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Mutations and deletions within the human CFHR gene cluster on chromosome 1 are associated with diseases, such as dense deposit disease, CFHR nephropathy or age-related macular degeneration. Resulting mutant CFHR proteins can affect complement regulation. Here we identify human CFHR2 as a novel alternative pathway complement regulator that inhibits the C3 alternative pathway convertase and terminal pathway assembly. CFHR2 is composed of four short consensus repeat domains (SCRs). Two CFHR2 molecules form a dimer through their N-terminal SCRs, and each of the two C-terminal ends can bind C3b. C3b bound CFHR2 still allows C3 convertase formation but the CFHR2 bound convertases do not cleave the substrate C3. Interestingly CFHR2 hardly competes off factor H from C3b. Thus CFHR2 likely acts in concert with factor H, as CFHR2 inhibits convertases while simultaneously allowing factor H assisted degradation by factor I.

The complement system is central to first-line defense against invading pathogens. However, excessive complement activation and/or the loss of complement regulation contributes to the development of autoimmune diseases, systemic inflammation, and thrombosis. One of the three pathways of the complement system, the alternative complement pathway, plays a vital role in amplifying complement activation and pathway signaling. Complement factor D, a serine protease of this pathway that is required for the formation of C3 convertase, is the rate-limiting enzyme. In this review, we discuss the function of factor D within the alternative pathway and its implication in both healthy physiology and disease. Because the alternative pathway has a role in many diseases that are characterized by excessive or poorly mediated complement activation, this pathway is an enticing target for effective therapeutic intervention. Nonetheless, although the underlying disease mechanisms of many of these complement-driven diseases are quite well understood, some of the diseases have limited treatment options or no approved treatments at all. Therefore, in this review we explore factor D as a strategic target for advancing therapeutic control of pathological complement activation.

The complement system plays a crucial role in retinal homeostasis. While the proteomic analysis of ocular tissues in diabetic retinopathy (DR) has shown the deposition of complement proteins, their exact role in the pathogenesis of DR is yet unclear. We performed a detailed investigation of the role of the complement system by evaluating the levels of major complement proteins including C3, C1q, C4b, Complement Factor B (CFB), and Complement Factor H (CFH) and their activated fragments from both the classical and alternative pathways in vitreous humor and serum samples from proliferative DR (PDR) patients and controls. Further, the expressions of complements and several other key pro- and anti-angiogenic genes in the serum and vitreous humor were analyzed in the blood samples of PDR and non-PDR (NPDR) patients along with controls without diabetes. We also assessed the pro-inflammatory cytokines and matrix metalloproteinases in the vitreous humor samples. There was a significant increase in C3 and its activated fragment C3bα' (110 kDa) along with a corresponding upregulation of CFH in the vitreous of PDR patients, which confirmed the increased activation of the alternative complement pathway in PDR. Likewise, a significant upregulation of angiogenic genes and downregulation of anti-angiogenic genes was seen in PDR and NPDR cases. Increased MMP9 activity and upregulation of inflammatory markers IL8 and sPECAM with a downregulation of anti-inflammatory marker IL-10 in PDR vitreous indicated the possible involvement of microglia in DR pathogenesis. Further, a significantly high C3 deposition in the capillary wall along with thickening of basement membranes and co-localization of CFH expression with CD11b activated microglial cells in diabetic retina suggested microglia as a source of CFH in diabetic retina. The increased CFH levels could be a feedback mechanism for arresting excessive complement activation in DR eyes. A gradual increase of and expression in retina with early to late changes in epiretinal membranes of DR patients indicated a major role for the alternative complement pathway in disease progression.

Dense deposit disease and glomerulonephritis with isolated C3 deposits are glomerulopathies characterized by deposits of C3 within or along the glomerular basement membrane. Previous studies found a link between dysregulation of the complement alternative pathway and the pathogenesis of these diseases. We analyzed the role of acquired and genetic complement abnormalities in a cohort of 134 patients, of whom 29 have dense deposit disease, 56 have glomerulonephritis with isolated C3 deposits, and 49 have primary membranoproliferative glomerulonephritis type I, with adult and pediatric onset. A total of 53 patients presented with a low C3 level, and 65 were positive for C3 nephritic factor that was significantly more frequently detected in patients with dense deposit disease than in other histological types. Mutations in CFH and CFI genes were identified in 24 patients associated with a C3 nephritic factor in half the cases. We found evidence for complement alternative pathway dysregulation in 26 patients with membranoproliferative glomerulonephritis type I. The complement factor H Y402H variant was significantly increased in dense deposit disease. We identified one at-risk membrane cofactor protein (MCP) haplotype for glomerulonephritis with isolated C3 deposits and membranoproliferative glomerulonephritis type I. Thus, our results suggest a critical role of fluid-phase alternative pathway dysregulation in the pathogenesis of C3 glomerulopathies as well as in immune complex–mediated glomerular diseases. The localization of the C3 deposits may be under the influence of MCP expression.

The structure of C3b in complex with factor I and a shortened version of factor H, along with functional analyses, leads to a mechanistic model for how regulators determine sequential cleavage events on C3b. The complement system labels microbes and host debris for clearance. Degradation of surface-bound C3b is pivotal to direct immune responses and protect host cells. How the serine protease factor I (FI), assisted by regulators, cleaves either two or three distant peptide bonds in the CUB domain of C3b remains unclear. We present a crystal structure of C3b in complex with FI and regulator factor H (FH; domains 1–4 with 19–20). FI binds C3b–FH between FH domains 2 and 3 and a reoriented C3b C-terminal domain and docks onto the first scissile bond, while stabilizing its catalytic domain for proteolytic activity. One cleavage in C3b does not affect its overall structure, whereas two cleavages unfold CUB and dislodge the thioester-containing domain (TED), affecting binding of regulators and thereby determining the number of cleavages. These data explain how FI generates late-stage opsonins iC3b or C3dg in a context-dependent manner, to react to foreign, danger or healthy self signals.

Oxidative stress and enhanced lipid peroxidation are linked to many chronic inflammatory diseases, including age-related macular degeneration (AMD). AMD is the leading cause of blindness in Western societies, but its aetiology remains largely unknown. Malondialdehyde (MDA) is a common lipid peroxidation product that accumulates in many pathophysiological processes, including AMD. Here we identify complement factor H (CFH) as a major MDA-binding protein that can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice. The CFH polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind MDA, indicating a causal link to disease aetiology. Our findings provide important mechanistic insights into innate immune responses to oxidative stress, which may be exploited in the prevention of and therapy for AMD and other chronic inflammatory diseases. Causes of age-related macular degeneration Age-related macular degeneration (AMD) is a leading cause of blindness in older people. A polymorphism in complement factor H (CFH) has been strongly associated with the disease, but the mechanism of the association has been unclear. Here it is shown that CFH binds specifically to the lipid peroxidation product, malondialdehyde, which builds up in AMD as a result of oxidative stress. Malondialdehyde and malondialdehyde-modified proteins induce inflammatory responses; CFH neutralizes this inflammatory potential both in vitro and in the mouse retina. A common CFH polymorphism associated with AMD leads to impaired binding to malondialdehyde, potentially explaining why homozygous individuals with this polymorphism have a 6–7-fold increased risk of developing the condition.

Borrelia , spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia , including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH 15-20  and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF. Key points • DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment • Borrelia spp. possesses immune evasion mechanisms, including human host complement • CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1 Graphical Abstract

Hepatocellular carcinoma (HCC) is difficult to detect, carries a poor prognosis, and is one of few cancers with an increasing yearly incidence. Molecular defects in complement factor H (CFH), a critical regulatory protein of the complement alternative pathway (AP), are typically associated with inflammatory diseases of the eye and kidney. Little is known regarding the role of CFH in controlling complement activation within the liver. While studying aging CFH-deficient (fH-/-) mice, we observed spontaneous hepatic tumor formation in more than 50% of aged fH-/- males. Examination of fH-/- livers (3-24 months) for evidence of complement-mediated inflammation revealed widespread deposition of complement-activation fragments throughout the sinusoids, elevated transaminase levels, increased hepatic CD8+ and F4/80+ cells, overexpression of hepatic mRNA associated with inflammatory signaling pathways, steatosis, and increased collagen deposition. Immunostaining of human HCC biopsies revealed extensive deposition of complement fragments within the tumors. Investigating the Cancer Genome Atlas also revealed that increased CFH mRNA expression is associated with improved survival in patients with HCC, whereas mutations are associated with worse survival. These results indicate that CFH is critical for controlling complement activation in the liver, and in its absence, AP activation leads to chronic inflammation and promotes hepatic carcinogenesis.

Age-related macular degeneration (AMD) is a common condition that leads to severe vision loss and dysregulation of the complement system is thought to be associated with the disease. To investigate associations of polymorphisms in AMD susceptibility genes with systemic complement activation, 2655 individuals were genotyped for 32 single nucleotide polymorphisms (SNPs) in or near 23 AMD associated risk genes. Component 3 (C3) and its catabolic fragment C3d were measured in serum and AMD staging was performed using multimodal imaging. The C3d/C3 ratio was calculated and associations with environmental factors, SNPs and various haplotypes of complement factor H (CFH) genes and complement factor B (CFB) genes were analyzed. Linear models were built to measure the influence of genetic variants on the C3d/C3 ratio. The study cohort included 1387 patients with AMD and 1268 controls. Higher C3d/C3 ratios were found for current smoker (p = 0.002), higher age (p = 1.56 × 10(-7)), AMD phenotype (p = 1.15 × 10(-11)) and the two SNPs in the C3 gene rs6795735 (p = 0.04) and rs2230199 (p = 0.04). Lower C3d/C3 ratios were found for diabetes (p = 2.87 × 10(-6)), higher body mass index (p = 1.00 × 10(-13)), the SNPs rs1410996 (p = 0.0001), rs800292 (p = 0.003), rs12144939 (p = 4.60 × 10(-6)) in CFH, rs4151667 (p = 1.01 × 10(-5)) in CFB and individual haplotypes in CFH and CFB. The linear model revealed a corrected R-square of 0.063 including age, smoking status, gender, and genetic polymorphisms explaining 6.3% of the C3d/C3 ratio. After adding the AMD status the corrected R-square was 0.067. In conclusion, none of the evaluated genetic polymorphisms showed an association with increased systemic complement activation apart from two SNPs in the C3 gene. Major genetic and non-genetic factors for AMD were not associated with systemic complement activation.

Activation of the complement cascade induces inflammatory responses and marks cells for immune clearance. In the central complement-amplification step, a complex consisting of surface-bound C3b and factor B is cleaved by factor D to generate active convertases on targeted surfaces. We present crystal structures of the pro-convertase C3bB at 4 angstrom resolution and its complex with factor D at 3.5 angstrom resolution. Our data show how factor B binding to C3b forms an open \"activation\" state of C3bB. Factor D specifically binds the open conformation of factor B through a site distant from the catalytic center and is activated by the substrate, which displaces factor D's self-inhibitory loop. This concerted proteolytic mechanism, which is cofactor-dependent and substrate-induced, restricts complement amplification to C3b-tagged target cells.