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Dysregulation of the alternative pathway of complement underlies the pathogenesis of C3 glomerulopathy (C3G). Because Factor H (FH) prevents excessive alternative pathway activity while Factor H-related protein 1 (FHR-1) is believed to enhance this response, we investigated the balance between FH and FHR-1 in C3G. To assess the role of FHR-1 in C3G pathogenicity, we used a multiplex ligation-dependent probe amplification to detect copy number variants in and enzyme linked immunosorbent assays to measure circulating protein levels in C3G patients compared to controls. Additionally, an C3b deposition assay was used to characterize the functional impact of FHR-1 on local complement activity. In this study, we confirm that copy number impacts C3G risk. In C3G patients with two copies of , the FHR-1:FH protein ratios are increased compared to controls; however, this increase is not disease specific. Rather, it is reflective of deteriorating renal function and was also observed in a second cohort of patients with chronic kidney disease from a variety of other causes. Functional studies showed that FHR-1 competes with FH to increase C3b deposition on mouse mesangial cell surfaces, an effect enhanced by heparan sulfate cleavage. Altogether, we show that as renal function declines, a change in the FHR-1:FH ratio combined with changes in heparan sulfate architecture increase complement activity. These findings suggest that complement activity may contribute to the chronic inflammation and progression of renal damage associated with chronic kidney disease.

The renal disorder C3 glomerulopathy with dense deposit disease (C3G-DDD) pattern results from complement dysfunction and primarily affects children and young adults. There is no effective treatment, and patients often progress to end-stage renal failure. A small fraction of C3G-DDD cases linked to factor H or C3 gene mutations as well as autoantibodies have been reported. Here, we examined an index family with 2 patients with C3G-DDD and identified a chromosomal deletion in the complement factor H-related (CFHR) gene cluster. This deletion resulted in expression of a hybrid CFHR2-CFHR5 plasma protein. The recombinant hybrid protein stabilized the C3 convertase and reduced factor H-mediated convertase decay. One patient was refractory to plasma replacement and exchange therapy, as evidenced by the hybrid protein quickly returning to pretreatment plasma levels. Subsequently, complement inhibitors were tested on serum from the patient for their ability to block activity of CFHR2-CFHR5. Soluble CR1 restored defective C3 convertase regulation; however, neither eculizumab nor tagged compstatin had any effect. Our findings provide insight into the importance of CFHR proteins for C3 convertase regulation and identify a genetic variation in the CFHR gene cluster that promotes C3G-DDD. Monitoring copy number and sequence variations in the CFHR gene cluster in C3G-DDD and kidney patients with C3G-DDD variations will help guide treatment strategies.

The complement component C3 is at the heart of the complement cascade. It is a complex protein, which generates different functional activated fragments (C3a, C3b, iC3b, C3c, C3d). C3b is a constituent of the alternative pathway C3 convertase (C3bBb), binds multiple regulators, and receptors, affecting thus the functioning of the immune system. The activated forms of C3 are a target for autoantibodies. This review focuses on the discovery, disease relevance, and functional consequences of the anti-C3b autoantibodies. They were discovered about 70 years ago and named immunoconglutinins. They were found after infections and considered convalescent factors. At the end of the twentieth century IgG against C3b were found in systemic lupus erythematosus and recently in lupus nephritis, correlating with the disease severity and flare. Cases of C3 glomerulopathy and immune complex glomerulonephritis were also reported. These antibodies recognize epitopes, shared between C3(H2O)/C3b/iC3b/C3c and have overt functional activity. They correlate with low plasmatic C3 levels in patients. , they increase the activity of the alternative pathway C3 convertase, without being C3 nephritic factors. They perturb the binding of the negative regulators Complement Receptor 1 and Factor H. The clear functional consequences and association with disease severity warrant further studies to establish the link between the anti-C3b autoantibodies and tissue injury. Comparative studies with such antibodies, found in patients with infections, may help to uncover their origin and epitopes specificity. Patients with complement overactivation due to presence of anti-C3b antibodies may benefit from therapeutic targeting of C3.

Leptospira, the causative agent of leptospirosis, interacts with several host molecules, including extracellular matrix components, coagulation cascade proteins, and human complement regulators. Here we demonstrate that acquisition of factor H (FH) on the Leptospira surface is crucial for bacterial survival in the serum and that these spirochetes, besides interacting with FH, FH related-1, and C4b binding protein (C4BP), also acquire FH like-1 from human serum. We also demonstrate that binding to these complement regulators is mediated by leptospiral immunoglobulin-like (Lig) proteins, previously shown to interact with fibronectin, laminin, collagen, elastin, tropoelastin, and fibrinogen. Factor H binds to Lig proteins via short consensus repeat domains 5 and 20. Competition assays suggest that FH and C4BP have distinct binding sites on Lig proteins.Moreover, FH and C4BP bound to immobilized Ligs display cofactor activity, mediating C3b and C4b degradation by factor I. In conclusion, Lig proteins are multifunctional molecules, contributing to leptospiral adhesion and immune evasion.

Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation.

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

Immune protection by the complement system depends on C3 cleavage by C3 convertases that is critical to all three activation pathways. Structural data on convertase formation in the classical pathway and on C3-substrate binding to convertases is lacking. We present the cryo-EM structures of the proconvertase (C4b2), convertase (C4b2b), and convertase-substrate complex (C4b2b-C3) of the classical pathway. The data show that C2 and C4b form proconvertases and convertases like factor B and C3b of the alternative pathway. Substrate C3 binds C4b of the convertase through two interfaces: one also found in the SCIN-inhibited C3bBb dimer, and another facilitated by conformational changes in C3. Bending of C3 and swinging of the C2 protease bring the C3-scissile loop into the active site. The second, charged, C4b-interaction site favors C3- substrate binding, but upon cleavage repels product C3b. Thus, a charge switch-over mechanism effects the catalytic turnover of the convertases producing opsonin C3b. The complement system depends on C3 cleavage that is critical to all three activation pathways, but currently structural data is lacking. Here authors present cryo-EM structures revealing a charge switch-over mechanism.

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.

Considerable advances have been achieved in the application of nanomaterials for immunotherapies, yet the precise immune effects induced by protein corona remain elusive. Here, we explore the formation mechanism and immune regulation process of protein corona in acute myeloid leukaemia (AML) mouse models using commercialized iron oxide nanoparticles (IONPs), with different surface modifications, including an FDA-approved variant. Using macrophages depleted or Complement Component 3 (C3) knockout mice, we demonstrate that carboxymethyl dextran-coated IONP (IONP-COOH) reduces leukaemia burden. Mechanistically, IONP-COOH indirectly binds to C3b after activating the complement alternative pathway, subsequently enhancing phagocytosis of macrophages and activating adaptive immunity mediated by complement corona. While aminated dextran-coated IONPs directly absorb C3b and activate the lectin pathway, leading to immune cell exhaustion. Our findings suggest that IONP-COOH may serve as an immune activator for AML treatment, offering a promising approach to developing therapeutic nanomaterials by leveraging surface chemistry to enhance immunotherapy. Iron oxide nanoparticles (IONP) are approved to treat iron deficiency and have been reported to reduce leukaemia in preclinical models. Here, the authors show that carboxymethyl dextran-coated IONP, but not other types of surface coating, activate the complement alternative pathway, enhancing macrophage phagocytosis and adaptive immunity to reduce leukaemia burden in mouse models.

Pentraxin 3 (PTX3), a long pentraxin and a humoral pattern recognition molecule (PRM), has been demonstrated to be protective against Aspergillus fumigatus, an airborne human fungal pathogen. We explored its mode of interaction with A. fumigatus, and the resulting implications in the host immune response. Here, we demonstrate that PTX3 interacts with A. fumigatus in a morphotype-dependent manner: (a) it recognizes germinating conidia through galactosaminogalactan, a surface exposed cell wall polysaccharide of A. fumigatus, (b) in dormant conidia, surface proteins serve as weak PTX3 ligands, and (c) surfactant protein D (SP-D) and the complement proteins C1q and C3b, the other humoral PRMs, enhance the interaction of PTX3 with dormant conidia. SP-D, C3b or C1q opsonized conidia stimulated human primary immune cells to release pro-inflammatory cytokines and chemokines. However, subsequent binding of PTX3 to SP-D, C1q or C3b opsonized conidia significantly decreased the production of pro-inflammatory cytokines/chemokines. PTX3 opsonized germinating conidia also significantly lowered the production of pro-inflammatory cytokines/chemokines while increasing IL-10 (an anti-inflammatory cytokine) released by immune cells when compared to the unopsonized counterpart. Overall, our study demonstrates that PTX3 recognizes A. fumigatus either directly or by interplaying with other humoral PRMs, thereby restraining detrimental inflammation. Moreover, PTX3 levels were significantly higher in the serum of patients with invasive pulmonary aspergillosis (IPA) and COVID-19-associated pulmonary aspergillosis (CAPA), supporting previous observations in IPA patients, and suggesting that it could be a potential panel-biomarker for these pathological conditions caused by A. fumigatus.