047550920 creator A5087378048 @default.
• W2047550920 date "2003-03-01" @default.
• W2047550920 modified "2023-09-28" @default.
• W2047550920 title "Mutagenesis of the γ-Carboxyglutamic Acid Domain of Human Factor VII to Generate Maximum Enhancement of the Membrane Contact Site" @default.
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