C4b-binding protein: Identification of binding sites and a possible function of the interaction with protein S

Joanna Webb

Research output: ThesisDoctoral Thesis (compilation)

Abstract

The subject of this thesis is plasma protein C4b-binding protein (C4BP). C4BP is an important regulator of the classical pathway of the complement system, a cascade-like system comprised of over 35 proteins, which partakes in the defence against micro-organisms and is involved of clearance of immune-complexes and apoptotic cells. C4BP contains two different types of subunits, seven identical alfa-chains, and one unique beta-chain, held together by disulphide bridging at the central core. Each chain is made up of repeating complement control protein (CCP) domains. The alfa-chains bind to complement protein C4b, and C4BP thereby regulates C4b-mediated reactions. We describe a positively charged cluster on the interface between CCP1 and 2 on the C4BP alfa-chain that is pivotal for binding of C4b and regulation of C4b-activity. In addition, the identified C4b-binding site is also demonstrated to be a heparin-binding site. The cluster was identified using homology 3D-modelling, based on the homology between C4BP and proteins with solved structure.

C4BP circulates in complex with vitamin K-dependent protein S, a cofactor in the anticoagulant system. Approximately 70% of protein S in plasma is bound to C4BP. It is only free protein S (thus the remaining 30%) that has cofactor function. The binding site for protein S on C4BP is fully contained in the beta-chain. We show that a large hydrophobic cluster on beta-chain CCP1 is crucial for binding of protein S, using site directed mutagenesis based on the homology 3D-model of the C4BP b-chain. CCP2 has previously been demonstrated to contribute to the C4BP-protein S interaction. We demonstrate that the role of CCP2 most likely is to direct the beta-chain and sterically facilitate binding of protein S to C4BP.

The complex formation between C4BP and protein S has remained an intriguing enigma. However, protein S contains a region rich with gamma-carboxylated Glu residues (the Gla-domain) that conveys a high affinity for negatively charged phospholipids to protein S. One of the first events during apoptosis is the transfer of phosphatidylserine from the inner leaflet of the cell membrane to the outer. We describe binding of C4BP to apoptotic Jurkat cells and neutrophils in the presence, but not absence, of protein S. Binding was calcium-dependent and mediated via the Gla-domain of protein S. Further, C4BP could still bind C4b also when attached to the apoptotic cell surface through protein S, suggesting that C4BP retained complement-controlling function and a physiological role for the C4BP-protein S complex formation. Apoptosis is characterised by a lack of inflammation in the surrounding tissues. Early complement components are important for rapid clearance of apoptotic cells, but subsequent complement activation and release of anaphylatoxins must be inhibited. Thus the presence of C4BP, a potent regulator of complement, on the surface of apoptotic cells, may be of outmost importance for control of inflammatory events. In contrast to C4BP, protein S could bind to apoptotic cells by itself. The role of protein S on the apoptotic cells surface without C4BP may be to control coagulation on the apoptotic cell surface.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Clinical Chemistry, Malmö
Supervisors/Advisors
  • [unknown], [unknown], Supervisor, External person
Award date2002 Oct 11
Publisher
Print ISBNs91-628-5340-6
Publication statusPublished - 2002

Bibliographical note

Defence details

Date: 2002-10-11
Time: 09:15
Place: Jubileumsaulan, entrance 59 U-MAS, Malmö

External reviewer(s)

Name: Meri, Seppo
Title: Professor
Affiliation: Department of Bacteriology and Immunology, University of Helsinki, Finland

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Article: I. Webb JH, Villoutreix BO, Dahlbäck B, and Blom AM. 2001.Localization of a hydrophobic binding site for anticoagulant protein S on the beta-chain of complement regulator C4b-binding protein. J Biol Chem 276: 4330-7.

Article: II. Webb JH, Villoutreix BO, Dahlbäck B, and Blom AM. 2002.Role of CCP2 of the C4b-binding protein beta-chain in protein S binding evaluated by mutagenesis and monoclonal antibodies. Submitted.

Article: III. Webb JH, Blom AM, and Dahlbäck B. 2002.Vitamin K-dependent protein S localizing complement regulator C4b-binding protein to the surface of apoptotic cells. J Immunol 169: 2580-6.

Article: IV. Webb JH, Blom AM, and Dahlbäck B. 2002.The binding of protein S and the protein S-C4BP complex to neutrophils is apoptosis-dependent. Submitted.

Article: V. Blom AM, Webb J, Villoutreix BO, and Dahlbäck B. 1999.A cluster of positively charged amino acids in the C4BP alfa-chain is crucial for C4b binding and factor I cofactor function. J Biol Chem 274: 19237-45.

Subject classification (UKÄ)

  • Medicinal Chemistry

Keywords

  • Clinical chemistry
  • apoptosis
  • binding sites
  • complement
  • C4b-binding protein
  • protein S
  • Klinisk kemi

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