The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related ( CFHR ) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.

Age-related macular degeneration (AMD) is the predominant cause of blindness in the industrialized world where destruction of the macula, i.e. the central region of the retina, results in loss of vision. AMD is preceded by the formation of deposits in the macula, which accumulate between the Bruch's membrane and the retinal pigment epithelium (RPE). These deposits are associated with complement-mediated inflammation and perturb retinal function. Recent genetic association studies have demonstrated that a common allele (402H) of the complement factor H (CFH) gene is a major risk factor for the development of AMD; CFH suppresses complement activation on host tissues where it is believed to bind via its interaction with polyanionic structures. We have shown previously that this coding change (Y402H; from a tyrosine to histidine residue) alters the binding of the CFH protein to sulfated polysaccharides. Here we demonstrate that the AMD-associated polymorphism profoundly affects CFH binding to sites within human macula. Notably, the AMDassociated 402H variant binds less well to heparan sulfate and dermatan sulfate glycosaminoglycans within Bruch's membrane when compared with the 402Y form; both allotypes exhibit a similar level of binding to the RPE. We propose that the impaired binding of the 402H variant to Bruch's membrane results in an overactivation of the complement pathway leading to local chronic inflammation and thus contributes directly to the development and/or progression of AMD. These studies therefore provide a putative disease mechanism and add weight to the genetic association studies that implicate the 402H allele as an important risk factor in AMD.

SUMMARY Activation of the complement system results in formation of membrane attack complexes (MACs), pores that disrupt lipid bilayers and lyse bacteria and other pathogens. Here, we present the crystal structure of the first assembly intermediate, C5b6, together with a cryo-electron microscopy reconstruction of a soluble, regulated form of the pore, sC5b9. Cleavage of C5 to C5b results in marked conformational changes, distinct from those observed in the homologous C3-to-C3b transition. C6 captures this conformation, which is preserved in the larger sC5b9 assembly. Together with antibody labeling, these structures reveal that complement components associate through sideways alignment of the central MAC-perforin (MACPF) domains, resulting in a C5b6-C7-C8β-C8α-C9 arc. Soluble regulatory proteins below the arc indicate a potential dual mechanism in protection from pore formation. These results provide a structural framework for understanding MAC pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage.

Complement factor H (CFH) regulates complement activation in host tissues through its recognition of polyanions, which mediate CFH binding to host cell surfaces and extracellular matrix, promoting the deactivation of deposited C3b. These polyanions include heparan sulfate (HS), a glycosaminoglycan (GAG) with a highly diverse range of structures, for which two regions of CFH (referred to as CCP6-8 and CCP19-20) have been implicated in HS binding. Mutations/polymorphisms within these GAG-binding sites have been associated with age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (aHUS). Here we demonstrate that CFH has tissue-specific binding properties, mediated through its two HS-binding regions. Our data shows that the CCP6-8 region of CFH binds more strongly to heparin (a highly sulfated form of HS) than CCPs19-20 and that their sulfate specificities are different. Furthermore, the HS-binding site in CCPs6-8, which is affected by the AMD-associated Y402H polymorphism, plays the principle role in host tissue recognition in the human eye, whilst the CCP19-20 region makes the major contribution to the binding of CFH in the human kidney. This helps provide a biochemical explanation for the genetic basis of tissue-specific diseases such as AMD and aHUS, and leads to a better understanding of the pathogenic mechanisms for these diseases of complement dysregulation.

The tight regulation of innate immunity on extracellular matrix (ECM) is a vital part of immune homeostasis throughout the human body and disruption to this regulation in the eye is thought to contribute directly to the progression of age-related macular degeneration (AMD). The plasma complement regulator factor H (FH) is believed to be the main regulator that protects ECM against damaging complement activation. However, here we demonstrate that a truncated form of FH, called factor-H like protein 1 (FHL-1), is the main regulatory protein in the layer of ECM under human retina, called Bruch’s membrane. Bruch’s membrane is a major site of AMD disease pathogenesis and where drusen, the hallmark lesions of AMD, form. We show that FHL-1 can passively diffuse through Bruch’s membrane, whereas the full sized, glycosylated, FH cannot. FHL-1 is largely bound to Bruch’s membrane through interactions with heparan sulfate and we show that the common Y402H polymorphism in the CFH gene, associated with an increased risk of AMD, reduces the binding of FHL-1 to this heparan sulfate. We also show that FHL-1 is retained in drusen while FH coats the periphery of the lesions, perhaps inhibiting their clearance. Our results identify a novel mechanism of complement regulation in the human eye, which highlights potential new avenues for therapeutic strategies.

IntroductionInflammation and complement activation are firmly implicated in the pathology of multiple sclerosis; however, the extent and nature of their involvement in specific pathological processes such as axonal damage, myelin loss and disease progression remains uncertain. This study aims to bring clarity to these questions.ResultsWe describe a detailed immunohistochemical study to localise a strategically selected set of complement proteins, activation products and regulators in brain and spinal cord tissue of 17 patients with progressive multiple sclerosis and 16 control donors, including 9 with central nervous system disease. Active, chronic active and chronic inactive multiple sclerosis plaques (35 in total) and non-plaque areas were examined.Multiple sclerosis plaques were consistently positive for complement proteins (C3, factor B, C1q), activation products (C3b, iC3b, C4d, terminal complement complex) and regulators (factor H, C1-inhibitor, clusterin), suggesting continuing local complement synthesis, activation and regulation despite the absence of other evidence of ongoing inflammation. Complement staining was most apparent in plaque and peri-plaque but also present in normal appearing white matter and cortical areas to a greater extent than in control tissue. C1q staining was present in all plaques suggesting a dominant role for the classical pathway. Cellular staining for complement components was largely restricted to reactive astrocytes, often adjacent to clusters of microglia in close apposition to complement opsonised myelin and damaged axons.ConclusionsThe findings demonstrate the ubiquity of complement involvement in multiple sclerosis, suggest a pathogenic role for complement contributing to cell, axon and myelin damage and make the case for targeting complement for multiple sclerosis monitoring and therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/2051-5960-2-53) contains supplementary material, which is available to authorized users.

SummaryMultiple sclerosis (MS) is a common inflammatory disease of the central nervous system with a poorly defined and complex immunopathogenesis. Although initiated by reactive T cells, persistent inflammation is evident throughout the disease course. A contribution from complement has long been suspected, based on the results of pathological and functional studies which have demonstrated complement activation products in MS brain and biological fluids. However, the extent and nature of complement activation and its contribution to disease phenotype and long-term outcome remain unclear. Furthermore, functional polymorphisms in components and regulators of the complement system which cause dysregulation, and are known to contribute to other autoimmune inflammatory disorders, have not been investigated to date in MS in any detail. In this paper we review evidence from pathological, animal model and human functional and genetic studies, implicating activation of complement in MS. We also evaluate the potential of complement components and regulators and their polymorphic variants as biomarkers of disease, and suggest appropriate directions for future research.

Background:There is increasing evidence of significant and dynamic systemic activation and upregulation of complement in multiple sclerosis (MS), which may contribute to disease pathogenesis.Objective:We aimed to investigate the pathological role of complement in MS and the potential role for complement profiling as a biomarker of MS disease state.Methods:Key components of the classical, alternative and terminal pathways of complement were measured in plasma and cerebrospinal fluid (CSF) of patients with MS in different clinical phases of disease and in matched controls.Results:Increased plasma levels of C3 (p<0.003), C4 (p<0.001), C4a (p<0.001), C1 inhibitor (p<0.001), and factor H (p<0.001), and reduced levels of C9 (p<0.001) were observed in MS patients compared with controls. Combined profiling of these analytes produced a statistical model with a predictive value of 97% for MS and 73% for clinical relapse when combined with selected demographic data. CSF-plasma correlations suggested that source of synthesis of these components was both systemic and central.Conclusion:These data provide further evidence of alterations in both local and systemic expression and activation of complement in MS and suggest that complement profiling may be informative as a biomarker of MS disease, although further work is needed to determine its use in distinguishing MS from its differential.