The α-Chains of C4b-binding Protein Mediate Complex Formation with Low Density Lipoprotein Receptor-related Protein
2002; Elsevier BV; Volume: 277; Issue: 4 Linguagem: Inglês
10.1074/jbc.m102293200
ISSN1083-351X
AutoresErik Westein, Cécile V. Denis, Bonno N. Bouma, Peter J. Lenting,
Tópico(s)Blood properties and coagulation
ResumoC4b-binding protein (C4BP) is a heparin-binding protein that participates in both the complement and hemostatic system. We investigated the interaction between C4BP and low density lipoprotein receptor-related protein (LRP), an endocytic receptor involved in the catabolism of various heparin-binding proteins. Both plasma-derived C4BP and recombinant C4BP consisting of only its α-chains (rC4BPα) bound efficiently to immobilized LRP, as determined by surface plasmon resonance analysis. Complementary, two distinct fragments of LRP, i.e. clusters II and IV, both associated to immobilized rC4BPα, and binding could be inhibited by the LRP antagonist receptor-associated protein. Further analysis showed that association of rC4BPα to LRP was inhibited by heparin or by anti-C4BP antibody RU-3B9, which recognizes the heparin-binding region of the C4BP α-chains. In cellular degradation experiments, LRP-expressing fibroblasts effectively degraded125I-labeled rC4BPα, whereas their LRP-deficient counterparts displayed a 4-fold diminished capacity of degrading125I-rC4BPα. Finally, initial clearance of C4BP in mice was significantly delayed upon co-injection with receptor-associated protein. In conclusion, our data demonstrate that the α-chains of C4BP comprise a binding site for LRP. We propose that LRP mediates at least in part the catabolism of C4BP and, as such, may regulate C4BP participation in complement and hemostatic processes. C4b-binding protein (C4BP) is a heparin-binding protein that participates in both the complement and hemostatic system. We investigated the interaction between C4BP and low density lipoprotein receptor-related protein (LRP), an endocytic receptor involved in the catabolism of various heparin-binding proteins. Both plasma-derived C4BP and recombinant C4BP consisting of only its α-chains (rC4BPα) bound efficiently to immobilized LRP, as determined by surface plasmon resonance analysis. Complementary, two distinct fragments of LRP, i.e. clusters II and IV, both associated to immobilized rC4BPα, and binding could be inhibited by the LRP antagonist receptor-associated protein. Further analysis showed that association of rC4BPα to LRP was inhibited by heparin or by anti-C4BP antibody RU-3B9, which recognizes the heparin-binding region of the C4BP α-chains. In cellular degradation experiments, LRP-expressing fibroblasts effectively degraded125I-labeled rC4BPα, whereas their LRP-deficient counterparts displayed a 4-fold diminished capacity of degrading125I-rC4BPα. Finally, initial clearance of C4BP in mice was significantly delayed upon co-injection with receptor-associated protein. In conclusion, our data demonstrate that the α-chains of C4BP comprise a binding site for LRP. We propose that LRP mediates at least in part the catabolism of C4BP and, as such, may regulate C4BP participation in complement and hemostatic processes. C4b-binding protein plasma-derived C4b-binding protein recombinant C4b-binding protein consisting of only its α-chains Dulbecco's modified Eagle's medium receptor-associated protein fused to glutathioneS-transferase low density lipoprotein receptor-related protein surface plasmon resonance heparan sulfate proteoglycan serum amyloid P component C4b-binding protein (C4BP)1 is a plasma protein, which serves as a regulator of the complement system (1Dahlback B. Thromb. Haemost. 1991; 66: 49-61Crossref PubMed Scopus (328) Google Scholar). C4BP binds the complement protein C4b, which results in enhancement of factor I-mediated degradation of C4b and inhibition of the classical pathway C3 convertase (C4b2a) complex (1Dahlback B. Thromb. Haemost. 1991; 66: 49-61Crossref PubMed Scopus (328) Google Scholar). In plasma, C4BP may serve as a carrier protein for at least two other plasma proteins: the vitamin K-dependent anticoagulant Protein S and serum amyloid P component (1Dahlback B. Thromb. Haemost. 1991; 66: 49-61Crossref PubMed Scopus (328) Google Scholar). In addition, C4BP may interact with coagulation factor VIII as well (2Koppelman S.J. van 't Veer C. Sixma J.J. Bouma B.N. Blood. 1995; 86: 2653-2660Crossref PubMed Google Scholar). The majority of the C4BP molecules (∼80%) consist of seven identical α-chains and a unique β-chain, whereas other isoforms lack either one of the α-chains or the β-chain (3Sanchez-Corral P. Criado G.O. Rodriguez D.C. J. Immunol. 1995; 155: 4030-4036PubMed Google Scholar). The β-chain is involved in the interaction with Protein S (4Hillarp A. Dahlback B. J. Biol. Chem. 1988; 263: 12759-12764Abstract Full Text PDF PubMed Google Scholar), and complex formation with serum amyloid P component (SAP) and complement proteins is mediated by the α-chains (5Schwalbe R.A. Dahlback B. Nelsestuen G.L. J. Biol. Chem. 1990; 265: 21749-21757Abstract Full Text PDF PubMed Google Scholar, 6Hessing M. van 't Veer C. Bouma B.N. J. Immunol. 1990; 144: 2632-2637PubMed Google Scholar). In addition, the α-chains have been found to contain binding sites for bacterial surface proteins from Streptococcus pyogenes (7Thern A. Stenberg L. Dahlback B. Lindahl G. J. Immunol. 1995; 154: 375-386PubMed Google Scholar) and heparin (8Hessing M. Vlooswijk R.A. Hackeng T.M. Kanters D. Bouma B.N. J. Immunol. 1990; 144: 204-208PubMed Google Scholar). The heparin interactive site, which overlaps with the C4b interactive site, encompasses a cluster of positively charged amino acids involving Arg residues at positions 39, 64, and 66 (9). The average plasma concentration of C4BP is ∼260 nm (150 μg/ml) (10Griffin J.H. Gruber A. Fernandez J.A. Blood. 1992; 79: 3203-3211Crossref PubMed Google Scholar), although its levels may increase up to 4-fold during inflammation, infection, or tissue damage (11Saeki T. Hirose S. Nukatsuka M. Kusunoki Y. Nagasawa S. Biochem. Biophys. Res. Commun. 1989; 164: 1446-1451Crossref PubMed Scopus (44) Google Scholar, 12Barnum S.R. Dahlback B. Complement Inflamm. 1990; 7: 71-77Crossref PubMed Scopus (48) Google Scholar). Plasma levels represent a balance between C4BP production and removal. At present, little is known concerning the molecular mechanisms that control the removal of C4BP from the circulation. The notion that C4BP is able to interact with heparin opens the possibility that C4BP may interact with heparan sulfate proteoglycans (HSPG) exposed at the cellular surface. Alternatively, C4BP may interact with cellular receptors like the low density lipoprotein receptor-related protein (LRP), which is known to recognize heparin-binding proteins. LRP, also known as the α2-macroglobulin receptor, is a member of the low density lipoprotein receptor family of endocytic receptors (13Gliemann J. Biol. Chem. 1998; 379: 951-964PubMed Google Scholar, 14Neels J.G. Horn I.R. van den Berg B.M. Pannekoek H. van Zonneveld A. Fibrinolysis Proteolysis. 1998; 12: 219-240Crossref Scopus (53) Google Scholar). It consists of a noncovalently linked heavy and light chain. The 85-kDa light chain comprises the transmembrane and cytoplasmic domains, whereas the ligand binding regions are located within the 515-kDa heavy chain (15Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. EMBO J. 1988; 7: 4119-4127Crossref PubMed Scopus (742) Google Scholar). The heavy chain contains four domains enriched in low density lipoprotein receptor class A domains, generally referred to as clusters I–IV. It has been reported that clusters II and IV play a prominent role in ligand binding to the receptor (16Neels J.G. van den Berg B.M. Lookene A. Olivecrona G. Pannekoek H. van Zonneveld A.J. J. Biol. Chem. 1999; 274: 31305-31311Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). LRP is widely distributed among tissues, such as liver, brain, and placenta, and is expressed in an array of cell types including parenchymal cells, Kupffer cells, neurons, astrocytes, smooth muscle cells, monocytes, and fibroblasts (17Zheng G. Bachinsky D.R. Stamenkovic I. Strickland D.K. Brown D. Andres G. McCluskey R.T. J. Histochem. Cytochem. 1994; 42: 531-542Crossref PubMed Scopus (288) Google Scholar). LRP has traditionally been reported as a receptor that is involved in hepatic clearance of numerous proteins (18Strickland D.K. Kounnas M.Z. Argraves W.S. FASEB J. 1995; 9: 890-898Crossref PubMed Scopus (249) Google Scholar), although recent studies demonstrate that LRP contributes to cellular signaling processes as well (19Willnow T.E. Nykjaer A. Herz J. Nat. Cell Biol. 1999; 1: E157-E162Crossref PubMed Scopus (190) Google Scholar). Ligands bound by LRP belong to a spectrum of structurally and functionally unrelated proteins (13Gliemann J. Biol. Chem. 1998; 379: 951-964PubMed Google Scholar, 14Neels J.G. Horn I.R. van den Berg B.M. Pannekoek H. van Zonneveld A. Fibrinolysis Proteolysis. 1998; 12: 219-240Crossref Scopus (53) Google Scholar, 18Strickland D.K. Kounnas M.Z. Argraves W.S. FASEB J. 1995; 9: 890-898Crossref PubMed Scopus (249) Google Scholar). These include apolipoproteins, lipases, proteinases, proteinase/inhibitor complexes, Kunitz-type inhibitors, matrix proteins, and several others. The mechanism by which binding to LRP is mediated varies between ligands. First, ligands (e.g.α2-macroglobulin/protease complexes) may bind directly from the circulation to LRP (20Jensen P.H. Moestrup S.K. Gliemann J. FEBS Lett. 1989; 255: 275-280Crossref PubMed Scopus (73) Google Scholar, 21Ashcom J.D. Tiller S.E. Dickerson K. Cravens J.L. Argraves W.S. Strickland D.K. J. Cell Biol. 1990; 110: 1041-1048Crossref PubMed Scopus (207) Google Scholar). Alternatively, binding may be promoted by so called accessory proteins. This possibility is exemplified by the urokinase-type plasminogen activator receptor, which facilitates internalization of urokinase complexed with its inhibitor plasminogen activator inhibitor-1 by LRP (22Kounnas M.Z. Henkin J. Argraves W.S. Strickland D.K. J. Biol. Chem. 1993; 268: 21862-21867Abstract Full Text PDF PubMed Google Scholar, 23Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonneveld A.J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar). Furthermore, LRP-mediated degradation may be preceded by sequestration of the ligands by HSPG. Examples hereof include β-amyloid precursor protein (24Kounnas M.Z. Moir R.D. Rebeck G.W. Bush A.I. Argraves W.S. Tanzi R.E. Hyman B.T. Strickland D.K. Cell. 1995; 82: 331-340Abstract Full Text PDF PubMed Scopus (448) Google Scholar), tissue factor pathway inhibitor (25Warshawsky I. Broze Jr., G.J. Schwartz A.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6664-6668Crossref PubMed Scopus (101) Google Scholar), activated factor IX (26Neels J.G. van den Berg B.M. Mertens K. ter Maat H. Pannekoek H. van Zonneveld A.J. Lenting P.J. Blood. 2000; 96: 3459-3465Crossref PubMed Google Scholar), and thrombospondin (27Mikhailenko I. Kounnas M.Z. Strickland D.K. J. Biol. Chem. 1995; 270: 9543-9549Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In the present study, we assessed binding of C4BP to LRP by surface plasmon resonance (SPR) employing purified components. Our data show that C4BP is able to interact with LRP with moderate affinity, and that binding involves the C4BP α-chain and the cluster II and IV regions of LRP. Furthermore, we found that LRP mediates the delivery of rC4BPα to the intracellular degradation pathway in mouse fibroblast cells, and that the initial clearance of C4BP in mice is delayed upon co-injection with the LRP-antagonist receptor-associated protein. Our data suggest that LRP contributes to the catabolism of the complement protein C4BP. The Biacore2000 biosensor system and reagents, including an amine-coupling kit and CM5 biosensor chips (research grade), were from Biacore AB (Uppsala, Sweden). Cell culture plates, Dulbecco's modified Eagle's medium (DMEM), DMEM/F-12 medium, fetal calf serum, penicillin, and streptomycin were from Invitrogen (Breda, The Netherlands). Unfractionated heparin was purchased from Sigma (Zwijndrecht, The Netherlands). Protein G-Sepharose was from Amersham Biosciences, Inc. Plasma-derived C4BP (pd-C4BP) and Protein S were purified as described (28Hessing M. Kanters D. Hackeng T.M. Bouma B.N. Thromb. Haemost. 1990; 64: 245-250Crossref PubMed Scopus (26) Google Scholar, 29Hackeng T.M. Hessing M. van 't Veer C. Meijer-Huizinga F. Meijers J.C. de Groot P.G. van Mourik J.A. Bouma B.N. J. Biol. Chem. 1993; 268: 3993-4000Abstract Full Text PDF PubMed Google Scholar). Recombinant C4BP, consisting of the α-chains but lacking the β-chain (rC4BPα), was produced using stably transfected baby hamster kidney cell lines, purified until homogeneity by immunoaffinity chromatography as reported previously (30van de Poel R.H. Meijers J.C. Rosing J. Tans G. Bouma B.N. Biochemistry. 2000; 39: 14543-14548Crossref PubMed Scopus (13) Google Scholar), and stored in 125 mm NaCl, 0.005% (v/v) Tween 20, 25 mm Hepes (pH 7.4) at −20 °C. Purified rC4BPα was labeled with Na125I (Amersham Biosciences, Inc.) using the IODO-GEN method (Pierce) as described (8Hessing M. Vlooswijk R.A. Hackeng T.M. Kanters D. Bouma B.N. J. Immunol. 1990; 144: 204-208PubMed Google Scholar). Free Na125I was removed by chromatography on a PD-10 column (Amersham Biosciences, Inc.) equilibrated in 125 mm NaCl, 0.005% (v/v) Tween 20, 25 mm Hepes (pH 7.4), and 125I-labeled rC4BPα was stored in small aliquots at −20 °C. Specific radioactivity was 4.0 (± 1.3) × 105 cpm/pmol rC4BPα (mean ± S.E.; n = 6). Each radiolabeled rC4BPα preparation was compared with unlabeled rC4BPα for binding to immobilized LRP employing SPR. In all cases labeled and unlabeled preparations displayed similar sensorgrams, demonstrating that both association and dissociation characteristics were unchanged upon labeling. On one occasion, this was investigated in more detail by determining affinity constants, which proved to be similar for radiolabeled and unlabeled rC4BPα (3.5 and 55.2 nmversus 5.6 and 66.2 nm, respectively). Purified full-length LRP (31Moestrup S.K. Gliemann J. J. Biol. Chem. 1991; 266: 14011-14017Abstract Full Text PDF PubMed Google Scholar) was kindly provided by Dr. S. K. Moestrup (University of Aarhus, Aarhus, Denmark). Receptor-associated protein fused to glutathioneS-transferase (GST-RAP) (32Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar) was prepared as described previously (33Lenting P.J. Neels J.G. van den Berg B.M. Clijsters P.P. Meijerman D.W. Pannekoek H. van Mourik J.A. Mertens K. van Zonneveld A.J. J. Biol. Chem. 1999; 274: 23734-23739Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Recombinant LRP fragments encompassing LRP cluster II and cluster IV were produced using stably transfected baby hamster kidney cell lines, which were kindly provided by Dr. H. Pannekoek (University of Amsterdam, Amsterdam, The Netherlands). Clusters were purified employing GST-RAP affinity chromatography as described (16Neels J.G. van den Berg B.M. Lookene A. Olivecrona G. Pannekoek H. van Zonneveld A.J. J. Biol. Chem. 1999; 274: 31305-31311Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and stored at 4 °C in 125 mm NaCl, 1 mmCaCl2, 0.005% (v/v) Tween 20, 25 mm Hepes (pH 7.4). Monoclonal antibody RU-3B9 (34Hessing 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) was purified from ascites using protein G-Sepharose as recommended by the manufacturer. RU-3B9 Fab fragments were prepared using the ImmunoPure Fab preparation kit (Pierce) as instructed. Bovine serum albumin (fraction V) was obtained from Sigma. Proteins were quantified by a BCA protein assay (Pierce) using albumin as a standard. Binding studies were performed employing a Biacore2000 biosensor system, and SPR analysis was done essentially as described (26Neels J.G. van den Berg B.M. Mertens K. ter Maat H. Pannekoek H. van Zonneveld A.J. Lenting P.J. Blood. 2000; 96: 3459-3465Crossref PubMed Google Scholar, 33Lenting P.J. Neels J.G. van den Berg B.M. Clijsters P.P. Meijerman D.W. Pannekoek H. van Mourik J.A. Mertens K. van Zonneveld A.J. J. Biol. Chem. 1999; 274: 23734-23739Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). LRP or rC4BPα was immobilized on a CM5 sensor chip at the indicated densities using the amine-coupling kit as instructed by the supplier. Routinely, a control channel was activated and blocked using the amine-coupling reagents in the absence of protein. Binding to coated channels was corrected for binding to noncoated channels (<5% of binding to coated channels). SPR analysis was performed in 125 mm NaCl, 0.005% (v/v) Tween 20, 25 mm Hepes (pH 7.4) at 25 °C at indicated flow rates. Regeneration of the surface of the LRP sensor chip was performed by subsequent application of 100 mmH3PO4 and 25 mm CaCl2. The rC4BPα sensor chip was regenerated using 100 mmH3PO4. For analysis of association and dissociation curves of the sensorgrams, BiaEvaluation software was used (Biacore AB). Interaction constants were determined by performing nonlinear global fitting of data corrected for bulk refractive index changes. Data were fitted to various models available within the software. For binding of rC4BPα to immobilized LRP, a model describing the interaction between rC4BPα and two independent binding sites (heterologous ligand, parallel reactions) was found to provide the best fit of the experimental data. Accuracy of the fits was judged from residual plots and statistical parameters employing previously described equations (35Horn I.R. van den Berg B.M. van der Meijden P.Z. Pannekoek H. van Zonneveld A.J. J. Biol. Chem. 1997; 272: 13608-13613Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Statistical significance of clearance data (see Fig. 4) and of association and dissociation rate constants (see Table I) were calculated using Student's unpaired ttest employing the InStat program (GraphPad Software, Inc.).Table IKinetic parameters for the binding of rC4BPα to LRP50 mm NaCl125 mmNaClp valuekon(app)(m−1 s−1) 1)4.0 (± 3.7) × 1057.0 (± 1.4) × 1050.16 2)6.4 (± 1.8) × 1051.4 (± 0.8) × 1050.024koff(app) (s−1) 1)2.5 (± 0.7) × 10−25.0 (± 2.8) × 10−20.14 2)3.6 (± 1.0) × 10−43.3 (± 1.9) × 10−40.24Kd(app) (nm) 1)62.5 (± 31.8)71.4 (± 42.5) 2)0.6 (± 0.2)2.4 (± 1.9)Association and dissociation of various concentrations rC4BPα to LRP (11 fmol/mm2) was assessed as described in the legend of Fig.1. Buffer consisted of 0.005% (v/v) Tween 20, 25 mm Hepes (pH 7.4) supplemented with either 50 or 125 mm NaCl. rC4BPα concentrations tested varied between 2 and 30 nm. The data obtained for all concentrations tested were analyzed to calculate apparent association rate constants (kon(app)) and apparent dissociation rate constants (koff(app)) as described using a two-site binding model (35Horn I.R. van den Berg B.M. van der Meijden P.Z. Pannekoek H. van Zonneveld A.J. J. Biol. Chem. 1997; 272: 13608-13613Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Each class of binding sites is referred to as 1 and 2, respectively. Apparent affinity constants (Kd(app)) were inferred from the ratiokoff(app):kon(app). Data are based on three to six measurements using four or five different concentrations for each measurement. Data represent the average (± S.D.). p values were calculated employing Student's unpaired t test. Open table in a new tab Association and dissociation of various concentrations rC4BPα to LRP (11 fmol/mm2) was assessed as described in the legend of Fig.1. Buffer consisted of 0.005% (v/v) Tween 20, 25 mm Hepes (pH 7.4) supplemented with either 50 or 125 mm NaCl. rC4BPα concentrations tested varied between 2 and 30 nm. The data obtained for all concentrations tested were analyzed to calculate apparent association rate constants (kon(app)) and apparent dissociation rate constants (koff(app)) as described using a two-site binding model (35Horn I.R. van den Berg B.M. van der Meijden P.Z. Pannekoek H. van Zonneveld A.J. J. Biol. Chem. 1997; 272: 13608-13613Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Each class of binding sites is referred to as 1 and 2, respectively. Apparent affinity constants (Kd(app)) were inferred from the ratiokoff(app):kon(app). Data are based on three to six measurements using four or five different concentrations for each measurement. Data represent the average (± S.D.). p values were calculated employing Student's unpaired t test. Cellular degradation of rC4BPα was examined using mouse fibroblast cell lines MEF-1 (American Tissue Culture Collection, CRL-2214), or their counterparts, which are genetically deficient for LRP, PEA-13 (American Tissue Culture Collection, CRL-2216). MEF-1 and PEA-13 cells have been isolated from embryos resulting from the mating of mice heterogenous for LRP gene disruption (36Willnow T.E. Herz J. J. Cell Sci. 1994; 107: 719-726Crossref PubMed Google Scholar). The MEF-1 cells express LRP endogenously, whereas PEA-13 cells have been demonstrated to contain two (via homologous recombination) disrupted alleles for the LRP gene. Cells were seeded at least 48 h before the experiment and grown to 90–95% confluence in 24-well plates in DMEM supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Before incubation, cells were extensively washed with DMEM/F-12 medium.125I-labeled rC4BPα was mixed with nonlabeled rC4BPα to a 1:1 molar ratio, and the mixture was then added to the cells in a final volume of 250 μl in incubation medium (DMEM/F-12 medium supplemented with 1% (w/v) bovine serum albumin and 2 mmCaCl2). Final concentration of rC4BPα was 100 nm. After a 1-h incubation at 4 °C, cells were washed three times with 500 μl of incubation medium to remove nonbound material. Subsequently, incubation was allowed to proceed at 37 °C in a volume of 250 μl. At indicated time points, samples were taken to determine the amount of degraded material. Degraded material is defined as the radioactivity that is soluble in 10% trichloroacetic acid. In all experiments a control was included in which the amount of degradation was examined in the absence of cells. We used 14–16-week- old C57BL/6J mice. Three to six mice were housed in each cage and fed a standard chow diet and water ad libitum. Mice were injected intravenously with pd-C4BP (10.9 mg/kg) alone or in combination with GST-RAP (30 mg/kg) into the tail vein. Blood samples were collected in polypropylene Eppendorf tubes, containing approximately 0.1 volume of 129 mm trisodium citrate, at indicated time points from anesthetized mice by retro-orbital venous plexus puncture. Plasma was prepared by centrifugation of the blood at 2500 × g for 20 min at room temperature. Residual C4BP was determined using an immunosorbent assay specific for human C4BP. For each time point, 3 mice were used. Binding of pd-C4BP to LRP was investigated by SPR analysis using purified components. An increase in response was observed when pd-C4BP (30 nm) was passed over immobilized LRP (7 and 11 fmol/mm2), demonstrating that pd-C4BP is able to associate with LRP (Fig.1A). As the highest response is observed at the highest density of LRP, binding appears to be dose-dependent. Replacement of pd-C4BP solution by buffer resulted in a gradual decline of the response, indicating that pd-C4BP dissociates from LRP and that binding is reversible (Fig.1A). In plasma, C4BP is able to associate with other plasma proteins, like Protein S and SAP. Our data therefore do not fully exclude that the observed response originates from traces of Protein S or SAP present within the pd-C4BP preparation. However, no additional response was observed upon subsequent injection of anti-Protein S or anti-SAP antibodies after injection of C4BP preparations (data not shown). In addition, no association of purified Protein S to immobilized LRP was detected (Fig. 1A). Apparently, the observed increase in response originates from binding of C4BP to LRP. To further characterize the interaction between C4BP and LRP, SPR analysis was performed employing purified recombinant C4BP that consists only of the α-chains (30van de Poel R.H. Meijers J.C. Rosing J. Tans G. Bouma B.N. Biochemistry. 2000; 39: 14543-14548Crossref PubMed Scopus (13) Google Scholar). As shown in Fig. 1B, recombinant rC4BPα (2–30 nm) efficiently associated to immobilized LRP (11 fmol/mm2) in a dose-dependent manner. Binding appeared to be similar for plasma-derived and recombinant C4BP. The interaction between rC4BPα and immobilized LRP was studied in more detail by assessment of the apparent association and dissociation rate constants, which are summarized in TableI. Experimental data were fitted most appropriately using a model describing the interaction of rC4BPα with two classes of binding sites (heterologous ligand, parallel interactions). The resulting apparent affinity constants (Kd(app)) values were 2.4 ± 1.9 nm and 71.4 ± 42.5 nm, respectively. These data demonstrate that LRP is able to bind the C4BP α-chain with moderate affinity in a reversible and dose-dependent manner. The observation that C4BP interacts with two different binding sites may suggest heterogeneity of LRP because of its immobilization. Alternatively, LRP may comprise distinct regions that are able to interact with C4BP α-chains. To identify LRP regions involved in binding C4BP α-chains, purified recombinant receptor fragments were used. These fragments, designated cluster II and IV, respectively, have been established to encompass the ligand binding domains of LRP (16Neels J.G. van den Berg B.M. Lookene A. Olivecrona G. Pannekoek H. van Zonneveld A.J. J. Biol. Chem. 1999; 274: 31305-31311Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). When either cluster (200 nm) was incubated with immobilized recombinant rC4BPα (10 fmol/mm2), reversible binding of cluster II and IV to rC4BPα was observed (Fig.2A). The specificity of the interaction was subsequently assessed by investigating the binding of cluster II or IV to immobilized rC4BPα in the presence of various concentrations of the LRP-antagonist GST-RAP. Indeed, GST-RAP (0–750 nm) efficiently interfered with binding of either cluster (150 nm) to immobilized rC4BPα (Fig. 2B). Thus, both recombinant LRP fragments encompassing the ligand binding domains, i.e. clusters II and IV, comprise a binding site for rC4BPα, and binding is inhibited in the presence of GST-RAP. The observation that LRP recognizes C4BP in a system employing purified components prompted us to investigate the contribution of LRP to the delivery of rC4BPα to the intracellular degradation pathway. This was addressed in experiments using mouse fibroblast cells genetically deficient for LRP (i.e. PEA-13 cells), or their counterparts expressing LRP endogenously (i.e. MEF-1 cells) (36Willnow T.E. Herz J. J. Cell Sci. 1994; 107: 719-726Crossref PubMed Google Scholar). Employing LRP-expressing MEF-1 cells, an increase in the amount of degraded 125I-labeled rC4BPα was observed in time (Fig. 3). However, when degradation was examined in the presence of 1 μm GST-RAP, the amount of 125I-labeled rC4BPα degraded was markedly reduced (Fig. 3), indicating that the degradation process involves a GST-RAP-sensitive receptor. In the presence of LRP-deficient PEA-13 cells, the amount of rC4BPα degraded was similar to that of MEF-1 cells in the presence of the LRP antagonist GST-RAP (Fig. 3). Together, these data strongly suggest that the cellular uptake and transport of 125I-rC4BPα to the intracellular degradation pathway involves a LRP-dependent pathway. To investigate whether LRP also contributes to the clearance of C4BPin vivo, human pd-C4BP (10.9 mg/kg) was injected intravenously into the tail vein of C57BL/6J mice in the absence or presence of GST-RAP (30 mg/kg). At indicated time points, blood samples were collected, and plasma was subsequently analyzed for residual C4BP content. As shown in Fig. 4, human pd-C4BP was cleared in mice in a biphasic manner with an initial half-life of ∼50 min, whereas, in the presence of GST-RAP, pd-C4BP was cleared at a slower rate (approximate half-life of 60 min). At the 15- and 30-min time points, the content of C4BP was significantly higher (p = 0.0026 and p = 0.0098, respectively) in the presence of GST-RAP than in the absence of this LRP-antagonist. Apparently, inhibition of GST-RAP-sensitive receptors, like LRP, is associated with a delay in initial clearance of C4BP. It has been reported that binding of the C4BP α-chains to C4b is strongly ionic strength-dependent, whereas binding of streptococcal M proteins to these α-chains is of more hydrophobic nature (9Blom A.M. Berggard K. Webb J.H. Lindahl G. Villoutreix B.O. Dahlback B. J. Immunol. 2000; 164: 5328-5336Crossref PubMed Scopus (69) Google Scholar). To investigate the nature of the α-chain/LRP interaction, association of rC4BPα (20 nm) to immobilized LRP (11 fmol/mm2) was examined in 0.005% (v/v) Tween 20, 25 mm Hepes (pH 7.4), supplemented with various concentrations of NaCl (25–150 mm). As shown in Fig.5, association of rC4BPα to LRP was sensitive to the concentration of NaCl. Optimal association was observed at 35 mm NaCl, whereas association was reduced over 6-fold at NaCl concentrations exceeding 100 mm. To further examine the effect of NaCl on the rC4BPα/LRP interaction, the apparent association and dissociation rate constants were determined employing 50 mm NaCl. As for the assessment of the rate constants in the presence of 125 mm NaCl, binding of rC4BPα to LRP involved two classes of binding sites (TableI). With regard to the calculated rate constants, no significant differences were found, except for the class 2 association rate constant. Class 2 kon(app) de
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