Andreas P. Sjöberg

The factor H variant associated with age-related macular degeneration (His-384) and the non-disease-associated form bind differentially to C-reactive protein, fibromodulin, DNA, and necrotic cells.

Recently, a polymorphism in the complement regulator factor H (FH) gene has been associated with age-related macular degeneration. When histidine instead of tyrosine is present at position 384 in the seventh complement control protein (CCP) domain of FH, the risk for age-related macular degeneration is increased. It was recently shown that these allotypic variants of FH, in the context of a recombinant construct corresponding to CCPs 6–8, recognize polyanionic structures differently, which may lead to altered regulation of the alternative pathway of complement. We show now that His-384, corresponding to the risk allele, binds C-reactive protein (CRP) poorly compared with the Tyr-384 form. We also found that C1q and phosphorylcholine do not compete with FH for binding to C-reactive protein. The interaction with extracellular matrix protein fibromodulin, which we now show to be mediated, at least in part, by CCP6–8 of FH, occurs via the polypeptide of fibromodulin and not through its glycosaminoglycan modifications. The Tyr-384 variant of FH bound fibromodulin better than the His-384 form. Furthermore, we find that CCP6–8 is able to interact with DNA and necrotic cells, but in contrast the His-384 allotype binds these ligands more strongly than the Tyr-384 variant. The variations in binding affinity of the two alleles indicate that complement activation and local inflammation in response to different targets will differ between His/His and Tyr/Tyr homozygotes.

Short leucine-rich glycoproteins of the extracellular matrix display diverse patterns of complement interaction and activation

The extracellular matrix consists of structural macromolecules and other proteins with regulatory functions. An important family of the latter class of molecules found in most tissues is the small leucine-rich repeat proteins (SLRPs). We have previously shown that the SLRP fibromodulin binds directly to C1q and activates the classical pathway of complement. In the present study we further examine the interactions between SLRPs and complement. Osteoadherin, like fibromodulin, binds C1q and activates the classical pathway strongly while moderate activation is seen in the terminal pathway. This can be explained by the interaction of fibromodulin and osteoadherin with factor H, a major soluble inhibitor of complement. Also, chondroadherin was found to bind C1q and activate complement, albeit to a lesser extent. Chondroadherin also binds factor H. We confirm published data showing that biglycan and decorin bind C1q but do not activate complement. In this study a similar pattern is seen for lumican although its affinity for C1q is lower than for biglycan and decorin. Furthermore, using electron microscopy and radiolabeled SLRPs, we demonstrate two different classes of SLRP binding sites on C1q, to head and stalk respectively, where only binding to the head appears to be activating. We propose a role for SLRPs in the regulation of complement activation in diseases involving the extracellular matrix, particularly those characterized by chronic inflammation such as rheumatoid arthritis, atherosclerosis, osteoarthritis and chronic obstructive lung disease.

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

C-reactive protein (CRP) is the major acute phase protein in humans. It has been shown that CRP interacts with factor H, an inhibitor of the alternative pathway of complement, and now we demonstrate binding of CRP to the fluid-phase inhibitor of the classical pathway, C4b-binding protein (C4BP). C4BP bound to directly immobilized recombinant CRP as well as CRP attached to phosphorylcholine. The binding was sensitive to ionic strength and was enhanced in the presence of calcium. C4BP lacking β-chain and protein S, which is a form of C4BP increasing upon inflammation, bound CRP with higher affinity than the C4BP-protein S complex. The binding could not be blocked with mAbs directed against peripheral parts of the α-chains of C4BP while the isolated central core of C4BP obtained by partial proteolytic digestion bound CRP, indicating that the binding site for CRP is localized in the central core of the C4BP molecule. Furthermore, we found complexes in serum from a patient with an elevated CRP level and trace amounts of CRP were also identified in a plasma-derived C4BP preparation. We were also able to detect C4BP-CRP complexes in solution and established that C4BP retains full complement regulatory activity in the presence of CRP. In addition, we found that C4BP can compete with C1q for binding to immobilized CRP and that it inhibits complement activation locally. We hypothesize that CRP limits excessive complement activation on targets via its interactions with both factor H and C4BP.

Complement inhibitor C4b-binding protein interacts directly with small glycoproteins of the extracellular matrix.

Components derived from cartilage have been suggested to maintain the inflammation in joints in arthritis. Small leucine-rich repeat proteins (SLRPs) are structural components of cartilage important in organizing the meshwork of extracellular matrix components. It has recently been shown that the SLRP fibromodulin interacts with complement initiator C1q, leading to complement activation. The complement response is limited since fibromodulin also interacts with the complement inhibitor factor H. We have now found that osteoadherin, chondroadherin, fibromodulin, and proline arginine-rich end leucine-rich repeat protein bind to the complement inhibitor C4b-binding protein (C4BP). Using direct binding assays with C4BP fragments and C4BP mutants lacking individual domains in combination with electron microscopy, we have demonstrated that mainly the central core of C4BP mediated binding to SLRPs. Binding of SLRPs to C4BP did not affect its ability to inhibit complement. Osteoadherin, fibromodulin, and chondroadherin, which bind C1q and activate complement, were found to cause significantly higher C9 deposition in C4BP-depleted serum compared with Igs, indicating that the level of complement activation initiated by SLRPs is regulated by simultaneous binding to C4BP. A similar dual binding of C1q and complement inhibitors was observed previously for other endogenous ligands (amyloid, prions, C-reactive protein, and apoptotic cells) but not for exogenous activators (bacteria-bound Igs). These interactions can be significant during inflammatory joint diseases, such as rheumatoid arthritis, where cartilage is degraded, and cartilage components are released into synovial fluid, where they can interact with factors of the complement system.

Native, amyloid fibrils and beta-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly.

Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and beta-oligomers) of recombinant human PrP (90-231 and 121-231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, beta-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that beta-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.