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• W2109672844 abstract "Human C4b-binding protein (C4BP) is a regulator of the complement system and plays an important role in the regulation of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for activated protein C. C4BP is a multimeric protein containing several identical α-chains and a single β-chain (C4BPβ), each chain being composed of short consensus repeats (SCRs). Previous studies have localized the protein S binding site to the NH2-terminal SCR (SCR-1) of C4BPβ. To further localize the protein S binding site, we constructed chimeras containing C4BPβ SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to tissue-type plasminogen activator. Binding assays of protein S with these chimeras indicated that SCR-2 contributes to the interaction of protein S with SCR-1, since the affinity of protein S for SCR-1+2 was up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay that measures protein S cofactor activity, we showed that cofactor activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no additional effect on SCR-1, and therefore the effect of SCR-2 was specific. In conclusion, β-chain SCR-2 contributes to the interaction of C4BP with protein S. Human C4b-binding protein (C4BP) is a regulator of the complement system and plays an important role in the regulation of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for activated protein C. C4BP is a multimeric protein containing several identical α-chains and a single β-chain (C4BPβ), each chain being composed of short consensus repeats (SCRs). Previous studies have localized the protein S binding site to the NH2-terminal SCR (SCR-1) of C4BPβ. To further localize the protein S binding site, we constructed chimeras containing C4BPβ SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to tissue-type plasminogen activator. Binding assays of protein S with these chimeras indicated that SCR-2 contributes to the interaction of protein S with SCR-1, since the affinity of protein S for SCR-1+2 was up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay that measures protein S cofactor activity, we showed that cofactor activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no additional effect on SCR-1, and therefore the effect of SCR-2 was specific. In conclusion, β-chain SCR-2 contributes to the interaction of C4BP with protein S. C4b-binding protein (C4BP) 1The abbreviations used are: C4BP, complement C4b-binding protein; C4BPβ, C4BP β-chain; APC, activated protein C; SCR, short consensus repeat; tPA, tissue plasminogen activator; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline. 1The abbreviations used are: C4BP, complement C4b-binding protein; C4BPβ, C4BP β-chain; APC, activated protein C; SCR, short consensus repeat; tPA, tissue plasminogen activator; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline. is an important regulator of the complement system (1Gigli I. Fujita T. Nussenzweig V. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6596-6600Crossref PubMed Scopus (250) Google Scholar, 2Fujita T. Nussenzweig V. J. Exp. Med. 1979; 150: 267-276Crossref PubMed Scopus (103) Google Scholar, 3Fujita T. Gigli I. Nussenzweig V. J. Exp. Med. 1978; 148: 1044-1051Crossref PubMed Scopus (193) Google Scholar, 4Nussenzweig V.J. Melton R. Methods Enzymol. 1981; 80: 124-133Crossref Scopus (11) Google Scholar, 5Scharfstein J. Ferreira A. Gigli I. Nussenzweig V. J. Exp. Med. 1978; 148: 207-222Crossref PubMed Scopus (212) Google Scholar). It accelerates C2a decay from the classical pathway C3-convertase (C4b2a) complex (1Gigli I. Fujita T. Nussenzweig V. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6596-6600Crossref PubMed Scopus (250) Google Scholar, 6Daha M.R. van Es L.A. J. Immunol. 1980; 125: 2051-2054PubMed Google Scholar) and promotes factor I-mediated degradation of C4b (1Gigli I. Fujita T. Nussenzweig V. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6596-6600Crossref PubMed Scopus (250) Google Scholar, 2Fujita T. Nussenzweig V. J. Exp. Med. 1979; 150: 267-276Crossref PubMed Scopus (103) Google Scholar, 3Fujita T. Gigli I. Nussenzweig V. J. Exp. Med. 1978; 148: 1044-1051Crossref PubMed Scopus (193) Google Scholar, 7Fujita T. Tamura N. J. Exp. Med. 1983; 157: 1239-1251Crossref PubMed Scopus (28) Google Scholar). C4BP also has a high affinity for anticoagulant vitamin K-dependent protein S, and together they form a noncovalent 1:1 stoichiometric complex (8Dahlbäck B. Thromb. Haemostasis. 1991; 66: 49-61Crossref PubMed Scopus (323) Google Scholar, 9Dahlbäck B. Frohm B. Nelsestuen G. J. Biol. Chem. 1990; 265: 16082-16087Abstract Full Text PDF PubMed Google Scholar, 10Dahlbäck B. Biochem. J. 1983; 209: 847-856Crossref PubMed Scopus (216) Google Scholar, 11Dahlbäck B. Stenflo J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2512-2516Crossref PubMed Scopus (296) Google Scholar). Binding of protein S to C4BP results in a decreased cofactor function of protein S for anticoagulant activated protein C (APC) in the degradation of coagulation factors Va and VIIIa (12Dahlbäck B. J. Biol. Chem. 1986; 261: 12022-12027Abstract Full Text PDF PubMed Google Scholar, 13Bertina R.M. van Wijngaarden A. Reinalda Poot J. Poort S.R. Bom V.J. Thromb. Haemostasis. 1985; 53: 268-272Crossref PubMed Scopus (97) Google Scholar, 14Comp P.C. Esmon C.T. New Engl. J. Med. 1984; 311: 1525-1528Crossref PubMed Scopus (441) Google Scholar). Complex formation between protein S and C4BP has no effect on the inhibition of complement activation. C4BP is a multimeric glycoprotein (M r 530,000–570,000), composed of six or seven identical α-chains, and approximately 80–85% of C4BP contains an additional single β-chain that binds protein S (8Dahlbäck B. Thromb. Haemostasis. 1991; 66: 49-61Crossref PubMed Scopus (323) Google Scholar, 15Griffin J.H. Gruber A. Fernández J.A. Blood. 1992; 79: 3203-3211Crossref PubMed Google Scholar, 16Hillarp A. Hessing M. Dahlbäck B. FEBS Lett. 1989; 259: 53-56Crossref PubMed Scopus (72) Google Scholar). In their COOH-terminal regions, the α- and β-chains contain cysteine residues that form the interchain disulfide bridges in the so-called core region (17Hillarp A. Dahlbäck B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1183-1187Crossref PubMed Scopus (72) Google Scholar). In electron microscopy studies, C4BP has an octopus-like appearance (18Dahlbäck B. Smith C.A. Müller-Eberhard H.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3461-3465Crossref PubMed Scopus (177) Google Scholar, 19Dahlbäck B. Müller-Eberhard H.J. J. Biol. Chem. 1984; 259: 11631-11634Abstract Full Text PDF PubMed Google Scholar, 20Hessing M. Kanters D. Heijnen H.F. Hackeng T.M. Sixma J.J. Bouma B.N. Eur. J. Immunol. 1991; 21: 2077-2085Crossref PubMed Scopus (10) Google Scholar). Under normal conditions, approximately 60% of total protein S is bound to C4BP, and 40% is free (21Garcı́a de Frutos P. Alim R.I. Härdig Y. Zöller B. Dahlbäck B. Blood. 1994; 84: 815-822Crossref PubMed Google Scholar). During an acute phase response, C4BP levels can increase up to 4-fold. Due to a mechanism of differential regulation of α- and β-chain expression, an increase of α-chains predominates during such an acute phase response, and hence free protein S is held at stable levels (21Garcı́a de Frutos P. Alim R.I. Härdig Y. Zöller B. Dahlbäck B. Blood. 1994; 84: 815-822Crossref PubMed Google Scholar). The α-chains (M r 70,000) are composed of eight homologous domains called short consensus repeats (SCRs) (17Hillarp A. Dahlbäck B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1183-1187Crossref PubMed Scopus (72) Google Scholar, 22Chung L.P. Bentley D.R. Reid K.B.M. Biochem. J. 1985; 230: 133-141Crossref PubMed Scopus (111) Google Scholar). SCRs are commonly found structures in complement regulatory proteins such as factor H and decay acceleration factor, in which the SCR units have complement C3b/C4b binding properties (23Reid K.B.M. Bentley D.R. Campbell R.D. Chung L.P. Sim R.B. Kristensen T. Tack B.F. Immunol. Today. 1986; 7: 230-234Abstract Full Text PDF PubMed Scopus (187) Google Scholar). However, noncomplement regulatory proteins have also been found containing SCR units such as β2 glycoprotein I and the β-subunit of coagulation factor XIII, in which the function of the SCR units are unknown (for reviews, see Refs. 23Reid K.B.M. Bentley D.R. Campbell R.D. Chung L.P. Sim R.B. Kristensen T. Tack B.F. Immunol. Today. 1986; 7: 230-234Abstract Full Text PDF PubMed Scopus (187) Google Scholar and 24Reid K.B.M. Day A.J. Immunol. Today. 1989; 10: 177-180Abstract Full Text PDF PubMed Scopus (264) Google Scholar). The β-chain (M r 45,000) is composed of three SCR units, and previous studies have shown the protein S binding site to be localized within the NH2-terminal SCR unit (SCR-1) of the β-chain (25Fernández J.A. Villoutreix B.O. Hackeng T.M. Griffin J.H. Bouma B.N. Biochemistry. 1994; 33: 11073-11078Crossref PubMed Scopus (21) Google Scholar, 26Fernández J.A. Griffin J.H. J. Biol. Chem. 1994; 269: 2535-2540Abstract Full Text PDF PubMed Google Scholar, 27Härdig Y. Rezaie A. Dahlbäck B. J. Biol. Chem. 1993; 268: 3033-3036Abstract Full Text PDF PubMed Google Scholar, 28Härdig Y. Dahlbäck B. J. Biol. Chem. 1996; 271: 20861-20867Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In this study, recombinant chimeras were constructed composed of each individual β-chain SCR unit and combinations of SCR units (SCR-1+2, SCR-1+3, and SCR-2+3) fused to the NH2 terminus of a modified tissue plasminogen activator (tPA) in which the serine residue was replaced by an alanine residue (29Meijers J.C.M. Mulvihill E.R. Davie E.W. Chung D.W. Biochemistry. 1992; 31: 4680-4684Crossref PubMed Scopus (73) Google Scholar). This inactive tPA module is well characterized and has been proven in previous studies to be a useful tool to investigate the function of protein domains (29Meijers J.C.M. Mulvihill E.R. Davie E.W. Chung D.W. Biochemistry. 1992; 31: 4680-4684Crossref PubMed Scopus (73) Google Scholar, 30Johanessen M. Diness V. Pingel K. Petersen L.C. Rao D. Lioubin P. O'Hara P. Mulvihill E. Thromb. Haemostasis. 1990; 63: 54-59Crossref PubMed Scopus (24) Google Scholar, 31Herwald H. Renne T. Meijers J.C.M. Chung D.W. Page J.D. Colman R.W. Müller-Esterl W. J. Biol. Chem. 1996; 271: 13061-13067Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The aim of this study was to investigate the role of each individual β-chain SCR unit in the interaction between protein S and C4BP. Studies using chimeric SCR-tPA constructs show that SCR-2 of C4BP β-chain is involved in the interaction of protein S with SCR-1. C4BP was immunopurified from human plasma as described by Hessing et al. (32Hessing M. Kanters D. Hackeng T.M. Bouma B.N. Thromb. Haemostasis. 1990; 64: 245-250Crossref PubMed Scopus (26) Google Scholar). Protein C was purified and activated as described previously (33Koedam J.A. Meijers J.C.M. Sixma J.J. Bouma B.N. J. Clin. Invest. 1988; 82: 1236-1243Crossref PubMed Scopus (191) Google Scholar). Protein S was purified from prothrombin concentrates as described by Hackeng et al.(34Hackeng T.M. Hessing M. van 't Veer C. Meijer-Huizinga F. Meijers J.C.M. de Groot P.G. van Mourik J.A. Bouma B.N. J. Biol. Chem. 1993; 268: 3993-4000Abstract Full Text PDF PubMed Google Scholar). Each individual β-chain SCR unit and adjacent SCR units (SCR-1+2 and SCR-2+3) were amplified using wild type recombinant C4BP β-chain. This construct was made by PCR amplification from a human liver cDNA library using oligonucleotides C4BPβF (5′-TTTGAATTCTGGGGAGAGGACTTTGATCAC-3′) and C4BPβR (5′-TTTGAATTCTATTACATCTGCTCAGCTGTA-3′). After amplification, the PCR product was cleaved with EcoRI (underlined) and cloned in EcoRI-cleaved expression vector pcDNA3 (Invitrogen, Leek, The Netherlands). The sequence and orientation of the amplified region of this construct was confirmed by dideoxy sequencing. This construct was designated pcDNA3-C4BPβ. PCR strategies for amplification of SCR units from pcDNA3-C4BPβ were based on the intron/exon organization of the C4BPβ gene as described (35Hillarp A. Pardo-Manuel F. Ruiz R.R. Rodrı́guez de Córdoba S. Dahlbäck B. J. Biol. Chem. 1993; 268: 15017-15023Abstract Full Text PDF PubMed Google Scholar) with the primers depicted in Table I. For the amplification of SCR-1+3, a modified C4BPβ was used that lacked SCR-2 (pcDNA3-C4BPβΔSCR-2 in Table I) that will be described elsewhere. 2R. H. L. van de Poel, J. C. M. Meijers, and B. N. Bouma, manuscript in preparation. After amplification, PCR products were cleaved withBglII/XhoI and cloned inBglII/XhoI-cleaved expression vector ZpL7 containing a modified tPA (30Johanessen M. Diness V. Pingel K. Petersen L.C. Rao D. Lioubin P. O'Hara P. Mulvihill E. Thromb. Haemostasis. 1990; 63: 54-59Crossref PubMed Scopus (24) Google Scholar). These chimeric SCR-tPA constructs were designated SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3 and SCR-2+3, respectively. The sequence of the amplified regions of all constructs was confirmed by dideoxy sequencing.Table IOligonucleotide sequences used for PCR amplificationThe template used for each PCR amplification is shown at the bottom of the table. Wild type recombinant C4BPβ is depicted as pcDNA3-C4BPβ. Recombinant C4BPβ lacking the second NH2-terminal SCR unit is depicted as pcDNA3-C4BPβΔSCR-2. In the left column, forward primers are denoted by the letter F, and backward primers are denoted by the letter R. In the second column, nucleotide sequences of the primers are shown. Boldface, BglII restriction site. Underlined,XhoI restriction site. On top of the six outmost right columns, the different β-chain SCR unit chimeras are depicted by numbers equal to the corresponding β-chain SCR units contained within the chimeras. Combinations of template DNA and primers used for amplification of the different chimeras are indicated by dots. Open table in a new tab The template used for each PCR amplification is shown at the bottom of the table. Wild type recombinant C4BPβ is depicted as pcDNA3-C4BPβ. Recombinant C4BPβ lacking the second NH2-terminal SCR unit is depicted as pcDNA3-C4BPβΔSCR-2. In the left column, forward primers are denoted by the letter F, and backward primers are denoted by the letter R. In the second column, nucleotide sequences of the primers are shown. Boldface, BglII restriction site. Underlined,XhoI restriction site. On top of the six outmost right columns, the different β-chain SCR unit chimeras are depicted by numbers equal to the corresponding β-chain SCR units contained within the chimeras. Combinations of template DNA and primers used for amplification of the different chimeras are indicated by dots. Transfection of baby hamster kidney cells was performed as described previously (36Meijers J.C.M. Davie E.W. Chung D.W. Blood. 1992; 79: 1435-1440Crossref PubMed Google Scholar). Expression of all constructs was performed in conditioned medium (CHO-II-SFM; Life Technologies, Inc., Paisley, U.K.), and harvested medium was stored at −20 °C until needed for further use. Purification of chimeric SCR-tPA constructs was performed as described previously (29Meijers J.C.M. Mulvihill E.R. Davie E.W. Chung D.W. Biochemistry. 1992; 31: 4680-4684Crossref PubMed Scopus (73) Google Scholar) using a monoclonal antibody against tPA. Concentrations of chimeric SCR-tPA constructs were determined using an ELISA system that determines tPA concentration (ImulyseTM tPA; Biopool, Umeå, Sweden). Purified constructs were applied to 10% SDS-PAGE under reducing and nonreducing conditions and stained by Coomassie Brilliant Blue or transferred to polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) for standard Western blotting procedures using sheep anti-tPA antibodies (1 μg/ml, Enzyme Research Laboratories Inc.) followed by a polyclonal peroxidase-conjugated antibody against sheep antibodies (Dako, Glostrup, Denmark). The binding of protein S to immobilized chimeric SCR-tPA constructs was performed as follows. Microtiter plates (96-well vinyl assay plates; catalog no. 2595, Costar, Cambridge, MA) were coated overnight at 4 °C with a polyclonal antibody against tissue plasminogen activator (5 μg/ml; ImulyseTM-tPA, Biopool AB) in coat buffer (15 mm Na2CO3.10 H2O, 35 mm NaHCO3, pH 9.6), 50 μl/well. Plates were washed three times with Tris-buffered saline (TBS; 50 mm Tris-HCl, pH 7.4, 150 mm NaCl) containing 0.1% (v/v) Tween 20. Plates were blocked for 2 h at 37 °C with TBS containing 3% (w/v) bovine serum albumin (blocking buffer), 100 μl/well. CHO-II-SFM supernatant derived from baby hamster kidney cells transfected with the constructs described above was then added to the wells and incubated for 2 h at 37 °C in blocking buffer containing 0.1% Tween 20, 50 μl/well. After washing three times with TBS containing 0.1% Tween 20, increasing concentrations of protein S were added to the wells and incubated for 2 h at 37 °C in blocking buffer containing 0.1% Tween 20 and 5 mm CaCl2, 50 μl/well. Plates were washed three times with TBS containing 0.1% Tween 20, and bound protein S was detected using a polyclonal peroxidase-conjugated antibody against protein S (1.3 g/liter IgG; Dako), 1:2000 diluted in blocking buffer containing 0.1% Tween 20 and 5 mm CaCl2, 50 μl/well. After washing three times with TBS containing 0.1% Tween 20, staining solution consisting of 0.4 mg/mlo-phenylenediamine and 0.002% H2O2in 100 mm phosphate, 50 mm citric acid buffer (pH 5.0) was added to the wells (100 μl/well). The reaction was stopped by adding 50 μl/well 1 mH2SO4, and absorbance was measured at 490 nm in a V max microtiter plate reader (Molecular Devices, Menlo Park, CA). Values were corrected for background absorbance. Microtiter plates were coated overnight at 4 °C with a polyclonal antibody against protein S (3 g/liter IgG; Dako), 1:1000 diluted in coat buffer, 50 μl/well. Plates were washed three times with TBS containing 0.1% Tween 20. Plates were blocked for 2 h at 37 °C with blocking buffer, 100 μl/well. Purified human protein S (1 μg/ml) was added to the wells and incubated in blocking buffer containing 0.1% Tween 20 and 5 mm CaCl2 for 2 h at 37 °C, 50 μl/well. Plates were washed three times with TBS containing 0.1% Tween 20, and increasing concentrations of purified chimeric SCR-tPA constructs were added and incubated for 2 h at 37 °C in blocking buffer containing 0.1% Tween 20 and 5 mm CaCl2, 50 μl/well. After washing the plates three times with TBS containing 0.1% Tween 20, sheep anti-tPA antibodies (1 μg/ml; Enzyme Research Laboratories Inc.) were added in blocking buffer containing 0.1% Tween 20 and 5 mmCaCl2, 50 μl/well. After 1 h of incubation at 37 °C, plates were washed three times with TBS containing 0.1% Tween 20. Bound sheep antibodies against tPA were detected by adding a polyclonal peroxidase-conjugated rabbit antibody against sheep antibodies (Dako), 1:1000 diluted in blocking buffer containing 0.1% Tween 20 and 5 mm CaCl2, 50 μl/well. After washing three times with TBS containing 0.1% Tween 20, the wells were developed and measured as described above. Values were corrected for background absorbance. A fluid phase binding assay was used to investigate the stoichiometry of the interaction between protein S and the SCR-tPA constructs that bound to protein S. For this assay, 5 mg of purified rabbit antibodies directed against human protein S (Dako) was coupled to 2.5 ml of CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. Fluid phase binding was performed by allowing increasing concentrations of SCR-tPA constructs to bind to 10 nm human protein S in a volume of 100 μl of TBS containing 5% bovine serum albumin and 10 mm CaCl2. Binding was performed overnight at 4 °C with constant rotation. Then 100 μl of rabbit anti-human protein S-Sepharose beads in TBS was added (final CaCl2concentration 5 mm). After 90 min of incubation with constant rotation at room temperature, total protein S was removed from the incubation mixture by spinning down the Sepharose beads in an Eppendorf centrifuge for 3 min at 14,000 rpm. The supernatant was analyzed with a polyclonal tPA ELISA (ImulyseTM-tPA, Biopool AB) for nonbound (free) SCR-tPA constructs. As a control, the supernatant was also analyzed with a polyclonal protein S ELISA to confirm that all of the protein S was precipitated by the Sepharose beads. In this polyclonal ELISA, rabbit anti-protein S antibodies were used as catching antibodies, and peroxidase-conjugated rabbit anti-protein S antibodies were used as detecting antibodies (antibodies from Dako). Microtiter plates were coated overnight at 4 °C with a monoclonal antibody against C4BPα (8C11) in coat buffer, 2 μg/ml, 50 μl/well. Plates were washed three times with TBS containing 0.1% Tween 20. Plates were blocked for 2 h at 37 °C with blocking buffer, 100 μl/well. Purified human C4BP (2 μg/ml) was incubated in blocking buffer for 1 h at 37 °C. Purified human protein S (0.5 nm) was preincubated with chimeric SCR-tPA constructs (0–400 nm) for 1 h at 37 °C in blocking buffer containing 5 mmCaCl2. After washing the plates three times with TBS containing 0.1% Tween 20, aliquots of 50 μl from the preincubation mixtures were applied to the plates and incubated for 2 h at 37 °C. After washing three times with TBS containing 0.1% Tween 20, bound protein S was detected using a polyclonal peroxidase-conjugated antibody against protein S (1.3 g/liter IgG; Dako), 1:2000 diluted in blocking buffer containing 0.1% Tween 20 and 5 mmCaCl2, 50 μl/well. After washing three times with TBS containing 0.1% Tween 20, the wells were developed and measured as described above. Values were corrected for background absorbance. Protein S cofactor activity was determined with an activated partial thromboplastin time-based assay using a KC-10A microcoagulometer (Amelung, Lemgo, Germany). For this assay, plasma deficient in protein S and C4BP was prepared by immunoadsorption as described previously (37Hackeng T.M. van 't Veer C. Meijers J.C.M. Bouma B.N. J. Biol. Chem. 1994; 269: 21051-21058Abstract Full Text PDF PubMed Google Scholar). Purified human protein S (160 nm) was preincubated for 30 min at 37 °C with serial dilutions of chimeric SCR-tPA constructs in TBS containing 0.3% (w/v) bovine serum albumin plus 3 mmCaCl2. Aliquots of 12.5 μl from the preincubation mixtures were added to a mixture of 25 μl of plasma deficient in protein S and C4BP and 12.5 μl of 240 nm activated protein C solution (final APC concentration 30 nm). After adding 25 μl of PTT reagent (Roche Molecular Biochemicals), coagulation was initiated by adding 25 μl of 25 mmCaCl2 (final volume 100 μl). In the range of protein S used in this assay (0–20 nm protein S), there was a linear relationship between clotting time and protein S concentration (data not shown). The protein S cofactor activity was expressed as a percentage of the maximum cofactor activity in the absence of chimeric SCR-tPA constructs. In order to study the role of each individual C4BPβ SCR unit in the interaction of C4BP with protein S, chimeras were constructed of β-chain SCR units fused to the NH2 terminus of a modified tPA. Baby hamster kidney cells were transfected with the expression vector containing the chimeric SCR-tPA constructs. Expression levels in the medium were detected using a tPA ELISA system and were 1–5 μg/ml after 3 days of culture. After purification of the chimeric constructs with an immobilized monoclonal antibody against tPA, the constructs were applied to 10% SDS-PAGE under reducing and nonreducing conditions and stained by Coomassie Brilliant Blue (Fig. 1, A and B) or transferred to polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) for standard Western blotting procedures using a polyclonal antibody against tPA (Fig. 2,A and B). The chimeric SCR-tPA constructs appeared as diffuse bands, which is probably caused by heterogeneous glycosylation of the proteins, because the β-chain of C4BP and the tPA module are both highly glycosylated (17Hillarp A. Dahlbäck B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1183-1187Crossref PubMed Scopus (72) Google Scholar, 29Meijers J.C.M. Mulvihill E.R. Davie E.W. Chung D.W. Biochemistry. 1992; 31: 4680-4684Crossref PubMed Scopus (73) Google Scholar). Constructs containing single β-chain SCR units had molecular weights of approximately 73,000 with the exception of SCR-3, which had an estimated molecular weight of 65,000. Constructs containing two β-chain SCR units had molecular weights of approximately 80,000. The chimeric SCR-tPA constructs were also detected by Western blotting using a polyclonal antibody against tPA (Fig. 2, A and B).Figure 2Immunoblot of chimeric SCR-tPA constructs. Purified chimeric SCR-tPA constructs (200 ng) were applied to 10% SDS-PAGE gel under nonreducing (A) and reducing conditions (B). Proteins were transferred to polyvinylidene difluoride membranes and visualized using polyclonal antibodies against tPA. Lane 1, SCR-1-tPA;lane 2, SCR-2-tPA; lane 3, SCR-3-tPA; lanes 4 and 8, molecular weight markers; lane 5, SCR-1+2-tPA;lane 6, SCR-1+3-tPA; lane 7, SCR-2+3-tPA; lane 9, tPA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The interaction of chimeric SCR-tPA constructs with protein S was investigated using a direct binding assay in which protein S was allowed to bind to immobilized chimeric SCR-tPA constructs (Fig. 3). The results are expressed as a percentage of maximum binding (B max) for each construct. Apparent dissociation constants of the binding of protein S to chimeric SCR-tPA constructs were 10.3 nm for SCR-1, 1.9 nm for SCR-1+2, and 13.7 nm for SCR-1+3, respectively. Protein S did not bind to SCR-2, SCR-3, SCR-2+3, or tPA. The apparent dissociation constant for the binding of protein S to plasma C4BP was 1.7 nm, which is identical to published values (10Dahlbäck B. Biochem. J. 1983; 209: 847-856Crossref PubMed Scopus (216) Google Scholar). This indicates that protein S has an affinity for SCR-1+ 2 that is comparable with the affinity for plasma C4BP, whereas the affinity of protein S for SCR-1 and SCR-1+3 was approximately 5 times lower. A binding assay was performed in which chimeric SCR-tPA constructs were allowed to bind to immobilized protein S. The results of the binding experiments of chimeric SCR-tPA constructs to immobilized protein S are presented in Fig. 4. Binding of each chimeric SCR-tPA construct is expressed as a percentage of B maxto protein S. The apparent dissociation constants for the binding of chimeric SCR-tPA constructs to protein S are 207.4 nm for SCR-1, 24.9 nm for SCR-1+2, and 124.0 nm for SCR-1+3, respectively. SCR-2, SCR-3, SCR-2+3, and tPA did not bind to protein S. The apparent dissociation constant for the binding of plasma-purified C4BP to protein S was 2.9 nm, which is in the range of previously published values between 2 and 5 nm(28Härdig Y. Dahlbäck B. J. Biol. Chem. 1996; 271: 20861-20867Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This implies that in this system, SCR-1+2 has an affinity for protein S approximately 5–10 times lower compared with plasma-purified C4BP. The stoichiometry of the interaction between protein S and the SCR-tPA constructs that bound to protein S was analyzed using a fluid phase binding assay. Increasing concentrations of SCR-tPA constructs were allowed to bind to protein S, after which total protein S was immunoprecipitated using rabbit anti-protein S antibodies coupled to Sepharose beads. After immunoprecipitation, no protein S could be detected in the supernatant using a polyclonal protein S ELISA. Free SCR-tPA in the supernatant was determined using a polyclonal tPA ELISA. Bound SCR-tPA was calculated by subtracting values for free SCR-tPA from the concentrations of SCR-tPA added. Values for bound SCR-tPA were plotted against free SCR-tPA (data not shown), and theB max of each SCR-tPA construct was calculated. The stoichiometry of the interaction of protein S with each protein S-binding SCR-tPA construct is presented in TableII as the ratio ofB max and the protein S concentration used. The values for B max/protein S displayed in Table IIare presented as means of two separate experiments. One protein S molecule was found to bind to 1.0 molecule of SCR-1, 1.0 molecule of SCR-1+2, and 1.1 molecules of SCR-1+3, respectively. These findings show that each SCR-tPA construct that is able to bind to protein S (SCR-1, SCR-1+2, and SCR-1+3) contains one binding site for protein S, confirming the existence of a single binding site for protein S on the β-chain of C4BP.Table IIOverview of apparent dissociation constants and concentrations of 50% inhibitionLigandApparentK D, binding of protein S to immobilized SCRs (Fig. 3)Apparent K D, binding of SCRs to immobilized protein S (Fig. 4)50% inhibition of binding of protein S to immobilized C4BP (Fig. 5)50% inhibition of protein S cofactor activity (Fig. 6)Stoichiometry (B max/protein S)nmnmnmnmSCR-110.3207.42123201.0SCR-1+21.924.922701.0SCR-1+313.7124.01792101.1Only the chimeric SCR-tPA constructs that bound to protein S are shown. The data presented are derived from the figures referred to in the table. The stoichiometry of the interaction of the SCR-tPA constructs with protein" @default.
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• W2109672844 date "1999-05-01" @default.
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• W2109672844 title "Interaction between Protein S and Complement C4b-binding Protein (C4BP)" @default.
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• W2109672844 doi "https://doi.org/10.1074/jbc.274.21.15144" @default.
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