C4b-binding protein binds to necrotic cells and DNA, limiting DNA release and inhibiting complement activation

Abstract

After cell death, via apoptosis or necrosis, the uptake of dead cells by neighboring cells or phagocytes prevents the release of intracellular content. An array of molecules, including initiation molecules of the complement system, are involved in marking dead cells for uptake. After binding of these molecules, complement activation takes place, which when uncontrolled might result in a proinflammatory state. In the current study we demonstrate that complement inhibitor, C4b-binding protein (C4BP), binds strongly to necrotic cells, irrespective of the cell type used or the method of induction. After binding of the C4BP–protein S (PS) complex to necrotic cells via PS-phosphatidylserine and C4BP-DNA interactions, C4BP-PS inhibits complement activation on these cells. C4BP binds DNA via a patch of positively charged amino acids, mainly on the second complement control domain of the C4BP α-chain (affinity constant: 190 nM). Furthermore, C4BP limits DNA release from necrotic cells and inhibits DNA-mediated complement activation in solution. The C4BP–necrotic cell interaction also occurs in vivo as necrotic areas of arteriosclerotic plaques and of various cancers stain strongly positive for C4BP. This study describes a novel mechanism in which C4BP limits the inflammatory potential of necrotic cells.

Annexin A2 and A5 Serve as New Ligands for C1q on Apoptotic Cells

“…The binding of C4BP, which circulates mainly in complex with protein S, to dying cells is mediated by interaction of protein S with PS (14) and to a much lesser extent via an interaction of the C4BP ␣-chains with DNA (16). In comparison, we showed recently that FH binds to annexin A2, DNA, and histones on the surface of apoptotic cells (17).…”

“…Double-stranded 25-mer oligonucleotides (G-C) 25 and (A-T) 25 were produced using equimolar amounts of single-stranded 5Ј-biotinylated 25-mer oligonucleotides (MWG) as described (16). Briefly, equal concentrations of G-C and A-T were coupled to the streptavidin sensor chip surface to a level of 300 response units (Biacore 2000, GE Healthcare).…”

Serum amyloid P component facilitates DNA clearance and inhibits plasmid transfection: implications for human DNA vaccine

“…15 For dot blot, all blots were incubated with 5 mg ml À1 purified proteins or 10% serum diluted with TSC buffer (10 mM TrisHCl, 150 mM NaCl, 2 mM CaCl 2 , pH 8.0) for 15 min and visualized with an SAP-specific antibody. For gel retardation analysis, linear plasmids (0.2 mg) and proteins were mixed in TSC buffer and then separated on 1% agarose gel in 40 mM Tris-acetate buffer (without EDTA, pH 8.0).…”

“…12,13 Thus, DNA clearance is essential for maintaining an internal homeostatic environment in human beings, especially after the intradermal or intramuscular injection of a large amount of plasmid DNA vaccine. Many molecules, such as serum amyloid P component (SAP), 14 C4b-binding protein, 15 mannosebinding lectin, 16 high-mobility group B proteins, 17 LL37 18 and heparan sulfate proteoglycan, 19 have been reported to have strong DNA-binding activity in the extracellular environment, especially in the serum. Some of these molecules may contribute to DNA clearance and, therefore, form barriers against the inflammation induced by exogenous DNA.…”

“…Some of these molecules may contribute to DNA clearance and, therefore, form barriers against the inflammation induced by exogenous DNA. 15 In this regard, we hypothesized that certain extracellular molecules may facilitate the clearance of extracellular DNA after plasmid inoculation and, subsequently, contribute to the low transfection efficiency of DNA vaccines in vivo, especially in the human beings.…”