Complement Control Protein Domains Research Articles

Molecular Interactions Required for Activation of Complement Component C2 Include Exosites Located on the Serine Protease Domain of C1s and Mannose-Binding Lectin Associated Protease-2.

The activation of the CP/LP C3 proconvertase complex is a key event in complement activation and involves cleavage of C4 and C2 by the C1s protease (classical pathway) or the mannose-binding lectin-associated serine protease (MASP)-2 (lectin pathway). Efficient cleavage of C4 by C1s and MASP-2 involves exosites on the complement control protein and serine protease (SP) domains of the proteases. The complement control protein domain exosite is not involved in cleavage of C2 by the proteases, but the role of an anion-binding exosite (ABE) on the SP domains of the proteases has (to our knowledge) never been investigated. In this study, we have shown that the ABE on the SP of both C1s and MASP-2 is crucial for efficient cleavage of C2, with mutant forms of the proteases greatly impaired in their rate of cleavage of C2. We have additionally shown that the site of binding for the ABE of the proteases is very likely to be located on the von Willebrand factor domain of C2, with the precise area differing between the enzymes: whereas C1s requires two anionic clusters on the von Willebrand factor domain to enact efficient cleavage of C2, MASP-2 apparently only requires one. These data provide (to our knowledge) new information about the molecular determinants for efficient activation of C2 by C1s and MASP-2. The enhanced view of the molecular events underlying the early stages of complement activation provides further possible intervention points for control of this activation that is involved in a number of inflammatory diseases.

Cryo-electron microscopy structures of human thyroid peroxidase (TPO) in complex with TPO antibodies.

Determination of the structure of the extracellular domain of human thyroid peroxidase (hTPO) by cryo-electron microscopy (cryo-EM) is described. TPO, purified to homogeneity was complexed with the human TPO monoclonal autoantibody 2G4 Fab and also with a mouse monoclonal TPO antibody 4F5 Fab (which competes with autoantibody binding to TPO). Both complexes were analysed by cryo-EM. The two structures (global resolution 3.92Å and 3.4Å for the 2G4 complex and 4F5 complex respectively) show TPO as a monomer with 4 domains; the N-terminal domain, the peroxidase domain (POD), the complement control protein like (CCP) domain and the epidermal growth factor like (EGF) domain which are all visible in the structures. The relative positions of the domains are fixed with a disulphide bond between cysteine residues Cys146 in the POD and Cys756 in the CCP domain preventing significant flexibility of the molecule. The entrance to the enzyme active site, the haem group and the calcium binding site are clearly visible on the opposite side of the TPO molecule from the 2G4 and 4F5 binding sites. Extensive interactions are seen between TPO and the two antibodies which both bind to distinct epitopes on the POD domain, including some residues in the immunodominant region B (IDR-B) mainly via different residues. However the epitopes of the two antibodies contain three shared TPO residues. This is the first high resolution structure of TPO to be reported and it should help guide development of new inhibitors of TPO enzyme activity for therapeutic applications.

A novel epidermal growth factor‐like domain protein 6 from Apostichopus japonicus mediates coelomocyte proliferation

Epidermal growth factor-like domain protein (EGFL) 6, a member of the epidermal growth factor (EGF) domain protein superfamily, has been reported to regulate various physiological processes, including cell adhesion, proliferation, migration and inflammation in mammals. Up to date, the study of invertebrate EGFL6 expression pattern and functional analysis of EGFL6 involved in proliferation is lacking. In this study, a novel EGFL6 homologue was identified from Apostichopus japonicus (designated as AjEGFL6) by rapid amplification of cDNA ends. The full-length cDNA of AjEGFL6 was 3915 bp, with a putative open reading frame of 2490 bp encoding 829 amino acid (aa) residue proteins. Structural analysis revealed that AjEGFL6 processed characteristic five EGF repeat domains and subclass of EGF_CA domain (89–338), UPF domain (504–607) and complement control protein (CCP, 755–808) domain. Multiple sequence alignment and phylogenetic analysis supported that AjEGFL6 belongs to a new member of the EGFL6 protein subfamily. Spatial expression analysis indicated that AjEGFL6 mRNA transcripts in the polian vesicle are expressed at a high level compared with that in coelomocytes. For Vibrio splendidus-challenged sea cucumbers, the peak expression of AjEGFL6 mRNAs in coelomocytes was detected at 24 h (12.29-fold), and it remained at high levels (1.52-fold) until 72 h. Functional investigation via MTT and EdU assays revealed that 60 and 120 ng of recombinant EGFL6 administration significantly upregulated cell viability by 19.72% and 46.13% respectively. Taken together, these findings suggest that AjEGFL6 may serve as a coelomocyte proliferation regulator in the sea cucumber.

Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation.

Components of the extracellular matrix (ECM), when exposed to body fluids may promote local complement activation and inflammation. Pathologic complement activation at the glomerular basement membrane and at the Bruch’s membrane is implicated in renal and eye diseases, respectively. Binding of soluble complement inhibitors to the ECM, including factor H (FH), is important to prevent excessive complement activation. Since the FH-related (FHR) proteins FHR1 and FHR5 are also implicated in these diseases, our aim was to study whether these FHRs can also bind to ECM components and affect local FH activity and complement activation. Both FH and the FHRs showed variable binding to ECM components. We identified laminin, fibromodulin, osteoadherin and PRELP as ligands of FHR1 and FHR5, and found that FHR1 bound to these ECM components through its C-terminal complement control protein (CCP) domains 4-5, whereas FHR5 bound via its middle region, CCPs 3-7. Aggrecan, biglycan and decorin did not bind FH, FHR1 and FHR5. FHR5 also bound to immobilized C3b, a model of surface-deposited C3b, via CCPs 3-7. By contrast, soluble C3, C3(H2O), and the C3 fragments C3b, iC3b and C3d bound to CCPs 8-9 of FHR5. Properdin, which was previously described to bind via CCPs 1-2 to FHR5, did not bind in its physiologically occurring serum forms in our assays. FHR1 and FHR5 inhibited the binding of FH to the identified ECM proteins in a dose-dependent manner, which resulted in reduced FH cofactor activity. Moreover, both FHR1 and FHR5 enhanced alternative complement pathway activation on immobilized ECM proteins when exposed to human serum, resulting in the increased deposition of C3-fragments, factor B and C5b-9. Thus, our results identify novel ECM ligands of FH family proteins and indicate that FHR1 and FHR5 are competitive inhibitors of FH on ECM and, when bound to these ligands, they may enhance local complement activation and promote inflammation under pathological conditions.

Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26).

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and HIV-1. However, its primary receptor portfolio remains controversial, potentially including sialic acid, coxsackie and adenovirus receptor (CAR), integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned on the basis of the inability to cocrystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domain CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild-type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (i.m.) injection but not after intranasal (i.n.) administration. Ad26 transduction was 10-fold lower than Ad5 transduction after intratumoral (i.t.) injection of CD46-expressing tumors. Ad26 transduction of liver was 1,000-fold lower than that ofAd5 after intravenous (i.v.) injection. These data demonstrate the use of CD46 by Ad26 in certain situations but also show that the receptor has little consequence by other routes of administration. Finally, i.v. injection of high doses of Ad26 into CD46 mice induced release of liver enzymes into the bloodstream and reduced white blood cell counts but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during coronavirus disease 2019 (COVID-19) vaccination with this serotype of adenovirus. IMPORTANCE The human species D Ad26 is being investigated as a low-seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection and its role in virus biology, vaccine efficacy, and, importantly, vaccine safety.

A novel C-type lectin activates the complement cascade in the primitive oyster Crassostrea gigas

The ancient origin of the lectin pathway of the complement system can be traced back to protochordates (such as amphioxus and tunicates) by the presence of components such as ficolin, glucose-binding lectin, mannose-binding lectin-associated serine protease (MASP), and C3. Evidence for a more primitive origin is offered in the present study on the Pacific oyster Crassostrea gigas. C3 protein in C. gigas (CgC3) was found to be cleaved after stimulation with the bacteria Vibrio splendidus. In addition, we identified a novel C-type lectin (defined as CgCLec) with a complement control protein (CCP) domain, which recognized various pathogen-associated molecular patterns (PAMPs) and bacteria. This protein was involved in the activation of the complement system by binding CgMASPL-1 to promote cleavage of CgC3. The production of cytokines and antibacterial peptides, as well as the phagocytotic ratio of haemocytes in CgCLec-CCP-, CgMASPL-1-, or CgC3-knockdown oysters, decreased significantly after V. splendidus stimulation. Moreover, this activated CgC3 participated in perforation of bacterial envelopes and inhibiting survival of the infecting bacteria. These results collectively suggest that there existed an ancient lectin pathway in molluscs, which was activated by a complement cascade to regulate the production of immune effectors, phagocytosis, and bacterial lysis.

Evaluating the Occurrence of Rare Variants in the Complement Factor H Gene in Patients With Early-Onset Drusen Maculopathy

Early-onset drusen maculopathy (EODM) is a severe disease and can lead to advanced macular degeneration early in life; however, genetic and phenotypic characteristics of individuals with EODM are not well studied. To identify genotypic and phenotypic characteristics of individuals with EODM. This case-control study collected data from the European Genetic Database from September 2004 to October 2019. A total of 89 patients with EODM diagnosed at 55 years or younger and 91 patients with age-related macular degeneration (AMD) diagnosed at 65 years or older were included. Coding regions of CFH, CFI, C3, C9, CFB, ABCA4, PRPH2, TIMP3, and CTNNA1 genes were sequenced, genetic risk scores (GRS) were calculated based on 52 AMD-associated variants, and phenotypic characteristics on color fundus photographs were analyzed comparing patients with EODM and AMD. GRS, frequency of rare genetic complement variants, and phenotypic characteristics. This case-control study included 89 patients with EODM (mean [SD] age, 51.8 [8.7] years; 58 [65.2%] were female) and 91 patients with AMD (mean [SD] age, 77.6 [6.1] years; 45 [49.5%] female). At a mean (SD) age of 56.4 (7.3) years, 40 of 89 patients with EODM (44.9%) were affected by geographic atrophy or choroidal neovascularization. A lower GRS was observed in patients with EODM compared with patients with AMD (1.03 vs 1.60; P = .002), and 27 of 89 patients with EODM (30.3%) carried rare variants in the CFH gene compared with 7 of 91 patients with AMD (7.7%). Carriership of a rare CFH variant was associated with EODM (odds ratio, 7.2; 95% CI, 2.7-19.6; P < .001). A large macular drusen area (more than 50% covered with drusen) was observed in patients with EODM (24 of 162 eyes [14.8%]) compared with patients with AMD (9 of 164 eyes [5.5%]) (odds ratio, 4.57; 95% CI, 1.5-14.1; P = .008). A large proportion of patients with EODM in this study carried rare CFH variants, with most of the identified CFH variants clustered in the first 7 complement control protein domains affecting factor H and factor H-like 1. Because EODM frequently leads to advanced macular degeneration at an early age and can result in many years of vision loss, this study supports targeting the complement system and sequencing the CFH gene in patients with EODM to improve genetic counseling and future treatments for AMD.

ANTI-CRA: A FAMILY STUDY OF A BRAZILIAN FAMILY IN MOLECULAR TIMES

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. 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. 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-Cr a specificity and other underlying antibodies were ruled out through adsorption and elution with R1R1, Jk(b-) and Fy(b-) allogeneic RBCs. This study reported for the first time a rare case of anti-Cr a 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-Cr a 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.

The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases.

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.

Tuning the Functionality by Splicing: Factor H and Its Alternative Splice Variant FHL-1 Share a Gene but Not All Functions.

The alternative pathway regulator Factor H-like protein 1 (FHL-1) is composed of the first 7 N-terminal complement control protein domains of Factor H (FH) and protects host surfaces from uncontrolled complement attack. Although FHL-1 shares the N-terminal regulatory domains with FH, it was thought to be a weaker regulator. Recently, the regulatory activity of FHL-1 was shown to be comparable to FH. Nonetheless, the question remained whether FHL-1 is an indispensable, unique regulator. The discovery that FHL-1 is the predominant regulator on Bruch’s membrane, a critical site for the onset and progression of age-related-macular degeneration (AMD), showed that FHL-1 is essential for complement regulation. A common single nucleotide polymorphism in FH/FHL-1 that predisposes for AMD underlines the important role of FHL-1 in this context. Reports that some cancer tissues specifically upregulate FHL-1 expression, thereby evading immune surveillance, suggests a pronounced regulatory activity of the splice variant. Several microorganisms specifically recruit FHL-1 to evade complement attack. From a phylogenetic point of view, FHL-1 appears much later than other complement regulators, which could imply a specific role that is possibly not systemic but rather tissue specific. This review focuses on the current knowledge of FHL-1 and its physiological and pathophysiological roles.

Novel structure of the N-terminal helical domain of BibA, a group B streptococcus immunogenic bacterial adhesin.

BibA, a group B streptococcus (GBS) surface protein, has been shown to protect the pathogen from phagocytic killing by sequestering a complement inhibitor: C4b-binding protein (C4BP). Here, the X-ray crystallographic structure of a GBS BibA fragment (BibA126-398) and a low-resolution small-angle X-ray scattering (SAXS) structure of the full-length N-terminal domain (BibA34-400) are described. The BibA126-398 fragment crystal structure displayed a novel and predominantly helical structure. The tertiary arrangement of helices forms four antiparallel three-helix-bundle-motif repeats, with one long helix from a bundle extending into the next. Multiple mutations on recombinant BibA34-400 delayed the degradation of the protein, and circular dichroism spectroscopy of BibA34-400 suggested a similar secondary-structure composition to that observed in the crystallized BibA126-398 fragment. A model was generated for the 92 N-terminal residues (BibA34-125) using structural similarity prediction programs, and a BibA34-400 model was generated by combining the coordinates of BibA34-126 and BibA126-398. The X-ray structure of BibA126-398 and the model of BibA34-400 fitted well into the calculated SAXS envelope. One possible binding site for the BibA N-terminal domain was localized to the N-terminal CCP (complement-control protein) domains of the C4BP α-chain, as indicated by the decreased binding of BibA to a ΔCCP1 C4BP α-chain mutant. In summary, it is suggested that the GBS surface protein BibA, which consists of three antiparallel α-helical-bundle motifs, is unique and belongs to a new class of Gram-positive surface adhesins.

A helminth-derived suppressor of ST2 blocks allergic responses.

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.

A Neuron-Specific Antiviral Mechanism Modulates the Persistent Infection of Rice Rhabdoviruses in Leafhopper Vectors.

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.

Spatially conserved motifs in complement control protein domains determine functionality in regulators of complement activation-family proteins

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.

Molecular and functional characterization of a mannose-binding lectin/ficolin-associated protein (MAp44) from Nile tilapia (Oreochromis niloticus) involved in the immune response to bacterial infection

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.

Expression and functional characterization of a mannose-binding lectin-associated serine protease-1 (MASP-1) from Nile tilapia (Oreochromis niloticus) in host defense against bacterial infection

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.

Loss-of-Function Mutations in the CFH Gene Affecting Alternatively Encoded Factor H-like 1 Protein Cause Dominant Early-Onset Macular Drusen

PurposeTo 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.DesignMulticenter case series, in vitro experimentation, and retrospective analysis of previously reported variants.ParticipantsSeven families with apparently autosomal dominant EOMD.MethodsPatients 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 MeasuresDetailed clinical phenotypes, genomic findings, in vitro characterization of mutation effect on protein function, and postulation of the pathomechanism underpinning EOMD.ResultsAll 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.ConclusionsEarly-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.

Cutting Edge: FHR-1 Binding Impairs Factor H-Mediated Complement Evasion by the Malaria Parasite Plasmodium falciparum.

Human complement is the first line of defense against invading pathogens, including the malaria parasite Plasmodium falciparum We previously demonstrated that human complement represents a particular threat for the clinically relevant blood stages of the parasite. To evade complement-mediated destruction, the parasites acquire factor H (FH) via specific receptors. We now report that the FH-related protein FHR-1 competes with FH for binding to the parasites. FHR-1, which is composed of five complement control protein domains with variable homology to FH but lacks C3b regulatory activity, accumulates on the surfaces of intraerythrocytic schizonts and free merozoites. Although binding of FH to schizont-infected RBCs and merozoites is increased in FHR-1-deficient human serum, the addition of recombinant FHR-1 decreases FH binding. The presence of FHR-1 consequently impairs C3b inactivation and parasite viability. We conclude that FHR-1 acts as a protective factor in human immunity by counteracting FH-mediated microbial complement evasion.

Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis

The vegetative insecticidal proteins (Vip), secreted by many Bacillus thuringiensis strains during their vegetative growth stage, are genetically distinct from known insecticidal crystal proteins (ICPs) and represent the second-generation insecticidal toxins. Compared with ICPs, the insecticidal mechanisms of Vip toxins are poorly understood. In particular, there has been no report of a definite receptor of Vip toxins to date. In the present study, we identified the scavenger receptor class C like protein (Sf-SR-C) from the Spodoptera frugiperda (Sf9) cells membrane proteins that bind to the biotin labeled Vip3Aa, via the affinity magnetic bead method coupled with HPLC-MS/MS. We then certified Vip3Aa protoxin could interact with Sf-SR-C in vitro and ex vivo. In addition, downregulation of SR-C expression in Sf9 cells and Spodoptera exigua larvae midgut reduced the toxicity of Vip3Aa to them. Coincidently, heterologous expression of Sf-SR-C in transgenic Drosophila midgut significantly enhanced the virulence of Vip3Aa to the Drosophila larvae. Moreover, the complement control protein domain and MAM domain of Sf-SR-C are involved in the interaction with Vip3Aa protoxin. Furthermore, endocytosis of Vip3Aa mediated by Sf-SR-C correlates with its insecticidal activity. Our results confirmed for the first time that Sf-SR-C acts as a receptor for Vip3Aa protoxin and provides an insight into the mode of action of Vip3Aa that will significantly facilitate the study of its insecticidal mechanism and application.

Divergent immune roles of two fucolectin isoforms in Apostichopus japonicus

The F-type lectin (fucolectin) family represents a new group with innate immunity. In this study, two fucolectin isoforms (designated as AjFTL-1 and AjFTL-2) were identified in sea cucumber (Apostichopus japonicus) through rapid amplification of cDNA ends. Full-length cDNAs of AjFTL-1 and AjFTL-2 measured 2134 and 1286 bp, encoding two secreted proteins comprising 317 and 181 amino acid residues, respectively. The signal peptide, l-fucose binding motif (“HX(26)RXDX(4)R/K”) and cation binding sequence motif (“h2DGx”) were conserved in AjFTL-1 and AjFTL-2. However, AjFTL-1 contains an additional complement control protein domain. Multiple sequence alignments supported that AjFTL-1 and AjFTL-2 are new members of the F-type lectin family. Tissues distribution analysis indicated that both AjFTL-1 and AjFTL-2 were widely expressed in all tested tissues, featuring differential expression patterns. Vibrio splendidus infection in vivo can significantly upregulate the mRNA transcripts of the two genes, with a larger magnitude observed in AjFTL-1. By contrast, lipopolysaccharide stimulation in vitro can markedly induce the expression level of AjFTL-2 but not that of AjFTL-1. Silencing AjFTL-2 by siRNA can suppress the AjNOS transcript, whereas injection of the recombinant protein of AjFTL-2 can significantly induce AjNOS expression. By contrast, the loss- and gain-of-function of AjFTL-1 caused no effect on the expression of AjNOS. Our present study provides evidence supporting that AjFTL-1 and AjFTL-2 play diverse roles in the innate immune defense of sea cucumbers toward bacterial infection.