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Iron (Fe) is considered as an essential micronutrient due to its diverse functions in living systems. However, regulation of free iron levels is essential because free Fe ions, in excess, induce biological toxicity through different routes, including production of reactive oxygen species. Ferritin proteins play a vital role in controlling free Fe ion homeostasis by sequestering excess iron in the body. Ferritins comprise an H subunit with a ferroxidase center and an L subunit with a Fe nucleation site. However, lower vertebrates such as fish harbor an additional subunit termed ferritin M, which shows the characteristic features of both H and L. In this study, two ferritin subunits (H and M) with ferroxidase centers were identified and characterized from red-lip mullet (Liza haematocheila). The open reading frames of red-lip mullet ferritin H (LhFerH) and ferritin M-like (LhFerM) subunits comprise 534 and 531 bps, which encode for putative polypeptides of 177 and 176 amino acids, respectively. LhFerH and LhFerM were found to retain well-conserved residues, including seven ferroxidase di-iron centers, characteristic domains, and signatures of their known homologs. We cloned the open reading frames of the two ferritin subunits to overexpress the corresponding proteins in Escherichia coli and subsequently demonstrated their iron sequestration activity along with antibacterial activity against E. coli using respective purified recombinant proteins in vitro. A basal expression analysis of two LhFer genes in selected tissues using qPCR assays showed pronounced expression in blood cells with respect to both genes. A relatively high expression level of LhFerH was also detected in muscle tissues. The expression level of LhFer in the head kidney was significantly up-regulated following lipopolysaccharides (LPS) and Lactococcus garvieae injection. The resulting gene expression pattern upon immune stimulation suggests that ferritin may contribute to the defense against harmful pathogen infection. Collectively, our results indicate that both LhFerH and LhFerM potentially participate in the homeostasis of free Fe ions and in the host immune defense of red-lip mullet.

Natural killer enhancing factor A (NKEF-A), also known as peroxiredoxin 1 (Prx1), is a well-known antioxidant involved in innate immunity. Although NKEF-A/Prx1 has been studied in different fish species, the present study broadens the knowledge of NKEF-A gene in terms of molecular structure, function, and immune responses in fish species. Hippocampus abdominalis NKEF-A (HaNKEF-A) cDNA encoded a putative protein of 198 amino acids containing a thioredoxin_2 domain, VCP motifs, and three conserved cysteine residues including peroxidatic and resolving cysteines. Amino acid sequence comparison and phylogenetic breakdown showed the higher sequence identity and closer evolutionary position of HaNKEF-A to those of other fish counterparts. A recombinant protein of HaNKEF-A was shown to i) protect supercoiled DNA against mixed catalyzed oxidation, ii) reduce insulin disulfide bonds, and iii) scavenge extracellular H2O2. Results of in vitro assays demonstrated the concentration dependent antioxidant function of recombinant HaNKEF-A. In addition, qPCR assessments revealed that the HaNKEF-A transcripts were constitutively expressed in fourteen tissues with the highest expression in liver. As an innate immune response, HaNKEF-A transcripts were up-regulated in liver post injection of LPS, Edwardsiella tarda, Streptococcus iniae, and polyinosinic-polycytidylic acid. Thus, HaNKEF-A can safeguards big-belly seahorse from oxidative damage and pathogenic infections. This study provides insight into the functions of NKEF-A/Prx1 in fish species.

Cathepsin Z (CTSZ) is lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. Hence, CTSZ is also acknowledged as an acute-phase protein in host immunity. In this study, we sought to identify the CTSZ homolog from disk abalone (AbCTSZ) and characterize it at the molecular, genomic, and transcriptional levels. AbCTSZ encodes a protein with 318 amino acids and a molecular mass of 36kDa. The structure of AbCTSZ reveals amino acid sequences that are characteristic of the signal sequence, pro-peptide, peptidase-C1 papain family cysteine protease domain, mini-loop, HIP motif, N-linked glycosylation sites, active sites, and conserved Cys residues. A pairwise comparison revealed that AbCTSZ shared the highest amino acid homology with its molluscan counterpart from Crassostrea gigas. A multiple alignment analysis revealed the conservation of functionally crucial elements of AbCTSZ, and a phylogenetic study further confirmed a proximal evolutionary relationship with its invertebrate counterparts. Further, an analysis of AbCTSZ genomic structure revealed seven exons separated by six introns, which differs from that of its vertebrate counterparts. Quantitative real time PCR (qPCR) detected the transcripts of AbCTSZ in early developmental stages and in eight different tissues. Higher levels of AbCTSZ transcripts were found in trochophore, gill, and hemocytes, highlighting its importance in the early development and immunity of disk abalone. In addition, we found that viable bacteria (Vibrio parahaemolyticus and Listeria monocytogenes) and bacterial lipopolysaccharides significantly modulated AbCTSZ transcription. Collectively, these lines of evidences suggest that AbCTSZ plays an indispensable role in the innate immunity of disk abalone.

Peroxiredoxins (Prxs) are ubiquitously expressed antioxidant proteins that can protect aerobic organisms from oxidative stress. Here, we characterized the HaPrx3 homolog at the molecular level from big-belly seahorse (Hippocampus abdominalis) and analyzed its functional activities. The coding sequence of HaPrx3 consists of 726 bp, which encodes 241 amino acids. The predicted molecular weight and theoretical isoelectric point (pI) of HaPrx3 was 26.20 kDa and 7.04, respectively. Multiple sequence alignments revealed that the arrangements of domains, catalytic triads, dimers, and decamer interfaces of HaPrx3 were conserved among Prx sequences of other organisms. According to the phylogenetic analysis, HaPrx3 is clustered with the teleost Prx3 subclade. The highest transcript level of HaPrx3 was detected in the ovary tissue among fourteen healthy fish tissues. The mRNA levels of HaPrx3 in blood and liver tissues were significantly (P

Reactive oxygen species, generated in all the aerobic organisms, can cause oxidative stress. Excessive ROS may become a source of carcinogen due to DNA damage, lipid peroxidation, cell injury, and cell death. In order to prevent these adverse effects of ROS, antioxidant enzymes have evolved in aerobic organisms. Catalase is a major antioxidant enzyme that breaks down excessive H 2 O 2 and inhibits apoptotic cell death. Here we molecularly characterized catalase from red-lip mullet. The cDNA sequence of LhCAT consists of an ORF of 1545 bp, which encodes a 527 amino acid peptide (~60 kDa). Based on bioinformatics analysis, LhCAT possesses a domain architecture characteristic of catalases, including a catalase proximal active site signature and a catalase proximal heme-ligand signature. It also has heme and NADPH binding sites homologous to previously described catalases. Pairwise alignment with its homologs revealed that LhCAT shares 95.1% identity with Oplegnathus fasciatus catalase and 97.4% similarity with Sparus aurata catalase. An uprooted phylogenetic tree demonstrated that LhCAT resides in a clade with catalases from other teleosts and exhibits a close relationship with Oplegnathus fasciatus catalase. Among twelve tissue types, we observed the highest LhCAT mRNA expression in the liver, followed by blood. Immune challenge by Lactococcus garvieae , or Poly I:C in the blood or spleen resulted in up-regulation at 24 h post injection. We also tested the antioxidant activity of recombinant LhCAT against hydrogen peroxide and found its optimal concentration to be 12.5 μg/mL. Collectively, these data suggested that LhCAT play an important role in antioxidant defense and immune response of red-lip mullet.

Interleukin-10 (IL-10) is a pleiotropic cytokine involved in the regulation of innate and adaptive immunity. In this study, IL-10 from big-belly seahorse (Hippocampus abdominalis) (HaIL-10) was characterized based on its molecular and functional aspects. The coding sequence of HaIL-10 is 570 bp in length and encodes a 189-amino acid residue protein (calculated molecular weight, 21.89 kDa). The deduced amino acid sequence comprises a typical signal peptide and a mature peptide domain sequence carrying four conserved Cys residues and two additional Cys residues specific to fish. Phylogenetic analysis indicated an evolutionary relationship between HaIL-10 and its counterparts in other vertebrates, with close clustering to the fish-specific homologs. Recombinant HaIL-10 (rHaIL-10) significantly reduced nitric oxide (NO) production by lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells in a concentration-dependent manner but had no effect on cell viability, suggestive of its involvement in immune response. The protein expressions of iNOS and COX-2 were significantly reduced by rHaIL-10 in LPS-induced murine macrophages RAW 264.7 cells. HaIL-10 mRNA expression was observed in all analyzed tissues, with the maximum expression being noted in the kidney and ovary. However, transcriptional levels of HaIL-10 were significantly higher in the blood, gill, and intestine upon in vivo induction with LPS, polyinosinic:polycytidylic acid [poly (I:C)], and Streptococcus iniae. To summarize, our findings help in the improved understanding of the biological functions of HaIL-10 and modulation of HaIL-10 mRNA expression in response to immune stress.

Natural killer enhancing factor A (NKEF-A), also known as peroxiredoxin 1 (Prx1), is a well-known antioxidant involved in innate immunity. Although NKEF-A/Prx1 has been studied in different fish species, the present study broadens the knowledge of NKEF-A gene in terms of molecular structure, function, and immune responses in fish species. Hippocampus abdominalis NKEF-A (HaNKEF-A) cDNA encoded a putative protein of 198 amino acids containing a thioredoxin_2 domain, VCP motifs, and three conserved cysteine residues including peroxidatic and resolving cysteines. Amino acid sequence comparison and phylogenetic breakdown showed the higher sequence identity and closer evolutionary position of HaNKEF-A to those of other fish counterparts. A recombinant protein of HaNKEF-A was shown to i) protect supercoiled DNA against mixed catalyzed oxidation, ii) reduce insulin disulfide bonds, and iii) scavenge extracellular H

Tumor necrosis factor alpha-induced protein 8-like 2 (TNFAIP8L2) is a newly described negative immune regulator, whose enigmatic biological functions are not clearly understood. In the present study, the TNFAIP8L2 homolog of rock bream ( Oplegnathus fasciatus ) was identified and characterized. The genomic composition of rock bream TNFAIP8L2 (~ 6.7 kb) represented a tripartite arrangement in which three exons are interrupted by two introns. The rock bream TNFAIP8L2 transcript (1974 bp) possessed a coding sequence of 561 bp encoding a peptide of 186 amino acids. The predicted rock bream TNFAIP8L2 protein was 21.1 kDa and revealed the typical features of known TNFAIP8L2 members including the DED-like domain. Rock bream TNFAIP8L2 was composed of six α-helices and demonstrated a distinct folding pattern of the TNFAIP8L2 family. It showed a certain degree of homology and phylogenetic relationship with the corresponding tilapia counterpart. Based on an interspecies genomic organizational comparison of TNFAIP8L2 orthologs, they could be classified into two classes, with teleost and non-teleost origin respectively. While teleost TNFAIP8L2 s manifest a tripartite arrangement, non-teleost counterparts demonstrate a dipartite structure suggesting the loss of an intron during the post-piscine speciation. Promoter proximal region of rock bream TNFAIP8L2 consisted of multiple immune responsive cis -regulatory elements. Analysis of basal transcription in eleven tissues revealed its constitutive expression in examined tissues with highest magnitude in the head kidney. The modulated temporal mRNA expression of rock bream TNFAIP8L2 in head kidney post-challenges with stimulants (LPS and poly I:C) and pathogens ( Streptococcus iniae and irido virus) was stimulant-specific. Additionally, a drastic down-regulation of rock bream TNFAIP8L2 mRNA level occurred in blood cells collected from experimentally injured animals, and it was accompanied by a contemporaneous down-regulation of cytokines, TNF- α and TGFβ3 . All these findings imply that rock bream TNFAIP8L2 is potentially responsible for immune and inflammatory modulation in rock bream.

Repertoires of proteins and small peptides play numerous physiological roles as hormones, antimicrobial peptides, and cellular signaling factors. The beta-thymosins are a group of small acidic peptides involved in processes such as actin sequestration, neuronal development, wound healing, tissue repair, and angiogenesis. Recent characterization of the beta thymosins as immunological regulators in invertebrates led to our identification and characterization of a beta-thymosin homologue (Tβ) from Haliotis discus discus . The cDNA possessed an ORF of 132 bp encoding a protein of 44 amino acids with a molecular mass of 4977 Da. The amino acid sequence shows high identity with another molluskan beta-thymosin and has a characteristic actin binding motif (LKKTET) and glutamyl donors. Phylogenetic analysis showed a close relationship with molluskan homologues, as well as its distinct identity and common ancestral origin. Genomic analysis revealed a 3 exon–2 intron structure similar to the other homologues. In silico promoter analysis also revealed significant transcription factor binding sites, providing evidence for the expression of this gene under different cellular conditions, including stress or pathogenic attack. Tissue distribution profiling revealed a ubiquitous presence in all the examined tissues, but with the highest expression in mantle and hemocyte. Immune challenge with lipopolysaccharide, poly I:C and Vibrio parahemolyticus induced beta-thymosin expression in gill and hemocytes, affirming an immune-related role in invertebrates.

The thioredoxin (Trx) system plays a significant role in cellular antioxidative defense by dismutating the surpluses of reactive oxygen species. Thus, the role of thioredoxin reductase (TrxR) cannot be ignored, owing to its participation in initiating the Trx enzyme cascade. Here, we report the identification and molecular characterization of a teleostean TrxR (RbTrxR-3) ortholog that showed high similarity with the TrxR-3 isoforms of other vertebrates. The complete RbTrxR-3 coding sequence comprised 1800 nucleotides, encoding a 600-amino acid protein with a predicted molecular mass of ~66 kDa. RbTrxR-3 consisted of 16 exons separated by 15 introns and had a total length of 12,658 bp. In silico analysis of the RbTrxR-3 protein sequence revealed that it possesses typical TrxR domain architecture. Moreover, using multiple sequence alignment and pairwise sequence alignment strategies, we showed that RbTrxR-3 has high overall sequence similarity to other teleostean TrxR-3 proteins, including highly conserved active site residues. Phylogenetic reconstruction of RbTrxR-3 affirmed its close evolutionary relationship with fish TrxR-3 orthologs, as indicated by its clustering pattern. RbTrxR-3 transcriptional analysis, performed using quantitative polymerase chain reaction (qPCR), showed that RbTrxR-3 was ubiquitously distributed, with the highest level of mRNA expression in the blood, followed by the gill, and liver. Live bacterial and viral stimuli triggered the modulation of RbTrxR-3 basal transcription in liver tissues that correlated temporally with that of its putative substrate, rock bream thioredoxin1 under the same conditions of pathogenic stress. Finally, resembling the typical function of TrxR protein, purified recombinant RbTrxR-3 showed detectable dose-dependent thiol reductase activity against 5,5'-dithiobis (2-nitrobenzoic) acid. Taken together, these results suggest that RbTrxR-3 plays a role in the host Trx system under conditions of oxidative and pathogenic stress.

Caspase 10 is an initiator caspase in death cascades of death receptor mediated apoptotic signaling. We identified and molecularly characterized a novel homolog of caspase 10 from black rockfish (Sebastes schlegelii) and designated as RfCasp10. The complete coding region of RfCasp10 was found to consist of 1659 bps, encoding a 553 amino acid protein with a predicted molecular mass of 61.7 kDa. The characteristic caspase family domain architecture, including death effecter domains (DEDs), was clearly identified in RfCasp10. Moreover, the RfCasp10 gene was found to contain 13 exons. Our pairwise sequence alignment confirmed the prominent sequence similarity of RfCasp10 with its fish homologs, and phylogenetic reconstruction affirmed its homology and substantial evolutionary relationship with known caspases 10 similitudes, in particular with those of teleosts. As detected by qPCR, RfCasp10 was markedly expressed in blood tissues under physiological conditions, whereas its expression was found to be upregulated under pathogenic stress, elicited by Streptococcus iniae and polyinosinic:polycytidylic acid in blood, liver, and spleen tissues. Collectively, our study suggests the plausible elicitation of RfCasp10 mediated apoptosis in immune relevant tissues of black rockfish as a host immune response to a bacterial or viral infection.

Interleukin 1β (IL-1β) and interleukin 8 (IL-8) are two major pro-inflammatory cytokines which play a central role in initiation of inflammatory responses against bacterial- and viral-infections. IL-1β is a member of the interleukin 1 family proteins and IL-8 is classified as a CXC-chemokine. In the current study, putative IL-1β and IL-8 counterparts were identified from a black rockfish transcriptomic database and designated as RfIL-1β and RfIL-8. The RfIL-1β cDNA sequence consists of 1140 nucleotides with a 759bp open reading frame (ORF) which encodes a 252 amino acid (aa) protein, whereas the RfIL-8 cDNA sequence (898bp) harbors a 300bp ORF encoding a 99 aa protein. Furthermore, the RfIL-1β aa sequence contains an IL-1 super family-like domain and an N-terminal IL-1 super family propeptide, while the amino acid sequence of RfIL-8 consists of a typical chemokine-CXC domain. Analysis of sequenced BAC clones containing RfIL-1β and RfIL-8 showed each gene to contain 4 exons interrupted by 3 introns. Pairwise comparison and phylogeny analysis of these cytokine sequences clearly revealed their closer relationship with other corresponding members of teleosts compared to birds and mammals. Constitutive differences in RfIL-1β and RfIL-8 mRNA expression were detected in a tissue-specific manner with the highest expression of each mRNA in spleen tissue. Two immune challenge experiments were conducted with Streptococcus iniae and polyinosinic:polycytidylic acid (poly I:C; a viral double stranded RNA mimic), and transcripts were quantified in spleen and peripheral blood cells. Significantly increased RfIL-1β and RfIL8 transcript levels were detected with almost similar profile patterns, further suggesting a putative involvement of these pro-inflammatory cytokines in the rockfish immunity.

Toll-like receptors (TLRs) are a large family of pattern recognition receptors, which are involved in triggering host immune responses against various pathogens by detecting their evolutionarily conserved pathogen associated molecular patterns (PAMPs). TLR21 is a non-mammalian type TLR, which recognizes unmethylated CpG DNA, and is considered as a functional homolog of mammalian TLR9. In this study, we attempted to identify and characterize a novel TLR21 counterpart from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at molecular level. The complete coding sequence of RbTLR21 was 2919bp in length, which encodes a polypeptide of 973 amino acids with a predicted molecular mass of 112kDa and a theoretical isoelectric point of 8.6. The structure of the deduced RbTLR21 protein is similar to that of the members of typical TLR family, and includes the ectodomain, which consists of 16 leucine rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. According to the pairwise sequence analysis data, RbTLR21 was homologous to that of the orange-spotted grouper (Epinephelus coioides) with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 is closely related to E. coioides TLR21. The RbTLR21 was ubiquitously expressed in all the tissues tested, but the highest expression was found in spleen. Additionally, upon stimulation with Streptococcus iniae, rock bream iridovirus (RBIV), and Edwardsiella tarda, RbTLR21 mRNA was significantly up-regulated in spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed an ortholog of the TLR21 family and may be important in mounting host immune responses against pathogenic infections.

Cell-to-cell contacts play a key role in multicellular systems and organisms. Fasciclin-1 (FAS-1) is a lipid-linked membrane associated glycoprotein that is a member of a newly recognized family of cell adhesion molecules sharing features with the immunoglobulins, cadherins, integrins, and selectins. Here, we report the identification and molecular characterization of a novel FAS-1 domain-containing cDNA from disk abalone (Haliotis discus discus), including its gene expression profile and immune response to bacterial stimuli and tissue injuries. Designated as Abfac1, the 909bp open reading frame (ORF) encodes 303 amino acid (aa) residues with a predicted molecular mass of 33kDa and isoelectric (pI) value of 4.9. The aa sequence contains two FAS-1 domains and three conserved regions, FRa motif, H-box, and FRb motif. Phylogenetic analysis showed the closest relation to Jellyfish cell adhesion protein. In healthy abalone, Abfac1 expression is highest in hepatopancreas followed by mantle and lowest in digestive gland. In immune-stimulated abalones, relative Abfac1 mRNA expression was increased in hemocytes by ~11-fold at 48h after the Vibrio parahaemolyticus infection, by 3.1-fold at 6h after the Listeria monocytogenes infection and by ~9-fold at 6h after the LPS injection. Similarly, tissue injuries caused significant increase of relative mRNA expression by 3.5-fold in hemocytes and by ~10-fold in mantle at 12h post-injury. These results suggest that the novel member of the FAS-1 domain-containing protein family, Abfac1, may be involved in immune response and cell adhesion in disk abalone.

Troponin C (TnC) is one of the subunits composing the troponin complex, which is primarily expressed in muscle tissue and plays a major role in regulating contractility. We have identified a novel TnC -like gene ( RpTnC ) from the Ruditapes philippinarum Manila clam. Sequence analysis indicated that RpTnC has a 450 bp coding sequence, encoding a 150 amino acid protein with a molecular mass of 17.4 kDa. The RpTnC protein consisted of four EF-hand motifs (I–IV), each with a Ca 2 + -binding site. In silico comparative analysis of protein sequence showed that only site IV, demonstrating a conserved stretch (DxDxSx 6 E), is functionally active for Ca 2 + -coordination. Moreover, RpTnC was homologically (61.3% identity) and phylogenetically closest to Japanese flying squid TnC. The mRNA expression analysis using quantitative real-time PCR revealed a differential basal-expression of RpTnC transcripts in six different clam tissues, with higher levels in adductor muscle and mantle. Intramuscular administration of CaCl 2 caused a prominent upregulation of RpTnC transcripts in adductor muscle (~ 5 fold). Collectively, our findings suggest that the TnC homolog of Manila clam identified in this study may be involved in important role(s) in clam physiology, mainly in its muscle tissues, and its transcription could be significantly influenced by increased Ca 2 + levels.