Structure-function relationships of C4b-binding protein
Eur. J. Immunol. 1991. 21: 2077-2085
Martin Hewing., Deon Kanters, Harry F. G. Heijnen., T h a n M. Hackeng, Jan J. Sixma and Bonno N. Bouma Department of Haematology, University Hospital Utrecht, Utrecht
2077
Structure-function studies on human C4b-binding protein using monoclonal antibodies* Human C4b-binding protein (C4BP) is a multimeric regulatory complement component interacting with vitamin K-dependent protein S and complement C4b. Using hybridoma technology, a panel of monoclonal antibodies (mAb) specific for intact human C4BP and its 160-kDa chymotryptic central core fragment were prepared to study the structure-function relationships of C4BP By Western blot analysis and competition experiments, four distinct groups of mAb were identified and mapped on the C4BP molecule. By rotary shadowing, spider-like images of C4BP-antibody complexes were obtained and immunoelectron microscopy provided some information on the stoichiometry of the antibody-C4BP interaction. Certain antibodies interacted with C4BP molecules only at a ratio of 1 : 1. Others formed complexes of two or more antibodies bound to homologous sites on the C4BP molecule. Using an enzyme-linked immunosorbent sandwich assay for the measurement of the complex formation between protein S and C4BP, mAb against the central core and the disulfide-linked p chain of C4BP were identified that inhibited the binding of protein S to C4BP In a binding assay using 1251-labeledC4BP and solid-phase C4b, the inhibitory effect of one group of anti-C4BP mAb on the binding of C4BP to C4b was demonstrated.
1 Introduction
of C4BP suggest that C4BP has a spider-like structure with seven elongated tentacles extending from a central, ringC4b-binding protein (C4BP) is a high-M, plasma protein like core [14, 151. Each of the a chains forms a tentacle with (concentration 200 pg/ml), which regulates the activation the N terminus located at the periphery of the tentacle, of the classical pathway of the C system [l-41. C4BP whereas the C termini are located in the central core of functions as a cofactor to factor I in the proteolytic C4BP [15-171. Multiple binding sites for C4b were located degradation of C4b and in addition accelerates the decay on the peripheral half of each tentacle [14], whereas one rate of the C4b2a complex 131. C4BP also interacts single binding site for protein S was localized near the noncovalently with one of the plasma proteins of the blood C-terminal disulfide-linked knot of the a chains [14, 181. In coagulation system, the vitamin K-dependent protein S 1988, Hillarp and Dahlback [19] demonstrated the exis[5].Protein S is an anticoagulant plasma protein function- tence of a new distinct disulfide-linked 45-kDa subunit (fi ing as a cofactor for activated protein C in the degradation chain) in C4BRwhich seemed to be important for protein S of coagulation factors Va and VIIIa [6-91. When C4BP binding. The p chain was sensitive to proteolysis by binds to protein S, it inhibits its anticoagulant cofactor chymotrypsin and, when cleaved, the protein S binding function [lo, 111.The binding of protein S to C4BP has no ability was lost. When occupied with protein S the p chain was protected from cleavage by chymotrypsin. The primary direct effect on the C4b-binding function of C4BP [12]. sequence of the p chain showed a high degree of similarity C4BP is composed of multiple, 70-kDa subunits ( a chains) with the a chain. The p chain consists of 235 amino acid linked by disulfide bridges and with identical N-terminal residues and the 175 N-terminal residues can be divided sequences [1, 5 , 131. Previous electron microscopic studies into three short consensus repeats (SCR) homologous to the eight SCR in the a chain of C4BP [20]. Recently, the existence of a subpopulation of C4BP molecules lacking (3 chain and protein S binding ability has been demonstrated [21,22]. In contrast, Suzuki and Nishioka [23] localized the [I 87721 proteins binding site on C4BP at a 2.5-kDa peptide * This work was supported by the Foundation for Medical corresponding to SeF7-Tyf17 located in the most Cterminal SCR unit of each a chain [24-261. Research (MEDIGON) grant no. 900-512-082.
*
Present address: Biotechnological Research Unit, Protein Chemistry Group, Organon Teknika, NL-5280 AB Boxtel, The Netherlands. Present address: Centre for Electron Microscopy, Medical Faculty. University of Utrecht, Utrecht, The Netherlands.
Correspondence: Martin Hessing, Biotechnological Research Unit, Protein Chemistry Group, Organon Teknika, Boseind 15, NL-5280 AB Boxtel, The Netherlands Abbreviation: C4BP: C4b-binding protein 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
Limited proteolysis of C4BP by chymotrypsin results in the cleavage of two peptide bonds, at Tyr395and T r ~ ~ ~ ~ , y i e l d i n g two major fragments: a 48-kDa fragment constituting the major portion of the extended tentacle and a 160-kDa fragment representing the central core of C4BP [15,16]. The C4b- and heparin-binding and factor I cofactor activity are located in the 48-kDa fragments [27,28] and Chung and Reid [24] tentatively assigned the C4b cofactor activity site for factor1 to residues 177-332 and the region for C4b
+
0014-2980/91/0909-2077$3.50 .25/0
2078
M. Hessing, D. Kanters, H. F. G . Heijnen et al.
binding to residues 332-395. The positioning of the C4bbinding site to the C-terminal half of the a chain is not compatible with the results obtained from the electron microscopy imagery of C4BP-C4b complexes [14, 151. The N-terminal end of the a' chain of C4b seems to be important for its interaction with C4BP [29, 301.
In the present study, we confirmed the localization of the protein S-binding site to the 160-kDa central core of C4BP and the C4b-binding sites on the 48-kDa arms, but in addition demonstrated the importance of the p chain for protein S binding. mAb against native C4BP and the central core were prepared and characterized by biochemical and electron microscopic methods. It was demonstrated that both mAb against the central core and mAb against the chain of C4BP were able to inhibit the binding of protein S to C4BP In addition, we found mAb that inhibited the binding of C4BP to C4b. Electron microscopy of various immune complexes permitted a preliminary localization of the relative positions of several epitopes of the mAb on the C4BP molecule.
Eur. J. Immunol. 1991. 21: 2077-2085 stopped by the addition of 3% (w/w) soybean trypsin inhibitor on ice [32]. C4 and C4b were separated on a mono Q ion-exchange column using FF'LC equipment and a linear gradient of NaCl(l50 to 500 mM NaCl, 20 ml). All proteins appeared to be homogeneous as judged by SDSPAGE. Protein concentrations were determined by measuring the absorbance at 280 nm. An absorbtivity ( l % , 1 cm) of 9.5, 14.1 and 14.3 was used for protein S [33], C4BP [171 and the purified mAb [34], respectively,whereas a value of 10.0 was assumed for the C4BP fragments. C4 antigen was measured with a nephelometer (Beckman, Palo Alto, CA) using the appropriate antiserum. Protein S and C4BP were radiolabeled with Na1251(Amersham Int., Amersham, GB) to a specific radioactivity of 1-7 pCi/yg (= 37 kBq-2.59 MBq/yg) using the Iodogen or Iodobead procedure (Pierce Chemical Co., Rockford, IL). Radiolabeled protein S and C4BP were gel filtered on a PD-10 disposable column (Pharmacia) equilibrated with 50 mM Tris, pH 7.4, 150 mM NaCl, in order to remove free iodine.
2.4 Isolation of C4BP fragments
2 Materials and methods 2.1 Materials Protein G-Sepharose 4W, CNBr-activated Sepharose, a mono Q, and a Superose 12 column and prestained molecular weight markers were obtained from Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden). All other chemicals obtained were of the best grade available.
2.2 Antisera Rabbit antiserum to protein S was prepared as described [31]. Affinity-purified rabbit antibodies directed against protein S were purified on a protein S-Sepharose column using standard procedures. Polyclonal antibodies for C4BP were obtained by multiple S.C.injections of purified C4BP in CFA in rabbits. The antiserum was judged monospecific by double immunodiffusion analysis and immunoblotting using purified C4BP and normal plasma. 2.3 Proteins
All proteins were of human origin. Protein S was purified as described [9]. C4BP was purified from a barium citrate precipitate of human plasma using anti-C4BP mAb affinity chromatography [22]. C4BP and C4BP-protein S complexes were separated with mono Q ion-exchange chromatography in 50 mM Tris-HC1, 100 mM NaCl, 2 mM EDTA, 0.02% NaN3, pH 7.4, and a linear gradient of NaCl(O.1 to 0.5 M) using fast protein liquid chromatography (FPLC) equipment. In some cases, C4BP was precipitated with 5% (w/v) PEG 6000, dissolved in 3 M guanidinium hydrochloride, S 0 m ~Tris-HC1, pH7.4, and gel filtered on a Superose 12 column (1.6 x SO cm, 15 mVh) equilibrated in the same buffer using FPLC equipment to remove bound protein S. Complement C4 was obtained from fresh frozen citrated plasma [29]. C4b was obtained by incubating C4 with 1% (w/w) trypsin for 1 min at 37 "C. The reaction was
Limited proteolysis of C4BP by chymotrypsin was essentially performed according to Hillarp and Dahlback [ 181. C4BP was incubated with chymotrypsin (7.5%, w/w, 2 h, 37 "C) in the presence of a 4-fold molar excess (as compared to C4BP) of human protein S to protect the protein Sbinding site. Protein S and C4BP were preincubated overnight at 37°C prior to the chymotrypsin digestion. The digestion was stopped by diisopropyl fluorophosphate (1 m ~ and ) the cleavage products were precipitated with 60% saturation of (NH&S04. The precipitate was dissolved in 3 M guanidinium hydrochloride, 50 mM Tris, pH 7.4, 1 mM PMSF and subsequently applied to a Superose 12 column (1.6 x 50 cm, 15 mlh) equilibrated in the same buffer using FPLC equipment. The fractions containing the 160-kDa central core were pooled according to the pattern on 3%-18% gradient SDS-PAGE (not shown).
2.5 Electrophoretic and immunochemical techniques SDS-PAGE was performed according to Laemmli [35].The gels were stained with Coomassie brilliant blue R-250 or with silver [36]. Reduction was performed by incubating C4BP for 5 min at 90°C with 5% (v/v) 2-ME, 10mM Tris-HC1, 3% (w/v) SDS, 0.01% (w/v) bromophenolblue, 10% (v/v) glycerol, pH 6.8.Western blotting was essentially performed as described [37] using a semi-dry electrophoretic transfer apparatus (Biolyon, Paris, France). Proteins transferred to Immobilon membranes (Millipore, Molsheim, France) were either stained with Coomassie blue or blocked with 50 mM Tris-HCI, pH 7.4, 150 mM NaCl, 5% nonfat dry milk [38], incubated in a Miniblotter 25 (Immunetics, Cambridge, MA) with the mAb and visualized with peroxidase-conjugated rabbit anti-mouse IgG (Dako, Glostrup, Denmark).
2.6 Production of mAb BALB/c mice were immunized by S.C.injection of 10 p.g of purified intact C4BP or its chymotryptic 160-kDa central
Eur. J. Immunol. 1991. 21: 2077-2085
Structure-function relationships of C4b-binding protein
core fragment in CFA and after 2 , 4 and 6 weeks they were boosted with the same amount of antigen in IFA. Three days after the final injection, spleen cells were fused with Ag8.653 myeloma cells. Fusion and hybridoma selection was performed according to standard procedures. Culture SN were screened for the presence of specific antibodies by ELISA or Western blotting, in which intact C4Be reduced C4BP or the isolated central core were used as antigen. Bound antibodies were detected with peroxidase-conjugated rabbit antibodies against mouse IgG. The cells yielding positive SN were selected and grown in protein Sand C4BP-deficient culture medium to avoid the interference of human and bovine proteins and C4BP in the '251-labeled protein S (1251-proteinS) binding assay. Protein S and C4BP deficient medium was prepared essentially as described [9]. Using ELISA for C4BP and protein S no detectable protein S and C4BP antigen (
Eur. J. Immunol. 1991. 21: 2077-2085
Martin Hewing., Deon Kanters, Harry F. G. Heijnen., T h a n M. Hackeng, Jan J. Sixma and Bonno N. Bouma Department of Haematology, University Hospital Utrecht, Utrecht
2077
Structure-function studies on human C4b-binding protein using monoclonal antibodies* Human C4b-binding protein (C4BP) is a multimeric regulatory complement component interacting with vitamin K-dependent protein S and complement C4b. Using hybridoma technology, a panel of monoclonal antibodies (mAb) specific for intact human C4BP and its 160-kDa chymotryptic central core fragment were prepared to study the structure-function relationships of C4BP By Western blot analysis and competition experiments, four distinct groups of mAb were identified and mapped on the C4BP molecule. By rotary shadowing, spider-like images of C4BP-antibody complexes were obtained and immunoelectron microscopy provided some information on the stoichiometry of the antibody-C4BP interaction. Certain antibodies interacted with C4BP molecules only at a ratio of 1 : 1. Others formed complexes of two or more antibodies bound to homologous sites on the C4BP molecule. Using an enzyme-linked immunosorbent sandwich assay for the measurement of the complex formation between protein S and C4BP, mAb against the central core and the disulfide-linked p chain of C4BP were identified that inhibited the binding of protein S to C4BP In a binding assay using 1251-labeledC4BP and solid-phase C4b, the inhibitory effect of one group of anti-C4BP mAb on the binding of C4BP to C4b was demonstrated.
1 Introduction
of C4BP suggest that C4BP has a spider-like structure with seven elongated tentacles extending from a central, ringC4b-binding protein (C4BP) is a high-M, plasma protein like core [14, 151. Each of the a chains forms a tentacle with (concentration 200 pg/ml), which regulates the activation the N terminus located at the periphery of the tentacle, of the classical pathway of the C system [l-41. C4BP whereas the C termini are located in the central core of functions as a cofactor to factor I in the proteolytic C4BP [15-171. Multiple binding sites for C4b were located degradation of C4b and in addition accelerates the decay on the peripheral half of each tentacle [14], whereas one rate of the C4b2a complex 131. C4BP also interacts single binding site for protein S was localized near the noncovalently with one of the plasma proteins of the blood C-terminal disulfide-linked knot of the a chains [14, 181. In coagulation system, the vitamin K-dependent protein S 1988, Hillarp and Dahlback [19] demonstrated the exis[5].Protein S is an anticoagulant plasma protein function- tence of a new distinct disulfide-linked 45-kDa subunit (fi ing as a cofactor for activated protein C in the degradation chain) in C4BRwhich seemed to be important for protein S of coagulation factors Va and VIIIa [6-91. When C4BP binding. The p chain was sensitive to proteolysis by binds to protein S, it inhibits its anticoagulant cofactor chymotrypsin and, when cleaved, the protein S binding function [lo, 111.The binding of protein S to C4BP has no ability was lost. When occupied with protein S the p chain was protected from cleavage by chymotrypsin. The primary direct effect on the C4b-binding function of C4BP [12]. sequence of the p chain showed a high degree of similarity C4BP is composed of multiple, 70-kDa subunits ( a chains) with the a chain. The p chain consists of 235 amino acid linked by disulfide bridges and with identical N-terminal residues and the 175 N-terminal residues can be divided sequences [1, 5 , 131. Previous electron microscopic studies into three short consensus repeats (SCR) homologous to the eight SCR in the a chain of C4BP [20]. Recently, the existence of a subpopulation of C4BP molecules lacking (3 chain and protein S binding ability has been demonstrated [21,22]. In contrast, Suzuki and Nishioka [23] localized the [I 87721 proteins binding site on C4BP at a 2.5-kDa peptide * This work was supported by the Foundation for Medical corresponding to SeF7-Tyf17 located in the most Cterminal SCR unit of each a chain [24-261. Research (MEDIGON) grant no. 900-512-082.
*
Present address: Biotechnological Research Unit, Protein Chemistry Group, Organon Teknika, NL-5280 AB Boxtel, The Netherlands. Present address: Centre for Electron Microscopy, Medical Faculty. University of Utrecht, Utrecht, The Netherlands.
Correspondence: Martin Hessing, Biotechnological Research Unit, Protein Chemistry Group, Organon Teknika, Boseind 15, NL-5280 AB Boxtel, The Netherlands Abbreviation: C4BP: C4b-binding protein 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
Limited proteolysis of C4BP by chymotrypsin results in the cleavage of two peptide bonds, at Tyr395and T r ~ ~ ~ ~ , y i e l d i n g two major fragments: a 48-kDa fragment constituting the major portion of the extended tentacle and a 160-kDa fragment representing the central core of C4BP [15,16]. The C4b- and heparin-binding and factor I cofactor activity are located in the 48-kDa fragments [27,28] and Chung and Reid [24] tentatively assigned the C4b cofactor activity site for factor1 to residues 177-332 and the region for C4b
+
0014-2980/91/0909-2077$3.50 .25/0
2078
M. Hessing, D. Kanters, H. F. G . Heijnen et al.
binding to residues 332-395. The positioning of the C4bbinding site to the C-terminal half of the a chain is not compatible with the results obtained from the electron microscopy imagery of C4BP-C4b complexes [14, 151. The N-terminal end of the a' chain of C4b seems to be important for its interaction with C4BP [29, 301.
In the present study, we confirmed the localization of the protein S-binding site to the 160-kDa central core of C4BP and the C4b-binding sites on the 48-kDa arms, but in addition demonstrated the importance of the p chain for protein S binding. mAb against native C4BP and the central core were prepared and characterized by biochemical and electron microscopic methods. It was demonstrated that both mAb against the central core and mAb against the chain of C4BP were able to inhibit the binding of protein S to C4BP In addition, we found mAb that inhibited the binding of C4BP to C4b. Electron microscopy of various immune complexes permitted a preliminary localization of the relative positions of several epitopes of the mAb on the C4BP molecule.
Eur. J. Immunol. 1991. 21: 2077-2085 stopped by the addition of 3% (w/w) soybean trypsin inhibitor on ice [32]. C4 and C4b were separated on a mono Q ion-exchange column using FF'LC equipment and a linear gradient of NaCl(l50 to 500 mM NaCl, 20 ml). All proteins appeared to be homogeneous as judged by SDSPAGE. Protein concentrations were determined by measuring the absorbance at 280 nm. An absorbtivity ( l % , 1 cm) of 9.5, 14.1 and 14.3 was used for protein S [33], C4BP [171 and the purified mAb [34], respectively,whereas a value of 10.0 was assumed for the C4BP fragments. C4 antigen was measured with a nephelometer (Beckman, Palo Alto, CA) using the appropriate antiserum. Protein S and C4BP were radiolabeled with Na1251(Amersham Int., Amersham, GB) to a specific radioactivity of 1-7 pCi/yg (= 37 kBq-2.59 MBq/yg) using the Iodogen or Iodobead procedure (Pierce Chemical Co., Rockford, IL). Radiolabeled protein S and C4BP were gel filtered on a PD-10 disposable column (Pharmacia) equilibrated with 50 mM Tris, pH 7.4, 150 mM NaCl, in order to remove free iodine.
2.4 Isolation of C4BP fragments
2 Materials and methods 2.1 Materials Protein G-Sepharose 4W, CNBr-activated Sepharose, a mono Q, and a Superose 12 column and prestained molecular weight markers were obtained from Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden). All other chemicals obtained were of the best grade available.
2.2 Antisera Rabbit antiserum to protein S was prepared as described [31]. Affinity-purified rabbit antibodies directed against protein S were purified on a protein S-Sepharose column using standard procedures. Polyclonal antibodies for C4BP were obtained by multiple S.C.injections of purified C4BP in CFA in rabbits. The antiserum was judged monospecific by double immunodiffusion analysis and immunoblotting using purified C4BP and normal plasma. 2.3 Proteins
All proteins were of human origin. Protein S was purified as described [9]. C4BP was purified from a barium citrate precipitate of human plasma using anti-C4BP mAb affinity chromatography [22]. C4BP and C4BP-protein S complexes were separated with mono Q ion-exchange chromatography in 50 mM Tris-HC1, 100 mM NaCl, 2 mM EDTA, 0.02% NaN3, pH 7.4, and a linear gradient of NaCl(O.1 to 0.5 M) using fast protein liquid chromatography (FPLC) equipment. In some cases, C4BP was precipitated with 5% (w/v) PEG 6000, dissolved in 3 M guanidinium hydrochloride, S 0 m ~Tris-HC1, pH7.4, and gel filtered on a Superose 12 column (1.6 x SO cm, 15 mVh) equilibrated in the same buffer using FPLC equipment to remove bound protein S. Complement C4 was obtained from fresh frozen citrated plasma [29]. C4b was obtained by incubating C4 with 1% (w/w) trypsin for 1 min at 37 "C. The reaction was
Limited proteolysis of C4BP by chymotrypsin was essentially performed according to Hillarp and Dahlback [ 181. C4BP was incubated with chymotrypsin (7.5%, w/w, 2 h, 37 "C) in the presence of a 4-fold molar excess (as compared to C4BP) of human protein S to protect the protein Sbinding site. Protein S and C4BP were preincubated overnight at 37°C prior to the chymotrypsin digestion. The digestion was stopped by diisopropyl fluorophosphate (1 m ~ and ) the cleavage products were precipitated with 60% saturation of (NH&S04. The precipitate was dissolved in 3 M guanidinium hydrochloride, 50 mM Tris, pH 7.4, 1 mM PMSF and subsequently applied to a Superose 12 column (1.6 x 50 cm, 15 mlh) equilibrated in the same buffer using FPLC equipment. The fractions containing the 160-kDa central core were pooled according to the pattern on 3%-18% gradient SDS-PAGE (not shown).
2.5 Electrophoretic and immunochemical techniques SDS-PAGE was performed according to Laemmli [35].The gels were stained with Coomassie brilliant blue R-250 or with silver [36]. Reduction was performed by incubating C4BP for 5 min at 90°C with 5% (v/v) 2-ME, 10mM Tris-HC1, 3% (w/v) SDS, 0.01% (w/v) bromophenolblue, 10% (v/v) glycerol, pH 6.8.Western blotting was essentially performed as described [37] using a semi-dry electrophoretic transfer apparatus (Biolyon, Paris, France). Proteins transferred to Immobilon membranes (Millipore, Molsheim, France) were either stained with Coomassie blue or blocked with 50 mM Tris-HCI, pH 7.4, 150 mM NaCl, 5% nonfat dry milk [38], incubated in a Miniblotter 25 (Immunetics, Cambridge, MA) with the mAb and visualized with peroxidase-conjugated rabbit anti-mouse IgG (Dako, Glostrup, Denmark).
2.6 Production of mAb BALB/c mice were immunized by S.C.injection of 10 p.g of purified intact C4BP or its chymotryptic 160-kDa central
Eur. J. Immunol. 1991. 21: 2077-2085
Structure-function relationships of C4b-binding protein
core fragment in CFA and after 2 , 4 and 6 weeks they were boosted with the same amount of antigen in IFA. Three days after the final injection, spleen cells were fused with Ag8.653 myeloma cells. Fusion and hybridoma selection was performed according to standard procedures. Culture SN were screened for the presence of specific antibodies by ELISA or Western blotting, in which intact C4Be reduced C4BP or the isolated central core were used as antigen. Bound antibodies were detected with peroxidase-conjugated rabbit antibodies against mouse IgG. The cells yielding positive SN were selected and grown in protein Sand C4BP-deficient culture medium to avoid the interference of human and bovine proteins and C4BP in the '251-labeled protein S (1251-proteinS) binding assay. Protein S and C4BP deficient medium was prepared essentially as described [9]. Using ELISA for C4BP and protein S no detectable protein S and C4BP antigen (
