Serum Amyloid P Component Binding to C4b-binding Protein

Abstract

Human C4b-binding protein (C4BP), which is a regulator of the classical complement pathway C3 convertase, forms high affinity complexes with anticoagulant protein S and with the pentraxin serum amyloid P component (SAP). SAP is a plasma protein present in all amyloid deposits. Recently, SAP was shown to inhibit the complement regulatory functions of C4BP. In this investigation, we have studied the structural requirements for the C4BP-SAP interaction. C4BP was subjected to chymotrypsin digestion, which yielded two major fragments corresponding to the central core (160 kDa) and to the cleaved-off tentacles (48 kDa). SAPSepharose specifically bound the 160-kDa fragment, suggesting that the central core of C4BP contains the binding site for SAP. In a quantitative affinity chromatography assay, the dissociation constants for binding of intact C4BP and of the 160-kDa central core fragment to SAP were found to be 30 and 70 nM, respectively. Recombinant C4BP composed of only ␣-chains bound SAP with similar affinity (K d ‫؍‬ 22 nM), whereas nonglycosylated recombinant ␣-chain C4BP (synthesized in the presence of tunicamycin) bound SAP with lower affinity (K d ‫؍‬ 126 nM). This suggests that the carbohydrate moiety of the central core of C4BP is important for binding of C4BP to SAP in contrast to the C4BP ␤-chain, which is not required. EDTA, heparin, and phosphorylethanolamine as well as a peptide comprising amino acids 27-39 of SAP were found to completely displace C4BP from the SAP matrix. Moreover, the immobilized SAP peptide bound C4BP in a reaction that, in contrast to the C4BP-SAP interaction, was not dependent on calcium.

The α7β0 Isoform of the Complement Regulator C4b-Binding Protein Induces a Semimature, Anti-Inflammatory State in Dendritic Cells

“…We have found that the CCP6 domain of C4BP (55), structural arrangements in the polymeric C4BP spatial conformation because of the presence of ProS either by an allosteric mechanism or by direct steric hindrance might: 1) preclude a direct interaction of the CCP6 domain from the a-chains with the putative "tolerogenesis" receptor on DCs, or 2) reduce the overall avidity of polymeric C4BP binding to the receptor through the CCP6 domain. In this respect, although the C4BP(b + )-ProS complex attached to apoptotic cells through ProS is still able to bind C4b through domains CCP1-3 of the a-chains (23), serum amyloid P binding to the central core linking the seven or eight chains impairs its factor I cofactor activity (56). Nevertheless, further structure-activity studies will be necessary to dissect the relationship between structure and function of polymeric C4BP on DCs.…”

Regulation of Complement Activation by C-Reactive Protein: Targeting of the Inhibitory Activity of C4b-Binding Protein

“…C4BP has previously been reported to bind to serum amyloid P component (SAP) (22), which is a member of the pentraxin family and has an overall structure strikingly similar to that of CRP. SAP is one of the major acute phase reactants in mice.…”

“…Recombinant C4BP (20), monomeric C4BP ␣-chains (21), and the C4BP central core region (22) were expressed or prepared as described. Recombinant C4BP (C4BPrec) and its truncated form composed of CCP1-8 were expressed in HEK 293 cells and purified by affinity chromatography using mAb 104 directed against CCP1 of the C4BP ␣-chain (20).…”

A Novel Interaction Between Type IV Pili ofNeisseria gonorrhoeaeand the Human Complement Regulator C4b-Binding Protein

“…First, we tested whether C4BP purified from plasma consisting of seven ␣-chains and one ␤-chain will bind pili with the same apparent affinity as the recombinant C4BP that is composed exclusively of ␣-chains. ␣-Chains are known to bind C4b (17), heparin (41), Bordetella pertussis (24), Streptococcus pyogenes (23), and serum amyloid P component (42), whereas ␤-chain binds anticoagulant protein S (18). We found that both forms of C4BP bound to pili with similar affinities, implying that ␣-chains confer the ability to bind gonococcal pili.…”