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Complement C4bC2 complex formation: an investigation by surface plasmon resonance.

A Laich1, R B Sim

  • 1MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU, Oxford, UK. alaich@bioch.ax.ac.uk

Biochimica Et Biophysica Acta
|May 9, 2001
PubMed
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Human complement proteins C4b and C2 form complexes, with binding dependent on salt concentration. This study quantizes C4bC2 complex affinity and explores potential cross-reactivity with complement factor B.

Area of Science:

  • Immunology
  • Biochemistry
  • Molecular Biology

Background:

  • The complement system is crucial for innate immunity.
  • Understanding protein interactions within the complement cascade is vital for therapeutic development.
  • The classical and alternative pathways of complement activation involve complex protein-binding events.

Purpose of the Study:

  • To investigate the complex formation between human complement proteins C4b and C2.
  • To determine the binding affinity (Kd) of the C4bC2 complex under various conditions.
  • To explore potential cross-reactivity between C4b and complement factor B (FB).

Main Methods:

  • Surface plasmon resonance (SPR) was used to immobilize C4b and measure its interaction with fluid-phase C2.
  • Binding kinetics were assessed at varying ionic strengths (30-830 mM NaCl) and in the presence/absence of MgCl2.

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  • Affinity was calculated using steady-state analysis, and interactions with complement factor B were also evaluated.

    • Main Results:

    • C4bC2 complex formation showed maximum binding at 30 mM NaCl, decreasing significantly above 300 mM NaCl.
    • The affinity (Kd) of the C4bC2 complex was determined to be 7.2x10(-8) M under physiological conditions.
    • C4bC2 binding occurred as a multiple-site event with Mg2+ and a single-site event in the presence of EDTA. Weak binding of C4b to complement factor B (Kd = 1.2x10(-6) M) was observed.

    Conclusions:

    • The binding of C2 to C4b is highly dependent on ionic strength and influenced by divalent cations.
    • The C4bC2 complex forms with high affinity, and its binding characteristics differ in the presence of Mg2+ versus EDTA.
    • The observed weak interaction between C4b and complement factor B suggests potential cross-reactivity between the classical and alternative complement pathways.