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Mutations in alpha-chain of C4BP that selectively affect its factor I cofactor function

Anna M Blom1, Bruno O Villoutreix, Björn Dahlbäck

  • 1Lund University Department of Clinical Chemistry, University Hospital Malmö, The Wallenberg Laboratory, S-205 02 Malmö, Sweden and INSERM U428, Université Paris V, Paris 75006, France. Anna.Blom@klkemi.mas.lu.se

The Journal of Biological Chemistry
|August 2, 2003
PubMed

Insights

C4b-binding protein (C4BP) mutants selectively lost cofactor activity for C4b and C3b cleavage. Despite normal binding, these mutants failed to induce conformational changes necessary for protease function, highlighting specific structural requirements for complement regulation.

Area of Science:

  • Immunology
  • Biochemistry
  • Molecular Biology

Background:

  • C4b-binding protein (C4BP) is a key regulator of complement activation, inhibiting all pathways.
  • C4BP functions as a cofactor for serine protease factor I (FI) in degrading complement factors C4b and C3b.
  • C4BP structure involves alpha-chains (8 complement control protein domains) and a beta-chain (3 CCP domains).

Purpose of the Study:

  • To investigate the role of specific mutations in C4BP's alpha-chain CCP3 domain on its cofactor activity.
  • To determine if altered cofactor activity correlates with changes in C4b/C3b binding affinity or conformational changes.

Main Methods:

  • Site-directed mutagenesis to create C4BP mutants (K126Q/K128Q and F144S/F149S) in the alpha-chain CCP3 domain.
  • Surface plasmon resonance (SPR) to measure binding affinity of mutants to C4b/C3b.
  • Assays to assess cofactor activity in C4b and C3b cleavage and inhibitory effects on C3-convertase.

Main Results:

  • Two C4BP mutants (K126Q/K128Q and F144S/F149S) selectively lost cofactor activity for C4b and C3b cleavage.
  • These mutants exhibited normal binding affinity for C4b/C3b and similar inhibitory effects on C3-convertase as wild-type C4BP.
  • The data suggest that C4b and C3b do not undergo the same conformational changes upon binding to the mutants as with wild-type C4BP.

Conclusions:

  • Specific residues within C4BP's alpha-chain CCP3 are critical for its cofactor function, independent of substrate binding affinity.
  • The loss of cofactor activity in mutants is attributed to impaired conformational changes in C4b/C3b upon binding.
  • These findings elucidate the mechanism of C4BP-mediated complement regulation and identify key structural determinants for cofactor activity.

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