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

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.

Abstract (Swedish)

Popular Abstract in Swedish

Komplementsystemet är ett system bestående av cirka 35 proteiner i blodet och på cellytor, som ingår i kroppens medfödda immunförsvar. Det är ett mycket viktigt men också explosivt system, potentiellt skadligt för kroppens egna celler, som kräver strikt reglering. Denna avhandlings huvudaktör, C4b-bindande protein (C4BP), nedreglerar komplementsystemet. C4BP har en ovanlig struktur, som påminner om en bläckfisk. C4BP består av sju identiska alfa-kedjor, och en unik beta-kedja. Varje kedja är uppbyggd av repeterande subenheter, complement control protein (CCP) domäner. Alfa-kedjorna består av åtta CCP domäner och beta-kedjan av tre. Alfa-kejorna binder C4b, C4BPs fysiologiska målmolekyl, och C4BP reglerar därmed C4b-beroende komplementreaktioner. Vi har visat att en grupp positivt laddade aminosyror mellan CCP1 och 2 på alfa-kedjorna binder C4b. Även heparin kunde binda på samma ställe.



C4BP cirkulerar i komplex med protein S, som är en viktig kofaktor i det antikoagulanta systemet. Ungefär 70% av allt protein S i plasma är i komplex med C4BP, och det förlorar då sin antikoagulanta funktion. Orsaken till varför C4BP och protein S bildar ett högaffint komplex i plasma har varit okänd. Protein S har dock en domän med modifierade glutaminsyror (Gla-domänen) med hög affinitet för negativt laddade fosfolipider. Celler som genomgår programmerad celldöd (apoptos) exponerar tidigt sådana fosfolipider. Dessutom aktiveras komplementsystemet av apoptotiska celler. Detta leder dock inte till någon inflammatorisk respons i vävnaden runt omkring vilket man kunde vänta. Det betdyer att något måste finnas i närheten av den apoptotiska cellen som nedreglerar komplementaktiveringen. Vi fann att C4BP kan binda till apoptotiska celler i närvaro, men inte frånvaro, av protein S. Bindningen medierades av Gla-domänen på protein S och var calcium-beroende. Dessutom kunde C4BP fortfarande binda C4b, vilket tyder på att C4BP behåller sin fysiologiska komplementreglerande funktion även när det är förankrat till den apoptotiska cellen via protein S , och pekar på en fysiologisk roll av komplexbildningen mellan protein S och C4BP.



Vi har också studerat var på C4BP som protein S binder. Det är sedan tidigare känt att protein S binder till beta-kedjan på C4BP. Vi har nu visat att en grupp hydrofoba aminsyror på CCP1 av C4BP beta-kedjan är oumbärliga för bindningen till protein S. Vidare tyder våra resultat på att rollen av CCP2 i interaktionen är att stabilsera CCP1 så att protein S kan binda, snarare än att vara direkt involverad i bindningen.

• author: Webb, Joanna
• supervisor: [unknown] [unknown]
• opponent: Professor Meri, Seppo, Department of Bacteriology and Immunology, University of Helsinki, Finland
• organization: Clinical Chemistry, Malmö (research group)
• publishing date: 2002
• type: Thesis
• publication status: published
• subject: Medicinal Chemistry
• keywords: Clinical chemistry, apoptosis, binding sites, complement, C4b-binding protein, protein S, Klinisk kemi
• pages: 120 pages
• defense location: Jubileumsaulan, entrance 59 U-MAS, Malmö
• defense date: 2002-10-11 09:15:00
• ISBN: 91-628-5340-6
• language: English
• LU publication?: yes
• additional info: 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.
• id: 1d3226df-6048-4198-9177-9e0997837fef (old id 464884)
• date added to LUP: 2016-04-04 11:19:23
• date last changed: 2018-11-21 21:04:04