CCP1-4 of the C4b-binding protein alpha-chain are required for factor I mediated cleavage of complement factor C3b
Anna M Blom1, Lena Kask, Björn Dahlbäck
1The Wallenberg Laboratory, Department of Clinical Chemistry, Lund University, University Hospital Malmö, S-205 02 Malmö, Sweden. anna.blom@klkemi.mas.lu.se
Insights
C4b-binding protein (C4BP) requires its N-terminal CCP domains, particularly CCP2 and 3, for C3b cleavage cofactor activity. Binding C3b involves a larger surface area on C4BP compared to C4b.
Area of Science:
- Immunology
- Complement System Biology
- Protein Structure-Function Analysis
Background:
- C4b-binding protein (C4BP) is a key regulator of the complement system, inhibiting the classical pathway.
- C4BP acts as a cofactor for Factor I (FI) in cleaving C3b, influencing the alternative pathway.
- The major plasma form of C4BP comprises seven alpha-chains and one beta-chain, with alpha-chains containing eight CCP domains.
Purpose of the Study:
- To determine the structural requirements of C4BP's alpha-chain for interacting with C3b.
- To elucidate the role of specific complement control protein (CCP) domains in C4BP's cofactor activity.
- To compare the binding characteristics of C3b and C4b to C4BP.
Main Methods:
- Utilized nineteen recombinant C4BP variants, including truncated, CCP-deleted, and alanine-mutated forms.
- Assessed C4BP's cofactor activity in C3b cleavage.
- Investigated C4BP's role in degrading surface-bound C3b and accelerating alternative C3-convertase decay.
Main Results:
- C4BP requires all four N-terminal CCP domains of the alpha-chain, with CCP2 and CCP3 being most critical for C3b cofactor activity.
- A positively charged amino acid cluster between CCP1 and CCP2 is essential for C3b binding.
- C3b binding to C4BP necessitates a larger molecular surface area compared to C4b binding.
- C4BP demonstrated cofactor activity for surface-bound C3b degradation and alternative C3-convertase decay, but required a 1,000-fold excess over Factor H.
Conclusions:
- The N-terminal CCP domains of C4BP's alpha-chain are crucial for its cofactor function in C3b cleavage.
- Distinct structural features, including a charged amino acid cluster, mediate C3b binding.
- C4BP's interaction with C3b is less efficient than with C4b, requiring significantly higher concentrations for alternative pathway inhibition compared to Factor H.
Abstract:
C4b-binding protein (C4BP) is a potent regulator of the complement system because it strongly inhibits the classical pathway of complement. Furthermore, C4BP serves as a cofactor to factor I (FI) in the cleavage of fluid phase C3b and can, therefore, influence the alternative pathway of complement. The major form of C4BP in plasma consists of seven identical alpha-chains and one beta-chain. Both types of subunits are composed of complement control protein (CCP) domains, eight such domains make up one alpha-chain. To elucidate the structural requirements for the interaction between C3b and the alpha-chain, nineteen recombinant C4BP variants were used: six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally four variants in which double alanine residues were introduced between CCPs. We found that C4BP requires all four N-terminal CCPs of the alpha-chain, with CCP2 and 3 being the most important, to act as a cofactor in the cleavage of C3b. Also, a cluster of positively charged amino acids on the interface between CCP1 and 2 is involved in the binding. Compared to the interaction with C4b, we conclude that binding of C3b to C4BP requires larger molecular surface on C4BP. We found that C4BP was able to act as cofactor in degradation of surface bound C3b and to accelerate decay of alternative C3-convertase. However, in both cases 1,000-fold molar excess of C4BP over factor H (FH), well known inhibitor of the alternative pathway, was required to obtain the same effect.
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