The amino-terminal module of the C4b-binding protein alpha-chain is crucial for C4b binding and factor I-cofactor function.
C4b-binding protein (C4BP) regulates the classical pathway C3-convertase of the complement system. Human C4BP is composed of seven identical subunits (alpha-chains) and one unique one (beta-chain). Both types of chains contain homologous repeats called complement control proteins (CCPs); the alpha-chain contains eight CCPs and the beta-chain three. Each alpha-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 alpha-chain for the complement-regulatory functions of C4BP. These recombinant proteins were composed of C4BP alpha-chains with one, two or three of the amino-terminal CCPs replaced by corresponding CCPs from the C4BP beta-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 alpha-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 alpha-chain. These results show that the amino-terminal CCP of the C4BP alpha-chain (CCP 1) is crucial for the C4b binding and factor I-cofactor activity.
Härdig
Y
Y
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, S-205 02 Malmö, Sweden.
Hillarp
A
A
Dahlbäck
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000925:Anticoagulants / Q000378:metabolism; D001667:Binding, Competitive; D015935:Complement C4b / Q000378:metabolism*; D017244:Complement Factor I / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D018604:Epitope Mapping; D006023:Glycoproteins; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008958:Models, Molecular; D011487:Protein Conformation; D017293:Protein S / Q000378:metabolism; D011951:Receptors, Complement / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D017293:Protein S; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015935:Complement C4b; D017244:Complement Factor I