C1q Domain-containing Proteins Research Articles

185 Maternal live yeast supplementation protects newly weaned piglets against lipopolysaccharide challenge by regulating immune-associated milk proteins

Abstract Sow milk serves as a crucial source of nutritional, immunological, and growth-promoting components for the piglets. Our previous studies established that maternal live yeast (LY) supplementation altered the expression of antioxidant and immune-regulatory genes in the intestinal mucosa of suckling piglets. The current study aimed to investigate the effects of maternal LY supplementation during late gestation and lactation on milk proteomic profile in dams and health of offspring under lipopolysaccharide (LPS) challenge. On d 77 of gestation, 40 sows were allocated to 2 dietary treatments: without (CON) or with LY supplementation at 0.05% of diet during gestation and 0.1% during lactation. Milk samples were collected on d 0, 10, and 18 post-partum (n = 6), respectively. Within 24 h postweaning, piglets from the CON and LY groups were intraperitoneally injected with sterile saline or LPS [25 μg/kg body weight (BW)] and divided into four groups (n = 8): 1) saline + CON (CS), 2) saline + LY (YS), 3) LPS + CON (CLPS), and 4) LPS + LY (YLPS). Data were analyzed in a 2 × 2 factorial arrangement with main effects of maternal diet and immunological challenge. Rectal temperature was measured hourly for 4 h post-injection after which piglets were euthanized. Results showed that LPS increased concentrations of serum tumor necrosis factor (TNF) -α and interlekin (IL) -10 (P < 0.05) and tended to increase IL-8 in the ileal mucosa of piglets (P < 0.10). At 3 h post-injection, YLPS piglets tended to have a lower rectal temperature than CLPS piglets (P < 0.10). YLPS piglets also had decreased protein levels of TNF-α in the ileal mucosa (P < 0.05), with a tendency for greater protein abundance of E-cadherin but decreased mRNA abundance of catalase (P < 0.10) in the jejunal mucosa at 4 h post-injection (P < 0.05). In addition, YLPS piglets had less mRNA abundance of TLR2 and TLR4 in the liver than CLPS piglets (P < 0.05). With shotgun proteomics analysis, we found that milk of LY sows had more abundant proteins associated with immunity, including immunoglobulin and complement protein (Ig-like and C1q domain-containing proteins) on d 0 and d 10. Insulin-like growth factor binding protein (IGFBP) 5 was also more abundant in the d 0 milk of LY sows, whereas prostaglandin synthase was greater in d 10 and d 18 milk of CON sows. Additionally, pathway analysis showed that proteins identified in the milk of CON sows have roles in neutrophil degranulation and IL-12 signaling and production, whereas pathways associated with regulation of complement system, IGF and DHCR24 signaling, were enriched in the milk of LY sows. These results suggest that maternal LY supplementation could protect newly weaned piglets from bacteria endotoxin effect by suppressing inflammatory response, improving expression of tight junction proteins in offspring, and altering immune-associated proteins in sow milk.

Invertebrate C1q Domain-Containing Proteins: Molecular Structure, Functional Properties and Biomedical Potential.

C1q domain-containing proteins (C1qDC proteins) unexpectedly turned out to be widespread molecules among a variety of invertebrates, despite their lack of an integral complement system. Despite the wide distribution in the genomes of various invertebrates, data on the structure and properties of the isolated and characterized C1qDC proteins, which belong to the C1q/TNF superfamily, are sporadic, although they hold great practical potential for the creation of new biotechnologies. This review not only summarizes the current data on the properties of already-isolated or bioengineered C1qDC proteins but also projects further strategies for their study and biomedical application. It has been shown that further broad study of the carbohydrate specificity of the proteins can provide great opportunities, since for many of them only interactions with pathogen-associated molecular patterns (PAMPs) was evaluated and their antimicrobial, antiviral, and fungicidal activities were studied. However, data on the properties of C1qDC proteins, which researchers originally discovered as lectins and therefore studied their fine carbohydrate specificity and antitumor activity, intriguingly show the great potential of this family of proteins for the creation of targeted drug delivery systems, vaccines, and clinical assays for the differential diagnosis of cancer. The ability of invertebrate C1qDC proteins to recognize patterns of aberrant glycosylation of human cell surfaces and interact with mammalian immunoglobulins indicates the great biomedical potential of these molecules.

A C1q domain-containing protein in Pinctada fucata contributes to the innate immune response and elimination of the pathogen

The C1q domain-containing proteins (C1qDCs) in bivalve mollusks primarily exist as the globular head C1q proteins (ghC1qs), for the N-terminal collagen domains were very rare in bivalves, although widespread in C1qDCs of vertebrates. In this work, the C1qDC protein with only a ghC1q domain (named as Pf-ghC1q) was identified from Pinctada fucata, and molecular characterization, gene expression, and functional studies were also conducted. The full-length cDNA sequence of Pf-ghC1q was 738 bp long, containing a signal peptide of 23 residues encoded. Pf-ghC1q was clustered with some C1qDCs from other invertebrates in the phylogenetic tree analysis, rather than vertebrates. Pf-ghC1q was detected in all tested tissues, including the mantle, hemocyte, digestive gland, gill, and adductor muscle. Moreover, the expression levels of Pf-ghC1q were up-regulated in all tested tissues after the challenge with Vibrio alginolyticus 4 h later. The expression level of Pf-ghC1q was inhibited by specific si-276, and the low level of Pf-ghC1q affected the phagocytosis efficiency of V. alginolyticus by hemocytes. These results indicated that Pf-ghC1q may participate in the target recognition of V. alginolyticus and the phagocytosis process in the immune response of P. fucata.

Comparative analysis of the Mercenaria mercenaria genome provides insights into the diversity of transposable elements and immune molecules in bivalve mollusks

BackgroundThe hard clam Mercenaria mercenaria is a major marine resource along the Atlantic coasts of North America and has been introduced to other continents for resource restoration or aquaculture activities. Significant mortality events have been reported in the species throughout its native range as a result of diseases (microbial infections, leukemia) and acute environmental stress. In this context, the characterization of the hard clam genome can provide highly needed resources to enable basic (e.g., oncogenesis and cancer transmission, adaptation biology) and applied (clam stock enhancement, genomic selection) sciences.ResultsUsing a combination of long and short-read sequencing technologies, a 1.86 Gb chromosome-level assembly of the clam genome was generated. The assembly was scaffolded into 19 chromosomes, with an N50 of 83 Mb. Genome annotation yielded 34,728 predicted protein-coding genes, markedly more than the few other members of the Venerida sequenced so far, with coding regions representing only 2% of the assembly. Indeed, more than half of the genome is composed of repeated elements, including transposable elements. Major chromosome rearrangements were detected between this assembly and another recent assembly derived from a genetically segregated clam stock. Comparative analysis of the clam genome allowed the identification of a marked diversification in immune-related proteins, particularly extensive tandem duplications and expansions in tumor necrosis factors (TNFs) and C1q domain-containing proteins, some of which were previously shown to play a role in clam interactions with infectious microbes. The study also generated a comparative repertoire highlighting the diversity and, in some instances, the specificity of LTR-retrotransposons elements, particularly Steamer elements in bivalves.ConclusionsThe diversity of immune molecules in M. mercenaria may allow this species to cope with varying and complex microbial and environmental landscapes. The repertoire of transposable elements identified in this study, particularly Steamer elements, should be a prime target for the investigation of cancer cell development and transmission among bivalve mollusks.

A Novel C1q Domain-Containing Protein Isolated from the Mollusk Modiolus kurilensis Recognizing Glycans Enriched with Acidic Galactans and Mannans.

C1q domain-containing (C1qDC) proteins are a group of biopolymers involved in immune response as pattern recognition receptors (PRRs) in a lectin-like manner. A new protein MkC1qDC from the hemolymph plasma of Modiolus kurilensis bivalve mollusk widespread in the Northwest Pacific was purified. The isolation procedure included ammonium sulfate precipitation followed by affinity chromatography on pectin-Sepharose. The full-length MkC1qDC sequence was assembled using de novo mass-spectrometry peptide sequencing complemented with N-terminal Edman’s degradation, and included 176 amino acid residues with molecular mass of 19 kDa displaying high homology to bivalve C1qDC proteins. MkC1qDC demonstrated antibacterial properties against Gram-negative and Gram-positive strains. MkC1qDC binds to a number of saccharides in Ca2+-dependent manner which characterized by structural meta-similarity in acidic group enrichment of galactose and mannose derivatives incorporated in diversified molecular species of glycans. Alginate, κ-carrageenan, fucoidan, and pectin were found to be highly effective inhibitors of MkC1qDC activity. Yeast mannan, lipopolysaccharide (LPS), peptidoglycan (PGN) and mucin showed an inhibitory effect at concentrations three orders of magnitude greater than for the most effective saccharides. MkC1qDC localized to the mussel hemal system and interstitial compartment. Intriguingly, MkC1qDC was found to suppress proliferation of human adenocarcinoma HeLa cells in a dose-dependent manner, indicating to the biomedical potential of MkC1qDC protein.

Identification and immune functional analysis of a novel C1q domain-containing protein in Yellow River Carp (Cyprinus carpio L.)

C1q domain-containing proteins (C1qDC) refer to a class of proteins, which contain the C1q domain. They play crucial roles in the immune response and recognition in different species, especially invertebrates and lower invertebrates. In this study, the full-length cDNA sequence of a novel C1qDC gene from Yellow River carp (Cyprinus carpio L.) (named as CcC1qDC) was identified and characterized. The results showed that the CcC1qDC was 958 bp in length, contains a 723 bp open reading frame (ORF) encoding a deduced protein of 241 amino acids with an estimated molecular weight of 27.2 kDa. The deduced amino acid sequence of CcC1qDC displayed a high identity to that of the common carp Cyprinus carpio (98.3%). The mRNA transcripts of CcC1qDC were ubiquitously expressed in different tissues of Yellow River carp, with a relatively high expression in the liver, gills, and spleen. The expression profile of CcC1qDC after LPS and poly I:C stimulations were also examined in Yellow River Carp by quantitative real-time PCR (qRT-PCR), and it was indicated that CcC1qDC expression levels were significantly up-regulated in the liver and spleen (p < 0.05), and down-regulated in kidney and head kidney (p < 0.05) after stimulations. Furthermore, rCcC1q showed an obvious bacterial agglutination activity against Aeromonas hydrophila and Bacillus subtilis, and the high binding activity to LPS and PGN in vitro. These results confirmed that CcC1qDC played an important role in immune recognition and response against pathogen infection in Yellow River Carp. This study could provide a theoretical basis for developing disease control strategies in aquaculture.

Hemolymph C1qDC promotes the phagocytosis of oyster Crassostrea gigas hemocytes by interacting with the membrane receptor β-integrin

Phagocytosis constitutes a conserved cellular process for multicellular animals to ingest or engulf other cells or particles, which is facilitated by the use of opsonins to bind foreign particles and interact with cell surface receptors. The invertebrate secreted C1q domain-containing proteins (C1qDCs) have been reported to exhibit opsonic activity, while the detailed mechanisms of opsonization still remain unclear. In the present study, a C1qDC (designated as CgC1qDC-5) with opsonic activity was identified from the hemolymph of oyster Crassostrea gigas. CgC1qDC-5 exhibited the ability to bind pathogen-associated molecular patterns (PAMPs) of lipopolysaccharides (LPS) and Lipid A. It could also bind and agglutinate Gram-negative bacteria Escherichia coli, Vibrio splendidus and Vibrio anguillarum, whereas the agglutinating activity could be inhibited by LPS. In addition, CgC1qDC-5 could enhance the phagocytosis of hemocytes toward E. coli, V. splendidus, and V. anguillarum. GST pull-down and surface plasmon resonance assays in vitro revealed that CgC1qDC-5 could interact with β-integrin (CgIntegrin). In vivo, CgC1qDC-5 was observed to bind hemocytes and co-localized with CgIntegrin on the cell membrane of hemocytes. Antibody-mediated blockage of CgIntegrin hindered the CgC1qDC-5-enhanced hemocytic phagocytosis. CgIntegrin also exhibited the ability to bind the Gram-negative bacteria E. coli, V. splendidus, V. anguillarum and Vibrio parahaemolyticus, and PAMP of LPS, but not Lipid A. A phagocytosis assay demonstrated that CgIntegrin could directly mediate phagocytosis toward bacteria as a phagocytic receptor. These results collectively suggested that CgC1qDC-5 could serve as an opsonin to recognize and bind bacteria, and subsequently interact with CgIntegrin on the hemocyte surface to enhance the CgIntegrin-mediated phagocytosis in oyster.

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.

The transcriptomic expression of pattern recognition receptors: Insight into molecular recognition of various invading pathogens in Oyster Crassostrea gigas

Pattern recognition receptors (PRRs) are essential in recognizing specific pathogen-associated molecular patterns (PAMPs) on microbes and triggering responses to eliminate the invading pathogens. Previous genomic studies have revealed a great number of PRR genes in the Pacific oyster Crassostrea gigas, a sessile and filter-feeder marine bivalve belonging to the phylum Mollusca. On the survey of PRRs in the assembly oyster reference genome version 9, a total of 1084 PRRs were identified, which were composed of at least 12 gene families. Some of the gene families were significantly expanded, including C-type lectins (CTLs), fibrinogen-related proteins (FREPs), scavenger receptor cysteine-rich repeat protein (SRCRs), leucine-rich repeat (LRR)-only proteins (LRRops), and especially C1q domain-containing proteins (C1qDCs). The transcriptomic profiles of these abundant PRRs in response to PAMP treatments were investigated by RNA-Seq using the SOLiD EZ BeadTM system. Compared to the control library, there were 6,655, 7,273, 7,593, 6,830, 6687 and 8250 differentially expressed genes in the haemocytes of oysters in response to lipopolysaccharide (LPS) stimulation for 6 h, 12 h and 24 h, and peptidoglycan (PGN), glucan (GLU) and poly I:C (IC) stimulation for 12 h, respectively. After stimulation for 12 h, there were 134, 97, 114 and 159 genes up-regulated in the LPS, PGN, GLU and IC library, respectively. Most of the gene families involved in immune response towards PAMPs were C1qDCs, CTLs and FREPs, while only a few members of LRR and immunoglobin-containing proteins (LRRIGs), retinoic acid-inducible gene I [RIG-I]-like receptors (RLRs) and Toll like receptors (TLRs) were up-regulated. After LPS stimulation, the expression level of 258 non-redundant PRR genes in oyster haemocytes increased significantly with different expression pattern, and most of them were C1qDCs, CTLs, LRRops and FREPs. The transcriptomic analyses indicated that there was a dynamic and orchestrated specific expression regulation of numerous PRR genes in response to pathogen invasion. The expanded PRR gene family members were differentiated with more specific functional responses to certain PAMPs rather than the versatile ones. Based on the different expression pattern during the LPS stimulation, the oyster PRRs could be assigned into three consecutive steps in the response against pathogen invading. All the results would provide useful information for future studies of oyster PRRs and deep insight into the researches on invertebrate innate immunity.

Uncovering the immunological repertoire of the carpet shell clam Ruditapes decussatus through a transcriptomic-based approach

The grooved carpet-shell clam Ruditapes decussatus is native to the Northern Atlantic and Mediterranean Sea and has a high commercial value. It is one of the main native bivalve species cultured in Europe. The main objective of the present study was to gain further insights into the immunological repertoire of R. decussatus through a transcriptomic approach. Pooled mantle samples of eight R. decussatus individuals were sequenced using Illumina platform. A total of 67 132 contigs with more than 800 bp were obtained. Manual annotation of these contigs revealed 146 immune-related genes. The gene families in which the highest number of immune-related genes was observed were: C1q domain-containing proteins (63), tumor necrosis factors (15) and toll-like receptors (TLRs, 10). A total of 5 359 putative single nucleotide polymorphisms (SNPs) were identified in the 146 immune-related genes. The density of SNPs ranged between 0.04 and 7.92 SNPs/100 bp. The highest and the lowest SNP density were observed in genes of the C1q domain-containing protein family. Due to the importance of TLRs in innate immunity, we focused our attention on these membrane receptors. Ten TLRs were identified based on protein domain organization. Phylogenetic analysis revealed that R. decussatus TLRs were diverse and only 3 showed orthology with TLRs of known immune functions in other bivalve species. Moreover, our analysis suggests that lineage restricted-expansions of TLRs occurred in all mollusc taxa analysed including in venerids.

Three novel C1q domain containing proteins from the disk abalone Haliotis discus discus: Genomic organization and analysis of the transcriptional changes in response to bacterial pathogens.

The globular C1q (gC1q) domain containing proteins, commonly referred as C1q domain containing (C1qDC) proteins, are an essential family of proteins involved in various innate immune responses. In this study, three novel C1qDC proteins were identified from the disk abalone (Haliotis discus discus) transcriptome database and designated as AbC1qDC1, AbC1qDC2, and AbC1qDC3. The cDNA sequences of AbC1qDC1, AbC1qDC2, and AbC1qDC3 consisted of 807, 1305, and 660bp open reading frames (ORFs) encoding 269, 435, and 220 amino acids (aa), respectively. Putative signal peptides and the N-terminal gC1q domain were identified in all three AbC1qDC proteins. An additional predicted motif region, known as the coiled coil region (CCR), was identified next to the signal sequence of AbC1qDC2. The genomic organization of the AbC1qDCs was determined using a bacterial artificial chromosome (BAC) library. It was found that the CDS of AbC1qDC1 was distributed among three exons, while the CDSs of AbC1qDC2 and AbC1qDC3 were distributed between two exons. Sequence analysis indicated that the AbC1qDC proteins shared <40% identity with other counterparts from different species. According to the neighbor-joining phylogenetic tree, the proteins were grouped within an invertebrate group with high evolutionary distances, which suggests that they are new members of the C1qDC family. Higher expression of AbC1qDC1 and AbC1qDC2 was detected in hepatopancreas, muscle, and mantle tissues compare to the other tissues analyzed, using reverse transcription, followed by quantitative real-time PCR (qPCR) using SYBR Green, whereas AbC1qDC3 was predominantly expressed in gill tissues, followed by muscles and the hepatopancreas. The temporal expression of AbC1qDC transcripts in gills after bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and lipopolysaccharide stimulation indicated that AbC1qDCs can be strongly induced by both Gram-negative and Gram-positive bacterial species with different response profiles. The results of this study suggest that AbC1qDCs are involved in immune responses against invading bacterial pathogens.

Four C1q domain-containing proteins involved in the innate immune response in Hyriopsis cumingii.

C1q is a key subcomponent of the complement C1 complex. This subcomponent contains a globular C1q (gC1q) domain with remarkable ligand binding properties. C1q domain-containing (C1qDC) proteins are composed of all proteins with a gC1q domain. C1qDC proteins exist in many invertebrates and recognize non-self-ligands. In our study, four C1qDC genes, namely, HcC1qDC1-HcC1qDC4, were identified from Hyriopsis cumingii. HcC1qDC1-HcC1qDC4 encode a protein of 224, 204, 305, and 332 amino acids, respectively. All C1qDC proteins consist of a gC1q domain at the C terminal. In addition to the gC1q domain, a coiled-coil region is found in HcC1qDC4. Multiple alignments and phylogenetic tree analysis revealed that the C1qDC proteins highly differ from one another. Tissue distribution analysis demonstrated that HcC1qDC1-HcC1qDC4 are widely distributed in hemocytes, hepatopancreas, gills, mantle, and foot. These C1qDC genes are regulated by bacteria to varying degrees. These recombinant HcC1qDC proteins exhibit a binding activity against different bacterial species. Our results may suggest the roles of HcC1qDC genes in anti-bacterial immune defense.

L-C1qDC-1, a novel C1q domain-containing protein from Lethenteron camtschaticum that is involved in the immune response

The C1q domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain at their C-terminus. These proteins are involved in various processes in vertebrates and are assumed to serve as important pattern recognition receptors in innate immunity in invertebrates. Here, a novel C1qDC protein from Lethenteron camtschaticum was identified and characterized (designated as L-C1qDC-1). After a partial cDNA sequence of L-C1qDC-1 was identified in a L. camtschaticum liver cDNA library, the full-length cDNA was obtained using 3′- and 5′-rapid amplification of cDNA ends (RACE). L-C1qDC-1 encodes 236 amino acids and contains a signal peptide, a collagen-like sequence with Gly-Xaa-Yaa repeats, and a C-terminal gC1q domain. The L-C1qDC-1 protein was primarily distributed in the gut, liver and supraneural body of L. camtschaticum and was also marginally detectable in leukocytes via real-time PCR and immunofluorescence assays. Furthermore, both immunoprecipitation and immunofluorescence results showed that in L. camtschaticum serum, L-C1qDC-1 could interact with variable lymphocyte receptor (VLR) B and displayed strong colocalization with cancer cell immune responses. These results indicated that the L-C1qDC-1 gene encodes a novel C1qDC protein that may play an important role in the immune responses of L. camtschaticum, providing clues for understanding the universal functions of C1qDC proteins in other species and suggesting that these proteins could serve as pattern recognition molecules in immunotherapy.

A novel multi-domain C1qDC protein from Zhikong scallop Chlamys farreri provides new insights into the function of invertebrate C1qDC proteins

The C1q domain containing (C1qDC) proteins are a family of proteins possessing globular C1q (gC1q) domains, and they rely on this domain to recognize various ligands such as PAMPs, immunoglobulins, ligands on apoptotic cell. In the present study, a novel multi-domain C1qDC protein (CfC1qDC-2) was identified from scallop Chlamys farreri, and its full length cDNA was composed of 1648 bp, encoding a signal peptide and three typical gC1q domains. BLAST analysis revealed significant sequence similarity between CfC1qDC-2 and C1qDC proteins from mollusks. Three gC1q domains were predicted in its tertiary structure to form a tightly packed bell-shaped trimer, and each one adopted a typical 10-stranded sandwich fold with a jelly-roll topology and contained six aromatic amino acids forming the hydrophobic core. The mRNA transcripts of CfC1qDC-2 were mainly detected in the tissues of hepatopancreas and gonad of adult scallops, and the expression level was up-regulated in hemocytes after stimulated by LPS, PGN and β-glucan. During the embryonic development of scallop, the mRNA transcripts of CfC1qDC-2 were presented in all the detected stages, and the expression level was up-regulated from D-hinged larvae and reached the highest at eye-spot larvae. The recombinant protein of MBP-CfC1qDC-2 (rCfC1qDC-2) could bind various PAMPs including LPS, PGN, LTA, β-glucan, mannan as well as polyI:C, and different microorganisms including three Gram-negative bacteria, three Gram-positive bacteria and two yeasts, as well as scallop apoptotic cells. Meanwhile, rCfC1qDC-2 could interact with human heat-aggregated IgG and IgM, and inhibit the C1q-dependent hemolysis of rabbit serum. All these results indicated that CfC1qDC-2 could recognize not only PAMPs as a PRR, but also the apoptotic cells. Moreover, the similar structures and functions shared by CfC1qDC-2 and complement C1q provided a new insight into the evolution of C1qDC proteins in complement system.

Transcriptional changes in Manila clam (Ruditapes philippinarum) in response to Brown Ring Disease

Brown Ring Disease (BRD) is a bacterial infection affecting the economically-important clam Ruditapes philippinarum. The disease is caused by a bacterium, Vibrio tapetis, that colonizes the edge of the mantle, altering the biomineralization process and normal shell growth. Altered organic shell matrices accumulate on the inner face of the shell leading to the formation of the typical brown ring in the extrapallial space (between the mantle and the shell). Even though structural and functional changes have been described in solid (mantle) and fluid (hemolymph and extrapallial fluids) tissues from infected clams, the underlying molecular alterations and responses remain largely unknown. This study was designed to gather information on clam molecular responses to the disease and to compare focal responses at the site of the infection (mantle and extrapallial fluid) with systemic (hemolymph) responses. To do so, we designed and produced a Manila clam expression oligoarray (15K Agilent) using transcriptomic data available in public databases and used this platform to comparatively assess transcriptomic changes in mantle, hemolymph and extrapallial fluid of infected clams. Results showed significant regulation in diseased clams of molecules involved in pathogen recognition (e.g. lectins, C1q domain-containing proteins) and killing (defensin), apoptosis regulation (death-associated protein, bcl-2) and in biomineralization (shell matrix proteins, perlucin, galaxin, chitin- and calcium-binding proteins). While most changes in response to the disease were tissue-specific, systemic alterations included co-regulation in all 3 tested tissues of molecules involved in microbe recognition and killing (complement-related factors, defensin). These results provide a first glance at molecular alterations and responses caused by BRD and identify targets for future functional investigations.

LjC1qDC-1, a novel C1q-domain containing protein from lampetra japonica involving in the immune response (INM2P.428)

Abstract The C1q domain containing (C1qDC) proteins are defined as a family of all proteins containing the globular C1q (gC1q) domain in the C-terminus. They participate in a series of immune responses depending on versatile gC1q domain which can bind a variety of self and non-self ligands. In this study, a novel C1qDC protein was identified from Lampetra japonica (designated as LjC1qDC-1). Its full-length cDNA was of 1662 bp with an open reading frame (ORF) of 711 bp encoding a polypeptide of 236 amino acids. The deduced protein contained a signal peptide, a collagen-like sequence with Gly-Xaa-Yaa repeats, and a typical gC1q domain. Real-time PCR analysis showed that the transcript of LjC1qDC-1 gene was distributed in various immune associated tissues of lamprey. This observation was further confirmed by FACS and confocal laser scanning microscope. In addition, LjC1qDC-1 interaction with specific VLR was analysised by co-immunoprecipitation and western blot. All these results indicated that LjC1qDC-1 gene identified may play an important role in the immune responses of lamprey, which provided a clue to understand the universal function of C1qDC proteins in different species.

A novel C1qDC protein acting as pattern recognition receptor in scallop Argopecten irradians

The C1q domain containing (C1qDC) proteins refer to a family of proteins containing the versatile charge pattern recognition globular C1q domain in the C-terminus, which could bind various ligands including PAMPs and trigger a serial of immune response. In this study, a novel C1qDC protein was identified from Argopecten irradians (designated as AiC1qDC-2). Its full-length cDNA was of 1062 bp with an open reading frame of 720 bp encoding a polypeptide of 240 amino acids containing a typical gC1q domain. This gC1q domain possessed the typical 10-stranded β-sandwich fold with a jelly-roll topology common to all C1q family members, and shared high homology with most of the other identified gC1q domains. The mRNA transcripts of AiC1qDC-2 were mainly detected in hepatopancreas, and also marginally detectable in mantle, gonad, adductor, gill and hemocytes. Its relative expression level in hemocytes was significantly up-regulated after challenges of fungi Pichia pastoris GS115 (P < 0.05), Gram-positive bacteria Micrococcus luteus (P < 0.05) and Gram-negative bacteria Vibrio anguillarum (P < 0.05). The recombinant protein of AiC1qDC-2 (rAiC1qDC-2) could bind various PAMPs, including LPS, PGN, polyI:C, mannan, β-1,3-glucan as well as Yeast-glucan, and displayed agglutinating activity to fungi P. pastoris GS115, Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli TOP10F′ as well as V. anguillarum. All these results indicated that AiC1qDC-2 could function as a pattern recognition receptor to recognize various PAMPs on different pathogens in the innate immune responses of scallop, and provided new clues to understand the role of invertebrate C1qDC proteins in the ancient complement system.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733 bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded β-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of C1qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by d-mannose and PGN but not by LPS, glucan or d-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway.

A novel C1q-domain-containing protein from Zhikong scallop Chlamys farreri with lipopolysaccharide binding activity

The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus. They are involved in various processes of vertebrates and supposed to be an important pattern recognition receptor in innate immunity of invertebrates. In this study, a novel member of C1q-domain-containing protein family was identified from Zhikong scallop Chlamys farreri (designated as CfC1qDC) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfC1qDC was of 777 bp, consisting of a 5′-terminal untranslated region (UTR) of 62 bp and a 3′ UTR of 178 bp with a polyadenylation signal sequence AATAAA and a poly (A) tail. The CfC1qDC cDNA encoded a polypeptide of 178 amino acids, including a signal peptide and a C1q-domain of 158 amino acids with the theoretical isoelectric point of 5.19 and the predicted molecular weight of 17.2 kDa. The C1q-domain in CfC1qDC exhibited homology with those in sialic acid binding lectin from mollusks and C1qDC proteins from higher vertebrates. The typical 10 β-strand jelly-roll folding topology structure of C1q-domain and the residues essential for effective packing of the hydrophobic core were well conserved in CfC1qDC. By fluorescent quantitative real-time PCR, mRNA transcripts of CfC1qDC were mainly detected in kidney, mantle, adductor muscle and gill, and also marginally detectable in hemocytes. In the bacterial challenge experiment, after the scallops were challenged by Listonella anguillarum, there was a significant up-regulation in the relative expression level of CfC1qDC and at 6 h post-injection, the mRNA expression reached the maximum level and was 4.55-fold higher than that of control scallops. Similarly, the expression of CfC1qDC mRNA in mixed primary cultures of hemocytes stimulated by lipopolysaccharides (LPS) was up-regulated and reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. In order to investigate its function, the cDNA fragment encoding the mature peptide of CfC1qDC was recombined and expressed in Escherichia coli BL21(DE3). The recombinant CfC1qDC protein displayed a significantly strong activity to bind LPS from E. coli, although no obvious antibacterial or agglutinating activity toward Gram-negative bacteria E. coli JM109, L. anguillarum and Gram-positive bacteria Micrococcus luteus was observed. These results suggested that CfC1qDC was absolutely a novel member of the C1qDC protein family and was involved in the recognition of invading microorganisms probably as a pattern recognition molecule in mollusk.

Bioinformatic identification of genes encoding C1q-domain-containing proteins in zebrafish

C1q is the first subcomponent of classical pathway in the complement system and a major link between innate and acquired immunities. The globular (gC1q) domain similar with C1q was also found in many non-complement C1q-domain-containing (C1qDC) proteins which have similar crystal structure to that of the multifunctional tumor necrosis factor (TNF) ligand family, and also have diverse functions. In this study, we identified a total of 52 independent gene sequences encoding C1q-domain-containing proteins through comprehensive searches of zebrafish genome, cDNA and EST databases. In comparison to 31 orthologous genes in human and different numbers in other species, a significant selective pressure was suggested during vertebrate evolution. Domain organization of C1q-domain-containing (C1qDC) proteins mainly includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 11 highly conserved residues within the C1q domain, among which 2 are invariant within the zebrafish gene set. A more extensive database searches also revealed homologous C1qDC proteins in other vertebrates, invertebrates and even bacterium, but no homologous sequences for encoding C1qDC proteins were found in many species that have a more recent evolutionary history with zebrafish. Therefore, further studies on C1q-domain-containing genes among different species will help us understand evolutionary mechanism of innate and acquired immunities.