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Purpose To characterize the molecular mechanism underpinning early-onset macular drusen (EOMD), a phenotypically severe subtype of age-related macular degeneration (AMD), in a subgroup of patients. Design Multicenter case series, in vitro experimentation, and retrospective analysis of previously reported variants. Participants Seven families with apparently autosomal dominant EOMD. Methods Patients underwent a comprehensive ophthalmic assessment. Affected individuals from families A, B, and E underwent whole exome sequencing. The probands from families C, D, F, and G underwent Sanger sequencing analysis of the complement factor H (CFH) gene. Mutant recombinant factor H like-1 (FHL-1) proteins were expressed in HEK293 cells to assess the impact on FHL-1 expression and function. Previously reported EOMD-causing variants in CFH were reviewed. Main Outcome Measures Detailed clinical phenotypes, genomic findings, in vitro characterization of mutation effect on protein function, and postulation of the pathomechanism underpinning EOMD. Results All affected participants demonstrated bilateral drusen. The earliest reported age of onset was 16 years (median, 46 years). Ultra-rare (minor allele frequency [MAF], ≤0.0001) CFH variants were identified as the cause of disease in each family: CFH c.1243del, p.(Ala415ProfsTer39) het; c.350+1G→T het; c.619+1G→A het, c.380G→A, p.(Arg127His) het; c.694C→T p.(Arg232Ter) het (identified in 2 unrelated families in this cohort); and c.1291T→A, p.(Cys431Ser). All mutations affect complement control protein domains 2 through 7, and thus are predicted to impact both FHL-1, the predominant isoform in Bruch’s membrane (BrM) of the macula, and factor H (FH). In vitro analysis of recombinant proteins FHL-1R127H, FHL-1A415f/s, and FHL-1C431S demonstrated that they are not secreted, and thus are loss-of-function proteins. Review of 29 previously reported EOMD-causing mutations found that 75.8% (22/29) impact FHL-1 and FH. In total, 86.2% (25/29) of EOMD-associated variants cause haploinsufficiency of FH or FHL-1. Conclusions Early-onset macular drusen is an underrecognized, phenotypically severe subtype of AMD. We propose that haploinsufficiency of FHL-1, the main regulator of the complement pathway in BrM, where drusen develop, is an important mechanism underpinning the development of EOMD in a number of cases. Understanding the molecular basis of EOMD will shed light on AMD pathogenesis given their pathologic similarities.

Highlights • Alpha-enolase, aldehyde reductase-I and dimethylargininase-I are SREAT autoantigens. • Molecular mimicry between thyroid and CNS autoantigens is hypothesized in SREAT. • Homology with TSH-R, Tg and TPO exists for 6, 27 and 47 of 46,809 CNS-proteins. • The above homologies are often in epitope-containing parts of thyroid autoantigens. • Most of the above proteins are expressed in CNS regions which are altered in SREAT., A few patients with Hashimoto’s thyroiditis or Graves’ disease develop a multiform syndrome of the central nervous system (CNS) termed Hashimoto’s encephalopathy or steroid-responsive encephalopathy associated with autoimmune thyroid disease (HE/SREAT). They have high levels of thyroid autoantibodies (TgAb, TPOAb and/or TSH-R-Ab) in blood and cerebrospinal fluid. Autoantibodies against alpha-enolase, aldehyde reductase-I (AKRIA) and/or dimethylargininase-I (DDAHI), proteins expressed in the CNS among other tissues, were detected in the blood and, when searched, in the cerebrospinal fluid of HE/SREAT patients. Recently, we reported that alpha-enolase, AKRIA and DDAHI share local sequence homology with each of the three autoantigens (TgAb, TPOAb, TSH-R-Ab), often in epitope-containing segments of the thyroid autoantigens. We hypothesized that there might be additional CNS-expressed proteins homologous to thyroid autoantigens, possibly overlapping known epitopes of the thyroid autoantigens. We used bioinformatic methods to address this hypothesis. Six, 27 and 47 of 46,809 CNS-expressed proteins share homology with TSH-R, Tg and TPO, respectively. The homologous regions often contain epitopes, and some match regions of thyroid autoantigens which have homology with alpha-enolase, AKRIA and/or DDAHI. Several of the aforementioned proteins are present in CNS areas that show abnormalities at neuroimaging in HE/SREAT patients. Furthermore, autoantibodies against some of the said six, 27 and 47 proteins were reported to be associated with a number of autoimmune diseases. Not only we validated our hypothesis, but we think that such a variety of potential CNS targets for thyroid Ab against epitopes contained in regions that have local homology with CNS proteins may explain the polymorphic phenotypes of HE/SREAT. Only when elevated amounts of these Ab are synthesized and trespass the blood-brain barrier, HE/SREAT appears. This might explain why HE/SREAT is so relatively rare.

The Sez6 family consists of Sez6, Sez6L, and Sez6L2. Its members are expressed throughout the brain and have been shown to influence synapse numbers and dendritic morphology. They are also linked to various neurological and psychiatric disorders. All Sez6 family members contain 2-3 CUB domains and 5 complement control protein (CCP) domains, suggesting that they may be involved in complement regulation. We show that Sez6 family members inhibit C3b/iC3b opsonization by the classical and alternative pathways with varying degrees of efficacy. For the classical pathway, Sez6 is a strong inhibitor, Sez6L2 is a moderate inhibitor, and Sez6L is a weak inhibitor. For the alternative pathway, the complement inhibitory activity of Sez6, Sez6L, and Sez6L2 all equaled or exceeded the activity of the known complement regulator MCP. Using Sez6L2 as the representative family member, we show that it specifically accelerates the dissociation of C3 convertases. Sez6L2 also functions as a cofactor for Factor I to facilitate the cleavage of C3b; however, Sez6L2 has no cofactor activity toward C4b. In summary, the Sez6 family are novel complement regulators that inhibit C3 convertases and promote C3b degradation.

Regulation of complement activation in the host cells is mediated primarily by the regulators of complement activation (RCA) family proteins that are formed by tandemly repeating complement control protein (CCP) domains. Functional annotation of these proteins, however, is challenging as contiguous CCP domains are found in proteins with varied functions. Here, by employing an in silico approach, we identify five motifs which are conserved spatially in a specific order in the regulatory CCP domains of known RCA proteins. We report that the presence of these motifs in a specific pattern is sufficient to annotate regulatory domains in RCA proteins. We show that incorporation of the lost motif in the fourth long-homologous repeat (LHR-D) in complement receptor 1 regains its regulatory activity. Additionally, the motif pattern also helped annotate human polydom as a complement regulator. Thus, we propose that the motifs identified here are the determinants of functionality in RCA proteins., Hina Ojha et al. show that the presence of five motifs in a specific pattern in CCP (complement control protein) domains is indicative of functional RCA (regulators of complement activation) proteins. This study suggests that an in silico regulatory RCA prediction tool CoReDo can be used to identify putative regulatory RCA proteins.

Mannose-binding lectin-associated serine protease-1 (MASP-1), a multifunctional serine protease, plays an important role in innate immunity which is capable of activating the lectin pathway of the complement system and also triggering coagulation cascade system. In this study, a MASP-1 homolog (OnMASP-1) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression and inflammation functional levels. The open reading frame (ORF) of OnMASP-1 is 2187 bp of nucleotide sequence encoding a polypeptide of 728 amino acids. The deduced amino acid sequence has 6 characteristic structures, including two C1r/C1s-Uegf-BMP domains (CUB), one epidermal growth factor domain (EGF), two complement control protein domains (CCP) and a catalytic serine protease domain (SP). Expression analysis revealed that the OnMASP-1 was highly expressed in the liver, and widely exhibited in other tissues containing intestine, spleen and kidney. In addition, the OnMASP-1 expression was significantly up-regulated in spleen and head kidney following challenges with Streptococcus agalactiae and Aeromonas hydrophila. The up-regulations of OnMASP-1 mRNA and protein expression were also demonstrated in hepatocytes and monocytes/macrophages in vitro stimulation with S. agalactiae and A. hydrophila. Recombinant OnMASP-1 protein was likely to participate in the regulation of inflammatory and migration reaction by monocytes/macrophages. These results indicated that OnMASP-1, playing an important role in innate immunity, was likely to involve in host defense against bacterial infection in Nile tilapia.

Background/case studies Cromer blood group system consists of 12 high-prevalence and three low-prevalence antigens. The molecular basis for the antigens is known, and the majority is the product of a single nucleotide change in the DAF gene and has been localized to one of the four complement control protein domains on the DAF protein. Antibodies to Cromer antigens are rarely encountered. This case reports a 72 year-oldAfrican descendent Brazilian woman with endometrioid adenocarcionoma had an antibody reactive with all red blood cells (RBCs) and cord RBCs. The direct antiglobulin test (DAT) and autocontrol were negative. She had 2 previous pregnancies and a previous transfusion 20 years ago. Siblings were recruited and a familiar study was performed. Serological and molecular studies were carried out to define the patient‘s phenotype and the antibody specificity. Methods Serological investigation was performed by hemagglutination in gel cards.Antibody identification was performed in the patient's serum with untreated and treated RBCs with papain, trypsin, α-chymotrypsin and 200 mM dithiothreitol (DTT).Antigen typing was performed for the most common antigens with commercial reagents and for high frequency antigens with sera from our collection coming from SCARF. Genomic DNA was used to amplify and sequence CROM exons. Results The patient‘s serum reacted with all panel cellsincluding phenotypically similar RBCs. Only the autologous control was non-reactive. Crossmatching was negative with RBCs of 2 siblings and positive with the other two. Antibody titration showed a titre of 64.The antigen was sensitive to papain, typsin and α-chymotrypsin-treatment but was resistant to 200 mM DTTwhich suggested the presence of a high-frequency antibody to an antigen of the Cromer system. Sequencing analysis of CROM revealed the patient and her two sisters with negative crossmatching were homozygous for the molecular change c.679G > Cin exon 6, which predict the Cr(a-) phenotype. DNA sequencing of the two other siblings with positive crossmatching showed this molecular change in heterozygous.The antibody was determined to be of anti-Cra specificity and other underlying antibodies were ruled out through adsorption and elution with R1R1, Jk(b-) and Fy(b-) allogeneic RBCs. Conclusion This study reported for the first time a rare case of anti-Cra in a Brazilian family. Due to the complexity of the Cromer system and the scarcity of anti-Cra and Cr(a-) RBCs, molecular analysis was essential for the conclusion of the case. Anti-Cra is rarely encountered and has already been related to moderate transfusion reaction but its clinical significance can be variable. Although no transfusions were requiredat this time, the family study was essential to find compatible donors.

Activation of proteins often involves conformational transitions, and these switches are often difficult to characterize in multidomain proteins. Full-length factor H (FH), consisting of 20 small consensus repeat domains (150 kD), is a complement control protein that regulates the activity of the alternative complement pathway. Different preparations of FH can also reduce the disulfide bonds linking large Von Willebrand factor (VWF) multimers into smaller, less adhesive forms. In contrast, commercially available purified FH (pFH) has little or no VWF reductase activity unless the pFH is chemically modified by either ethylenediaminetetraacetic acid (EDTA) or urea. We used atomic force microscopy single molecule force measurements to investigate different forms of FH, including recombinant FH and pFH, in the presence or absence of EDTA and urea, and to correlate the conformational changes to its activities. We found that the FH conformation depends on the method used for sample preparation, which affects the VWF reductase activity of FH.

C4b Binding Protein (C4BP) is a major fluid phase inhibitor of the classical and lectin pathways of the complement system. Complement inhibition is achieved by binding to and restricting the role of activated complement component C4b. C4BP functions as a co-factor for factor I in proteolytic inactivation of both soluble and cell surface-bound C4b, thus restricting the formation of the C3-convertase, C4b2a. C4BP also accelerates the natural decay/dissociation of the C3 convertase. This makes C4BP a prime target for exploitation by pathogens to escape complement attack, as seen in Streptococcus pyogenes or Flavivirus. Here, we examined whether C4BP can act on its own in a complement independent manner, against pathogens. C4BP bound H1N1 and H3N2 subtypes of Influenza A Virus (IAV) most likely via multiple sites in Complement Control Protein (CCP) 1-2, 4-5, and 7-8 domains of its α-chain. In addition, C4BP CCP1-2 bound H3N2 better than H1N1. C4BP bound three IAV envelope proteins: Haemagglutinin (~70 kDa), Neuraminidase (~55 kDa), and Matrix protein 1 (~25kDa). C4BP suppressed H1N1 subtype infection into the lung epithelial cell line, A549, while it promoted infection by H3N2 subtype. C4BP restricted viral entry for H1N1 but had the opposite effect on H3N2, as evident from experiments using pseudo-typed viral particles. C4BP downregulated mRNA levels of pro-inflammatory IFN-α, IL-12, and NFκB in the case of H1N1, while it promoted a pro-inflammatory immune response by upregulating IFN- α, TNF-α, RANTES, and IL-6 in the case of H3N2. We conclude that C4BP differentially modulates the efficacy of IAV entry, and hence, replication in a target cell in a strain-dependent manner, and acts as an entry inhibitor for H1N1. Thus, CCP containing complement proteins such as factor H and C4BP may have additional defense roles against IAV that do not rely on the regulation of complement activation.

Heme oxygenase is a cytoprotective enzyme with strong antioxidant and anti-apoptotic properties. Its cytoprotective role is mainly attributed to its enzymatic activity, which involves the degradation of heme to biliverdin with simultaneous release of carbon monoxide (CO). Recent studies uncovered a new cytoprotective role for heme oxygenase-1 (HO-1) by identifying a regulatory role on the complement control protein decay-accelerating factor. This is a key complement regulatory protein preventing dysregulation or overactivation of complement cascades that can cause kidney injury. Cell-specific targeting of HO-1 induction may, therefore, be a novel approach to attenuate complement-dependent forms of kidney disease. Published version

TGF-β is a secreted signaling protein involved in many physiological processes: organ development, production and maintenance of the extracellular matrix, as well as regulation of the adaptive immune system. As a cytokine, TGF-β stimulates the differentiation of CD4+T-cells into regulatory T-cells (Tregs) that act to promote peripheral immune tolerance. The murine parasiteHeligmosomoides polygyrustakes advantage of this pathway to induce inducing Foxp3+Tregsin a similar manner using a TGF-β mimic (TGM), comprised of five tandem complement control protein (CCP) domains, designated D1-D5. Despite having no structural homology to TGF-β or to TGF-β family proteins, TGM binds directly to the TGF-β type I and type II receptors, TβRI and TβRII. To further investigate, NMR titration, and SPR and ITC binding experiments were performed, showing that TGM-D2, with the aid of D1, binds TβRI and TGM-D3 binds TβRII. Competition ITC experiments showed that TGM-D3 competes with TGF-β for binding to TβRII, consistent with TGM-D3-induced NMR chemical shift perturbations of TβRII which aligned with the solvent inaccessible areas of TβRII upon binding TGF-β. Thus, TGM-D3 binds to the same edged β-strand of TβRII that is used to bind TGF-β. Competition ITC experiments demonstrated that TGM-D1D2 and TGF-β3:TβRII compete for binding to TβRI, while TGM-D2-induced NMR chemical shift perturbation of TβRI showed that TGM-D2 binds to the same pre-helix extension of TβRI as does the TGF-β/TβRII binary complex. The solution structure of TGM-D3 revealed that while it has the overall structure of a CCP domain, TGM-D3 has an insertion in the hypervariable loop uncommon to CCP domains. These findings suggest that parasitic TGM, despite its lack of structural similarity to TGF-β, evolved to take advantage of the binding regions of the mammalian TGF-β type I and type II receptors. The structure of this TGM domain, along with the predicted structure of otherH. polygyrussecreted proteins reported in the literature, suggest that TGM is part of a larger family of evolutionarily-adapted immunomodulatory CCP-containing proteins.

The Sez6 family consists of Sez6, Sez6L, and Sez6L2. Its members are expressed throughout the brain and have been shown to influence synapse numbers and dendritic morphology. They are also linked to various neurological and psychiatric disorders. All Sez6 family members contain 2-3 CUB domains and 5 complement control protein (CCP) domains, suggesting that they may be involved in complement regulation. We show that all Sez6 family members inhibit C3 deposition by the classical and alterative pathways with varying degrees of efficacy. For the classical pathway, Sez6 is a strong inhibitor, Sez6L2 is a moderate inhibitor, and Sez6L is a weak inhibitor. Using Sez6L2 as the representative family member, we show that it specifically deactivates C3 convertases by accelerating the decay or dissociation of the C3 convertase components. Sez6L2 also deactivates C3 convertases of the alternative pathway by serving as a cofactor for Factor I to facilitate the cleavage of C3b. However, Sez6L2 has no cofactor activity toward C4b. In summary, the Sez6 family are novel complement regulators that inhibit C3 convertases.

Factor H exists as a 155,000 dalton, extended protein composed of twenty small domains which is flexible enough that it folds back on itself. Factor H regulates complement activation through its interactions with C3b and polyanions. Three binding sites for C3b and multiple polyanion binding sites have been identified on Factor H. In intact Factor H these sites appear to act synergistically making their individual contributions difficult to distinguish. Recombinantly expressed fragments of human Factor H were examined using surface plasmon resonance (SPR) for interactions with C3, C3b, iC3b, C3c, and C3d. Eleven recombinant proteins of lengths from one to twenty domains were used to show that the three C3b-binding sites exhibit 100-fold different affinities for C3b. The N-terminal site [complement control protein (CCP) domains 1-6] bound C3b with a K d of 0.08 μM and this interaction was not influenced by the presence or absence of domains 7 and 8. Full length Factor H similarly exhibited a K d for C3b of 0.1 μM. Unexpectedly, the N-terminal site (CCP 1-6) bound native C3 with a K d of 0.4 μM. The C-terminal domains (CCP 19-20) exhibited a K d of 1.7 μM for C3b. We localized a weak third C3b binding site in the CCP 13-15 region with a K d estimated to be ~15 μM. The C-terminal site (CCP 19-20) bound C3b, iC3b, and C3d equally well with a K d of 1 to 2 μM. In order to identify and compare regions of Factor H that interact with polyanions a family of 18 overlapping three domain recombinant proteins spanning the entire length of Factor H were expressed and purified. Immobilized heparin was used as a model polyanion and SPR confirmed the presence of heparin binding sites in CCP 6-8 (K d 1.2 μM) and in CCP 19-20 (4.9 μM) and suggested the existence of a weak third polyanion binding site in the center of Factor H (CCP 11-13). Our results unveil the relative contributions of different regions of Factor H to its regulation of complement, and may contribute to the understanding of how defects in certain Factor H domains lead to disease.

The IL-33-ST2 pathway is an important initiator of type 2 immune responses. We previously characterised the HpARI protein secreted by the model intestinal nematode Heligmosomoides polygyrus, which binds and blocks IL-33. Here, we identify H. polygyrus Binds Alarmin Receptor and Inhibits (HpBARI) and HpBARI_Hom2, both of which consist of complement control protein (CCP) domains, similarly to the immunomodulatory HpARI and Hp-TGM proteins. HpBARI binds murine ST2, inhibiting cell surface detection of ST2, preventing IL-33-ST2 interactions, and inhibiting IL-33 responses in vitro and in an in vivo mouse model of asthma. In H. polygyrus infection, ST2 detection is abrogated in the peritoneal cavity and lung, consistent with systemic effects of HpBARI. HpBARI_Hom2 also binds human ST2 with high affinity, and effectively blocks human PBMC responses to IL-33. Thus, we show that H. polygyrus blocks the IL-33 pathway via both HpARI which blocks the cytokine, and also HpBARI which blocks the receptor.

Many plant rhabdoviruses are neurotropic and can persistently infect the central nervous system (CNS) of their insect vectors without causing significant cytopathology. The mechanisms by which the insect CNS resists infection by plant rhabdoviruses are largely unknown. Here, we report that the neural factor Hikaru genki homolog of the leafhopper Nephotettix cincticeps (NcHig) limits the spread of the nucleorhabdovirus rice yellow stunt virus (RYSV) in vector CNS. NcHig is predominantly expressed in the CNS of N. cincticeps, and the knockdown of NcHig expression by RNA interference enhances RYSV infection of the CNS. Furthermore, immuno-blockade of NcHig function by microinjection of N. cincticeps with NcHig antibody also enhances viral infection of the CNS. Thus, we conclude that the neuron-specific factor NcHig can control RYSV propagation in the CNS. Interestingly, we find the Hig homolog of the leafhopper Recilia dorsalis also has antiviral activity during the persistent infection of the cytorhabdovirus rice stripe mosaic virus (RSMV) in vector CNS. We further determine that RYSV and RSMV matrix proteins specifically interact with the complement control protein (CCP) domains of Higs. Thus, the matrix protein-binding ability of Hig is potentially essential for its antiviral activity in rice leafhoppers. Our results demonstrate an evolutionarily conserved antiviral mechanism for Hig to mediate the persistent infection of rice rhabdoviruses in the CNS of leafhopper vectors.

Copyright © 2020 Murugaiah, Varghese, Saleh, Tsolaki, Alrokayan, Khan, Collison, Sim, Nal, Al-Mohanna and Kishore. The complement system is an ancient innate immune defence mechanism that can recognise molecular patterns on the invading pathogens. Factor H, as an inhibitor of the alternative pathway, down-regulates complement activation on the host cell surface. Locally synthesised factor H at the site of infection/injury, including lungs, can act as a pattern recognition molecule without involving complement activation. Here, we report that factor H, a sialic acid binder, interacts with influenza A virus (IAV) and modulates IAV replication, as observed by an upregulation of matrix protein 1 (M1) expression in H3N2-infected A549 cells, while downregulating M1 in H1N1 subtype-infected cells. Far-western blot revealed that factor H binds hemagglutinin (HA, ~70kDa), neuraminidase (NA, ~60kDa), and M1 (~25kDa). IAV-induced transcriptional levels of IFN-α, TNF-α, IL-12, IL-6, IFN-α, while RANTES were reduced following factor H treatment for the H1N1 subtype at 6 h post-infection. However, for the H3N2 subtype, mRNA levels of these pro-inflammatory cytokines were enhanced. Recombinant form of vaccinia virus complement control protein (VCP), which like factor H, contains CCP modules and has complement-regulatory activity, mirrored the results obtained with factor H. Both factor H (25%) and VCP (45%) were found to reduce luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles. Factor H (50%) and VCP (30%) enhanced the luciferase reporter activity for H3N2, suggesting an entry inhibitory role of factor H and VCP against H1N1, but not H3N2. Thus, factor H can modulate IAV infection and inflammatory response independent of its complement-related functions. King Saud University, Riyadh, International Scientific Partnership Programme (ISPP) ISPP-145.

Complement system is the first line of human defence against intruding pathogens and is recognized as a potentially useful therapeutic target. Human malaria parasite Plasmodium employs a series of intricate mechanisms that enables it to evade different arms of immune system, including the complement system. Here, we show the expression of a multi-domain Plasmodium Complement Control Protein 1, PfCCp1 at asexual blood stages and its binding affinity with C3b as well as C4b proteins of human complement cascade. Using a biochemical assay, we demonstrate that PfCCp1 binds with complement factors and inhibits complement activation. Active immunization of mice with PfCCp1 followed by challenge with Plasmodium berghei resulted in the loss of biphasic growth of parasites and early death in comparison to the control group. The study also showed a role of PfCCp1 in modulating Toll-like receptor (TLR)-mediated signalling and effector responses on antigen-presenting cells. PfCCp1 binds with dendritic cells that down-regulates the expression of signalling molecules and pro-inflammatory cytokines, thereby dampening the TLR2-mediated signalling; hence acting as a potent immuno-modulator. In summary, PfCCp1 appears to be an important component of malaria parasite directed immuno-modulating strategies that promote the adaptive fitness of pathogens in the host.

The complement system is one of the major effector mechanisms of immune system, playing essential roles in both the innate and adaptive immune responses. In the present study, the counterparts of vertebrate complement components were identified by screening the sequenced genome of Crassostrea gigas , resulting in the identification of 792 gene models containing complement-related domains. The transcriptome of haemocytes at 6, 12 and 24 h post lipopolysaccharides (LPS) stimulation showed differential expression of 77 C1q domain containing proteins, 53 C-type lectins and 42 fibrinogen-related proteins. mRNAs encoding 18 serine protease domain-containing (SPC) proteins, 4 MACPF-domain containing proteins and 11 C3 receptor-like proteins were up-regulated upon LPS stimulation, and CgC3 mRNA was significantly increased at 12 h. The presence of CgC3 was confirmed in cell free plasma and was present in three subunit chains as expected for the processed mature protein. The complement related PRRs with coiled coil regions and SPC proteins with CUB domains may function in the activation of CgC3, whereas, the C3-like receptors with integrin-α/β domain mediated the phagocytosis of C3-labled pathogens. These PRRs appear to serve as opsonins to promote phagocytosis of opsonized pathogens. The overall results suggested the existence of a potential multi-component complement system in C. gigas .

β2-glycoprotein I (β2GPI) is an abundant multi-domain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as Antiphospholipid Syndrome (APS). Previous studies have shown that β2GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to β2GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate β2GPI's structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of β2GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating β2GPI's physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.

Factor H–related protein (FHR) 1 is one of the five human FHRs that share sequence and structural homology with the alternative pathway complement inhibitor FH. Genetic studies on disease associations and functional analyses indicate that FHR-1 enhances complement activation by competitive inhibition of FH binding to some surfaces and immune proteins. We have recently shown that FHR-1 binds to pentraxin 3. In this study, our aim was to investigate whether FHR-1 binds to another pentraxin, C-reactive protein (CRP), analyze the functional relevance of this interaction, and study the role of FHR-1 in complement activation and regulation. FHR-1 did not bind to native, pentameric CRP, but it bound strongly to monomeric CRP via its C-terminal domains. FHR-1 at high concentration competed with FH for CRP binding, indicating possible complement deregulation also on this ligand. FHR-1 did not inhibit regulation of solid-phase C3 convertase by FH and did not inhibit terminal complement complex formation induced by zymosan. On the contrary, by binding C3b, FHR-1 allowed C3 convertase formation and thereby enhanced complement activation. FHR-1/CRP interactions increased complement activation via the classical and alternative pathways on surfaces such as the extracellular matrix and necrotic cells. Altogether, these results identify CRP as a ligand for FHR-1 and suggest that FHR-1 enhances, rather than inhibits, complement activation, which may explain the protective effect of FHR-1 deficiency in age-related macular degeneration.

Serine protease cleaved-complement component 4 (C4) at sessile loop, which is significant for completion of lectin and classical complement pathways at the time of infections. The co-crystalized structure of C4 with Mannose-binding protein-associated serine protease 2 (MASP2) provided the structural and functional aspects of its interaction and underlined the C4 activation by MASP2. The same study also revealed the significance of complement control protein (CCP) domain through mutational study, where mutated CCP domain led to the inhibition of C4 activation. However, the interaction of trypsin serine domain with C4α sessile loop revealed another aspect of C4 activation. The human C4 cleavage by Trypsin (Tryp) in a control manner was explored but not yet revealed the identification of cleaved fragments. Hence, the present study investigated the Tryp mediated C4 activation using computational approach (protein-protein docking and molecular dynamics simulation) by comparing with the co-crystalized structure of C4-MASP2. Docking result identified the crucial interacting residues Gly219, Gln178, and Asn102 of Tryp catalytic pocket which were interacting with Arg756 and Glu759 (sessile loop) of α-Chain (C4) in a similar manner to C4-MASP2 co-crystallized complex. Moreover, MD simulation results and mutational study underlined the conformational rearrangements in the C4 due to the Tryp interaction. Comparative analysis of C4 alone, C4-Tryp, and C4-MASP2 revealed the impact of Tryp on C4 was similar as MASP2. These studies designate the role of sessile loop in the interaction with serine domain, which could be useful to understand the various interactions of C4 with other complement components.

The pestivirus bovine viral diarrhea virus (BVDV) is known to bind to the CD46 molecule, which subsequently promotes entry of the virus. Mapping of the BVD-virion-binding site has shown that two peptides, 66EQIV69 and 82GQVLAL87, located on antiparallel beta sheets in the most distal complement control protein module (CCP1), provide the attachment platform. In the present study, we reveal new CD46-encoding transcripts that are predicted to encode CCP1-containing soluble forms. Further, we show that the serum of most adult cattle contains soluble CD46 (sCD46) and that a recombinant soluble isoform neutralizes BVDV infectivity in an in vitro assay. We have then established an ELISA for determination of plasma sCD46 in a large cohort of animals. Overall, serum sCD46 amounts to 8 ± 18 ng/mL (mean ± SD, n = 440), with a IC [95-105] ranging from 6,4 to 9,8 ng/mL and extreme values ​​between 0 and 178 ng/mL. We found that sCD46 is not detectable in fetal and neonatal sera and that its plasma concentration increases progressively up to adulthood. We also detected high- and low-sCD46 performers and show that this phenotype does not depend of environment. As modern rearing techniques make it possible to disseminate genetically-determined phenotypes very quickly in a population, a large-scale study examining whether high-sCD46 animals provide epidemiological protection against BVDV infection and transmission should be undertaken.

Novel therapies are urgently needed to combat the global threat of multidrug-resistant pathogens. Complement forms an important arm of innate defenses against infections. In physiological conditions, complement activation is tightly controlled by soluble and membrane-associated complement inhibitors, but must be selectively activated on invading pathogens to facilitate microbial clearance. Many pathogens, including Neisseria gonorrhoeae and N. meningitidis, express glycans, including N-acetylneuraminic acid (Neu5Ac), that mimic host structures to evade host immunity. Neu5Ac is a negatively charged 9-cabon sugar that inhibits complement, in part by enhancing binding of the complement inhibitor factor H (FH) through C-terminal domains (19 and 20) on FH. Other microbes also bind FH, in most instances through FH domains 6 and 7 or 18-20. Here we describe two strategies to target complement activation on Neisseriae. First, microbial binding domains of FH were fused to IgG Fc to create FH18-20/Fc (binds gonococci) and FH6,7/Fc (binds meningococci). A point mutation in FH domain 19 eliminated hemolysis caused by unmodified FH18-20, but retained binding to gonococci. FH18-20/Fc and FH6,7/Fc mediated complement-dependent killing in vitro and showed efficacy in animal models of gonorrhea and meningococcal bacteremia, respectively. The second strategy utilized CMP-nonulosonate (CMP-NulO) analogs of sialic acid that were incorporated into LOS and prevented complement inhibition by physiologic CMP-Neu5Ac and resulted in attenuated gonococcal infection in mice. While studies to establish the safety of these agents are needed, enhancing complement activation on microbes may represent a promising strategy to treat antimicrobial resistant organisms.

Complement factor B/C2 family (Bf/C2F) proteins are core complement system components in vertebrates that are absent in invertebrates and have been lost by numerous species, raising evolutionary questions. At least 3 duplication events have occurred from Cnidaria (ancestor) to mammals. Type II Bf/C2 genes appeared during separation of Proterostomia and Deuterostomes. The second event occurred during separation of vertebrates and invertebrates, yielding type II-2 Bf/C2. The third event occurred when jawed and jawless fish were separated, eventually producing Bf and C2 genes. Herein, we report the second mollusc Sinonovacula constricta Bf/C2-type gene (ScBf). ScBf is similar to Ruditapes decussatus Bf-like because both lack the first complement control protein module at the N terminus present in mammalian Bf/C2 proteins. Uniquely, the Ser protease (SP) module at the C terminus of ScBf is ∼50 aa longer than in other complement factor B/C2-type (Bf/C2T) proteins, and is Glu-rich. Bf/C2T proteins in molluscs lack the catalytic Ser in the SP module. Surprisingly, ScBf regulates rabbit erythrocyte agglutination, during which it is localized on the erythrocyte surface. Thus, ScBf may mediate the agglutination cascade and may be an upstream regulator of this process. Our findings provide new insight into the origin of the Bf/C2F.-Peng, M., Li, Z., Niu, D., Liu, X., Dong, Z., Li, J. Complement factor B/C2 in molluscs regulates agglutination and illuminates evolution of the Bf/C2 family.

The complement system is highly efficient in targeting pathogens, but lack of its apposite regulation results in host-cell damage, which is linked to diseases. Thus, complement activation is tightly regulated by a series of proteins, which primarily belong to the regulators of complement activation (RCA) family. Structurally, these proteins are composed of repeating complement control protein (CCP) domains where two to four successive domains contribute to the regulatory functions termed decay-accelerating activity (DAA) and cofactor activity (CFA). However, the precise constitution of the functional units and whether these units can be joined to form a larger composition with dual function have not been demonstrated. Herein, we have parsed the functional units for DAA and CFA by constructing chimeras of the decay-accelerating factor (DAF) that exhibits DAA and membrane cofactor protein (MCP) that exhibits CFA. We show that in a four-CCP framework, a functional unit for each of the regulatory activities is formed by only two successive CCPs wherein each participates in the function, albeit CCP2 has a bipartite function. Additionally, optimal activity requires C-terminal domains that enhance the avidity of the molecule for C3b/C4b. Furthermore, by composing a four-CCP DAF-MCP chimera with robust CFA (for C3b and C4b) and DAA (for classical and alternative pathway C3 convertases), named decay cofactor protein, we show that CCP functional units can be linked to design a dual-activity regulator. These data indicate that the regulatory determinants for these two biological processes are distinct and modular in nature.

The lectin pathway of the complement system has a pivotal role in the defense against infectious organisms. Mannose-binding lectin/ficolin-associated protein (MAp44), a multifunctional complement regulator, regulates the complement activation by competing with MASP-1, MASP-2 and MASP-3 for MBL and ficolin binding sites. In this study, we described the identification and functional characterization of a MAp44 homologue (OnMAp44) from Nile tilapia (Oreochromis niloticus) at molecular, cellular and protein levels. The open reading frame (ORF) of OnMAp44 is 1140 bp of nucleotide sequence encoding a polypeptide of 379 amino acids. The deduced amino acids sequence has four characteristic structures, including two C1r/C1s-Uegf-BMP domains (CUB), one epidermal growth factor domain (EGF) and one complement control protein domains (CCP). Expression analysis revealed that the OnMAp44 was highly expressed in liver, and widely existed in other examined tissues. In addition, the OnMAp44 expression was significantly up-regulated in spleen and head kidney following challenges with Streptococcus agalactiae and Aeromonas hydrophila. The up-regulations of OnMAp44 mRNA and protein expression were also observed in hepatocytes and monocytes/macrophages in vitro stimulation with S. agalactiae and A. hydrophila. Recombinant OnMAp44 protein was able to participate in the regulation of inflammation and migration reaction. Taken together, the results indicated that OnMAp44 was likely to involve in the immune response to bacterial infection in Nile tilapia.

Influenza A virus (IAV) is a major causative agent of respiratory tract infection in humans. Human IAVs cause seasonal epidemics, whilst avian IAV strains sporadically cross the host range barrier leading to zoonotic infections. The complement system is a component of the innate immune system that acts to remove pathogens including IAVs. Many bacterial pathogens and several viral pathogens have been reported to actively target components of the complement activation pathway as a defensive strategy. The aim of this project is to understand if there is an interaction between IAV and co-factor H, a critical inhibitor of the complement system and what effect any interaction may have on influenza replication. Using purified human co-factor H protein and purified human (H1N1 and H3N2) and avian (H9N2 and H5N3) IAVs we have demonstrated using ELISA assays that an interaction does occur between co-factor H and all the IAV strains tested. Far western blot analysis suggests that the interaction is with the viral Hemagglutinin (HA), which is responsible for attachment and entry of IAV to cells. Interestingly the interaction has divergent effects on the replication of the IAV strains; enhancing human H1N1 replication and restricting H3N2 replication in A549 and THP-1 cells whilst having no effect on the replication of avian H5N3 or H9N2 viruses. Understanding IAV interaction with the complement pathway could enhance our ability to produce effective vaccines.

Human thyroid peroxidase (TPO), is an important enzyme responsible for the biosynthesis of thyroid hormones and is a major autoantigen in autoimmune thyroid diseases (AITDs) such as the destructive Hashimoto’s thyroiditis. Although the structure of TPO has yet to be determined, its extracellular domain consists of three regions that exhibit a high degree of sequence similarity to domains of known three-dimensional structure: the myeloperoxidase (MPO)-like domain, complement control protein (CCP)-like domain, and epidermal growth factor (EGF)-like domain. Homology models of TPO can therefore be constructed, providing some structural context to its known function, as well as facilitating the mapping of regions that are responsible for its autoantigenicity. In this review, we highlight recent progress in this area, in particular how a molecular modelling approach has advanced the visualisation and interpretation of epitope mapping studies for TPO, facilitating the dissection of the interplay between TPO protein structure, function, and autoantigenticity.