From the Department of Plasma Protein Technology, Central
Laboratory of the Netherlands Red Cross Blood Transfusion Service,
Amsterdam, The Netherlands and the Sir William Dunn
School of Pathology, University of Oxford, Oxford, United Kingdom
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ABSTRACT |
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Recently, we established that mutations at calcium-binding sites within the first epidermal growth factor (EGF)-like domain of activated factor IX affect its interaction with factor VIIIa (Lenting, P. J., Christophe, O. D., ter Maat, H., Rees, D. J. G., and Mertens, K. (1996) J. Biol. Chem. 271, 25332-25337). In the present study, we have investigated the functional role of residue Glu78, which is not involved in calcium binding. Glu78 is also located in the first EGF-like domain and, when mutated to Lys, is associated with severe hemophilia B. Because Glu78 is conserved in related vitamin K-dependent proteins, it is difficult to understand how a mutation at this position is associated with factor IX-specific function. In this study, we addressed the hypothesis that Glu78 exerts its biological activity by interacting with another residue. One candidate was found to be the second EGF-like domain residue, Arg94, which is also associated with severe hemophilia B when mutated. We constructed a series of mutants that included mutations at position 78 alone (Glu78 to Lys/Glu78 to Asp) or at both positions 78 and 94 (Glu78 to Lys and Arg94 to Asp). The functional parameters of immunopurified and activated mutants were compared with normal activated factor IX. Mutants were indistinguishable from normal factor IXa in cleaving the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide or activating factor X in the absence of factor VIIIa. In contrast, in the presence of factor VIIIa, factor IXa Glu78 to Asp and factor IXa Glu78 to Lys/Arg94 to Asp were stimulated to the same extent as normal factor IXa, whereas factor IXa Glu78 to Lys was markedly less stimulated (140-fold versus 2,000-fold). This suggests that residues 78 and 94 should carry an opposite charge for a normal interaction of factor IXa to factor VIIIa. This hypothesis was confirmed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy (Ser558-Gln565) or light chain (Glu1811-Lys1818) and in direct binding studies. We propose that residues 78 and 94 link both EGF-like domains and thereby maintain the integrity of the factor VIII light chain binding site.
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INTRODUCTION |
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Factor IX (FIX)1 is a
vitamin K-dependent procoagulant zymogen with a central
role in blood coagulation. In the intrinsic and extrinsic blood
coagulation pathways, FIX is converted to activated FIX (FIXa) by
factor XIa and the complex factor VIIa-tissue factor, respectively, in
the presence of calcium ions (1). Activated FIX consists of an
N-terminal light chain containing a Gla module with several
-carboxylated glutamic acid residues, a short hydrophobic stack, two
domains that share considerable homology with the epidermal growth
factor (EGF), and a C-terminal heavy chain consisting of the catalytic
domain (2-4). These two chains are linked via a disulfide bridge.
FIXa is a serine protease that, in complex with calcium ions and the cofactor factor VIIIa (FVIIIa), activates factor X (FX) on procoagulant membranes (5). The rate of FX activation by FIXa in this complex is markedly enhanced in the presence of FVIIIa (6, 7). The physiological importance of FVIII and FIX is apparent from the notion that deficiency or dysfunction of either of the two proteins is associated with bleeding disorders known as hemophilia A and B, respectively (8). FIXa and FVIIIa assemble into the FXa-activating complex through several interactive sites (9, 10). Recently, we have shown that the light chain of FVIIIa contains a high affinity binding site for FIXa between residues Glu1811 and Lys1818 (11). Furthermore, it has been established that the sequence Ser558-Gln565 of the FVIIIa heavy chain interacts with FIXa (10).
Both the heavy and the light chain of FIXa have been proposed to be involved in FVIIIa binding (12, 13), although the precise locations of the interactive sites have not been elucidated yet. The FIXa heavy chain has been implicated by the finding that the enhancement of FIXa proteolytic activity by FVIIIa is inhibited by a monoclonal antibody against the FIXa heavy chain (12). We have obtained evidence that the FIXa light chain also interacts with the light chain of FVIII (13). In support of this notion, a monoclonal anti-FIX light chain antibody was found to abolish the binding of FIXa to FVIII light chain (13). Moreover, FVIII light chain binds to the light chain of FIXa in ligand blotting studies (13), while FIXa light chain proteolytic fragments interfere with FVIIIa-induced conformational changes in the FIXa active site (14). Finally, the effect of most of the FIX light chain mutations that combine moderate or severe hemophilia B (less than 5% biological activity compared with normal FIX) with normal FIX protein levels (15) are located in the first EGF-like domain (16, 17). A recent analysis of the crystal structure of the isolated first EGF-like domain of FIX revealed that a subset of these residues (Asp47, Gln50, and Asp64) is also associated with calcium binding (18).
By using recombinant FIX variants mutated at position 64 we have demonstrated recently that the mechanism by which calcium binding to the first EGF-like domain contributes to FIX activity is complex (19) and involves amidolytic activity, proteolytic activity in the absence of FVIIIa, and the interaction with the FVIII light chain (19). The first EGF-like domain of FIX also comprises residues that, when mutated, are associated with severe hemophilia B, although by a calcium-independent mechanism (16). These residues include Glu78, which has previously been described as being associated with a FVIIIa-dependent defect (15, 16).
In the present study, we have addressed the contribution of the residue 78 to FIXa function employing mutant recombinant FIX. The immunopurified mutants were compared with normal FIX with regard to a number of functional parameters including enzymatic activity and FVIII binding. This approach revealed that Glu78, in concert with Arg94, is crucial for the interaction with the FVIII light chain, presumably because these residues link the two EGF-like domains by an electrostatic interaction.
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EXPERIMENTAL PROCEDURES |
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Materials--
L--Phosphatidyl-L-serine,
L-
-phosphatidylcholine, heparin grade I-A, and vitamin
K1 were obtained from Sigma. Cell factories (6000 cm2) were from Nunc A/S (Roskilde, Denmark). Dulbecco's
modified Eagle's medium, fetal calf serum, Geneticin G-418 sulfate,
penicillin, streptomycin, fungizone, restriction enzymes, and DNA
modifying enzymes were obtained from Life Technologies, Inc. (Breda,
The Netherlands). Pfu polymerase was purchased from
Stratagene (Cambridge, UK). Oligonucleotide primers were from
Perkin-Elmer (Gouda, The Netherlands). The Sequenase kit was obtained
from Amersham Life Sciences (Breda, The Netherlands). Protein
A-Sepharose CL4B, Q-Sepharose FF, and CNBr-Sepharose CL4B were from
Pharmacia LKB Biotechnology AB (Uppsala, Sweden). Microtiter plates
(Immulon) were from Dynatech (Plockingen, Germany).
CH3SO2-D-Leu-Gly-Arg-p-nitroanilide
(CH3SO2-LGR-pNA; product name CBS 31.39) was
from Diagnostica Stago (Asnières, France).
Plasmid Constructions--
Plasmids encoding FIX E78K and FIX
E78D were previously described (16). Site-directed mutagenesis was
performed using the plasmid encoding FIX E78K as a template to
construct the plasmid encoding FIX E78K/R94D. The two mutagenesis
primers were as follows (mismatched bases are underlined):
5-AAGAATGGCGACTGCGAGCAG-3
as the sense primer and
5
-CTGCTCGCAGTCGCCATTCTT-3
as the antisense primer.
The resulting construct was verified by sequencing.
Proteins-- The monoclonal anti-FIX antibodies CLB-FIX 2 and CLB-FIX 14 have been described previously (9, 11). Polyclonal antibodies against FIX were obtained as described (9). The monoclonal anti-FVIII antibodies CLB-CAg 12, CLB-CAg 69, and CLB-CAg 117 against FVIII-LC have been described elsewhere (20, 21). Antibodies were purified employing Protein A-Sepharose as recommended by the manufacturer. Antibodies were conjugated with horseradish peroxidase as described (22). The human FVIII and FVIII light chain were purified as outlined previously (9). FX was purified as described (23).
Synthetic Peptides-- Peptides encompassing residues Ser558-Gln565 and Glu1804-Lys1818 from human FVIII, representing FIXa binding sites on the FVIII heavy chain (10) and FVIII light chain (11), respectively, were prepared as described (9, 11). Both peptides were more than 90% pure as determined by high performance liquid chromatography analysis, and their identity was confirmed by mass spectrometry analysis (Eurosequence B.V., Groningen, The Netherlands).
Recombinant FIX-- Madin-Darby canine kidney cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 (µg/ml) streptomycin. Transfection was performed using the calcium phosphate coprecipitation method with previously described plasmids encoding for FIX E78K, FIX E78D, or FIX E78K/R94D. Briefly, following selection of transfected cells with medium containing geneticin at 800 µg/ml, individual clones were isolated and propagated in selective medium to obtain stable cell lines. Production of FIX was monitored by measuring FIX antigen by enzyme-linked immunosorbent assay (see below), and cell lines producing the appropriate amounts of antigen were selected for large scale production in 1-liter cell factories as outlined previously (19).
Purification of FIX-- Normal plasma-derived FIX (pd-FIX) was prepared from a concentrate of human prothrombin, FIX, and FX (24) by immunoaffinity chromatography (13). Normal recombinant FIX (wt-FIX) and mutants thereof were purified from concentrated conditioned medium by the same immunoaffinity step as described previously (19). If present, residual contaminants were removed by Q-Sepharose FF chromatography in 0.1 M NaCl, 5% (v/v) glycerol, and 50 mM Tris-HCl (pH 7.4), using a linear NaCl gradient (0.1-1 M) for elution. The specific antigen of the purified recombinant proteins varied between 150 and 250 units/mg. SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining revealed one single band for all recombinant FIX species, which migrated with the same Mr as purified human plasma FIX. This indicates that no propeptide-containing unprocessed FIX was present. As previously reported, this expression system yields recombinant FIX proteins with normal calcium-dependent properties and similar activities for recombinant wt-FIX and pd-FIX (16, 19). Activated pd-FIX and recombinant FIX were prepared as described previously by incubating FIX with human FXIa, obtained from Enzyme Research Laboratories (13). After purification, the FIXa concentration was determined by active site titration employing the active site titrant antithrombin (13). Purified antithrombin and human serum albumin (HSA) were from the Division of Products of the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service.
Protein Concentrations-- Protein was quantified by the method of Bradford (25), using HSA as a standard. FIX antigen was quantified by enzyme-linked immunosorbent assay employing a previously described method (13). FVIII activity was assayed by a spectrophotometric assay employing bovine coagulation factors (Coatest FVIII, Chromogenix, Mölndal, Sweden). The amount of FVIII present in 1 ml of human plasma (1 unit/ml) was assumed to correspond to 0.35 nM.
Hydrolysis of
CH3SO2-LGR-pNA--
Cleavage of
CH3SO2-LGR-pNA was assayed as described (13) in
2 mg/ml HSA, 0.1 M NaCl, 5 mM
CaCl2, 0.05 M Tris (pH 8.4) at 37 °C.
Kinetic parameters of substrate hydrolysis were determined employing
substrate concentrations ranging from 0 to 15 mM at an
enzyme concentration of 300 nM. The release of
p-nitroanilide was monitored at 405 nm, and the absorbance
values were converted into molar concentrations using a molar
extinction coefficient of 9.65 × 103
M1 cm
1 for
p-nitroanilide and a path length of 0.35 cm for a 100-µl volume.
FX Activation-- FXa formation was determined as described previously (26) with slight modifications. Briefly, phospholipid vesicles (PS and PC, 50%/50%) and calcium ions were preincubated for 10 min at 37 °C in silicone-treated glass tubes in 60% of the final volume before the sequential addition (with 30-s intervals) of FIXa and FX. The reaction was started by the addition of FX. After various incubation times, subsamples were drawn and assayed for FXa formation employing the chromogenic substrate S-2222 (Chromogenix AB, Mölndal, Sweden). During the measuring period, less than 5% of the FX was converted into FXa, and FXa formation was linear. Conversion of substrate was monitored at 405 nm, and active site-titrated FXa was used to relate substrate hydrolysis to FXa concentrations. In experiments using various FVIII concentrations (0-2 nM), unactivated FVIII was added to the FX-activating mixture containing FIXa (0.1 nM) and thrombin (5 nM) but no FX. After 1 min of incubation, FXa formation was initiated by the addition of FX (0.2 µM). FXa formation was quantified as described above, and initial rates were calculated from measurements from the initial 3 min of incubation.
Binding Assays-- Binding of normal or recombinant FIXa to immobilized FVIII light chain and calculation of binding parameters were performed as described (9).
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RESULTS |
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Recombinant Proteins--
To investigate the potential role of the
charge at position 78 on FIXa function, we have constructed two FIX
variants, encoding the mutants FIX E78K and FIX E78D, respectively. In
the FIX E78K mutant, the acidic residue is replaced by a basic residue,
whereas the FIX E78D mutant conserves an acidic residue at position 78. These mutants were expressed in Madin-Darby canine kidney cells and
isolated from the culture medium by immunoaffinity chromatography. Preliminary characterization of the purified mutants indicated that FIX
E78D had retained biological activity to a significant extent, whereas
FIX E78K apparently lacked biological activity (results not shown).
These data are in agreement with previous reports that established FIX
E78K as a dysfunctional protein associated with severe hemophilia B
(15) and low activity (16). Both purified mutants could be completely
converted to the FIXa form (Mr 48,000) using
FXIa under the same condition as normal plasma-derived FIX or
recombinant wt-FIX (16, 19) (see "Experimental Procedures"). The
final preparations of activated mutant and normal FIX were more than
90% active as assessed by active site titration by antithrombin.
Amidolytic Activity-- We have previously demonstrated that replacement of Asp64 within the first EGF-like domain decreased the amidolytic activity of FIXa in the presence of calcium ions (19). To investigate the potential role of the side chain at position 78, we first tested the mutants FIXa E78K and FIXa E78D for amidolytic activity toward the synthetic substrate CH3SO2-LGR-pNA in the presence of calcium ions. As shown in Fig. 1, the kinetic parameters for CH3SO2-LGR-pNA hydrolysis of FIXa E78K and FIXa E78D were similar to those of normal FIXa. Apparently, whereas exchange of the side chain at position 64 alters the amidolytic activity of FIXa in the presence of calcium ions (19), replacement of Glu78 by Asp or Lys has no effect on the ability of FIXa to hydrolyze small synthetic substrate in the same conditions. Thus, residue 78 is not involved in the calcium-dependent amidolytic activity of FIXa.
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FX Activation in the Absence of FVIIIa-- To further characterize the potential role of residue 78 in FIXa function, the proteolytic activity of mutant FIXa E78K and FIXa E78D was evaluated toward the macromolecular substrate FX. In the presence of calcium ions, phospholipid vesicles, and various concentrations of FX, both mutant molecules were able to activate FX at the same rate as normal FIXa (Fig. 2). Kinetic parameters for the activation of FX demonstrated that exchange of the charge of the side chain at position 78 affects neither the apparent Km nor the apparent kcat values. The calculated parameters were the same for both molecules and as such were similar to previous data obtained with different preparations of pd-FIXa or recombinant wt-FIXa (9, 19). The data of Fig. 2 demonstrate that the proteolytic activity of FIXa is not affected by replacement of Glu78, indicating that the interaction of FIXa with a phospholipid-FX complex does not involve this residue. Apparently, mutant FIXa molecules are indistinguishable from normal FIXa with regard to enzymatic activity.
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FX Activation in the Presence of FVIIIa-- Since proteolytic activity as such is independent of residue 78, the apparent dysfunction observed in our preliminary characterization of FIX E78K should be at the FVIIIa-dependent level. Therefore, we examined the effect of FVIIIa on the FX activation by mutant FIXa, using various concentrations of FVIIIa. Indeed, as shown in Fig. 3, FX activation by FIXa E78K is strongly reduced at a range of FVIIIa concentrations as compared with normal FIXa. However, the activity of FIXa E78K toward FX is still dependent on the FVIIIa concentration (Fig. 3). FVIIIa stimulated FX activation by normal FIXa approximately 2,000-fold, whereas mutant FIXa E78K was only stimulated 140-fold. The stimulation factor represents the ratio of FX activation rates, in the presence and absence of FVIIIa (0.35 nM), at a FX concentration of 0.2 µM. In contrast, the activity of FIXa E78D was enhanced by FVIII in a saturable and dose-dependent manner and to the same extent as that of normal FIXa (Fig. 3). These results show that conservation of an acidic residue at position 78 (mutant FIXa E78D) does not affect FVIIIa-dependent FX activation by FIXa. In contrast, mutant FIXa E78K displays a defect in FX activation that is exclusively manifest in the presence of FVIIIa. These findings suggest that the charge of the residue at position 78 contributes to the interaction with FVIII light chain.
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Binding of FIXa to FVIII Light Chain-- To further investigate the selective defect of FIXa E78K in the binding to the FVIII light chain, equilibrium binding studies were performed employing immobilized FVIII light chain. As shown in Fig. 4, FIXa E78D was similar to normal FIXa in binding to the FVIII light chain. The calculated binding parameters were the same for both molecules, and as such were similar to previous data obtained with different preparations of pd-FIXa or recombinant wt-FIXa (9, 19). In contrast, FVIII light chain binding of the dysfunctional mutant FIXa E78K was significantly reduced (see Fig. 4). The FVIII light chain binding capacity of FIXa E78K is conserved, while the affinity of the dysfunctional FIXa E78K for FVIII light chain is significantly reduced. These data might suggest that residue 78 is part of the binding site of the FVIII light chain and that in particular its charge is involved in FVIII binding. At the same time, however, this seems unlikely because Glu78 and its flanking sequences are conserved in other vitamin K-dependent coagulation factors, including FX (27), FVII (28), and Protein C (29). One alternative explanation to our experimental findings would be that Glu78 interacts with another residue, presumably with an opposite charge, which could also contribute to FVIII light chain binding. Residue Arg94 was selected as an appropriate candidate for a variety of reasons (see "Discussion").
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Functional Study of Recombinant FIXa E78K/R94D-- To address the mechanism by which Glu78 contributes to FVIII light chain binding, the plasmid encoding the double mutant FIX E78K/R94D was constructed. In comparison with normal FIX, this mutant carries the reversed charge at both position 78 and 94 (see "Experimental Procedures"). After purification and activation (see "Experimental Procedures"), the mutant FIXa E78K/R94D was found to be indistinguishable from normal FIXa as well as FIXa E78K and FIXa E78D with regard to enzymatic activity toward small substrates in the presence of calcium ions and toward FX in the presence of calcium ions and phospholipids (not shown). However, as shown in Fig. 3, FVIIIa enhanced the activity of FIXa E78K/R94D to nearly the same extent as normal FIXa. These results show that the introduction of the R94D mutation into the FIXa E78K mutant restores the FVIIIa-dependent FX activation by FIXa to approximately 80% of the normal level. Since correction of FX activation suggests that also FVIII binding had been restored, FVIIIa-FIXa interaction was further studied by inhibition studies employing synthetic peptides. Two peptides encompassing the primary structure of FIXa interactive sites in the heavy chain (A2 domain) and the light chain (A3 domain) of FVIII were used. Both peptides, Ser558-Gln565 and Lys1804-Lys1818, respectively, are known to interfere with FX activation in a noncompetitive manner (10, 11). As listed in Table I, peptide Ser558-Gln565 inhibited FXa formation by all mutants and normal FIXa to the same extent. This suggests that neither residue 78 nor residue 94 is involved in the interaction of FIXa with the FVIII heavy chain. In contrast, peptide Lys1804-Lys1818 inhibited FXa formation by dysfunctional FIXa E78K 3-fold less efficiently than normal FIXa, whereas FXa formation by FIXa E78D or FIXa E78K/R94D was inhibited to the same extent as normal FIXa. To further investigate whether the introduction of the mutation R94D in FIXa E78K abolishes the defect in binding to the FVIII light chain, equilibrium binding studies were performed employing immobilized FVIII light chain. As shown in Fig. 4, the presence of the mutation R94D in FIXa E78K restores nearly to a normal level the affinity of FIXa E78K/R94D to the FVIII light chain, while the affinity of the dysfunctional FIXa E78K was significantly reduced. These results confirm that the introduction of the mutation R94D in FIXa E78K abolishes the defect in binding to the FVIII light chain.
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DISCUSSION |
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EGF-like modules have been found in proteins associated with blood coagulation, fibrinolysis, activation of the complement, cell adhesion, and developmental determination of embryonic cells (for reviews, see Refs. 4 and 31). It is presumed that this structure is involved in general biological roles such as ligand receptor or protein-protein interactions (31). The FIXa light chain contains two EGF-like domains. In this study, we focused our investigations on the potential role of these two domains in FIXa binding to FVIIIa. Defective FIX molecules with mutations in the first and second EGF-like domains, as present in the blood of patients with severe hemophilia B, have shed light on the possible contribution of these motifs in FIXa-FVIIIa interaction and thus in FVIIIa-dependent FX activation by FIXa (15). Some residues, including Asp47, Asp49, Gln50, Asp64, and Glu78 in the first EGF-like domain and Gln92 and Arg94 in the second EGF-like domain, are potential sites involved in FVIIIa binding. The first EGF-like domain of FIX contains one high affinity binding site for calcium ions (32). A recent crystal structure analysis of this domain revealed that residues 47, 50, and 64 are involved in calcium binding (18). Recently, we have established that replacement of the side chain at position 64, thus abolishing calcium-binding to the first EGF-like domain, affects several functions of FIXa, including the FIXa-FVIII light chain interaction (19). In the present study, we investigated the role of residue 78, which does not involve calcium binding. To this end, the mutant FIX molecules FIX E78K and FIX E78D were constructed, and the immunopurified proteins were compared with normal FIX in a number of functional parameters.
We first demonstrated that, unlike residue 64 (19), residue 78 is not involved in enzymatic activity of FIXa toward small substrates in the presence of calcium ions (Fig. 1) or FX (Fig. 2). However, while FIXa E78D activity toward FX is stimulated by FVIIIa to the same extent as normal FIXa, FIXa E78K activity is only slightly stimulated (Fig. 3). These findings suggest that the charge of the side chain at position 78 specifically affects the interaction of FIXa with FVIIIa. More detailed analysis revealed that this is due to an abnormal interaction of FIXa E78K with FVIII light chain, as assessed from direct binding studies (Fig. 4) and from competition studies employing two synthetic peptides (Table I). Furthermore, we resolved how the charge of residue 78 contributes to the binding of FIXa to FVIII light chain. Because the FIXa binding site in the FVIII A3 domain is rich in basic residues (11), it seems conceivable that the acidic residue Glu78 is in direct interaction with the FVIII light chain. One argument against this view, however, is the notion that Glu78 and its surrounding sequence (Gly-Phe-Glu-Gly-Lys) is identical in FX (27), and highly similar in FVII (Ala-Phe-Glu-Gly-Arg; Ref. 28) and Protein C (Gly-Trp-Glu-Gly-Arg; Ref. 29). The apparent involvement of the non-FIX-specific residue Glu78 in a FIX-specific function such as FVIII binding may be explained by Glu78 being in interaction with another residue, which should have the potential of stabilizing a FIX-specific site involved in FVIII light chain binding and which should be FIX-specific. The Glu78 counterpart should share the same function, and consequently mutation at this position should result in a severe hemophilia B phenotype as well. First, Arg94 is conserved in the FIX sequence of other mammalian sequences (33-36), whereas other related human vitamin K-dependent coagulation factors have Asp (FX, Ref. 27) or Gly (FVII and Protein C; Refs. 28 and 29) in this position. Second, mutation at this FIX-specific position is associated with severe hemophilia B (see data base in Ref. 15). Finally, the recently published crystal structure demonstrated that the distance of Glu78 from the Gla domain or the heavy chain eliminates any possible contact with residues within one of these domains, while residue Arg94 located in the second EGF-like domain is close to Glu78 in porcine FIXa (Ref. 30; see Fig. 5).
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Therefore, we have addressed the possibility that a compensatory mutation at position 94 into FIXa E78K would restore the FVIIIa-dependent FX activation. Indeed, the activity toward FX of the double-mutant, FIXa E78K/R94D, is stimulated by FVIIIa to nearly the same extent as normal FIXa (Fig. 3). The apparently normal interaction of FIXa E78K/R94D with FVIIIa was confirmed from direct binding studies (Fig. 4) and employing synthetic peptides that comprise the FIXa-binding sequences on FVIII light and heavy chains (Table I). Thus, our results strongly support the theory that within normal FIXa, residue Glu78 is in functional association with residue Arg94.
Previously, it has been established that the two EGF-like domains in FIX interact weakly with each other (37). Our data suggest that the bridge formed by residues 78 and 94 links both EGF-like domains and that this interaction is crucial for the integrity of the FVIII light chain binding site on the FIXa light chain and thus for FVIIIa-dependent FX activation. The observation that the natural variant R94S, which cannot form a salt bridge with residue 78, has a very low activity (38) is consistent with our conclusion. However, we cannot exclude the possibility that the overall electrostatic environment in the contact region between both EGF-like domains may provide alternative possibilities of contact between the two modules. Determination of the precise nature of the interaction would require a high resolution three-dimensional structure of the complete FIXa light chain in a calcium ion-bound conformation.
Our findings would explain why chimeric recombinant FIXa in which the first EGF-like domain has been replaced by the corresponding domain of FX (39) or FVII (40) retain normal or increased biological activity, respectively, whereas chimeric FIX with the two EGF-like domains of FX possesses only 4% normal biological activity (39). Indeed, inspection of the known amino acid sequence of EGF-like domains from each of the above mentioned proteins shows that the first two chimeras possess the residues Glu78 and Arg94, whereas the chimera with the two EGF-like domains of FX possesses residues Glu78 and Asp94. In the latter case, no salt bridge (required for FVIIIa binding) exists between the two residues. In addition, our model for the interaction of the two EGF-like domains would also explain the notion that a fragment of FIX that contains the two EGF-like domains inhibits FVIII-dependent blood coagulation (41).
It is noteworthy that the same strategy has revealed that electrostatic interactions are also predominant in the binding between tissue-type plasminogen activator and plasminogen activator inhibitor-1 (42). By mutagenesis of basic to acidic residues in the enzyme and vice versa in the inhibitor, Madison et al. (42) succeeded in restoring the interaction between both proteins. In the present study, we noted a similar type of interaction between two modules within the same protein.
It is known that EGF-like domains in blood coagulation proteins have
features that set them apart from those in other molecules, not only
structurally but also in terms of function. For example, they do not
contain the conserved amino acids present in EGF and tumor growth
factor- that are necessary for these proteins to express growth
factor activity. Our study demonstrates that EGF-like domains of
vitamin K-dependent proteins are not just spacers between the Gla region and the protease part but can also serve as an important
functional element involved in the interaction with its natural
cofactor. Two possibilities may be considered to explain our findings.
First, the interaction between residues 78 and 94 could permit a
specific conformation of FIX and thereby a specific spatial alignment
of the molecule with the two FIXa-binding sites on FVIII (10, 11). This
specific contact between both domains then allows the correct exposure
of the FVIII light chain binding site on one of the EGF-like modules.
Second, our findings could also suggest that the association of two
similar motifs, encoded by separate exons, might form one single
binding site. This association may allow the formation of the FVIII
light chain binding site, which could be composed of several specific
FIX residues dispersed among these two domains. Further studies will be
necessary to distinguish between these two possibilities and to
determine the location of the FVIII light chain binding site on the FIX
light chain more precisely.
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FOOTNOTES |
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* This research was supported by the Human Capital and Mobility Program of the European Community, by a grant from the Fondation pour la Recherche Médicale (to O. D. C.), and by The Netherlands Organization for Scientific Research Grant 902-26-152 (to J. A. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Plasma Protein Technology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. Tel.: 31-20-5123120; Fax: 31-20-5123680.
1 The abbreviations used are: FIX, factor IX; FIXa, activated factor IX; pNA, p-nitroanilide; CH3SO2-LGR-pNA, CH3SO2-D-leucyl-L-glycyl-L-arginyl-p-nitroanilide; EGF, epidermal growth factor; FVII, factor VII; FVIII, factor VIII; FVIIIa, activated factor VIII; FX, factor X; FXIa, activated factor XI; wt-FIX, wild-type factor IX; pd-FIX, plasma-derived factor IX; HSA, human serum albumin.
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REFERENCES |
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