J. Biochem. 108, 654-662 (1990)
Monoclonal Antibody (VII-M31) to Bovine Factor VII: A Specific Epitope in the 7-Carboxyglutamic Acid Domain1 Shouichi Higashi,* Shun-ichiro Kawabata,* Hitoshi Nishimura,* Hideyuki Funasaki,' Shuzou Ohyama,** Seiji Miyamoto,** Akinobu Funatsu,** and Sadaaki Iwanaga* 2 'Department of Biology, Faculty of Science, Kyushu University 33, Higashi-ku, Fukuoka, Fukuoka 812; and "The Chemo-Sero-Therapeutic Research Institute 668, Kumamoto, Kumamoto 860
A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized. Antibody VII-M31 inhibited the activations of both factors IX and X catalyzed by factor Vila in the presence of tissue factor, phospholipids, and Ca2+. It possessed a strong affinity for factor VII in the presence of 5 mM Ca2+ (Ka = 1.12x 10~'°M). The immunoblotting test of other bovine proteins with the antibody, such as prothrombin, factor X, factor IX, protein C, protein S, and protein Z, in addition to human factor VII, revealed that it recognizes only a Ca2+-dependent epitope in bovine factor VII. Furthermore, this antibody VII-M31 covalently coupled with Affi-Gel allowed a simple and rapid purification of bovine factor VII. To localize the antigenic site in factor VII, various segments including a y-carboxyglutamic acid (Gla)-domainless protein, a Gla-domain peptide and the fragments isolated from the lysyl endopeptidase digest, were prepared. Among them, the isolated Gla-domain peptide and Gla-domainless factor VII were no longer recognized by antibody VII-M31, indicating that the sequence around the cleavage site by a-chymotrypsin is required for the interaction between the antibody and factor VII. In accordance with this result, the antibody bound specifically to a Gla-containing peptide corresponding to the NH2-terminal 23-50 residues of factor VII, which contains the chymotryptic cleavage site. These results suggest that the specific epitope of this antibody is localized in the carboxy-terminal 28 residues of the Gla-domain constituting the amino-terminal portion of bovine factor VII.
Factor Vila is a vitamin K-dependent plasma serine protease, of which the proteolytic activity is dramatically enhanced in the presence of tissue factor, Ca2+ and phospholipids (1, 2). Tissue factor exists in subendothelium (3), and when the vessel wall is injured, a membrane-bound tissue factor is exposed to the blood stream and can bind to factor Vila. The resulting tissue factor-factor Vila complex activates factor IX or factor X, leading to the initiation of the extrinsic clotting pathway. The zymogen factor VII is also able to bind with tissue factor with almost the same affinity as that of factor Vila (4). Although formation of a tissue factor-factor VTI/VIIa complex is essential as the initial step in the extrinsic clotting cascade, the detailed molecular mechanism of interaction between tissue factor and factor Vll/VIIa is still unclear. In the previous paper (5), we reported the complete amino acid sequence of bovine factor VII at the protein level. In the course of these 1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Portions of this paper (part of "MATERIALS AND METHODS") are presented in miniprint at the end of the paper. 2 To whom correspondence should be addressed. Abbreviations: Gla, y-carboxyglutamicacid; EGF, epidermal growth factor; Z-Arg-ONb, iV-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester; BSA, bovine serum albumin; ELISA, enzyme-linked immunoabsorbent assay; PTH, phenylthiohydantoin; TBS-1, 50 mM TrisHC1, pH 8.0, containing 0.1 M NaCl and 0.01% NaN3; TBS-2, 20 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl; TBS-3, 20 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl.
studies, we discovered the existence of a unique trisaccharide (Xyl2 -Glc) O-glycosidically linked to a serine residue in the first epidermal growth factor (EGF)-like domain of factor VII, in addition to those of factor IX and protein Z (6, 7). This finding led us to investigate whether the first EGF-like domain containing the carbohydrate moiety, which has a higher probability of being exposed on the surface of factor VII, contributes to the interaction of this factor with tissue factor. As the first step for this study, we prepared here monoclonal antibodies to bovine factor VII, since such antibodies are useful probes for surface determinants of proteins. Although a monoclonal antibody which recognizes specifically the first EGF-like domain could unfortunately not be generated, a single monoclonal antibody, named antibody VII-M31, available for a simple and rapid purification of bovine factor VII was developed. In this report, we describe the characterization of antibody VII-M31 and the identification of the specific epitope in factor VII recognized by this antibody. MATERIALS AND METHODS Purification of Proteins—All the procedures were carried out at 4°C. Monoclonal antibody VII-M31 was purified from ascites fluid of mice by ammonium sulfate precipitation (50% saturation) followed by either DEAE-Sepharose fast flow or Affi-Gel-protein A chromatography. Factor VII-
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J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/108/4/654/800962 by University of Otago user on 18 January 2019
Received for publication, May 25, 1990
A Specific Epitope in Bovine Factor VII
Preparation of Gla-Domain and Gla-Domainless Factor VII—Gla-domain and Gla-domainless factor VII were prepared from bovine factor VII by the method of Morita and Jackson (18). Protein concentration of each product was determined by amino acid analysis. Isolation of Peptide Fragments from Factor VII—Bovine factor VII was first S-aminoethylated and digested with lysyl endopeptidase, as described previously (6). The digest was separated by reversed-phase HPLC on a Cosmosil 3Cis (4.6x100 mm) column (6). One-twentieth volume aliquots of each peak after HPLC were coated on microtiter plates and their reactivities with antibody VII-M31 were tested by ELISA, as described in the miniprint section. Preparation of Fab' Fragment from Monoclonal Antibody VILM31— Antibody VII-M31 (50 mg) was digested with pepsin at an enzyme/substrate ratio of 1/50 (w/w) at 37°C for 8 h, in a total of 17 ml of 0.1 M sodium citrate, pH 3.5. The reaction was terminated by extensive dialysis against 10 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl. The resulting F(ab)'2 fragment was partially reduced by incubating it with 2-mercaptoethanol (final 9 mM) at 37°C for 1 h under N2 gas. Then, S-amidomethylation was performed by addition of iodoacetamide (final 11 mM) in ice for 1 h under N2 gas. Excess iodoacetamide was removed by dialysis against 50 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl and 0.01% NaN3 (TBS-1). Effect of Monoclonal Antibody VII-M31 on the Activation of Factors IX and X by Vila- Tissue Factor Complex —Ninety microliters of TBS-1 containing 0.1 mg/ml BSA, 10 ix\ of factor Vila (7.1 nM), 10 /x\ of 0.1 M CaCl2, and 10 //I of various concentrations of antibody VII-M31 or its Fab' fragment were mixed and incubated at room temperature for 60 min. Then, 10 n 1 of rabbit brain cephalin and 10 y. 1 of tissue factor (79.4 nM) were added and incubation was continued at 37°C. After 5 min, 10 ^1 of factor X (22.4 /*M) was added and the reaction mixture was incubated at 37°C for 2 min. The reaction of factor X activation was terminated by addition of 30 ^1 of 0.1 M EDTA. Twenty-microliter aliquots of the reaction mixture were added to the substrate solution, which consisted of 600//lof TBS-1 containingO.l mg/ml BSA and 10^1 of 5mM iV-benzyloxycarbonylL- pyroglutamyl- Gly-Arg-4- methylcoumaryl- 7 - amide in Vol. 108, No. 4, 1990
iV.AT-dimethylformamide, and released 7-amino-4-methylcoumarin was measured fluorometrically with excitation at 380 nm and emission at 440 nm. The factor IX activation assay was carried out using an ester substrate, iV-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester (ZArg-ONb). Various concentrations of antibody VII-M31 or its Fab' fragment in a volume of 30 n 1 were mixed with 34 JLI\ of factor Vila (7.1 nM) and 10//1 of 0.1 M CaCl2, and incubated at room temperature. After 60 min, 276 //I of factor IX (6.36 pM), 40 /i\ of tissue factor (79.4 nM), and 10 //I of rabbit brain cephalin were added and incubation was continued at 37°C for 120 min, then the reaction was terminated by adding 5 0 n \ of 0.1 M EDTA. Forty microliter aliquots of the reaction mixture were added to 435 /xl of TBS-1 and the reaction was started by adding 25 //I of 80 mM Z-Arg-ONb in AT,iV-dimethylformamide. After incubation at 37°C for 30 min, the reaction was terminated by adding 100 jul of 20% acetic acid. The amount of the released p-nitrobenzyl alcohol was determined using reversed-phase HPLC. This new assay method for factor IXa will be published in elsewhere (manuscript in preparation). Effect of Monoclonal Antibody VII-M31 on the Esterase Activity of Vila-Tissue Factor Complex—Twenty-seven microliters of factor Vila (170 nM), 300//I of various concentrations of antibody VII-M31 or its Fab' fragment and 20 /* 1 of 0.1 M CaCl2 were mixed and incubated at room temperature. One hour later, 10 /^l of rabbit brain cephalin and 10 //I of tissue factor (79.4 nM) were added, and then 25jul of 80 mM Z-Arg-ONb was added. The esterase activity of Vila-tissue factor complex after incubation at 37°C for 20 min was measured by the same method as those used for factor IXa assay. Kinetic Properties of Monoclonal Antibody VII-M31 —The dissociation constants (IQ) for antibody VII-M31 to factor VII and its derivatives were determined according to the method of Kimoto using microtiter plates (19). Microtiter plates were coated with 2.5 pi g/well of factor VII or 1.25 jt/g/well of S-aminoethylated factor VII in 20 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl (TBS-2), and 5 mM CaCl2, or 62 pmol/well of an ELISA-positive fragment, K-31. Then, various concentrations of antibody VII-M31 in TBS-2 containing 1% BSA, 0.05% Tween 20, and 5 mM CaCl2 were added and incubated overnight at 4°C (A-plate) and at room temperature for 1 h (B-plate). The ELISA was done in the same way as those described under "Supplemental Materials." The method of Scatchard analysis (20) was used to determine the K^ value for antibody VII-M31 to factor VII or its derivatives. Immunoblotting—Using 10 ng each of samples, SDSPAGE was carried out according to the method of Laemmli (21). After electrophoresis, gels were transferred to nitrocellulose membranes by using a semidry electroblot apparatus (Trans-Blot, Bio-Rad) at 15 V for 30 min in 25 mM Tris-192mM glycine buffer, pH 8.3, containing 20% methanol. Then, the membranes were blocked with 20 mM Tris-HCl, pH7.5, containing 0.5 M NaCl (TBS-3), 5 mM CaCl2, and 1% (w/w) non-fat dry milk at room temperature for 60 min. The membranes were washed three times with TBS-3 containing 0.05% Tween 20 and incubated overnight with antibody VII-M31 (10.4//g/ml) in TBS-3 containing 1% non-fat dry milk and 5 mM CaCl2 at room temperature. After being washed three times, membranes were incubat-
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deficient plasma was prepared from bovine plasma by the method of Radcliffe and Nemerson (2). Bovine factor VII was purified from the eluate of barium citrate adsorption of bovine plasma by DEAE-Sepharose CL-6B column chromatography (5) and immuno-affinity chromatography on a column of Affi-Gel 10 covalently coupled to antibody VII-M31. The conditions of this final purification step are shown in Fig. 9. Other proteins, such as bovine prothrombin, factors IX and X and proteins S, Z, and C from bovine plasma were purified as described previously by Hashimoto et al. (8). Tissue factor was purified from bovine brain acetone powder using a modification of the method of Broze et al. (9). Protein concentrations were determined by absorbance measurements at 280 nm. Extinction coefficients (£2*0) and molecular weights used were, respectively: factor VH/VIIa, 12.9 and 54,000 (10, 11); factor IX, 14.9 and 55,400 {12, 13); factor X, 12.4 and 55,000 (14, 15); and murine IgG, 14.0 and 150,000 (16). As for tissue factor, the concentration was determined by the dyebinding method with BSA as the standard (17).
655
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RESULTS Screening of Monoclonal Antibodies—Hybridomas producing monoclonal antibodies against factor VII were isolated. After screening assay as described in "MATERIALS AND METHODS," two clones, named VII-M218 and VIIM31, were selected for further investigation. Of these clones, the purified antibody from clone VTI-M218 bound not only factor VII but also other vitamin K-dependent
1 2
3
plasma proteins including prothrombin, factor LX, factor X, protein C, protein S, and protein Z, as revealed by the immunoblotting method (data not shown). This result suggests that antibody VTI-M218 recognizes a common antigenic site in these proteins. Because of this conservative specificity, further investigation of antibody VH-M218 was not performed. On the other hand, the purified monoclonal antibody from clone VTI-M31 bound with only bovine factor VTI and did not cross-react with other vitamin K-dependent proteins, or human factor VII, as shown in Fig. 1, A and B. Interestingly, the antibody VLT-M31 had still the ability to bind factor VII after treatment with SDS and reduction with 2-mercaptoethanol. Moreover, this monoclonal antibody showed a single band on SDS-PAGE (data not shown) and was identified as IgGl by radial immunodiffusion for murine irnmunoglobulins. Based on these results, further characterization of the antibody VH-M31 was performed. Effects of Antibody VH-M.31 on the Activation of Factors X and IX by Vila- Tissue Factor Complex—In the screening assay of hybridoma, we found that antibody VH-M31
4 5 6 7 8
a E (£ 200
400
600
800
1000 1200
VII-M3i/vlla, mol/mol
30K100
B
20K—11 S
Z
C
XKtt/ll HVII
12
3
4
5 6
1 7 8
60
CD
B 75K50K39K27K17KFig. 1. Cross-reactivity of antibody VII-M31 with vitamin Kdependent plasma proteins. Prothrombin (II) and proteins S (S), C (C), and Z (Z), and factors X (X), and IX (LX), and VII (bVH) from bovine source and human factor VH (hVTI) were subjected to SDS-PAGE (A) and the protein bands were detected by immunoblotting (B) against antibody VII-M31.
I *° CD E S.
20 n 100 200 300 Vll-M31/Vlla, mol/mol
400
500
Fig. 2. Effect of antibody VII-M31 on the activations of factor X (A) and factor LX (B) by factor Vila-tissue factor complex. A: Factor X (220 pmol) was incubated with factor Vila (0.071 pmol), tissue factor (0.79 pmol) and various concentrations of antibody VTI-M31 in the presence of Ca1+ and phospholipids. The factor Xa activity generated was measured using N'-benzyloxycarbonyl-Lpyroglutamyl-Gly-Arg-4-methylcoumaryl-7-amide as a substrate. B: Factor K (1.75 nmol) was incubated with factor Vila (0.24 pmol), tissue factor (3.1 pmol) and various concentrations of antibody VTI-M31 in the presence of Ca1+ and phospholipids. The factor LX activity generated was measured using Z-Arg-ONb as a substrate. In each case, the activities of factor Xa and factor LXa generated in the absence of antibody VH-M31 were set as 100%. J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/108/4/654/800962 by University of Otago user on 18 January 2019
ed at room tempature for 2 h with a 3,000-fold diluted horseradish peroxidase-conjugated goat anti-mouse IgG (H + L). After extensive washing, membranes were subjected to color development in TBS-3 containing 0.5 mg/ml 4-chloro-l-naphthol and 0.1% H 2 O 2 . Amino Acid and Amino-Terminal Sequence Analyses —Samples were hydrolyzed at 110'C for 20 h in 6 M HC1 in evacuated tubes. Following evaporation, hydrolysates were analyzed by a PICO TAG system (Waters, Millipore, Milford, MA), according to the method of Heinrikson and Meredith (22). Sequence analyses were performed using an Applied Biosystems 477A gas-phase sequencer. PTHderivatives were identified with an Applied Biosystems 120A PTH-analyzer with an on-line system, as described in detail previously (5).
657
A Specific Epitope in Bovine Factor VII 2.0
E
c o o>
Proteins, pmol/well
[metal ion], mM Fig. 3. Effect of divalent metal ions on the binding of antibody VII-M31 with factor VII. Factor VII (0.06 pmol/well) was coated with the microtiter plates and incubated with antibody VII-M31 (3.45 pmol/well) in the presence of various concentrations of CaClj (•), MgCli (O), and MnCl, (A). The amount of bound antibody was measured by the ELISA method, as described under "MATERIALS AND METHODS.'
Fig. 5. Binding of antibody VH-M31 with factor VH, Gladomain derived from factor VII, and Gla-domainless factor VII. Microtiter plates were coated with various concentrations of factor VTI (•), Gla-domain derived from factor VTI (O), and Gla-domainless factor VTI (A) and incubated with 10.4 pmol/well of antibody VTI-M31 in the presence of 5 mM CaCl2 at room temperature for 1 h. Binding of the antibody with each protein was monitored by the ELISA method.
VII " VIKGD) -
0
3 15 30
0 3 15 30
I
min—I
Lmin—*
Fig. 4. SDS-PAGE of a-chymotryptic digest of factor VII and its immunoblotting with antibody VII-M31. Factor VTI was digested with a-chymotrypsin at an enzyme/substrate ratio of 1/ 1,000 (w/w) at 20'C, and the aliquots at the indicated times were taken and subjected to SDS-PAGE (A) and to immunoblotting (B). VH, factor VH; VH (-GD), Gla-domainless factor VII.
inhibits the plasma clotting activity mediated by a VTIatissue factor complex. To investigate this property, we first examined the effect of antibody VTI-M31 on the activation of factor LX or factor X, both of which are natural substrates of Vila-tissue factor complex. The antibody VTI-M31 inhibited the activations of factor X (Fig. 2A) and factor LX (Fig. 2B) in a dose-dependent manner. The half-maximal inhibitions were observed in the presence of a 40- fold molar excess of antibody VTI-M31 to factor Vila for the factor IX activation, and an 80-fold molar excess of the antibody for the factor X activation. This antibody did not have any effect on the esterase activity of factor Vila toward Z-Arg-ONb. We also prepared the Fab' fragment derived from antibody VH-M31 and carried out similar inhibition experiments to examine the effect of the smaller Vol. 108, No. 4, 1990
2.0
B
E c 3 1.0
J ill
0
Peptlde No.
JUtitlU
Fig. 6. HFLC separation of lysyl endopeptidase digest derived from S-aminoethylated factor VH (A) and ELISA of the isolated peptide fragments (B). The lysyl endopeptidase digest of S-aminoethylated factor VII was applied to a Cosmosil 3Ci, column and eluted at aflowrate of 0.5 ml/min with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. The column eluate was monitored at 214 nm (solid line) and the broken line shows the percentage of acetonitrile in the elution medium. Major peaks after HPLC were subjected to ELISA in the presence of 5 mM CaCU.
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u.O
S. Higashi et al.
658
NH,-terminal sequences ofpeptides K-31 and K-43. K-43 K-31 Cycle PTH Yield PTH Yield No. amino acid a mi no acid (pmol) (pmol) Ala Ser 208 63 1 Asn 96 Phe 425 2 122 Gly ND' 3 ND 75 Phe 4 Ala 407 94 Leu 5 — 64 ND His 6 — ND ND 7 Leu 42 He 217 8 64 Phe 217 Leu 9 126 Pro 10 43 Arg Position 1-50 23-50 'ND, not detectable.
J.U —-—
„--—
A 2.0
E c o
0.5
O)
0.4
k s
N
2"
1.0
0]
Kd • 1.12llO'M (-)
/ ( nn
o. 08
0.0
0.2
0.09
0.4
0 10 B, nM
XN 0.11
0. 12
0.8
0.6
VII-M31,nM 2.0
B 1.8
TABLE II.
E c o
16• \
CD 1.4
•
.
u. 1.0
/
1.2 •
o.a
\
0.7 K
"tfo* 1.0 0.05
0.10
0.03
0.15
O.OS 0 . 0 7
O.OS O.OD
B, nM
0.20
0.25
VII-M31,nM
E c o O)
o
0
1
VII-M31, nM [metal ion], mM Fig. 7. Effect of divalent metal ions on the binding of antibody Vn-M31 with peptide K-31. K-31 (50 pmol/well) was coated with the microtiter plates and incubated with antibody VII-M31 (0.33 pmol/well) in the presence of various concentrations of CaCl, (•), MgCl, (O), and MnCl2 (*). The amount of bound antibody was measured by the ELISA method, as described under "MATERIALS AND METHODS.'
Fig. 8. Specific binding of antibody VII-M31 with factor VH (A), S-aminoethylated factor VII (B), or peptide K-31 (C) and their Scatchard analyses (insets). Factor VH (2.5/
Monoclonal Antibody (VII-M31) to Bovine Factor VII: A Specific Epitope in the 7-Carboxyglutamic Acid Domain1 Shouichi Higashi,* Shun-ichiro Kawabata,* Hitoshi Nishimura,* Hideyuki Funasaki,' Shuzou Ohyama,** Seiji Miyamoto,** Akinobu Funatsu,** and Sadaaki Iwanaga* 2 'Department of Biology, Faculty of Science, Kyushu University 33, Higashi-ku, Fukuoka, Fukuoka 812; and "The Chemo-Sero-Therapeutic Research Institute 668, Kumamoto, Kumamoto 860
A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized. Antibody VII-M31 inhibited the activations of both factors IX and X catalyzed by factor Vila in the presence of tissue factor, phospholipids, and Ca2+. It possessed a strong affinity for factor VII in the presence of 5 mM Ca2+ (Ka = 1.12x 10~'°M). The immunoblotting test of other bovine proteins with the antibody, such as prothrombin, factor X, factor IX, protein C, protein S, and protein Z, in addition to human factor VII, revealed that it recognizes only a Ca2+-dependent epitope in bovine factor VII. Furthermore, this antibody VII-M31 covalently coupled with Affi-Gel allowed a simple and rapid purification of bovine factor VII. To localize the antigenic site in factor VII, various segments including a y-carboxyglutamic acid (Gla)-domainless protein, a Gla-domain peptide and the fragments isolated from the lysyl endopeptidase digest, were prepared. Among them, the isolated Gla-domain peptide and Gla-domainless factor VII were no longer recognized by antibody VII-M31, indicating that the sequence around the cleavage site by a-chymotrypsin is required for the interaction between the antibody and factor VII. In accordance with this result, the antibody bound specifically to a Gla-containing peptide corresponding to the NH2-terminal 23-50 residues of factor VII, which contains the chymotryptic cleavage site. These results suggest that the specific epitope of this antibody is localized in the carboxy-terminal 28 residues of the Gla-domain constituting the amino-terminal portion of bovine factor VII.
Factor Vila is a vitamin K-dependent plasma serine protease, of which the proteolytic activity is dramatically enhanced in the presence of tissue factor, Ca2+ and phospholipids (1, 2). Tissue factor exists in subendothelium (3), and when the vessel wall is injured, a membrane-bound tissue factor is exposed to the blood stream and can bind to factor Vila. The resulting tissue factor-factor Vila complex activates factor IX or factor X, leading to the initiation of the extrinsic clotting pathway. The zymogen factor VII is also able to bind with tissue factor with almost the same affinity as that of factor Vila (4). Although formation of a tissue factor-factor VTI/VIIa complex is essential as the initial step in the extrinsic clotting cascade, the detailed molecular mechanism of interaction between tissue factor and factor Vll/VIIa is still unclear. In the previous paper (5), we reported the complete amino acid sequence of bovine factor VII at the protein level. In the course of these 1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Portions of this paper (part of "MATERIALS AND METHODS") are presented in miniprint at the end of the paper. 2 To whom correspondence should be addressed. Abbreviations: Gla, y-carboxyglutamicacid; EGF, epidermal growth factor; Z-Arg-ONb, iV-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester; BSA, bovine serum albumin; ELISA, enzyme-linked immunoabsorbent assay; PTH, phenylthiohydantoin; TBS-1, 50 mM TrisHC1, pH 8.0, containing 0.1 M NaCl and 0.01% NaN3; TBS-2, 20 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl; TBS-3, 20 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl.
studies, we discovered the existence of a unique trisaccharide (Xyl2 -Glc) O-glycosidically linked to a serine residue in the first epidermal growth factor (EGF)-like domain of factor VII, in addition to those of factor IX and protein Z (6, 7). This finding led us to investigate whether the first EGF-like domain containing the carbohydrate moiety, which has a higher probability of being exposed on the surface of factor VII, contributes to the interaction of this factor with tissue factor. As the first step for this study, we prepared here monoclonal antibodies to bovine factor VII, since such antibodies are useful probes for surface determinants of proteins. Although a monoclonal antibody which recognizes specifically the first EGF-like domain could unfortunately not be generated, a single monoclonal antibody, named antibody VII-M31, available for a simple and rapid purification of bovine factor VII was developed. In this report, we describe the characterization of antibody VII-M31 and the identification of the specific epitope in factor VII recognized by this antibody. MATERIALS AND METHODS Purification of Proteins—All the procedures were carried out at 4°C. Monoclonal antibody VII-M31 was purified from ascites fluid of mice by ammonium sulfate precipitation (50% saturation) followed by either DEAE-Sepharose fast flow or Affi-Gel-protein A chromatography. Factor VII-
654
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/108/4/654/800962 by University of Otago user on 18 January 2019
Received for publication, May 25, 1990
A Specific Epitope in Bovine Factor VII
Preparation of Gla-Domain and Gla-Domainless Factor VII—Gla-domain and Gla-domainless factor VII were prepared from bovine factor VII by the method of Morita and Jackson (18). Protein concentration of each product was determined by amino acid analysis. Isolation of Peptide Fragments from Factor VII—Bovine factor VII was first S-aminoethylated and digested with lysyl endopeptidase, as described previously (6). The digest was separated by reversed-phase HPLC on a Cosmosil 3Cis (4.6x100 mm) column (6). One-twentieth volume aliquots of each peak after HPLC were coated on microtiter plates and their reactivities with antibody VII-M31 were tested by ELISA, as described in the miniprint section. Preparation of Fab' Fragment from Monoclonal Antibody VILM31— Antibody VII-M31 (50 mg) was digested with pepsin at an enzyme/substrate ratio of 1/50 (w/w) at 37°C for 8 h, in a total of 17 ml of 0.1 M sodium citrate, pH 3.5. The reaction was terminated by extensive dialysis against 10 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl. The resulting F(ab)'2 fragment was partially reduced by incubating it with 2-mercaptoethanol (final 9 mM) at 37°C for 1 h under N2 gas. Then, S-amidomethylation was performed by addition of iodoacetamide (final 11 mM) in ice for 1 h under N2 gas. Excess iodoacetamide was removed by dialysis against 50 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl and 0.01% NaN3 (TBS-1). Effect of Monoclonal Antibody VII-M31 on the Activation of Factors IX and X by Vila- Tissue Factor Complex —Ninety microliters of TBS-1 containing 0.1 mg/ml BSA, 10 ix\ of factor Vila (7.1 nM), 10 /x\ of 0.1 M CaCl2, and 10 //I of various concentrations of antibody VII-M31 or its Fab' fragment were mixed and incubated at room temperature for 60 min. Then, 10 n 1 of rabbit brain cephalin and 10 y. 1 of tissue factor (79.4 nM) were added and incubation was continued at 37°C. After 5 min, 10 ^1 of factor X (22.4 /*M) was added and the reaction mixture was incubated at 37°C for 2 min. The reaction of factor X activation was terminated by addition of 30 ^1 of 0.1 M EDTA. Twenty-microliter aliquots of the reaction mixture were added to the substrate solution, which consisted of 600//lof TBS-1 containingO.l mg/ml BSA and 10^1 of 5mM iV-benzyloxycarbonylL- pyroglutamyl- Gly-Arg-4- methylcoumaryl- 7 - amide in Vol. 108, No. 4, 1990
iV.AT-dimethylformamide, and released 7-amino-4-methylcoumarin was measured fluorometrically with excitation at 380 nm and emission at 440 nm. The factor IX activation assay was carried out using an ester substrate, iV-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester (ZArg-ONb). Various concentrations of antibody VII-M31 or its Fab' fragment in a volume of 30 n 1 were mixed with 34 JLI\ of factor Vila (7.1 nM) and 10//1 of 0.1 M CaCl2, and incubated at room temperature. After 60 min, 276 //I of factor IX (6.36 pM), 40 /i\ of tissue factor (79.4 nM), and 10 //I of rabbit brain cephalin were added and incubation was continued at 37°C for 120 min, then the reaction was terminated by adding 5 0 n \ of 0.1 M EDTA. Forty microliter aliquots of the reaction mixture were added to 435 /xl of TBS-1 and the reaction was started by adding 25 //I of 80 mM Z-Arg-ONb in AT,iV-dimethylformamide. After incubation at 37°C for 30 min, the reaction was terminated by adding 100 jul of 20% acetic acid. The amount of the released p-nitrobenzyl alcohol was determined using reversed-phase HPLC. This new assay method for factor IXa will be published in elsewhere (manuscript in preparation). Effect of Monoclonal Antibody VII-M31 on the Esterase Activity of Vila-Tissue Factor Complex—Twenty-seven microliters of factor Vila (170 nM), 300//I of various concentrations of antibody VII-M31 or its Fab' fragment and 20 /* 1 of 0.1 M CaCl2 were mixed and incubated at room temperature. One hour later, 10 /^l of rabbit brain cephalin and 10 //I of tissue factor (79.4 nM) were added, and then 25jul of 80 mM Z-Arg-ONb was added. The esterase activity of Vila-tissue factor complex after incubation at 37°C for 20 min was measured by the same method as those used for factor IXa assay. Kinetic Properties of Monoclonal Antibody VII-M31 —The dissociation constants (IQ) for antibody VII-M31 to factor VII and its derivatives were determined according to the method of Kimoto using microtiter plates (19). Microtiter plates were coated with 2.5 pi g/well of factor VII or 1.25 jt/g/well of S-aminoethylated factor VII in 20 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl (TBS-2), and 5 mM CaCl2, or 62 pmol/well of an ELISA-positive fragment, K-31. Then, various concentrations of antibody VII-M31 in TBS-2 containing 1% BSA, 0.05% Tween 20, and 5 mM CaCl2 were added and incubated overnight at 4°C (A-plate) and at room temperature for 1 h (B-plate). The ELISA was done in the same way as those described under "Supplemental Materials." The method of Scatchard analysis (20) was used to determine the K^ value for antibody VII-M31 to factor VII or its derivatives. Immunoblotting—Using 10 ng each of samples, SDSPAGE was carried out according to the method of Laemmli (21). After electrophoresis, gels were transferred to nitrocellulose membranes by using a semidry electroblot apparatus (Trans-Blot, Bio-Rad) at 15 V for 30 min in 25 mM Tris-192mM glycine buffer, pH 8.3, containing 20% methanol. Then, the membranes were blocked with 20 mM Tris-HCl, pH7.5, containing 0.5 M NaCl (TBS-3), 5 mM CaCl2, and 1% (w/w) non-fat dry milk at room temperature for 60 min. The membranes were washed three times with TBS-3 containing 0.05% Tween 20 and incubated overnight with antibody VII-M31 (10.4//g/ml) in TBS-3 containing 1% non-fat dry milk and 5 mM CaCl2 at room temperature. After being washed three times, membranes were incubat-
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deficient plasma was prepared from bovine plasma by the method of Radcliffe and Nemerson (2). Bovine factor VII was purified from the eluate of barium citrate adsorption of bovine plasma by DEAE-Sepharose CL-6B column chromatography (5) and immuno-affinity chromatography on a column of Affi-Gel 10 covalently coupled to antibody VII-M31. The conditions of this final purification step are shown in Fig. 9. Other proteins, such as bovine prothrombin, factors IX and X and proteins S, Z, and C from bovine plasma were purified as described previously by Hashimoto et al. (8). Tissue factor was purified from bovine brain acetone powder using a modification of the method of Broze et al. (9). Protein concentrations were determined by absorbance measurements at 280 nm. Extinction coefficients (£2*0) and molecular weights used were, respectively: factor VH/VIIa, 12.9 and 54,000 (10, 11); factor IX, 14.9 and 55,400 {12, 13); factor X, 12.4 and 55,000 (14, 15); and murine IgG, 14.0 and 150,000 (16). As for tissue factor, the concentration was determined by the dyebinding method with BSA as the standard (17).
655
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RESULTS Screening of Monoclonal Antibodies—Hybridomas producing monoclonal antibodies against factor VII were isolated. After screening assay as described in "MATERIALS AND METHODS," two clones, named VII-M218 and VIIM31, were selected for further investigation. Of these clones, the purified antibody from clone VTI-M218 bound not only factor VII but also other vitamin K-dependent
1 2
3
plasma proteins including prothrombin, factor LX, factor X, protein C, protein S, and protein Z, as revealed by the immunoblotting method (data not shown). This result suggests that antibody VTI-M218 recognizes a common antigenic site in these proteins. Because of this conservative specificity, further investigation of antibody VH-M218 was not performed. On the other hand, the purified monoclonal antibody from clone VTI-M31 bound with only bovine factor VTI and did not cross-react with other vitamin K-dependent proteins, or human factor VII, as shown in Fig. 1, A and B. Interestingly, the antibody VLT-M31 had still the ability to bind factor VII after treatment with SDS and reduction with 2-mercaptoethanol. Moreover, this monoclonal antibody showed a single band on SDS-PAGE (data not shown) and was identified as IgGl by radial immunodiffusion for murine irnmunoglobulins. Based on these results, further characterization of the antibody VH-M31 was performed. Effects of Antibody VH-M.31 on the Activation of Factors X and IX by Vila- Tissue Factor Complex—In the screening assay of hybridoma, we found that antibody VH-M31
4 5 6 7 8
a E (£ 200
400
600
800
1000 1200
VII-M3i/vlla, mol/mol
30K100
B
20K—11 S
Z
C
XKtt/ll HVII
12
3
4
5 6
1 7 8
60
CD
B 75K50K39K27K17KFig. 1. Cross-reactivity of antibody VII-M31 with vitamin Kdependent plasma proteins. Prothrombin (II) and proteins S (S), C (C), and Z (Z), and factors X (X), and IX (LX), and VII (bVH) from bovine source and human factor VH (hVTI) were subjected to SDS-PAGE (A) and the protein bands were detected by immunoblotting (B) against antibody VII-M31.
I *° CD E S.
20 n 100 200 300 Vll-M31/Vlla, mol/mol
400
500
Fig. 2. Effect of antibody VII-M31 on the activations of factor X (A) and factor LX (B) by factor Vila-tissue factor complex. A: Factor X (220 pmol) was incubated with factor Vila (0.071 pmol), tissue factor (0.79 pmol) and various concentrations of antibody VTI-M31 in the presence of Ca1+ and phospholipids. The factor Xa activity generated was measured using N'-benzyloxycarbonyl-Lpyroglutamyl-Gly-Arg-4-methylcoumaryl-7-amide as a substrate. B: Factor K (1.75 nmol) was incubated with factor Vila (0.24 pmol), tissue factor (3.1 pmol) and various concentrations of antibody VTI-M31 in the presence of Ca1+ and phospholipids. The factor LX activity generated was measured using Z-Arg-ONb as a substrate. In each case, the activities of factor Xa and factor LXa generated in the absence of antibody VH-M31 were set as 100%. J. Biochem.
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ed at room tempature for 2 h with a 3,000-fold diluted horseradish peroxidase-conjugated goat anti-mouse IgG (H + L). After extensive washing, membranes were subjected to color development in TBS-3 containing 0.5 mg/ml 4-chloro-l-naphthol and 0.1% H 2 O 2 . Amino Acid and Amino-Terminal Sequence Analyses —Samples were hydrolyzed at 110'C for 20 h in 6 M HC1 in evacuated tubes. Following evaporation, hydrolysates were analyzed by a PICO TAG system (Waters, Millipore, Milford, MA), according to the method of Heinrikson and Meredith (22). Sequence analyses were performed using an Applied Biosystems 477A gas-phase sequencer. PTHderivatives were identified with an Applied Biosystems 120A PTH-analyzer with an on-line system, as described in detail previously (5).
657
A Specific Epitope in Bovine Factor VII 2.0
E
c o o>
Proteins, pmol/well
[metal ion], mM Fig. 3. Effect of divalent metal ions on the binding of antibody VII-M31 with factor VII. Factor VII (0.06 pmol/well) was coated with the microtiter plates and incubated with antibody VII-M31 (3.45 pmol/well) in the presence of various concentrations of CaClj (•), MgCli (O), and MnCl, (A). The amount of bound antibody was measured by the ELISA method, as described under "MATERIALS AND METHODS.'
Fig. 5. Binding of antibody VH-M31 with factor VH, Gladomain derived from factor VII, and Gla-domainless factor VII. Microtiter plates were coated with various concentrations of factor VTI (•), Gla-domain derived from factor VTI (O), and Gla-domainless factor VTI (A) and incubated with 10.4 pmol/well of antibody VTI-M31 in the presence of 5 mM CaCl2 at room temperature for 1 h. Binding of the antibody with each protein was monitored by the ELISA method.
VII " VIKGD) -
0
3 15 30
0 3 15 30
I
min—I
Lmin—*
Fig. 4. SDS-PAGE of a-chymotryptic digest of factor VII and its immunoblotting with antibody VII-M31. Factor VTI was digested with a-chymotrypsin at an enzyme/substrate ratio of 1/ 1,000 (w/w) at 20'C, and the aliquots at the indicated times were taken and subjected to SDS-PAGE (A) and to immunoblotting (B). VH, factor VH; VH (-GD), Gla-domainless factor VII.
inhibits the plasma clotting activity mediated by a VTIatissue factor complex. To investigate this property, we first examined the effect of antibody VTI-M31 on the activation of factor LX or factor X, both of which are natural substrates of Vila-tissue factor complex. The antibody VTI-M31 inhibited the activations of factor X (Fig. 2A) and factor LX (Fig. 2B) in a dose-dependent manner. The half-maximal inhibitions were observed in the presence of a 40- fold molar excess of antibody VTI-M31 to factor Vila for the factor IX activation, and an 80-fold molar excess of the antibody for the factor X activation. This antibody did not have any effect on the esterase activity of factor Vila toward Z-Arg-ONb. We also prepared the Fab' fragment derived from antibody VH-M31 and carried out similar inhibition experiments to examine the effect of the smaller Vol. 108, No. 4, 1990
2.0
B
E c 3 1.0
J ill
0
Peptlde No.
JUtitlU
Fig. 6. HFLC separation of lysyl endopeptidase digest derived from S-aminoethylated factor VH (A) and ELISA of the isolated peptide fragments (B). The lysyl endopeptidase digest of S-aminoethylated factor VII was applied to a Cosmosil 3Ci, column and eluted at aflowrate of 0.5 ml/min with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. The column eluate was monitored at 214 nm (solid line) and the broken line shows the percentage of acetonitrile in the elution medium. Major peaks after HPLC were subjected to ELISA in the presence of 5 mM CaCU.
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u.O
S. Higashi et al.
658
NH,-terminal sequences ofpeptides K-31 and K-43. K-43 K-31 Cycle PTH Yield PTH Yield No. amino acid a mi no acid (pmol) (pmol) Ala Ser 208 63 1 Asn 96 Phe 425 2 122 Gly ND' 3 ND 75 Phe 4 Ala 407 94 Leu 5 — 64 ND His 6 — ND ND 7 Leu 42 He 217 8 64 Phe 217 Leu 9 126 Pro 10 43 Arg Position 1-50 23-50 'ND, not detectable.
J.U —-—
„--—
A 2.0
E c o
0.5
O)
0.4
k s
N
2"
1.0
0]
Kd • 1.12llO'M (-)
/ ( nn
o. 08
0.0
0.2
0.09
0.4
0 10 B, nM
XN 0.11
0. 12
0.8
0.6
VII-M31,nM 2.0
B 1.8
TABLE II.
E c o
16• \
CD 1.4
•
.
u. 1.0
/
1.2 •
o.a
\
0.7 K
"tfo* 1.0 0.05
0.10
0.03
0.15
O.OS 0 . 0 7
O.OS O.OD
B, nM
0.20
0.25
VII-M31,nM
E c o O)
o
0
1
VII-M31, nM [metal ion], mM Fig. 7. Effect of divalent metal ions on the binding of antibody Vn-M31 with peptide K-31. K-31 (50 pmol/well) was coated with the microtiter plates and incubated with antibody VII-M31 (0.33 pmol/well) in the presence of various concentrations of CaCl, (•), MgCl, (O), and MnCl2 (*). The amount of bound antibody was measured by the ELISA method, as described under "MATERIALS AND METHODS.'
Fig. 8. Specific binding of antibody VII-M31 with factor VH (A), S-aminoethylated factor VII (B), or peptide K-31 (C) and their Scatchard analyses (insets). Factor VH (2.5/
