A molluscan calreticulin ortholog from Haliotis discus discus: Molecular characterization and transcriptional evidence for its role in host immunity.

Calreticulin (CALR), a Ca(2+) binding chaperone of the endoplasmic reticulum (ER) and mainly involved in Ca(2+) storage and signaling. In this study, we report the molecular characterization and immune responses of CALR homolog from disk abalone (AbCALR). The full length AbCALR cDNA (1837 bp) had an ORF of 1224 bp. According to the multiple alignments analysis, N- and P-domains were highly conserved in all the selected members of CALRs. In contrast, the C-domain which terminated with the characteristic ER retrieval signal (HDEL) was relatively less conserved. The phylogenetic analysis showed that all the selected molluscan homologs clustered together. Genomic sequence of AbCALR revealed that cDNA sequence was dispersed into ten exons interconnected with nine introns. AbCALR mRNA expression shows the significant (P < 0.05) up-regulation of AbCALR transcripts in hemocytes upon bacterial (Listeria monocytogenes and Vibrio parahaemolyticus), viral (Viral haemorrhagic septicaemia virus; VHSV) and immune stimulants (LPS and poly I:C) challenges at middle and/or late phases. These results collectively implied that AbCALR is able to be stimulated by pathogenic signals and might play a potential role in host immunity.

Two carboxypeptidase counterparts from rock bream (Oplegnathus fasciatus): molecular characterization, genomic arrangement and immune responses upon pathogenic stresses.

Carboxypeptidases (CPs) are proteases that hydrolyze C-terminal peptide bonds. They are involved in regulating the complement system of the immune system. Here, we report the molecular characterization and immune response of two carboxypeptidases, named carboxypeptidase A (Rb-CPA) and carboxypeptidase N1 (Rb-CPN1), from rock bream. The genomic sequence of Rb-CPA contains 12 exons interrupted by 11 introns, while the genomic sequence of Rb-CPN1 has 9 exons and 8 introns. The cDNA sequence of Rb-CPA encodes a 421-amino-acid (AA) polypeptide (48kDa), and the cDNA of Rb-CPN1 encodes a 448-AA polypeptide (51kDa). The amino acid sequences of Rb-CPA and Rb-CPN1 were found to harbor two characteristic Zn-binding signature domains and a peptidase-M14 Zn carboxypeptidase site. Pairwise analysis revealed that Rb-CPA and Rb-CPN1 had the highest identity with the corresponding proteins from Anoplopoma fimbria (87.6%) and Dicentrarchus labrax (96.9%), respectively. qPCR results indicated that Rb-CPA and Rb-CPN1 were constitutively expressed mainly in the kidney, heart, liver, and head kidney. Both genes were transcriptionally regulated in the liver upon challenge with pathogenic bacteria (Streptococcus iniae, Edwardsiella tarda), rock bream iridovirus (RBIV), and the immune modulators polyinosinic:polycytidylic acid and lipopolysaccharide. Taken together, our findings suggest that Rb-CPA and Rb-CPN1 have immune-related functions in rock bream.

Genomic characterization of interferon regulatory factor 5 from rock bream (Oplegnathus fasciatus) and its role in antiviral defense.

The interferon regulatory factor 5 (IRF5) is a key mediator of the Toll-like receptor (TLR)7 and TLR8 signaling pathways. In this study, we describe the identification of IRF5 (Rb-IRF5) from rock bream fish (Oplegnathus fasciatus) and its characteristics features at the genomic and expression levels. The full-length Rb-IRF5 sequence was identified from a cDNA library and its genomic sequence was obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The genomic sequence is comprised of 8 exons interrupted by 7 introns. The complete coding sequence of Rb-IRF5 is 1497 bp in length and encodes for 498 amino acids. The putative Rb-IRF5 protein consists of 3 important conserved domains: a DNA-binding domain (DBD) at the N-terminus, an IRF-associated domain (IAD), and a virus-activated domain (VAD) at the C-terminus. Based on pairwise sequence analysis, the highest sequence similarity/identity for Rb-IRF5 was observed with the IRF5 gene from turbot fish (>87%) and Japanese flounder (83%). Several important putative transcription factor-binding sites shared by the IRF gene family, including the NF-κB, Ap-1, IRF-1, and ICSBP/ISRE sites, were found in the 5' flanking region of Rb-IRF5. The predicted tertiary structure of the dimerized IAD and VAD of the Rb-IRF5 protein resembled that of its orthologs from humans. In healthy rock bream, the highest constitutive expression of Rb-IRF5 was detected in the liver. After iridovirus and polyinosinic-polycytidylic acid (poly(I:C)) challenge, Rb-IRF5 expression was significantly induced in the head kidney. Furthermore, rock bream recombinant type I interferon (Rb-IFN1) was also found to be an efficient inducer of Rb-IRF5 in a head kidney primary cell culture model. Upon IRF5 transfection, rock bream Mx (Rb-Mx), interferon I (Rb-IFN1) and tumor-necrosis factor α (Rb-TNFα) genes get significantly upregulated in rock bream heart cells. The findings of the present study explain the involvement of Rb-IRF5 in the induction of interferons and pro-inflammatory cytokines and thereby provide a model for how IRF5 modulates immune responses against viral infections in rock bream.

Three complement component 1q genes from rock bream, Oplegnathus fasciatus: genome characterization and potential role in immune response against bacterial and viral infections.

Complement component 1q (C1q) is a subcomponent of the C1 complex and the key protein that recognizes and binds to a broad range of immune and non-immune ligands to initiate the classical complement pathway. In the present study, we identified and characterized three novel C1q family members from rock bream, Oplegnathus fasciatus. The full-length cDNAs of C1q A-like (RbC1qAL), C1q B-like (RbC1qBL), and C1q C-like (RbC1qCL) consist of 780, 720 and 726 bp of nucleotide sequence encoding polypeptides of 260, 240 and 242 amino acids, respectively. All three RbC1qs possess a leading signal peptide and collagen-like region(s) (CLRs) in the N-terminus, and a C1q domain at the C-terminus. The C1q characteristic Gly-X-Y repeats are present in all three RbC1qs, while the CLR-associated sequence that enhances phagocytic activity is present in RbC1qAL ((49)GEKGEP(54)) and RbC1qCL ((70)GEKGEP(75)). Moreover, the coding region was distributed across six exons in RbCqAL and RbC1qCL, but only five exons in RbC1qBL. Phylogenetic analysis revealed that the three RbC1qs tightly cluster with the fish clade. All three RbC1qs are most highly expressed in the spleen and liver, as indicated by qPCR tissue profiling. In addition, all three are transcriptionally responsive to immune challenge, with liver expression being significantly up-regulated in the early phase of infection with intact, live bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus) and in the late phase of exposure to purified endotoxin (lipopolysaccharide). These data collectively suggest that the RbC1qs may play defense roles as an innate immune response to protect the rock bream from bacterial and viral infections.

Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): molecular characterization and transcriptional response upon immune stimulation.

Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. Proteinase inhibitors play a key role in regulating the activity of the respective proteinases. Among serine proteinase inhibitors, kazal-type proteinase inhibitors (KPIs) are widely found in mammals, avians, and a variety of invertebrates. In this study, we describe the identification of a kazal-type serine proteinase inhibitor (Ab-KPI) from the disk abalone, Haliotis discus discus, which is presumably involved in innate immunity. The full-length cDNA of Ab-KPI includes 600 bp nucleotides with an open reading frame (ORF) encoding a polypeptide of 143 amino acids. The deduced amino acid sequence of Ab-KPI contains a putative 17-amino acid signal peptide and two tandem kazal domains with high similarity to other kazal-type SPIs. Each kazal domain consists of reactive site (P1) residue containing a leucine (L), and a threonine (T) located in the second amino acid position after the second conserved cysteine of each domain. Temporal expression of Ab-KPI was assessed by real time quantitative PCR in hemocytes and mantle tissue following bacterial and viral hemorrhagic septicemia virus (VHSV) challenge, and tissue injury. At 6 h post-bacterial and -VHSV challenge, Ab-KPI expression in hemocytes was increased 14-fold and 4-fold, respectively, compared to control samples. The highest up-regulations upon tissue injury were shown at 9 h and 12 h in hemocytes and mantle, respectively. The transcriptional modulation of Ab-KPI following bacterial and viral challenges and tissue injury indicates that it might be involved in immune defense as well as wound healing process in abalone.

Genomic characterization and transcriptional evidence for the involvement of complement component 7 in immune response of rock bream (Oplegnathus fasciatus).

The complement component 7 (C7) is the central mediator of pathogenic attack at the membrane surface and its binding to the C5b-7 complex triggers cytolytic signaling. In this study, C7 of rock bream (Oplegnathus fasciatus) was identified (Rb-C7) and characterized at the genomic level. The Rb-C7 gene contains 18 exons and 17 introns and is composed of a 2490 bp complete open reading frame (ORF). The encoded polypeptide (830 amino acids) contains a number of well-conserved C7 signature domains. Important putative transcription factor binding sites, including those for NF-κB, SP-1, C/EBP, AP-1 and OCT-1, are present in the 5'-flanking region of Rb-C7. Phylogenetic analysis revealed a close proximity of Rb-C7 with the orthologues in tilapia and Japanese flounder. Quantitative real-time PCR (qPCR) analysis confirmed constitutive Rb-C7 expression throughout all the examined tissue of healthy rock bream, with highest expression in liver. In immune challenge experiment, Rb-C7 expression was up-regulated in head kidney and liver in response to Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide and rock bream iridovirus (RBIV). Furthermore, significant increases of both intracellular expression level and the number of Rb-C7-expressing cells were detected by in situ hybridization assay in head kidney and liver tissues upon E. tarda infection. These results suggested that Rb-C7 is lytic pathway gene in complement system and its transcriptional regulation may be an important immune response in pathogenic defense mechanism of rock bream.

Genomic characterization and expression analysis of complement component 8α and 8ß in rock bream (Oplegnathus fasciatus).

The complement component 8α and 8ß are glycoproteins that mediate formation of the membrane attack complex (MAC) on the surface of target cells. Full-length complement C8α (Rb-C8α) and C8ß (Rb-C8ß) sequences were identified from a cDNA library of rock bream (Oplegnathus fasciatus), and their genomic sequences were obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The Rb-C8α gene contains 64bp of 5'-UTR, open reading frame (ORF) of 1794bp, which encodes a polypeptide of 598 amino acids, 212bp of 3'-UTR. The Rb-C8ß gene contains 5'-UTR of 27bp, open reading frame (ORF) of 1761bp, which encodes a polypeptide of 587 amino acids, 3'-UTR of 164bp. Rb-C8α consists of 11 exons interrupted by 10 introns and Rb-C8ß consists of 12 exons interrupted by 11 introns. Sequence analysis revealed that both Rb-C8α and Rb-C8ß contain thrombospondin type-1, a low-density lipoprotein receptor domain class A, membrane attack complex/perforin (MACPF) domain and epidermal growth factor like domain. The promoter regions of both genes contain important putative transcription factor binding sites including those for NF-κB, SP-1, C/EBP, AP-1, and OCT-1. Rb-C8α and Rb-C8ß showed the highest amino acid identity of 62% and 83% to rainbow trout C8α and Japanese flounder C8ß respectively. Quantitative real-time PCR analysis confirmed that Rb-C8α and Rb-C8ß were constitutively expressed in all examined tissues, isolated from healthy rock bream, with highest expression occurring in liver. Pathogen challenge, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus led to up regulation of Rb-C8α and Rb-C8ß in liver. Positive regulations upon bacterial and viral challenges, and high degree of evolutionary relationship to respective orthologues, confirmed that Rb-C8α and Rb-C8ß important immune genes, likely involved in the complement system lytic pathway of rock bream.