The role of heparan sulfate as determining pathogenic factor in complement factor H-associated diseases

Complement factor H (FH) systemically inhibits excessive complement activation in the microenvironment of host cells, but for instance not on microbes. This self-recognition is mediated by two binding sites that recognize distinctly sulfated heparan sulfate (HS) domains. The interaction with HS not only concentrates FH on host cells, but directly affects its activity, evoking novel models of conformational activation. Genetic aberrations in the HS-binding domains systemically disturb the protective function of FH, yet the resulting loss of complement control affects mainly ocular and renal tissues. Recent results suggest that the specific expression of HS domains in these tissues restricts the interaction of HS to a single binding site within FH. This lack of redundancy could predispose eyes and kidneys to complement-mediated damage, making HS a central determinant for FH-associated diseases.     

Complement factor I in health and disease

Factor I (FI) is a crucial inhibitor controlling all complement pathways due to its ability to degrade activated complement proteins C3b and C4b in the presence of cofactors such as factor H, C4b-binding protein, complement receptor 1 or CD46. Complete deficiency of FI, which is synthesized mainly in the liver is rare and leads to complement consumption resulting in recurrent severe infections, glomerulonephritis or autoimmune diseases. Incomplete FI deficiency is in turn associated with atypical haemolytic uremic syndrome, a severe disease characterized by thrombocytopenia, microangiopathic haemolytic anaemia and acute renal failure. Structurally, FI is a 88 kDa heterodimer of a heavy chain consisting of one FI-membrane attack complex (FIMAC) domain, one CD5 domain and two low-density lipoprotein receptor domains (LDLr), and a light chain which is a serine protease domain (SP), linked to the heavy chain by a disulfide bond. FI cleaves its in vivo substrates C3b and C4b only in the presence of cofactors, it shows poor enzymatic activity towards synthetic substrates tested so far and it has no natural inhibitor.     

Inhibiting the C5-C5a receptor axis

Activation of the complement system is a major pathogenic event that drives various inflammatory responses in numerous diseases. All pathways of complement activation lead to cleavage of the C5 molecule generating the anaphylatoxin C5a and, C5b that subsequently forms the terminal complement complex (C5b-9). C5a exerts a predominant pro-inflammatory activity through interactions with the classical G-protein coupled receptor C5aR (CD88) as well as with the non-G protein coupled receptor C5L2 (GPR77), expressed on various immune and non-immune cells. C5b-9 causes cytolysis through the formation of the membrane attack complex (MAC), and sub-lytic MAC and soluble C5b-9 also possess a multitude of non-cytolytic immune functions. These two complement effectors, C5a and C5b-9, generated from C5 cleavage, are key components of the complement system responsible for propagating and/or initiating pathology in different diseases, including paroxysmal nocturnal hemoglobinuria, rheumatoid arthritis, ischemia-reperfusion injuries and neurodegenerative diseases. Thus, the C5-C5a receptor axis represents an attractive target for drug development. This review provides a comprehensive analysis of different methods of inhibiting the generation of C5a and C5b-9 as well as the signalling cascade of C5a via its receptors. These include the inhibition of C5 cleavage through targeting of C5 convertases or via the C5 molecule itself, as well as blocking the activity of C5a by neutralizing antibodies and pharmacological inhibitors, or by targeting C5a receptors per se. Examples of drugs and naturally occurring compounds used are discussed in relation to disease models and clinical trials. To date, only one such compound has thus far made it to clinical medicine: the anti-C5 antibody eculizumab, for treating paroxysmal nocturnal hemoglobinuria. However, a number of drug candidates are rapidly emerging that are currently in early-phase clinical trials. The C5-C5a axis as a target for drug development is highly promising for the treatment of currently intractable major human diseases.     

Molecular characterization of two novel cases of complete complement inhibitor Factor I deficiency

Factor I (FI) is the major complement inhibitor that degrades activated complement components C3b and C4b in the presence of specific cofactors. Complete FI deficiency results in secondary complement deficiency due to uncontrolled spontaneous alternative pathway activation. In this study we describe two unrelated patients with complete FI deficiency and undetectable alternative complement pathway activity. Both patients had experienced recurrent infections and arthralgia/arthritis. In one patient, analysis of genomic DNA revealed deletion of two adenine nucleotides in exon 2 of the CFI gene (c.133-134delAA), causing a frame shift and premature STOP codon/termination in the FIMAC (FI-membrane attack complex) domain (p.K45SfsX11). The other patient carried an A>T substitution in exon 6 (c.866A>T) encoding the LDLr2 (low density lipoprotein receptor) domain (p.D289V), resulting in an aspartic acid to valine change. Both patients were homozygous for the mutations while their healthy parents were heterozygous carriers. The mutations were introduced into recombinant FI, causing lack of FI expression and secretion upon transient transfection. Mutation p.K45SfsX11 theoretically allows expression of a 55 amino acid fragment of FI that lacks the serine protease domain, preventing proteolytic activity. In contrast, aspartic acid D289 is crucial for folding of FI. This report describes the molecular and functional consequences of two novel mutations of FI, providing a unique insight into the pathogenesis of complete FI deficiency in these patients.     

An ELISA assay with two monoclonal antibodies allows the estimation of free factor H and identifies patients with acquired deficiency of this complement regulator

Complement factor H (FH) serum levels can be affected by the presence of immune complexes of FH with autoantibodies like in autoimmune forms of atypical haemolytic uraemic syndrome (aHUS) or with C3b in homozygous factor I (FI) deficiency. These complexes reduce the amount of free functional circulating FH. In this study we aimed to determine whether FH levels measurement is disturbed in some pathological conditions and to establish a method for quantifying free and total FH in serum. For that purpose, FH levels were measured in serum samples from aHUS patients having anti-FH autoantibodies or mutations in FH gene, in patients with homozygous FI deficiency, and in healthy controls. Two anti-FH monoclonal antibodies, OX24 and A229, recognizing different functional regions in FH, were used as capture antibodies in an ELISA assay. In the control group and in the group of patients with FH mutations, the FH levels obtained with the two monoclonal antibodies were similar. In patients with anti-FH autoantibodies or with homozygous FI deficiency, however, FH levels measured with both antibodies were significantly different. As these patients had complexes of FH with autoantibodies or C3b, we interpreted that OX24 was detecting total FH and A229 was recognising free FH. Therefore, quantification of FH in plasma using these two monoclonal antibodies provides not only total FH level but also gives an estimation of how much FH circulates free and is thus available to properly control complement activation.     

Viral regulators of complement activation: Structure,function and evolution

The complement system surveillance in the host is effective in controlling viral propagation. Consequently, to subvert this effector mechanism, viruses have developed a series of adaptations. One among these is encoding mimics of host regulators of complement activation (RCA) which help viruses to avoid being labeled as ‘foreign’ and protect them from complement-mediated neutralization and complement-enhanced antiviral adaptive immunity. In this review, we provide an overview on the structure, function and evolution of viral RCA proteins.