Protein S binding in relation to the subunit composition of human C4b-binding protein

The human regulatory complement component C4b-binding protein (C4BP) circulates in plasma either as a free protein or in a bimolecular complex with the vitamin K-dependent protein S. The major form of C4BP is composed of 7 identical, disulfide-linked 70 kDa subunits (alpha-chains), the arrangement of which gives the C4BP molecule a spider-like appearance. Recently, we identified a unique 45 kDa subunit (beta-chain) in C4BP. We have now isolated a subpopulation of C4BP, which does not bind protein S. This C4BP species, which had a molecular weight slightly lower than that of the predominant form, was found to lack the beta-chain. Another lower molecular weight form of C4BP was also purified. It contained the beta-chain and was efficient in binding protein S. Its subunit composition was judged to comprise six alpha-chains and one beta-chain. These results indicate C4BP in plasma to be heterogeneous at a molecular level vis-a-vis subunit composition and/or protein S binding ability and provide support for the concept that the beta-chain of C4BP contains the single protein S binding site.     

Solution-phase equilibrium binding interaction of human protein S with C4b-binding protein.

Solution-phase equilibrium binding studies of human protein S (HPS) and C4b-binding protein (C4BP) were undertaken using purified components. Free C4BP was measured in solutions at equilibrium by using HPS immobilized on a solid phase, coupled with an antibody detection system. Disruption of the solution-phase equilibrium was minimized by using a brief (15 min) exposure to the solid-phase HPS. These studies yielded an equilibrium dissociation constant (Kd) approximately 6 x 10(-10) M and a stoichiometry of approximately 1.7 molecules of HPS bound to each molecule of C4BP. This Kd is between 27-fold and 930-fold lower than previously published values obtained by using solid-phase and nonequilibrium methods. Equilibrium was achieved in solutions containing low nanomolar concentrations of both HPS and C4BP in less than or equal to 1 h at 37 degrees C, suggesting a rapid association rate constant for the interaction. Thrombin cleavage of HPS had no effect on the observed binding parameters. The binding interaction between HPS and C4BP appears to be partly calcium dependent, since in the presence of EDTA the Kd was increased to about 6 x 10(-9) M, with no change in the stoichiometry. This high-affinity binding interaction between HPS and C4BP, whose Kd is more than 500-fold lower than the proteins' plasma concentrations, heightens the apparent physiologic importance of complex formation.     

Both G-type domains of protein S are required for the high-affinity interaction with C4b-binding protein.

Anticoagulant protein S interacts with the complement regulatory protein C4b-binding protein (C4BP) via its sex-hormone-binding globulin (SHB6)-like region, which contains two globular (G) domains. Similar G domains are found in Gas6, a protein homologous to protein S, which is not known to bind C4BP or to have any anticoagulant activity. To determine the relative importance of the two G domains in protein S for C4BP protein binding, three recombinant protein S chimeras were produced having either of the two globular domains, or the whole SHB6-like globulin region, replaced by corresponding parts from Gas6. The chimeras were tested for binding to immobilized C4BP using surface-plasmon-resonance technology and microtiter plate-based assays. In both systems, chimeras containing either only globular domains G1 or G2 from protein S were found to bind C4BP. Binding was stimulated by Ca2+ in a manner similar to that found for wild-type protein S. The affinities for C4BP of both chimeras containing individual G domains from protein S, were lower than that of wild-type protein S. Chimera II, containing the G1 domain from protein S, consistently bound C4BP more efficiently than chimera I, which had the protein S-derived G2 domain. The chimera containing the whole SHB6-like globulin region from Gas6 interacted considerably more weakly with C4BP. Our results demonstrate that both G domains of protein S are involved in the interaction between protein S and C4BP and that full affinity binding is dependent on contributions from both domains.     

Structural requirements of anticoagulant protein S for its binding to the complement regulator C4b-binding protein

The vitamin K-dependent anticoagulant protein S binds with high affinity to C4b-binding protein (C4BP), a regulator of complement. Despite the physiological importance of the complex, we have only a patchy view of the C4BP-binding site in protein S. Based on phage display experiments, protein S residues 447-460 were suggested to form part of the binding site. Several experimental approaches were now used to further elucidate the structural requirements for protein S binding to C4BP. Peptides comprising residues 447-460, 451-460, or 453-460 of protein S were found to inhibit the protein S-C4BP interaction, whereas deletion of residues 459-460 from the peptide caused complete loss of inhibition. In recombinant protein S, each of residues 447-460 was mutated to Ala, and the protein S variants were tested for binding to C4BP. The Y456A mutation reduced binding to C4BP approximately 10-fold, and a peptide corresponding to residues 447-460 of this mutant was less inhibitory than the parent peptide. A further decrease in binding was observed using a recombinant variant in which a site for N-linked glycosylation was moved from position 458 to 456 (Y456N/N458T). A monoclonal antibody (HPSf) selective for free protein S reacted poorly with the Y456A variant but reacted efficiently with the other variants. A second antibody, HPS 34, which partially inhibited the protein S-C4BP interaction, reacted poorly with several of the Ala mutants, suggesting that its epitope was located in the 451-460 region. Phage display analysis of the HPS 34 antibody further identified this region as its epitope. Taken together, our results suggest that residues 453-460 of protein S form part of a more complex binding site for C4BP. A recently developed three-dimensional model of the sex hormone-binding globulin-like region of protein S was used to analyze available experimental data.     

beta-Hydroxyaspartic acid and beta-hydroxyasparagine residues in recombinant human protein S are not required for anticoagulant cofactor activity or for binding to C4b-binding protein.

Among the vitamin K-dependent plasma proteins, only protein S contains the post-translationally modified amino acid erythro-beta-hydroxyasparagine (Hyn). Protein S also contains erythro-beta-hydroxyaspartic acid (Hya). The function of these unusual amino acids, located in the epidermal growth factor-like domains, is unknown. To determine if these post-translational modifications contribute to the functional integrity of human protein S (HPS), recombinant human protein S lacking Hya and Hyn (rHPSdesHya/Hyn) was purified from the medium of human kidney 293 cells that were transfected with HPS cDNA and grown in the presence of the hydroxylase inhibitor 2,2'-dipyridyl. Solution-phase equilibrium binding studies revealed that rHPSdesHya/Hyn binds C4b-binding protein (C4BP) in a manner indistinguishable from recombinant HPS and plasma-derived HPS, exhibiting a Kd in the presence of 2 mM CaCl2 of approximately 0.7 nM and a Kd in the presence of 4 mM EDTA approximately 10-fold higher. In a purified component system, rHPSdesHya/Hyn displayed normal anticoagulant cofactor activity in the activated protein C-catalyzed inactivation of coagulation factor Va bound in the prothrombinase complex. In addition, digestion of rHPSdesHya/Hyn with thrombin in the presence of EDTA appeared normal, and 2 mM CaCl2 prevented the cleavage. Together these results suggest that the post-translational modifications of Asn and Asp residues are not necessary for the macromolecular or Ca2+ interactions associated with the anticoagulant and C4BP binding characteristics of HPS.     

Assembly of protein S and C4b-binding protein on membranes

The interaction of protein S with membranes and subsequent combination with complement C4b-binding protein (C4BP) was studied. Protein S interacted with phospholipid vesicles in a calcium-dependent manner typical of other vitamin K-dependent proteins. Association of C4BP with protein S showed no apparent selectivity for membrane-bound or solution phase protein S. When bound to the membrane, the protein complexes projected out from the vesicle surface and induced vesicle radius changes of 11.4 nm for tightly packed protein S alone and 17.5 nm for the protein S-C4BP complex. Due to a low density of the protein S-C4BP on the membrane at saturation, the actual projection of this complex out from the membrane surface would be much greater than 17.5 nm. A low saturation density suggested that the protein complex had a large two-dimensional hydrodynamic radius in the plane of the membrane that prevented tight packing of protein. In the presence of calcium, the protein-protein interaction was rapid (ka greater than or equal to 1.10(6) M-1 s-1) and had very high affinity (KD less than or equal to 10(-10) M). The dissociation rate was slow with an estimated rate constant of less than or equal to 2.10(-4) s-1 at 25 degrees C. Protein-protein interaction was much slower in the absence of calcium with an estimated association rate constant of only 2.10(4) M-1 s-1. Consequently, the protein-protein interaction was greatly enhanced by calcium. The very high affinity interaction between protein S and C4BP suggested specificity and an important function for the protein S-C4BP complex in blood. In this regard it was important that C4BP which was bound to protein S on the phospholipid surface could interact with complement protein C4b. These results suggested that protein S may serve an important role in localizing C4BP to negatively charged phospholipid. This would provide regulation of complement activation at sites where the coagulation system is activated such as on the surface of activated platelets.     

Binding site for vitamin K-dependent protein S on complement C4b-binding protein

Half of the protein S in plasma is present as a complex with a C4b-binding protein (C4bp), a complement component (Mr 570,000). In this study, the protein S-binding site on C4bp was examined by using monoclonal anti-C4bp-IgGs. C4bp was cleaved by chymotryptic digestion into seven NH2-terminal arm fragments (Mr 48,000) and a COOH-terminal core fragment (Mr 160,000). The COOH-terminal fragment inhibited the cofactor activity of protein S and its binding to C4bp in a dose-dependent manner. A monoclonal anti-C4bp-IgG (MFbp16), which binds to the COOH-terminal fragment, inhibited the binding of protein S to C4bp. The chymotryptic digest of the reduced and carboxymethylated COOH-terminal fragment was subjected to MFbp16-Sepharose 4B column affinity chromatography, and a peptide of Mr 2,500 was obtained. Protein S bound to the Mr 2,500 peptide, and this binding was inhibited by C4bp in a dose-dependent manner. The sequence of this peptide corresponded to Ser447-Tyr467 near the COOH terminus of the C4bp subunit. MFbp16, which bound to Mr 570,000 C4bp (C4bp-high), did not bind to Mr 510,000 C4bp (C4bp-low) in human plasma that does not form a complex with protein S. This suggests that C4bp-low lacks the protein S-binding site present in the COOH-terminal region of C4bp-high. Since C4bp-low also dissociates into identical subunits when reduced, the interchain disulfide bond region that links the seven subunits of C4bp appears to be closer to the NH2-terminal end than the protein S-binding site.     

Binding of protein S to C4b-binding protein. Mutagenesis of protein S.

Protein S and C4b-binding protein (C4BP) form a tight complex (Kd approximately 0.6 nM) the physiologic purpose of which is unknown. The participation of protein S in this complex was investigated using site-specific mutagenesis. Normal recombinant human protein S (rHPS) and five specifically mutated protein S analogs were expressed in transformed human kidney 293 cells and the following properties were characterized: solution-phase C4BP binding, ability to be cleaved by thrombin, ability to act as a cofactor in the activated protein C-catalyzed inactivation of factor Va, and gamma-carboxyglutamic acid content. In some cases, beta-hydroxyaspartic acid plus beta-hydroxyasparagine content was also determined. Binding studies indicated that while clearly important for a high affinity interaction, the amino acid sequence Gly605-Ile614 identified by Walker (Walker, F J. (1989) J. Biol. Chem. 264, 17645-17648) does not account for all the binding energy of the HPS-C4BP interaction. All mutants perturbed in this region or lacking it altogether displayed reduced C4BP binding, and some retained anticoagulant cofactor function. Neither human factor X nor human steroid-binding protein had any measurable ability to compete with plasma HPS for C4BP binding. Furthermore, bovine protein S and a rHPS analog with bovine sequence from Gly597-Trp629 bound to human C4BP with the same affinity as did HPS, and both proteins substituted effectively for HPS as a cofactor for activated protein C in an otherwise human anticoagulation system. Together these results suggest that optimal binding of protein S to C4BP requires the putative alpha-helix Gly605-Ile614, as well as other undetermined regions of protein S, and that the regions of HPS responsible for C4BP binding and activated protein C cofactor function are structurally isolated.     

Inhibition of cofactor activity of protein S by a complex of protein S and C4b-binding protein. Evidence for inactive ternary complex formation between protein S, C4b-binding protein, and activated protein C

To elucidate the mechanism by which C4b-binding protein inhibits the cofactor activity of protein S for anticoagulant-activated protein C, the interactions between protein S, activated protein C, and C4b-binding protein were studied using solid-phase enzyme immunoassays. Both activated protein C and C4b-binding protein bound to protein S fixed to microplate wells. C4b-binding protein did not inhibit the binding of activated protein C to protein S, nor did activated protein C inhibit the binding of C4b-binding protein to protein S. Activated protein C bound to a protein S-C4b-binding protein complex which was cross-linked with a chemical reagent as well as it bound to free protein S. Protein S-C4b-binding protein complex competitively inhibited activated protein C-binding to free protein S and also the cofactor activity of free protein S. Immunoblotting analysis showed ternary complex formation with protein S, C4b-binding protein, and activated protein C in the liquid phase by treatment with the cross-linking reagent. These findings suggest that the protein S-C4b-binding protein complex inhibits the cofactor activity of free protein S probably by inhibition of functionally active protein S-activated protein C complex formation by the apparent competitive formation of an inactive ternary complex with protein S, C4b-binding protein, and activated protein C.     

Structural requirements for the intracellular subunit polymerization of the complement inhibitor C4b-binding protein

C4b-binding protein (C4BP), an important inhibitor of complement activation, has a unique spider-like shape. It is composed of six to seven identical alpha-chains with or without a single beta-chain, the chains being linked by disulfide bridges in their C-terminal parts. To elucidate the structural requirements for the assembly of the alpha-chains, recombinant C4BP was expressed in HEK 293 cells. The expressed C4BP was found to contain six disulfide-linked alpha-chains. Pulse-chase analysis demonstrated that the recombinant C4BP was rapidly synthesized in the cells and the polymerized C4BP appeared in the medium after 40 min. The alpha-chains were polymerized in the endoplasmic reticulum (ER) already after 5 min chase. The polymerization process was unaffected by blockage of the transport from the ER to the Golgi mediated by brefeldin A or low temperature (10 degrees C). The C-terminal part of the alpha-chain (57 amino acids), containing 2 cysteine residues and an amphiphatic alpha-helix region, was required for the polymerization. We constructed and expressed several mutants of C4BP that lacked the cysteine residues and/or were truncated at various positions in the C-terminal region. Gel filtration analysis of these variants demonstrated the whole alpha-helix region to be required for the formation of stable polymers of C4BP, which were further stabilized by the formation of disulfide bonds.     

Localization of a hydrophobic binding site for anticoagulant protein S on the beta -chain of complement regulator C4b-binding protein

C4b-binding protein (C4BP) is a plasma glycoprotein involved in regulation of the complement system. C4BP consists of seven alpha-chains and one unique beta-chain, all constructed of repeating complement control protein (CCP) modules. The beta-chain, made up of three CCPs, binds tightly to vitamin K-dependent protein S, a cofactor to anticoagulant activated protein C. When bound to C4BP, protein S loses its activated protein C cofactor function. In this study, we have mutated potentially important amino acids located at the surface of CCP1 of the beta-chain to probe the protein S-C4BP interaction. The substitutions were designed after analysis of a homology-based three-dimensional structure of the beta-chain and were L27T/F45Q, I16S/V18S, V31T/I33N, I16S/V18S/V31T/I33N, L38S/V39S, and K41E/K42E. The mutants were expressed in a prokaryotic system, purified using an N-terminal His-tag, refolded using an oxido-shuffling system, and tested in several assays for their ability to bind protein S. Our data define Ile(16), Val(18), Val(31), and Ile(33) as crucial for protein S binding, with secondary effects from Leu(38) and Val(39). In addition, Lys(41) and Lys(42) contribute slightly to the interaction. Our results further confirm that surface hydrophobicity analysis may be used to identify ligand recognition sites.     

Characterization of the interaction between human protein S and C4b-binding protein (C4bp)

C4b-binding protein, C4bp, is a regulatory factor of the complement system and is also known to be a binding protein of vitamin K-dependent coagulation factor, protein S. Whereas the C4b-binding site is known to be located in the middle part of the subunit chains of C4bp, the location and properties of protein S-binding site are uncertain. Therefore, we have examined the characteristics of the interaction between human protein S and C4bp. Proteolysis of C4bp-protein S complex with chymotrypsin yielded N-terminal-derived 48-kDa fragments of C4bp subunit chains and a C-terminal-derived 160-kDa core fragment of C4bp, to which protein S was still bound. This result suggested that the protein S-binding site is located in the core domain of C4bp. Gel filtration of guanidine-treated C4bp-protein S complex in the absence of guanidine resulted in the separation of C4bp and protein S. Binding assay with 125I-labeled protein S showed that the guanidine-treated C4bp lacked the protein S-binding activity. This result suggests that the protein S-binding site in C4bp is denatured irreversibly by guanidine treatment and therefore seems to be dependent on a specific conformation of C4bp. The C4bp-binding site of protein S was lost upon thrombin treatment, suggesting that the N-terminal thrombin-sensitive region of protein S may be related to the C4bp-binding site. Although free protein S was susceptible to chymotrypsin, leukocyte elastase, and cathepsin G, C4bp-bound protein S was found to be resistant to these proteases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of the alpha 3-fragment of complement C4 for the binding with C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the complement system. C4BP functions as a cofactor to factor 1 in the degradation of C4b and accelerates the decay rate of the C4b2a complex. Previously, we have demonstrated that monoclonal antibodies (C4-2 and 9) directed against the alpha'-chain of C4b inhibit the binding of C4b to C4BP. In order to identify the structural domain of C4b that binds C4BP, proteolytic fragments of C4 were generated with trypsin and Staphylococcus aureus V8 protease. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and amino acid sequence analysis of the proteolytic fragments reactive with the anti-C4 mAb's revealed that the residues Ala738-Arg826 of the alpha 3-fragment of C4b are important for the interaction with C4BP.     

The binding site of human C4b-binding protein on complement C4 is localized in the alpha'-chain

C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. We now demonstrate that C4b contains a binding site for C4BP, which is localized on the alpha'-chain of C4b. SDS-PAGE of C C4 and C4b both under reducing and nonreducing conditions was followed by a radiolabeled C4BP ligand blotting procedure. It was demonstrated that the C4BP binding site on C4b is localized on the alpha'-chain. In addition, we found C4BP binding to the alpha-chain of C4, which suggests that the binding site for C4BP becomes available after reduction of the C4 molecule. Direct binding of C4BP to the alpha- and alpha'-chains of C4 and C4b was demonstrated in a radio-labeled C4BP binding assay with the reduced and alkylated isolated chains. mAb against the alpha'-chain of C4b were prepared, characterized, and evaluated for their ability to block the binding of 125I-C4BP to C4b. Two mAb specific for the alpha'-chain of C4b were found that completely abolished C4BP binding to intact C4b. Other mAb recognizing both the alpha- and alpha'-chain of C4 and C4b demonstrated only minor inhibitory effect on the binding of C4BP to C4b. In conclusion, we have localized the C4BP binding site on the alpha'-chain of C4b and have demonstrated that this binding can be inhibited by mAb specific for the alpha'-chain.     

Role of CCP2 of the C4b-binding protein beta-chain in protein S binding evaluated by mutagenesis and monoclonal antibodies.

Complement regulator C4b-binding protein (C4BP) and the anticoagulant vitamin K-dependent protein S form a high affinity complex in human plasma. C4BP is composed of seven alpha-chains and a unique beta-chain, each chain comprising repeating complement control protein (CCP) modules. The binding site for protein S mainly involves the first of the three beta-chain CCPs (CCP1). However, recently it has been suggested that CCP2 of the beta-chain also contributes to the binding of protein S. To elucidate the structural background for the involvement of CCP2 in the protein S binding, several recombinant beta-chain CCP1-2 variants having mutations in CCP2 were expressed and tested for protein S binding. Mutations were chosen based on analysis of a homology model of the beta-chain and included R60A/R101A, D66A, L105A, F114A/I116A and H108A. All mutant proteins bound equally well as recombinant wild type to protein S. Several monoclonal antibodies against the beta-chain CCP2 were raised and their influence on protein S binding characterized. Taken together, the results suggest that the role of CCP2 in protein S binding is to orient and stabilize CCP1 rather than to be directly part of the binding site.     

Ribosomal components required for binding protein S1 to the 30 S subunit of Escherichia coli.

30 S subunits of Escherichia coli ribosomes washed with 3 m-NH₄C1 lose proteins S2, S3, S9, S10, S14, S20 and S21, as well as their ability to bind S1 with high affinity (Laughrea and Moore, 1978). Binding activity is restored when the split proteins are added back to the protein-deficient cores. Here we show that, among the split proteins, S9 is by far the most effective in restoring S1 binding capability to 3 m-NH₄Cl cores.     

Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein

Vitamin K-dependent protein S exists in two forms in plasma, as free protein and in a bimolecular, noncovalent complex with the regulatory complement protein C4b-binding protein (C4BP). The effects of C4BP on the protein Ca cofactor activity of protein S were studied in a plasma system and in a system using purified components from both human and bovine origin. Bovine protein S was found to interact with human C4BP with a 5-fold higher affinity than that observed for the interaction between human protein S and human C4BP. The binding of protein S, from either species, to human C4BP results in the loss of the protein Ca cofactor function. In bovine plasma, protein S could be totally complexed by the addition of human C4BP, with a concomitant total loss of protein Ca cofactor activity. The addition of purified human C4BP to human plasma resulted in only partial loss of protein Ca cofactor activity and the plasma protein S was not completely complexed. Human protein S functioned as a cofactor to human protein Ca, but not to bovine protein Ca, whereas bovine protein S demonstrated very little species specificity and functioned as a cofactor both with human and bovine protein Ca. The species specificity of the protein Ca-protein S interaction was useful in elucidating the effect of C4BP in the plasma system. In the system with purified bovine components, protein S was required for the degradation of factor Va by low concentrations of protein Ca, whereas in the system with human components protein Ca alone, even when added at very low concentrations, exhibited potential to degrade factor Va, and the presence of protein S only enhanced the reaction rate approximately 5-fold. In both these systems, the stimulating effect of protein S on factor Va degradation by protein Ca was completely lost when protein S bound to C4BP.     

Human C4b-binding protein has overlapping, but not identical, binding sites for C4b and streptococcal M proteins

Many strains of Streptococcus pyogenes bind C4b-binding protein (C4BP), an inhibitor of complement activation. The binding is mediated by surface M proteins in a fashion that has been suggested to mimic the binding of C4b. We have previously shown that a positively charged cluster at the interface between complement control protein domains 1 and 2 of C4BP alpha-chain is crucial for the C4b-C4BP interaction. To extend this observation, and to investigate the interaction with M proteins, we constructed and characterized a total of nine mutants of C4BP. We identified a key recognition surface for M proteins that overlaps with the C4b binding site because substitution of R64 and H67 by Gln dramatically reduces binding to both ligands. However, the analysis of all mutants indicates that the binding sites for C4b and M proteins are only overlapping, but not identical. Furthermore, M proteins were able to displace C4BP from immobilized C4b, whereas C4b only weakly affected binding of C4BP to immobilized M proteins. We found that the molecular mechanisms involved in these two interactions differ because the binding between M proteins and C4BP is relatively insensitive to salt in contrast to the C4BP-C4b binding. In addition, six mAbs directed against the alpha-chain interfered with C4b-C4BP interaction, whereas only two of them efficiently inhibited binding of C4BP to M proteins. Collectively, our results suggest that binding between C4b and C4BP is governed mostly by electrostatic interactions, while additional noncovalent forces cause tight binding of C4BP to streptococcal M proteins.     

Evidence that C4b-binding protein is an acute phase protein

C4b-binding protein is a regulatory factor for both complement and coagulation systems. We found that a human hepatoma cell line, Hep G2, was capable of synthesizing C4b-binding protein and that the secretion of C4b-binding protein was enhanced by interleukin-6 and tumor necrosis factor, which are known to be modulators of acute phase proteins. In addition, the plasma content of C4b-binding protein was found to increase in patients of acute pneumonia. These results suggest that C4b-binding protein is an acute phase protein.