C4b-binding protein : studies on its interactions with C4b, protein S and serum amyloid P component

Sammanfattning: C4b-binding protein (C4BP) is an acute-phase plasma glycoprotein which regulates the complement system through its affinity for C4b. In addition, C4BP binds to the anticoagulant protein S and to serum amyloid P component (SAP). Protein S is a vitamin K-dependent protein which looses its anticoagulant properties when binding to C4BP. C4BP is composed of two types of subunits, the a-chain and the b-chain. The major form of C4BP in human plasma is composed of eight disulphide-linked chains, seven a-chains and one b-chain, whereas a minor portion lacks the b-chain. Both types of chains contain internally homologous repeats called short consensus repeats (SCRs). The aim of this work has been to study the structure-function relationship of C4BP. By constructing cDNA clones encoding various truncated or chimeric C4BP molecules and expressing these recombinant proteins in either bacterias or mammalian cells, we have been able to localise regions involved in the interactions between C4BP and its ligands. We found that the amino-terminal SCR, SCR1, of the a-chain is crucial for the C4b binding and factor I-cofactor function of C4BP whereas neither the b-chain nor the N-linked carbohydrates are involved. The entire binding site for protein S was found to be contained within SCR1 of the C4BP b-chain and not to involve the N-linked carbohydrates. The binding site for SAP was localised to the central core fragment (SCR8 and the carboxy-terminal region of the a-chains) obtained after digestion of C4BP by chymotrypsin. Removal of the N-linked carbohydrates of C4BP led to a lower affinity for SAP. Furthermore, we have developed an immunological assay for the determination of the concentration of b-chain-containing forms of C4BP. Using this assay, we found that it is mainly the concentration of the form of C4BP without the b-chain that increases during the acute phase which suggests the expression of the a- and b-chains to be differentially regulated during the acute-phase response thereby ensuring a stable level of free anticoagulant active protein S. Finally, we have shown that polymerisation of C4BP is independent of the b-chain, the N-linked carbohydrates and of the three most amino-terminal SCRs of the a-chain.