Crystal structure of Trichinella spiralis calreticulin and the structural basis of its complement evasion mechanism involving C1q.

Helminths produce calreticulin (CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth Trichinella spiralis produces calreticulin (TsCRT), which binds C1q to inhibit activation of the complement classical pathway. Here, we used x-ray crystallography to resolve the structure of truncated TsCRT (TsCRTΔ), the first structure of helminth-derived CRT. TsCRTΔ was observed to share the same binding region on C1q with IgG based on the structure and molecular docking, which explains the inhibitory effect of TsCRT on C1q-IgG-initiated classical complement activation. Based on the key residues in TsCRTΔ involved in the binding activity to C1q, a 24 amino acid peptide called PTsCRT was constructed that displayed strong C1q-binding activity and inhibited C1q-IgG-initiated classical complement activation. This study is the first to elucidate the structural basis of the role of TsCRT in immune evasion, providing an approach to develop helminth-derived bifunctional peptides as vaccine target to prevent parasite infections or as a therapeutic agent to treat complement-related autoimmune diseases.

Identification of potential C1-binding sites in the immunoglobulin CL domains.

Immunoglobulin G (IgG) molecules that bind antigens on the membrane of target cells spontaneously form hexameric rings, thus recruiting C1 to initiate the complement pathway. However, our previous report indicated that a mouse IgG mutant lacking the Cγ1 domain activates the pathway independently of antigen presence through its monomeric interaction with C1q via the CL domain, as well as Fc. In this study, we investigated the potential interaction between C1q and human CL isoforms. Quantitative single-molecule observations using high-speed atomic force microscopy revealed that human Cκ exhibited comparable C1q binding capabilities with its mouse counterpart, surpassing the Cλ types, which have a higher isoelectric point than the Cκ domains. Nuclear magnetic resonance and mutation experiments indicated that the human and mouse Cκ domains share a common primary binding site for C1q, centred on Glu194, a residue conserved in the Cκ domains but absent in the Cλ domains. Additionally, the Cγ1 domain, with its high isoelectric point, can cause electrostatic repulsion to the C1q head and impede the C1q-interaction adjustability of the Cκ domain in Fab. The removal of the Cγ1 domain is considered to eliminate these factors and thus promote Cκ interaction with C1q with the potential risk of uncontrolled activation of the complement pathway in vivo in the absence of antigen. However, this research underscores the presence of potential subsites in Fab for C1q binding, offering promising targets for antibody engineering to refine therapeutic antibody design.

Galectin-3 - A novel ligand of complement protein C1q.

In the present study we report a novel interaction of human C1q, a primary activator of the Complement system, with human Galectin-3 (Gal-3). We investigated the potential recognition between C1q and Gal-3 on a solid hydrophobic surface by ELISA, by fluorescence spectroscopy, molecular docking and molecular dynamics (MD). The data showed that C1q and Gal-3 had a pronounced affinity for protein-protein interaction and supramolecular binding, locating the binding sites within the globular domains of C1q (gC1q) and on the backside of the carbohydrate recognition domain (CRD) of Gal-3. Fluorescence spectroscopy gave quantitative assessment of the recognition with KD value of 0.04 µM. MD analysis showed that when the active AAs of the two proteins interacted, electrostatic attraction, aided by a large number of hydrogen bonds, was dominant for the stabilization of the complex. When the contact of C1q and Gal-3 was not limited to active residues, the complex between them was stabilized mainly by Van der Waals interactions and smaller in number but stronger hydrogen bonds. This is the first report analyzing the interaction of Gal-3 with C1q, which could open the way to new applications of this protein-protein complex.

Serum complement proteins rather than inflammatory factors is effective in predicting psychosis in individuals at clinical high risk.

Immunological/inflammatory factors are implicated in the development of psychosis. Complement is a key driver of inflammation; however, it remains unknown which factor is better at predicting the onset of psychosis. This study aimed to compare the alteration and predictive performance of inflammation and complement in individuals at clinical high risk (CHR). We enrolled 49 individuals at CHR and 26 healthy controls (HCs). Twenty-five patients at CHR had converted to psychosis (converter) by the 3-year follow-up. Inflammatory cytokines, including interleukin (IL)-1ß, 6, 8, 10, tumor necrosis factor-alpha (TNF-alpha), macrophage colony-stimulating factor levels, and complement proteins (C1q, C2, C3, C3b, C4, C4b, C5, C5a, factor B, D, I, H) were measured by enzyme-linked immunosorbent assay at baseline. Except for TNF- alpha, none of the inflammatory cytokines reached a significant level in either the comparison of CHR individuals and HC or between CHR-converters and non-converters. The C5, C3, D, I, and H levels were significantly lower (C5, p = 0.006; C3, p = 0.009; D, p = 0.026; I, p = 0.016; H, p = 0.019) in the CHR group than in the HC group. Compared to non-converters, converters had significantly lower levels of C5 (p = 0.012) and C5a (p = 0.007). None of the inflammatory factors, but many complement factors, showed significant correlations with changes in general function and symptoms. None of the inflammatory markers, except for C5a and C5, were significant in the discrimination of conversion outcomes in CHR individuals. Our results suggest that altered complement levels in the CHR population are more associated with conversion to psychosis than inflammatory factors. Therefore, an activated complement system may precede the first-episode of psychosis and contribute to neurological pathogenesis at the CHR stage.

Understanding the contextual functions of C1q and LAIR-1 and their applications.

The importance of the complement component C1q has been highlighted by its involvement in autoimmunity, infection, inflammatory diseases, and tumors. The unique tulip-like structure of C1q has both a collagen-like stalk (C1q tail) and heterotrimeric globular head (gC1q), each with different binding specificities, and the binding of these components to their respective receptors leads to functional complexities in the body and bridges innate and adaptive immunity. This review describes the fundamental roles of C1q in various microenvironments and focuses on the importance of the interactions of C1q and its receptors with the inhibitory receptor LAIR-1 in maintaining homeostasis. Current therapeutic opportunities modulating LAIR-1 are also discussed.

The collagen structure of C1q induces wound healing by engaging discoidin domain receptor 2.

BACKGROUND: C1q has been reported to reveal complement-independent roles in immune and non-immune cells. C1q binds to its specific receptors to regulate distinct functions that rely on the environment and cell types. Discoidin domain receptor 2 (DDR2) is activated by collagen and functions in wound healing by controlling matrix metalloproteinase (MMP) expression. Since C1q exhibits a collagen-like structure, we hypothesized that C1q might engage DDR2 to regulate wound healing and extracellular matrix (ECM) remodeling. METHODS: Cell-based assay, proximity ligation assay, ELISA, and surface plasmon analysis were utilized to investigate DDR2 and C1q binding. We also investigate the C1q-mediated in vitro wound healing ability using the human fibrosarcoma cell line, HT1080. RESULTS: C1q induced the phosphorylation of DDR2, p38 kinase, and ERK1/2. C1q and DDR2 binding improved cell migration and induced MMP2 and MMP9 expression. DDR2-specific shRNA reduced C1q-mediated cell migration for wound healing. CONCLUSIONS: C1q is a new DDR2 ligand that promotes wound healing. These findings have therapeutic implications in wound healing-related diseases.

Molecular mechanism of calcium induced trimerization of C1q-like domain of otolin-1 from human and zebrafish.

The C1q superfamily includes proteins involved in innate immunity, insulin sensitivity, biomineralization and more. Among these proteins is otolin-1, which is a collagen-like protein that forms a scaffold for the biomineralization of inner ear stones in vertebrates. The globular C1q-like domain (gC1q), which is the most conserved part of otolin-1, binds Ca2+ and stabilizes its collagen-like triple helix. The molecular details of the assembly of gC1q otolin-1 trimers are not known. Here, we substituted putative Ca2+-binding acidic residues of gC1q otolin-1 with alanine to analyse how alanine influences the formation of gC1q trimers. We used human and zebrafish gC1q otolin-1 to assess how evolutionary changes affected the function of the protein. Surprisingly, the mutated forms of gC1q otolin-1 trimerized even in the absence of Ca2+, although they were less stable than native proteins saturated with Ca2+. We also found that the zebrafish gC1q domain was less stable than the human homologue under all tested conditions and became stabilized at higher concentrations of Ca2+, which showed that specific interactions leading to the neutralization of the negative charge at the axis of a gC1q trimer by Ca2+ are required for the trimers to form. Moreover, human gC1q otolin-1 seems to be optimized to function at lower concentrations of Ca2+, which is consistent with reported Ca2+ concentrations in the endolymphs of fish and mammals. Our results allow us to explain the molecular mechanism of assembly of proteins from the C1q superfamily, the modulating role of Ca2+ and expand the knowledge of biomineralization of vertebrate inner ear stones: otoliths and otoconia.

Novel globular C1q domain-containing protein (PmC1qDC-1) participates in shell formation and responses to pathogen-associated molecular patterns stimulation in Pinctada fucata martensii.

The C1q protein, which contains the globular C1q (gC1q) domain, is involved in the innate immune response, and is found abundantly in the shell, and it participates in the shell formation. In this study, a novel gC1q domain-containing gene was identified from Pinctada fucata martensii (P. f. martensii) and designated as PmC1qDC-1. The full-length sequence of PmC1qDC-1 was 902 bp with a 534 bp open reading frame (ORF), encoding a polypeptide of 177 amino acids. Quantitative real-time PCR (qRT-PCR) result showed that PmC1qDC-1 was widely expressed in all tested tissues, including shell formation-associated tissue and immune-related tissue. PmC1qDC-1 expression was significantly high in the blastula and gastrula and especially among the juvenile stage, which is the most important stage of dissoconch shell formation. PmC1qDC-1 expression was located in the outer epithelial cells of mantle pallial and mantle edge and irregular crystal tablets were observed in the nacre upon knockdown of PmC1qDC-1 expression at mantle pallial. Moreover, the recombined protein PmC1qDC-1 increased the rate of calcium carbonate precipitation. Besides, PmC1qDC-1 expression was significantly up-regulated in the mantle pallial at 6 h and was significantly up-regulated in the mantle edge at 12 h and 24 h after shell notching. The expression level of PmC1qDC-1 in mantle edge was significantly up-regulated at 48 h after LPS stimulation and was significantly up-regulated at 12 h, 24 h and 48 h after poly I:C stimulation. Moreover, PmC1qDC-1 expression was significantly up-regulated in hemocytes at 6 h after lipopolysaccharide (LPS) and poly I:C challenge. These findings suggest that PmC1qDC-1 plays a crucial role both in the shell formation and the innate immune response in pearl oysters, providing new clues for understanding the shell formation and defense mechanism in mollusk.

Headless C1q: a new molecular tool to decipher its collagen-like functions.

Complement component C1q, a soluble defense collagen, is the recognition protein of the classical complement pathway. C1q is able to recognize and interact with multiple targets and, via the subsequent activation of its cognate serine proteases C1r and C1s, initiates the complement cascade. C1q is made up of six ABC heterotrimers each containing two different functional regions, an N-terminal collagen-like region (CLR) and a C-terminal globular region (GR). These heterotrimers assemble via their N-terminal regions, resulting in the characteristic 'bouquet-like' shape of C1q with an N-terminal bundle of collagen fibers with six diverging stems each exhibiting a C-terminal globular head. The GRs are responsible for the versatile recognition of multiple C1q targets, whereas the CLRs trigger immune response through interacting with several cellular or soluble partners. We report here the generation of the first recombinant form of human C1q without its recognition globular heads. The noncollagenous domain 2 (nc2) of type IX collagen has been substituted for the C1q GR in order to control the correct registering of the collagen triple helices of C1q chains A, B, and C. The resulting CLR_nc2 recombinant protein produced in stably transfected EXPI293 mammalian cells was correctly assembled and folded, as demonstrated by mass spectrometry, mass photometry, and electron microscopy experiments. Its interaction properties were investigated using surface plasmon resonance analysis with known CLR ligands: the tetramer of C1r and C1s dimers and MBL-associated protein MAp44. Comparison with the interaction properties of native serum-derived C1q and CLR revealed that recombinant CLR_nc2 retains C1q CLR functional properties.

Glycine 236 in the Lower Hinge Region of Human IgG1 Differentiates FcγR from Complement Effector Function.

Abs of the IgG isotype mediate effector functions like Ab-dependent cellular cytotoxicity and Ab-dependent cellular phagocytosis by Fc interactions with FcγRs and complement-dependent cytotoxicity upon IgG-Fc binding to C1q. In this study, we describe the crucial role of the highly conserved dual glycines at position 236-237 in the lower hinge region of human IgG, including the lack of one glycine as found in IgG2. We found several permutations in this region that either silence or largely abrogate FcγR binding and downstream FcγR effector functions, as demonstrated by surface plasmon resonance, Ab-dependent cellular phagocytosis, and Ab-dependent cellular cytotoxicity assays. Although the binding regions of FcγRs and C1q on the IgG-Fc largely overlap, IgG1 with a deletion of G236 only silences FcγR-mediated effector functions without affecting C1q-binding or activation. Several mutations resulted in only residual FcγRI binding with differing affinities that are either complement competent or silenced. Interestingly, we also found that IgG2, naturally only binding FcγRIIa, gains binding to FcγRI and FcγRIIIa after insertion of G236, highlighting the crucial importance of G236 in IgG for FcγR interaction. These mutants may become invaluable tools for FcγR-related research as well as for therapeutic purposes in which only complement-mediated functions are required without the involvement of FcγR.

Molecular and functional analysis of PmC1qDC in nacre formation of Pinctada fucata martensii.

The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus which hold the potential function in the shell formation as shell matrix proteins. In this study, a C1qDC protein was identified and characterized in pearl oyster (Pinctada fucata martensii) (PmC1qDC) to explore its function in nacre formation. The PmC1qDC-deduced protein sequence carried a typical globular C1q (gC1q) domain that possessed the typical 10-stranded ß-sandwich fold with a jelly-roll topology common to all C1qDC family members and shared high homology with other gC1q domains. Homologous analysis of PmC1qDC presented it contained conserved secondary structure and Phe135, Phe155, Tyr166, Phe173, Tyr181, Phe183, and Phe256 amino acid residues. Expression pattern analysis showed that PmC1qDC expressed in all the detected tissues and exhibited a significantly higher expression level in nacre formation-associated tissues. After the shell notching, the expression level of PmC1qDC showed significantly up-regulation after 12 h in the central zone of mantle (MC). PmC1qDC expression significantly decreased in the MC after RNA interference (RNAi). Furthermore, disordered crystals with evident rough surface and irregular crystal tablets were observed in the nacre after RNAi. Results suggested that PmC1qDC affects the shell nacre formation, which is significant to improve the pearl production of pearl oyster.

C1q A08 Is a Half-Cryptic Epitope of Anti-C1q A08 Antibodies in Lupus Nephritis and Important for the Activation of Complement Classical Pathway.

To investigate the fine epitope(s) of anti-C1q A08 antibodies and their roles in complement activation in lupus nephritis, C1q A08 and related peptides with various amino acid sequences around A08 were synthesized. Anti-C1q A08 antibodies from 10 lupus nephritis patients were purified from plasmapheresis samples, and four monoclonal antibodies against C1q A08 were screened and identified from mouse hybridoma cells, to study the fine epitope(s) of C1q A08 using ELISA and Biolayer Interferometry (BLI). The biofunction of anti-C1q A08 antibodies for complement classical pathway activation was investigated by C3 activation assay. Anti-C1q A08 antibodies and anti-C1q antibodies were also detected in the sera of female BALB/C mice immunized by C1q A08 peptides. None of the anti-C1q A08 antibodies, which were affinity purified from the 10 lupus nephritis patients, could bind intact C1q coated on microtitre plates, neither could the anti-C1q antibodies bind to C1q A08 peptides coupled on resin, indicating that the human anti-C1q antibodies and anti-C1q A08 antibodies may recognize different epitopes of C1q. One of the four C1q A08 mAbs (32-4) bound to the six amino acids of N-terminus of C1q A08, while another C1q A08 mAb (17-9) bound to eight or 10 amino acids of C-terminus of A08. The third and fourth C1q A08 mAb (1A12 and 4B11) bound to the whole sequence of A08. Only 32-4 mAb bound to the intact C1q coating on an ELISA plate, whereas 17-9 mAb, 1A12 mAb, and 4B11 mAb could not. However, using a BLI assay, 17-9 mAb, 1A12 mAb, and 4B11 mAb, but not 32-4 mAb, could bind to intact C1q. Furthermore, 1A12 mAb and 4B11 mAb, but not 32-4 and 17-9 mAb, could inhibit the activation of complement classical pathway. Anti-C1q A08 antibodies were detected in all the female BALB/C mice in the experimental group but not in the control group. Two out of six in the experimental group developed anti-C1q antibodies. C1q A08 is a half-cryptic epitope of C1q involving N-terminal six amino acids of C1q A08, and this is important to the activation of a complement classical pathway, and some anti-C1q A08 antibodies were able to prevent this process. Epitope spreading of C1q occurred in the mice immunized with C1q A08 peptides.

Transient pentameric IgM fulfill biological function-Effect of expression host and transfection on IgM properties.

Recombinant production of IgM antibodies poses a special challenge due to the complex structure of the proteins and their not yet fully elucidated interactions with the immune effector proteins, especially the complement system. In this study, we present transient expression of IgM antibodies (IgM617, IgM012 and IgM012_GL) in HEK cells and compared it to the well-established stable expression system in CHO cells. The presented workflow investigates quality attributes including productivity, polymer distribution, glycosylation, antibody structure and activation of the classical complement pathway. The HEK293E transient expression system is able to generate comparable amounts and polymer distribution as IgM stably produced in CHO. Although the glycan profile generated by HEK293E cells contained a lower degree of sialylation and a higher portion of oligomannose structures, the potency to activate the complement cascade was maintained. Electron microscopy also confirmed the structural integrity of IgM pentamers produced in HEK293E cells, since the conventional star-shaped structure is observed. From our studies, we conclude that the transient expression system provides an attractive alternative for rapid, efficient and high-throughput production of complex IgM antibodies with slightly altered post-translational modifications, but comparable structure and function.

Antibody-mediated complement activation in pathology and protection.

Antibody-dependent complement activity is associated not only with autoimmune morbidity, but also with antitumor efficacy. In infectious disease, both recombinant monoclonal antibodies and polyclonal antibodies generated in natural adaptive responses can mediate complement activity to protective, therapeutic or disease-enhancing effect. Recent advances have contributed to the structural resolution of molecular complexes involved in antibody-mediated complement activation, defining the avid nature of participating interactions and pointing to how antibody isotype, subclass, hinge flexibility, glycosylation state, amino acid sequence and the contextual nature of the cognate antigen/epitope are all factors that can determine complement activity through impact on antibody multimerization and subsequent recruitment of complement component 1q. Beyond the efficiency of activation, complement activation products interact with various cell types that mediate immune adherence, trafficking, immune education and innate functions. Similarly, depending on the anatomical location and extent of activation, complement can support homeostatic restoration or be leveraged by pathogens or neoplasms to enhance infection or promote tumorigenic microenvironments, respectively. Advances in means to suppress complement activation by intravenous immunoglobulin (IVIG), IVIG mimetics and complement-intervening antibodies represent proven and promising exploratory therapeutic strategies, while antibody engineering has likewise offered frameworks to enhance, eliminate or isolate complement activation to interrogate in vivo mechanisms of action. Such strategies promise to support the optimization of antibody-based drugs that are able to tackle emerging and difficult-to-treat diseases by improving our understanding of the synergistic and antagonistic relationships between antibody mechanisms mediated by Fc receptors, direct binding and the products of complement activation.

Recombinant C1q variants modulate macrophage responses but do not activate the classical complement pathway.

Complement protein C1q plays a dual role in a number of inflammatory diseases such as atherosclerosis. While in later stages classical complement pathway activation by C1q exacerbates disease progression, C1q also plays a beneficial role in early disease. Independent of its role in complement activation, we and others have identified a number of potentially beneficial interactions of C1q with phagocytes in vitro, including triggering phagocytosis of cellular and molecular debris and polarizing macrophages toward an anti-inflammatory phenotype. These interactions may also be important in preventing autoimmunity. Here, we characterize variants of recombinant human C1q (rC1q) which no longer initiate complement activation, through mutation of the C1r2C1s2 interaction site. For insight into the structural location of the site of C1q that is important for interaction with phagocytes, we investigated the effect of these mutations on phagocytosis and macrophage inflammatory polarization, as compared to wild-type C1q. Phagocytosis of antibody coated sheep erythrocytes and oxidized LDL was measured in human monocytes and monocyte-derived macrophages (HMDM) respectively that had interacted with rC1q wild-type or variants. Secreted levels of cytokines were also measured in C1q stimulated HMDM. All variants of C1q increased phagocytosis in HMDM compared to controls, similar to native or wild-type rC1q. In addition, levels of certain pro-inflammatory cytokines and chemokines secreted by HMDM were modulated in cells that interacted with C1q variants, similar to wild-type rC1q and native C1q. This includes downregulation of IL-1α, IL-1ß, TNFα, MIP-1α, and IL-12p40 by native and rC1q in both resting and M1-polarized HMDM. This suggests that the site responsible for C1q interaction with phagocytes is independent of the C1r2C1s2 interaction site. Further studies with these classical pathway-null variants of C1q should provide greater understanding of the complement-independent role of C1q, and allow for potential therapeutic exploitation.

EDTA Treatment for Overcoming the Prozone Effect and for Predicting C1q Binding in HLA Antibody Testing.

The Luminex-based single antigen bead (SAB) assay is widely used to detect HLA antibody in transplant recipients. However, one limitation of the SAB assay is the prozone effect, which occurs mostly as a result of complement interference. We investigated the efficacy of EDTA treatment for overcoming the prozone effect and predicting C1q binding of HLA antibody. We subjected 27 non-treated (naïve) and EDTA-treated serum samples from highly sensitized patients to IgG-SAB assays, and we confirmed the prozone effect in 53% and 31% of class I and class II antibody tests, respectively, after EDTA treatment. When we conducted additional assays after dithiothreitol treatment and serum dilution, EDTA was the most efficacious in eliminating the prozone effect. Reducing the prozone effect by EDTA treatment strengthened the correlation between IgG mean fluorescence intensity (MFI) and C1q MFI values (ρ=0.825) as compared with the naïve sera (ρ=0.068). Although C1q positivity was dependent on the concentration of HLA antibody in EDTA-treated sera, the correlations varied individually. Overall, our results confirmed the efficacy of EDTA treatment for overcoming the prozone effect. EDTA treatment showed a positive effect on the correlation between IgG MFI and C1q MFI values.

Interaction of C1q With Pentraxin 3 and IgM Revisited: Mutational Studies With Recombinant C1q Variants.

Pentraxins and complement defense collagens are soluble recognition proteins that sense pathogens and altered-self elements, and trigger immune responses including complement activation. PTX3 has been shown to interact with the globular recognition domains (gC1q) of the C1q protein of the classical complement pathway, thereby modulating complement activity. The C1q-PTX3 interaction has been characterized previously by site-specific mutagenesis using individual gC1q domains of each of the three C1q chains. The present study is aimed at revisiting this knowledge taking advantage of full-length recombinant C1q. Four mutations targeting exposed amino acid residues in the gC1q domain of each of the C1q chains (LysA200Asp-LysA201Asp, ArgB108Asp-ArgB109Glu, TyrB175Leu, and LysC170Glu) were introduced in recombinant C1q and the interaction properties of the mutants were analyzed using surface plasmon resonance. All C1q mutants retained binding to C1r and C1s proteases and mannose-binding lectin-associated serine proteases, indicating that the mutations did not affect the function of the collagen-like regions of C1q. The effect of these mutations on the interaction of C1q with PTX3 and IgM, and both the PTX3- and IgM-mediated activation of the classical complement pathway were investigated. The LysA200Asp-LysA201Asp and LysC170Glu mutants retained partial interaction with PTX3 and IgM, however they triggered efficient complement activation. In contrast, the ArgB108Asp-ArgB109Glu mutation abolished C1q binding to PTX3 and IgM, and significantly decreased complement activation. The TyrB175Leu mutant exhibited decreased PTX3- and IgM-dependent complement activation. Therefore, we provided evidence that, in the context of the full length C1q protein, a key contribution to the interaction with both PTX3 and IgM is given by the B chain Arg residues that line the side of the gC1q heterotrimer, with a minor participation of a Lys residue located at the apex of gC1q. Furthermore, we generated recombinant forms of the human PTX3 protein bearing either D or A at position 48, a polymorphic site of clinical relevance in a number of infections, and observed that both allelic variants equally recognized C1q.

A novel C1q domain containing protein in black rockfish (Sebastes schlegelii) serves as a pattern recognition receptor with immunoregulatory properties and possesses binding activity to heat-aggregated IgG.

C1q-domain-containing (C1qDC) proteins, which are involved in a series of immune responses, are important pattern recognition receptors in innate immunity in vertebrates and invertebrates. Functional studies of C1qDC proteins in vertebrates are scarce. In the present study, a C1qDC protein (SsC1qDC) from the teleost black rockfish (Sebastes schlegelii) was identified and examined at expression and functional levels. The open reading frame of SsC1qDC is 636 bp, and the predicted amino acid sequence of SsC1qDC shares 62%-69% overall identity with the C1qDC proteins of several fish species. SsC1qDC possesses conserved C1qDC features, including a signal sequence and a C1q domain. SsC1qDC was expressed in different tissues and its expression was up-regulated by bacterial and viral infection. Recombinant SsC1qDC (rSsC1qDC) exhibited apparent binding activities against PAMPs including LPS and PGN. rSsC1qDC had antibacterial activity against Vibrio parahaemolyticus, and was able to enhance the phagocytic activity of macrophages towards Vibrio anguillarum. rSsC1qDC interacted with human heat-aggregated IgG. Furthermore, in the presence of rSsC1qDC, fish exhibited enhanced resistance against bacterial infection. Collectively, these results indicated that SsC1qDC serves as a pattern recognition receptor and plays a vital role in the defense system of black rockfish.

Identification of a novel C1q complement component in razor clam Sinonovacula constricta and its role in antibacterial activity.

The serum complement component C1q mediates a variety of immune regulatory functions. Herein, we identified a globular head C1q (ghC1q) gene in razor clam Sinonovacula constricta. The complete Sc-ghC1q gene was 872 bp long included an 81 bp 5'-untranslated region (UTR), a 95 bp 3'-UTR with a poly(A) tail, and an open reading frame (ORF) of 696 bp. The mRNA expression of Sc-ghC1q was upregulated in hepatopancreas and hemocytes. After Staphylococcus aureus or Vibrio anguillarum challenge, Sc-ghC1q mRNA transcript abundance was significantly upregulated in hemolymph. Recombinant Sc-ghC1q protein could bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and it could agglutinate both Gram-positive and Gram-negative bacteria. Additionally, flow cytometry revealed that Sc-ghC1q strongly promoted phagocytosis in hemocytes. Together, these results demonstrated that Sc-ghC1q played an important role in innate immunity in S. constricta.

Characterization and functional analysis of a novel gC1qR in the swimming crab Portunus trituberculatus.

The receptor for the globular head of complement component C1q, gC1qR, is a multifunctional and multiligand binding protein with a crucial role in host defense. In the present study, a full-length cDNA sequence of a gC1qR homolog (PtgC1qR) in Portunus trituberculatus was identified. PtgC1qR was a 268-amino-acid polypeptide with a conserved MAM33 domain and a mitochondrial targeting sequence in the first 56 amino acids. The transcripts of PtgC1qR were detected in all examined tissues with the highest level detected in the hepatopancreas. Compared with other early embryonic stages, PtgC1qR was highly expressed in the fertilized eggs and embryos at the cleavage stage, which suggest PtgC1qR may be a maternal gene. The transcripts of PtgC1qR in hemocytes exhibited time-dependent response expression pattern after challenged with bacteria (Vibrio alginolyticus, Micrococcus luteus) and fungi (Pichia pastoris). Moreover, the recombinant PtgC1qR (rPtgC1qR) exhibited strong antibacterial activity and microbial-binding activity, suggesting its crucial role in immune defense and recognition. Further phenoloxidase (PO) assay showed that rPtgC1qR could suppress the crab PO activity in vitro in a dose-dependent manner, and it could result in nearly 100% inhibition of PO activity under the concentration of 11.65 µM. Knockdown of PtgC1qR could significantly enhance the expression of serine protease related genes (PtSP1-3 and PtSPH), proPO-associated genes (PtproPO and PtPPAF) and C3-like genes (Ptα2M1 and PtTEP). However, the phagocytosis related genes (PtMyosin, PtRab5 and PtArp) and Ptα2M2 were significantly down-regulated in the PtgC1qR silenced crabs. These findings together demonstrate that PtgC1qR might function in crab immune response via its antibacterial activity, immune recognition or regulating the proPO system, complement pathway and phagocytosis.