scholarly journals The amino-terminal module of the C4b-binding protein α-chain is crucial for C4b binding and factor I-cofactor function

1997 ◽  
Vol 323 (2) ◽  
pp. 469-475 ◽  
Author(s):  
Ylva HÄRDIG ◽  
Andreas HILLARP ◽  
Björn DAHLBÄCK
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Binding Protein ◽  
Protein S ◽  
Chimeric Proteins ◽  
Amino Terminal ◽  
Factor I ◽  
Cofactor Activity ◽  
Α Chain ◽  
Β Chain

C4b-binding protein (C4BP) regulates the classical pathway C3-convertase of the complement system. Human C4BP is composed of seven identical subunits (α-chains) and one unique one (β-chain). Both types of chains contain homologous repeats called complement control proteins (CCPs); the α-chain contains eight CCPs and the β-chain three. Each α-chain contains a binding site for C4b although the detailed localization of this binding site is not known. We have used three different chimeric proteins, originally designed to localize the protein S-binding site on C4BP, to demonstrate the importance of the amino-terminal part of the α-chain for the complement-regulatory functions of C4BP. These recombinant proteins were composed of C4BP α-chains with one, two or three of the amino-terminal CCPs replaced by corresponding CCPs from the C4BP β-chain. Furthermore, seven different monoclonal antibodies were raised against C4BP and characterized using the recombinant chimeric proteins. Whereas all three recombinant chimeras bind protein S with the same affinity as plasma-purified C4BP, none of them bound to C4b. Three of the antibodies, which were found to bind to α-chain CCP 1 and CCP 2, completely inhibited the binding of plasma-purified C4BP to immobilized C4b. In addition, two of these antibodies totally blocked the factor I-cofactor activity of C4BP in a C4b-degradation assay. The binding site for one of the monoclonal antibodies was also studied using electron microscopy where it was confirmed that this antibody bound to the amino-terminal tip of the α-chain. These results show that the amino-terminal CCP of the C4BP α-chain (CCP 1) is crucial for the C4b binding and factor I-cofactor activity.

BINDING SITE OF VITAMIN K-DEPENDENT PROTEIN S ON C4b-BINDING PROTEIN

1987 ◽  
Author(s):  
K Suzuki ◽  
J Nishioka ◽  
H Kusumoto ◽  
Y Deyashiki
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Disulfide Bonds ◽  
Protein C ◽  
Binding Protein ◽  
Gel Filtration ◽  
Activated Protein C ◽  
Protein S ◽  
Cofactor Activity ◽  
Carboxyl Terminal

Protein S, a cofactor for activated protein C, reversibly complexes with a regulatory complement component C4b-binding protein (C4bp) in plasma. In plasma of patients with congenital protein S deficiency, most protein S exists as a complex with C4bp, which has no cofactor activity. C4bp (Mw 550,000) is composed of approximately seven subunits with Mw 75,000 which are linked by disulfide bonds near the carboxy1-terminus. We report here about the complex formation between protein S and C4bp particularly on the binding site of protein S on C4bp molecule. Protein S and C4bp were purified from human plasma. Seventeen mouse monoclonal antibodies against C4bp were prepared. Chymotrypsin-digested C4bp was separated on gel filtration into a fragment with Mw 160,000 derived from the carboxyl-terminal core of the intact C4bp and fragments with Mw 48,000 from the amino-terminus. The carboxy1-terminal fragment with Mw 160,000 was found to be composed of approximately seven polypeptides with Mw 25,000, which were linked by disulfide bonds.The experiments using these fragments and the monoclonal antibodies showed that: (1) Protein S bound not only to the intact C4bp, but also to the fragment with Mw 160,000. (2) The fragment with Mw 160,000 inhibited the binding of protein S to C4bp, but the fragment with Mw 48,000 did not. (3) One of the seventeen monoclonal antibodies blocked the inhibition of C4bp on the cofactor activity of protein S. (4) This antibody inhibited C4bp binding to protein S. (5) The antibody bound to the fragment with Mw 160,000. Based on these results, protein S was suggested to lose its cofactor activity for activated protein C by binding to the carboxyl-terminal core of C4bp where seven subunits are linked by disulfide bonds.

scholarly journals Localization of a Hydrophobic Binding Site for Anticoagulant Protein S on the β-Chain of Complement Regulator C4b-binding Protein

2000 ◽  
Vol 276 (6) ◽  
pp. 4330-4337 ◽  
Author(s):  
Joanna H. Webb ◽  
Bruno O. Villoutreix ◽  
Björn Dahlbäck ◽  
Anna M. Blom
Keyword(s):  
Binding Site ◽  
Binding Protein ◽  
Protein S ◽  
Hydrophobic Binding ◽  
Complement Regulator ◽  
Β Chain

scholarly journals Structural and functional studies of complement inhibitor C4b-binding protein

2002 ◽  
Vol 30 (6) ◽  
pp. 978-982 ◽  
Author(s):  
A. M. Blom
Keyword(s):  
Amino Acids ◽  
Computer Modelling ◽  
Binding Protein ◽  
Protein S ◽  
Classical Pathway ◽  
Complement Factor ◽  
Charged Amino Acids ◽  
Α Chain ◽  
Β Chain ◽  
Positively Charged

C4b-binding protein (C4BP) is a potent inhibitor of the classical pathway of the complement system. This large plasma glycoprotein consists of seven identical α-chains and a unique β-chain held together by disulphide bridges. Both types of subunits are composed almost exclusively of complement control protein domains (CCPs). Using homology-based computer modelling and mutagenesis of recombinant proteins we have localized binding sites for several ligands of C4BP: complement factor C4b, heparin and vitamin K-dependent anticoagulant protein S (PS). We found that C4b requires CCP1–3 of the α-chain for binding. The interaction is ionic in nature and mediated by a cluster of positively charged amino acids present on the interface between CCP1 and CCP2 of the α-chain. Loss of C4b-binding resulted in a loss of all inhibitory functions of C4BP within the classical pathway of complement. Binding of heparin required CCPs 1–3 of the α-chain, with CCP2 being the most important, as well as the cluster of positively charged amino acids involved in binding of C4b. The interaction between C4BP and PS is of very high affinity and conveyed by a cluster of surface exposed hydrophobic amino acids localized on CCP1 of the β-chain. Furthermore, C4BP is captured on the surface of several pathogens, which may contribute to their serum resistance and pathogenicity. We have localized interaction of C4BP with Neisseria gonorrhoeae, Bordetella pertussis, Streptococcus pyogenes and Escherichia coli to various regions of the α-chain.

scholarly journals Role of CCP2 of the C4b-binding protein β-chain in protein S binding evaluated by mutagenesis and monoclonal antibodies

2002 ◽  
Vol 270 (1) ◽  
pp. 93-100 ◽  
Author(s):  
Joanna H. Webb ◽  
Bruno O. Villoutreix ◽  
Björn Dahlbäck ◽  
Anna M. Blom
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Protein ◽  
Protein S ◽  
Β Chain

scholarly journals Degradation of human complement component C4b in the presence of the C4b-binding protein-protein S complex

1983 ◽  
Vol 209 (3) ◽  
pp. 857-863 ◽  
Author(s):  
B Dahlbäck ◽  
B Hildebrand
Keyword(s):  
Binding Sites ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Fluid Phase ◽  
Protein S ◽  
Factor I ◽  
Haemolytic Assay ◽  
Dependent Protein ◽  
Molecular Weight Form ◽  
Human Complement Component

Vitamin K-dependent protein S and the higher-molecular-weight form of C4b-binding protein (C4bp-high) interact, forming a 1:1 complex with a KD of approx. 1×10(-7) M [Dahlbäck (1983) Biochem. J. 209, 847-856]. In the present study the effect of protein S on the degradation of C4b by Factor I (C3b inactivator) and C4bp was investigated both in fluid phase and on cell surfaces, with the use of highly purified components. Fluid-phase degradation of C4b was monitored on sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis, and the effect on surface-bound C4b was estimated by haemolytic assay. No effect of protein S could be demonstrated in any of the systems used. Thus, although bound to C4bp, protein S is neither involved in, nor does it affect, the interaction between C4bp and C4b. This indicates that the binding sites on the C4bp molecule for protein S and for C4b are independent and different.

Monoclonal Antibodies Directed to the Calcium-Free Conformation of Human Protein S

1989 ◽  
Vol 62 (02) ◽  
pp. 708-714 ◽  
Author(s):  
Santica Marcovina ◽  
Raffaella Coppola ◽  
Carla Valsecchi ◽  
Adele Zoppo ◽  
Cecilia Gelfi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Protein ◽  
Antigenic Site ◽  
Fluid Phase ◽  
Protein S ◽  
Human Protein ◽  
Sephadex Column ◽  
Sds Page ◽  
Igg1 Subclass ◽  
Sepharose 4B

SummaryFour mouse hybridomas secreting monoclonal antibodies specific for human protein S (PS) have been generated. The antibodies, all of the IgG1 subclass, were designated S2, S3, S8, and S10. In a fluid phase radioimmunoassay, the binding of monoclonal antibodies to PS was about 30% greater in the presence of EDTA and totally inhibited in presence of Ca2+. Using the same technique, we performed displacement curves of 125I-labeled PS by purified PS, thrombin-cleaved PS, normal plasma, plasma from a patient on warfarin therapy, and plasma from a patient with no free PS and only PS bound to C4b-binding protein. The slopes of the curves show that the monoclonal antibodies reacted equally with all the tested forms of PS indicating that the antigenic site(s) to which the monoclonal antibodies are directed are present and exposed in free and bound PS, in thrombin-cleaved PS, and in the coumarin form of the protein. Each EDTA-dependent antibody, immobilized on Sepharose 4B-CNBr was used to purify PS from the barium citrate-absorbed, ammonium sulphate-soluble fraction of plasma. The fraction eluted from the immunoabsorbent with a buffer containing 4 mmol/1 CaCl2 and analysed by SDS-PAGE, contained two bands, one migrating with conventionally purified PS and the other with purified C4b-binding protein. Homogeneous PS was obtained by chromatography of the barium citrate absorbate on a DEAE-Sephadex column. The protein peak containing the bulk of PS was subsequently applied to the immunoadsorbent and eluted with 4 mmol/1 CaCl2. These studies demonstrate that EDTA-dependent monoclonal antibodies can simplify the isolation of PS from human plasma.

MONOCLONAL ANTIBODIES TO HUMAN PROTEIN S AND C4b-BINDING PROTEIN

1987 ◽  
Author(s):  
Martin Hessing ◽  
Joost C M Meijers ◽  
Jan A van Mourik ◽  
Bonno N Bouma
Keyword(s):  
Monoclonal Antibodies ◽  
Complex Formation ◽  
Binding Protein ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Thrombin Cleavage ◽  
Prolonged Aptt ◽  
Acid Domain ◽  
A Minor

Protein S (PS) circulates in plasma both free and in reversible association with the complement component C4b-binding protein (C4bp). Only free PS is functional as a cofactor for activated protein C (APC). Cleavage of PS by thrombin at a site near the r-carboxyglutamic acid domain is associated with a loss of cofactor activity. This may be a control mechanism for the anticoagulant activity of APC. These observations led us to investigate the role of C4bp and thrombin in the regulation of PS. Complex formation between purified PS and C4bp was studied in plasma and in a system with purified components. 125I-labeled PS was first incubated with either C4bp or citrated plasma and then subjected to polyacrylamide gelelectrophoresis in the absence of SDS. The formation of the C4bp-PS complex in plasma and in the purified system was demonstrated by autoradiography. Crossed immuno-electrophoresis using an antiserum against PS was performed in the presence of 8 mM EDTA. Human citrated plasma showed two precipitin peaks. Free PS migrated rapidly in the first dimension, whereas the C4bp-PS complex was just anodal to the application slot. The addition of C4bp to either plasma or purified PS resulted in the disappearance of the free PS peak and an increase of the slower migrating peak. The effect of purified C4bp on the PS-cofactor function of APC was studied in citrated plasma. The prolongation of the APTT induced by the addition of APC could be inhibited by the addition of increasing amounts of C4bp. Monoclonal antibodies to PS and C4bp were prepared and characterized. The monoclonal antibodies to either PS or C4bp did not block the complex formation between and PS, as was demonstrated by dot blotting of C4bp with 125I-PS and agarose gelelectrophoresis followed by Western blotting. Three out of 7 monoclonal antibodies to PS did not detect PS after thrombin cleavage on an immunoblot after non-reduced SDS polyacrylamide gelelectrophoresis. These 3 antibodies gave a significant shortening of the prolonged APTT induced by the addition of APC to normal plasma, indicating that these monoclonals inhibited the cofactor function of PS. The other 4 monoclonals to PS that did detect PS after thrombin cleavage on an immunoblot, gave only a minor inhibition of the PS cofactor function.

scholarly journals Expression and characterization of a recombinant C4b-binding protein lacking the β-chain

1995 ◽  
Vol 308 (3) ◽  
pp. 795-800 ◽  
Author(s):  
Y Härdig ◽  
P García de Frutos ◽  
B Dahlbäck
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Expression System ◽  
Activated Protein C ◽  
Protein S ◽  
High Molecular Mass ◽  
Eukaryotic Expression ◽  
Beta Chain ◽  
Factor I ◽  
Dependent Protein

C4b-binding protein (C4BP) is a high-molecular-mass glycoprotein which contains binding sites for complement component C4b, anti-coagulant vitamin K-dependent protein S and serum amyloid P component (SAP). The major form of C4BP in plasma is composed of seven identical alpha-chains and a single beta-chain. We have expressed full-length cDNA for the alpha-chain in a eukaryotic expression system and characterized functional properties of non-beta-chain-containing C4BP. During synthesis, recombinant alpha-chains polymerized into two different high-molecular-mass C4BP forms which were composed of seven or eight alpha-chains. Recombinant C4BP bound C4(H2O) (used instead of C4b) equally as well as native C4BP, functioned equally as well as factor I cofactor in the degradation of C4(H2O) and bound to SAP. In contrast, the recombinant C4BP did not bind protein S and therefore did not inhibit the ability of protein S to function as a cofactor to activated protein C. Tunicamycin treatment of the transfected cells prevented N-linked glycosylation, but did not affect polymerization of the alpha-chains into a high-molecular-mass C4BP. The non-glycosylated C4BP had comparable properties to glycosylated C4BP in several functional assays. These results demonstrate polymerization of C4BP alpha-chains to be independent both of the beta-chain and of the N-linked carbohydrates. Moreover, N-linked carbohydrates and the beta-chain were neither required for the ability of C4BP to bind C4b and to function as factor I cofactor nor for the interaction with SAP.

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