CCP1–4 of the C4b-binding protein α-chain are required for factor I mediated cleavage of complement factor C3b

Abstract

C4b-binding protein (C4BP) is a potent regulator of the complement system because it strongly inhibits the classical pathway of complement. Furthermore, C4BP serves as a cofactor to factor I (FI) in the cleavage of fluid phase C3b and can, therefore, influence the alternative pathway of complement. The major form of C4BP in plasma consists of seven identical α-chains and one β-chain. Both types of subunits are composed of complement control protein (CCP) domains, eight such domains make up one α-chain. To elucidate the structural requirements for the interaction between C3b and the α-chain, nineteen recombinant C4BP variants were used: six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally four variants in which double alanine residues were introduced between CCPs. We found that C4BP requires all four N-terminal CCPs of the α-chain, with CCP2 and 3 being the most important, to act as a cofactor in the cleavage of C3b. Also, a cluster of positively charged amino acids on the interface between CCP1 and 2 is involved in the binding. Compared to the interaction with C4b, we conclude that binding of C3b to C4BP requires larger molecular surface on C4BP. We found that C4BP was able to act as cofactor in degradation of surface bound C3b and to accelerate decay of alternative C3-convertase. However, in both cases 1000-fold molar excess of C4BP over factor H (FH), well known inhibitor of the alternative pathway, was required to obtain the same effect.

Introduction

The human complement system comprises at least 35 plasma and membrane bound proteins involved in efficient activation and tight regulation of this component of host defense (Muller-Eberhard, 1975). The complement system can be activated in three ways, the classical pathway is initiated by clustered antibodies bound to their targets, the mannan-binding lectin pathway begins due to recognition of certain saccharides (Matsushita and Fujita, 1992) and the alternative pathway is started by autoactivation of unstable complement factor C3 and its subsequent binding to activating pathogen surfaces.

The potentially harmful complement system must be carefully regulated and, therefore, several soluble and membrane bound proteins are dedicated to this function. Factor I (FI) is a serine proteinase contributing to down regulation of the three pathways of the complement (Sim and Laich, 2000). FI degrades activated forms of complement factors C3 (C3b) and C4 (C4b) only when they are bound to a suitable cofactor protein. The cofactors are mostly composed of complement control protein (CCP) domains and belong to a gene family of the regulators of complement activation (Hourcade et al., 1992). Factor H (FH) is a cofactor in the cleavage of C3b, whereas complement receptor 1 (CR1) and membrane cofactor protein (MCP) are able to serve as cofactors in the cleavage of both C3b and C4b. C4b-binding protein (C4BP) is known to regulate the lectin (Suankratay et al., 1999) and the classical pathways of complement. C4BP acts as a cofactor to FI in the inactivation of both fluid phase and cell-bound C4b (Scharfstein et al., 1978). Furthermore, C4BP prevents assembly of the C3-convertase (C4b2a) and accelerates natural decay of C2a from the complex (Gigli et al., 1979). C4BP is a large plasma glycoprotein (>500 kDa) consisting of several polypeptides: seven identical α-chains and a unique β-chain (Hillarp and Dahlback, 1988, Scharfstein et al., 1978). The α- and β-chains are held together by disufide bridges and are composed of eight and three CCP domains, respectively (Villoutreix et al., 1998). As shown by electron microscopy and biochemical studies, C4BP consists of a 160 kDa central core and several extended tentacles, which can be released by limited proteolysis with chymotrypsin (Dahlback and Muller Eberhard, 1984, Dahlback et al., 1983). We have found recently that the whole binding site for C4b encompasses CCP1–3 of the α-chain (Blom et al., 2001a). Furthermore, we have identified several positively charged amino acids located on the interface between CCP1 and 2 that are important for binding of C4b and functional activity of C4BP in the classical pathway of complement (Blom et al., 1999, Blom et al., 2000b).

FH efficiently down regulates the alternative pathway of complement (Zipfel et al., 1999). FH acts as a cofactor to FI in the degradation of C3b and it accelerates decay of the alternative C3-convertase (C3bBb). Some reports suggested that activities of C4BP and FH may be partially overlapping and that their functions are not restricted to only one pathway of complement activation. Interestingly, C4BP was originally purified as FI cofactor in the cleavage of C3b (Nagasawa and Stroud, 1977). However, these observations were later dismissed and attributed to a cross-contamination between the two proteins that were both purified from plasma. We decided to investigate the ability of C4BP to regulate alternative pathway of complement in more detail and to compare it with FH. Furthermore, we wanted to determine structural requirements for the interaction between C4BP and C3b.