Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H

The amino acid sequence of the region of bovine factor H containing the C3b binding site has been derived from sequencing overlapping cDNA clones. A cDNA sequence encoding 669 amino acids was obtained. Like human and mouse factor H the sequence can be arranged into a number of internally homologous units (CPs), each of which is about 60 amino acids long and is based on a framework of four conserved cysteine residues. Bovine factor H is of the same molecular mass as human and mouse factor H, and is therefore likely to be composed of 20 contiguous CPs. Comparisons with human and mouse factor H indicate that the partial bovine sequence encodes CPs 2–12 inclusive of bovine factor H. Bovine factor H binds to human ammonia-treated C3 (causing thiolester cleavage) [C3(NH3)] and promotes the cleavage of human C3(NH3) in the presence of bovine factor I. Other studies indicate that CPs 2–5 of human factor H encompass the C3b binding and factor I cofactor activity site. Multiple sequence alignments of human factor H, mouse factor H (which also interacts with human C3b) and bovine factor H with CP modules whose structures have been determined experimentally, have been used to predict residues in the hypervariable loops of CPs 2–5 and to identify residues of potential importance in human C3 binding and factor I cofactor activity. Leu-17 and Gly-20 of CP 2, Ser-17, Ala-19, Glu-21, Asp-23 and Glu-25 of CP 3 and Lys-18 of CP 4 are all conserved between the three species. It may be that CPs 3 and 4 interact with C3(NH3) directly, whilst CPs 2 and 5 maintain the correct orientation for CPs 3 and 4 to interact.

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

Biochemical Journal ◽

10.1042/bj3230469 ◽

1997 ◽

Vol 323 (2) ◽

pp. 469-475 ◽

Cited By ~ 44

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.

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Functional and antigenic properties of complement receptor type 2, CR2.

Journal of Experimental Medicine ◽

10.1084/jem.165.5.1424 ◽

1987 ◽

Vol 165 (5) ◽

pp. 1424-1429 ◽

Cited By ~ 24

Author(s):

K Mitomo ◽

T Fujita ◽

K Iida

Keyword(s):

Factor H ◽

Regulatory Proteins ◽

Complement Receptor ◽

Supporting Evidence ◽

Complement Cascade ◽

Factor I ◽

Antigenic Properties ◽

Membrane Bound ◽

Cofactor Activity

This is the first report demonstrating that C3d receptor (CR2) has functional activity in regulating complement cascade. Purified CR2 was examined for its cofactor activity in factor I-mediated cleavage of membrane-bound iC3b. CR2 plus C3b inactivator (I) released C3c from EA 125I-iC3b, and the release was inhibited when CR2 was preincubated with OKB7 monoclonal anti-CR2. Furthermore, immunoelectroblotting analysis showed crossreactivity of CR2 with 57H anti-CR1. These results indicate that CR2 has functional and antigenic similarity to CR1, thus providing a supporting evidence for placement of CR2 as a member of the recently defined gene family of C3- and C4-regulatory proteins composed of CR1, C4-binding protein, and factor H.

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Functional properties of membrane cofactor protein of complement

Biochemical Journal ◽

10.1042/bj2640581 ◽

1989 ◽

Vol 264 (2) ◽

pp. 581-588 ◽

Cited By ~ 107

Author(s):

T Seya ◽

J P Atkinson

Keyword(s):

Complement System ◽

Fluid Phase ◽

Factor H ◽

Classical Pathway ◽

Membrane Cofactor Protein ◽

Cleavage Reaction ◽

Factor I ◽

Thioester Bond ◽

Cofactor Activity ◽

The Complement System

Membrane cofactor protein (MCP or gp45-70) of the complement system is a cofactor for factor I-mediated cleavage of fluid-phase C3b and C3b-like C3, which opens the thioester bond. In the present study the activity of MCP was further characterized. Unexpectedly, in the absence of factor I, MCP stabilized the alternative- and, to a lesser extent, the classical-pathway cell-bound C3 convertases and thereby enhanced C3b deposition. Soluble MCP, if added exogenously, hardly functioned as cofactor for the cleavage of erythrocyte-bound C3b to iC3b; i.e. its activity, compared with the cofactor activity of factor H, was inefficient, since less than 10% of the bound C3b was MCP-sensitive. Further, exogenously added soluble MCP was also a weak cofactor for the cleavage of C3b bound to zymosan. Likewise, factor I, in the presence of cells bearing MCP, cleaved fluid-phase C3b inefficiently. These results imply that MCP has very little extrinsic cofactor activity for factor I. In contrast, exogenously added MCP and factor I mediated efficient cleavage of erythrocyte-bound C3b if the concentration of Nonidet P40 was sufficient to solubilize the cells. Interestingly, soluble MCP and factor I degraded C3b attached to certain solubilized acceptor membrane molecules more readily than others. The cleavage reaction of fluid-phase and cell-bound C3b by soluble MCP and factor I produced iC3b, but no C3c and C3dg. These and prior data indicate that soluble MCP has potent cofactor activity for fluid-phase C3b or C3b bound to solubilized molecules, but acts inefficiently towards C3b on other cells. This functional profile is unique for a C3b/C4b binding protein and, taken together with its wide tissue distribution, suggests an important role for MCP in the regulation of the complement system.

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