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A cluster of positively charged amino acids in the C4BP alpha-chain is crucial for C4b binding and factor I cofactor
A M Blom1, J Webb, B O Villoutreix
1The Wallenberg Laboratory, Department of Clinical Chemistry, Lund University, University Hospital Malmö, S-205 02 Malmö, Sweden.
Insights
C4b-binding protein (C4BP) regulates the complement pathway. Researchers identified key amino acids in C4BP essential for binding C4b and aiding its degradation, revealing a crucial site for complement system regulation.
Area of Science:
- Immunology
- Biochemistry
- Structural Biology
Background:
- C4b-binding protein (C4BP) is a key regulator of the classical complement pathway.
- C4BP acts as a cofactor for factor I in degrading C4b, a central component of complement.
- Previous studies suggested a positively charged amino acid cluster in C4BP's alpha-chain modules 1 and 2 is involved in C4b binding.
Purpose of the Study:
- To investigate the role of specific amino acids in C4BP's C4b binding and cofactor activity.
- To characterize the functional impact of mutations within the predicted C4b binding site.
Main Methods:
- Expression and functional analysis of three C4BP mutants (R39Q, R64Q/R66Q, R39Q/R64Q/R66Q).
- Assay of C4b binding affinity using immobilized C4b.
- Evaluation of cofactor activity in factor I-mediated C4b degradation.
Main Results:
- Mutant C4BP proteins showed significantly reduced affinity for C4b (15- to 140-fold lower).
- The identified C4b binding site also functions as a heparin binding site.
- Mutants exhibited impaired cofactor activity for C4b degradation, particularly the R39Q/R64Q/R66Q mutant.
- Specific peptide bond cleavage sites in C4b were differentially affected by the mutations.
Conclusions:
- A cluster of amino acids within C4BP is critical for C4b binding.
- This binding site is essential for C4BP's cofactor activity in complement regulation.
- The findings provide structural insights into the mechanism of complement control by C4BP.
Abstract:
C4b-binding protein (C4BP) is a regulator of the classical complement pathway, acting as a cofactor to factor I in the degradation of C4b. Computer modeling and structural analysis predicted a cluster of positively charged amino acids at the interface between complement control protein modules 1 and 2 of the C4BP alpha-chain to be involved in C4b binding. Three C4BP mutants, R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q, were expressed and assayed for their ability to bind C4b and to function as factor I cofactors. The apparent affinities of R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q for immobilized C4b were 15-, 50-, and 140-fold lower, respectively, than that of recombinant wild type C4BP. The C4b binding site demonstrated herein was also found to be a specific heparin binding site. In C4b degradation, the mutants demonstrated decreased ability to serve as factor I cofactors. In particular, the R39Q/R64Q/R66Q mutant was inefficient as cofactor for cleavage of the Arg937-Thr938 peptide bond in C4b. In contrast, the factor I mediated cleavage of Arg1317-Asn1318 bond was less affected by the C4BP mutations. In conclusion, we identify a cluster of amino acids that is part of a C4b binding site involved in the regulation of the complement system.