Characterization and Functional Analysis of Voltage‐Dependent Anion Channel 1 (VDAC1) from Orange‐Spotted Grouper (Epinephelus coioides)

The voltage‐dependent anion channel (VDAC) is a highly conserved integral protein of mitochondria in different eukaryotic species. It forms a selective channel in the mitochondrial outer membrane that serves as the controlled pathway for small metabolites and ions. In this study, a VDAC gene, EcVDAC1, was isolated from orange‐spotted grouper (Epinephelus coioides). The EcVDAC1 exhibits ubiquitous expression in various tissues of orange‐spotted grouper and is upregulated in liver, gill, and spleen after stimulation with lipopolysaccharides (LPS). Subcellular localization analysis shows that the EcVDAC1 protein colocalized with the mitochondria. A caspase‐3 assay demonstrates that overexpression of the EcVDAC1 induced apoptotic cell death in fathead minnow cells. The data presented in this study provide new information regarding the relationship between LPS and the EcVDAC1 gene, suggesting that the fish VDAC1 gene may play an important role in antibacterial immune response.     

Identification and characterization of interferon regulatory factor-1 from orange-spotted grouper (<Emphasis Type="Italic">Epinephelus coioides</Emphasis>)

Interferon-regulatory factor 1 (IRF-1) is the first member of IRF family, which is involved in many biological processes such as immune response, antiviral defense, cell growth regulation, and apoptosis. In this study, an IRF-1 gene, EcIRF-1, was isolated and characterized from orange-spotted grouper (Epinephelus coioides). The full-length cDNA of EcIRF-1 is 1,730 bp, including an open reading frame of 906 bp, a 5′-terminal untranslated region (5′-UTR) of 153 bp, and a 3′-UTR of 671 bp. The EcIRF-1 gene consists of 10 exons and 9 introns, spanning over approximate 4.3 kb of genomic sequence. The 5′-UTR sequence contains an exon and an intron, and the 3′-UTR sequence is included in the last exon. Expression analysis by real-time PCR reveals that the EcIRF-1 gene is ubiquitously expressed in various healthy fish tissues, whereas its expression is upregulated in vivo in response to polyinosinic–polycytidylic acid or lipopolysaccharide stimulation. Subcellular localization analysis shows the EcIRF-1 is an intranuclearly localized and immobile protein in the cultured fish cells. Data presented in this paper provide an important base to further understand EcIRF-1 gene function and its regulation associated with interferon immune system in orange-spotted grouper.     

Molecular cloning,characterization of one key molecule of teleost innate immunity from orange-spotted grouper (Epinephelus coioides): Serum amyloid A

The orange-spotted grouper (Epinephelus coioides), a favorite marine food fish, is widely cultured in China and Southeast Asian countries. However, little is known about its acute phase response (APR) caused by viral diseases. Serum amyloid A (SAA) is a major acute phase protein (APP). In this study, a new SAA homologous (EcSAA) gene was cloned from grouper, E. coioides, by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA sequence of SAA was 508 bp and contained a 363 bp open reading frame (ORF) coding for a protein of 121 aa. Similar to other fish known SAA genes, the EcSAA gene contained four exons and three introns. Quantitative real-time PCR analysis revealed that EcSAA mRNA is predominately expressed in liver and gill of grouper. Furthermore, the expression of EcSAA was differentially up-regulated in liver after infection with Staphyloccocus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Recombinant EcSAA (rEcSAA) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-EcSAA serum preparation. The rEcSAA fusion protein was demonstrated to bind to all tested bacteria and yeast, and inhibit the replication of SGIV. Overexpression of EcSAA in grouper spleen (GS) cells could also inhibit the replication of SGIV. These results suggest that EcSAA may be an important molecule in the innate immunity of grouper.     

Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish.     

Molecular cloning and expression analysis of interferon-gamma-inducible-lysosomal thiol reductase gene in orange-spotted grouper, Epinephelus coioides

In mammals, interferon-gamma-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. In this study a cDNA containing the orange-spotted grouper GILT (OsgGILT) coding sequence has been cloned and its complete sequence determined. The full-length cDNA of OsgGILT gene is 1066 bp nucleotides (nt) encoding a protein of 260 amino acids (aa), with a putative molecular weight of 28.7 kDa. The deduced OsgGILT possesses the typical structural feature of mammalian GILT, including an active-site CXXC motif, a GILT signature sequence CQHGX(2)ECX(2)NX(4)C, and 10 conserved cysteines. The result of real-time PCR showed that OsgGILT mRNA was expressed in heart, liver, brain, gill, kidney and muscle and more highly expressed in spleen. The OsgGILT expression is obviously up-regulated in spleen and kidney after induction with LPS, these results suggest that OsgGILT may be involved in the immune response to LPS challenge in orange-spotted grouper.     

Cloning of an orange-spotted grouper Epinephelus coioides heat shock protein 90AB (HSP90AB) and characterization of its expression in response to nodavirus

The heat shock proteins (HSPs) family which consists of HSP90, HSP70, and low molecular mass HSPs are involved in chaperone activity. Here, we report the cloning and characterization of HSP90AB gene from orange-spotted grouper, Epinephelus coioides. The full-length of grouper HSP90AB was 727 amino acids and possessed an ATPase domain as well as an evolutionarily conserved molecular chaperone. The HSP90AB-green fluorescent protein fusion protein was evenly distributed in the cytoplasm. Immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) analyses indicated that the expression of grouper HSP90AB was marginally increased following nodavirus infection. Grouper E. coioides that received HSP90 inhibitor geldanamycin (GA) showed an increase in HSP90AB expression and growth of nodavirus supporting nodavirus replication.     

The expression of two novel orange-spotted grouper (Epinephelus coioides) TNF genes in peripheral blood leukocytes, various organs, and fish larvae

The tumour necrosis factor (TNF) super-family is a group of important cytokines involved in inflammation, apoptosis, cell proliferation, and the general stimulation of the immune system. The TNF gene has been cloned in some bony fish; however, its counterparts are still unidentified in the majority of fish species. In this study, we cloned gTNF-1 and gTNF-2 from the orange-spotted grouper (Epinephelus coioides), an economically important farmed fish. Both genes include 4 exons and 3 introns and encoded 253 and 241 amino acid proteins with a molecular weight of approximately 27 and 26 kDa, respectively. The identity of the putative amino acid sequences between gTNF-1 and gTNF-2 was only 38%. The positions of cysteine residues, a protease cleavage site, and a transmembrane domain sequence derived from gTNF-1 and gTNF-2 were similar to those in other fish and mammalian TNF-α. The mRNA expression levels of the 2 gTNF molecules were evaluated in unstimulated/stimulated peripheral blood leukocytes, various organs, and fish larvae. Following lipopolysaccharide (LPS) treatment, gTNF-2 was expressed at higher levels, was up-regulated more quickly, and was more sensitive to the immune response than gTNF-1. gTNF-1 was constitutively expressed in the thymus, brain, and spleen, but it was also expressed in the heart, head kidney, and trunk kidney after LPS stimulation. gTNF-2 was constitutively expressed in the thymus, head kidney, trunk kidney, spleen, and intestine; further, gTNF-2 was highly expressed in all organs post-LPS stimulation. Finally, the gTNF expression levels were evaluated at various developmental stages in grouper larvae. A higher variation of gTNF expression levels was observed in fish larvae from a contaminated hatchery. This study revealed the different expression patterns of gTNF-1 and gTNF-2. In addition, gTNF-2 was more sensitive to pathogens than gTNF-1; therefore, it may be an appropriate marker for pathogen invasion and the evaluation of the larval rearing environment.     

A genome-wide association study on growth traits in orange-spotted grouper (<Emphasis Type="Italic">Epinephelus coioides</Emphasis>) with RAD-seq genotyping

The orange-spotted grouper, Epinephelus coioides, is one of the most popular fish in China and Southeast Asian countries because of its important economic value. However, molecular mechanism underlying the growth of orange-spotted grouper has never been fully understood. Herein, we performed a genome-wide association study (GWAS) on a natural population of 198 individuals aiming to screen the whole genome of orange-spotted grouper for identification of growth-related loci by restrictionsite associated DNA sequencing. In this research, 261,366 single nucleotide polymorphisms (SNPs) were developed, in which 110 SNPs were identified to be correlated with growth and 20 SNPs were further confirmed to be associated with both body weight and total length. From these identified SNPs, we annotated a total of 34 genes, including adgrb2, csnkza1, cers5, col22a1, creb5, dnd1, dzank1, dnai1, npy2r, fat3, lrrk2, lrp5, map3k9, and so on. Among these candidate genes, npy2r (neuropeptide Y receptor Y2) was reported to play a critical role in growth of the orange-spotted grouper. In addition, population structure, principal component analysis, kinship matrix and linkage disequilibrium were examined to verify the accuracy and reliability of our GWAS results. Our data will also provide a valuable genetic resource for further marker-assisted selection program to improve growth quality in groupers.     

Stimulatory effects of chitinase on growth and immune defense of orange-spotted grouper (Epinephelus coioides)

Chitinase, belonging to either family 18 or family 19 of the glycosylhydrolases, hydrolyze chitin into oligosaccharides. In the present study, the cDNA fragment encoding orange-spotted grouper (Epinephelus coioides) chitinase1 was subcloned into pPIC3.5K vector and expressed in Pichia pastoris GS115. The results showed that a band with the size of about 53 kDa could be detected by SDS-PAGE and Western blot. The recombinant protein of grouper chitinase1 (rgChi1) was added into the fish diet containing shrimp shell chitin for feeding experiment lasting 8 weeks. The weight of orange-spotted grouper, fed with diets containing rgChi1 at 0, 5, 10 and 20 μg/g was calculated on the 2nd, 4th, 6th and 8th weeks, and difference in growth rates was first observed in the 6th week of the feeding period and it kept until the end of the feeding experiment. At the end of 8 weeks feeding trial, the percent weight gain (PWG), growth rate (GR) and specific growth rate (SGR) of fish fed with 10 and 20 μg rgChi1/g feed were significantly higher compared to the control group. The neuropeptide Y (NPY), growth-hormone-releasing hormone (GHRH), growth-hormone (GH), interleukin-1beta (IL-1β), cyclooxygenase-2 (COX-2), superoxide dismutase (SOD) (Cu/Zn) and SOD (Mn) mRNA expression of fish fed with diet containing 10 μg/g or/and 20 μg/g rgChi1 were obviously higher than the control group. The lysozyme (LZM) and total SOD activity of fish fed with diet containing rgChi1 at 10 and 20 μg/g were significantly higher than that of the control. The aspartate aminotransferase (AST)/glutamic oxalacetic transaminases (GOT) activity in 20 μg/g group decreased compared to the control group. These results indicated that the grouper chitinase1 was successfully produced using the P. pastoris expression system and the recombinant protein had obvious effects on growth and immune defense. The mRNA expression and protein secretion of grouper chitinase1 and chitinase2 were significantly stimulated in spleen in response to bacterial lipopolysaccharide (LPS) challenge, strongly suggesting the existence of an innate pathway for local defense against chitin-containing organisms. Moreover, the pathogen such as Escherichia coli and Staphylococcus aureus could be inhibited by the recombinant protein of grouper chitinase1 to a certain extent.     

Orange-spotted grouper (Epinephelus coioides) toll-like receptor 22: molecular characterization, expression pattern and pertinent signaling pathways

The toll-like receptors (TLRs) are an important gene family in host innate immunologic surveillance. The TLR22 gene is an essential member of the TLRs that is only found in aquatic animals and has been detected in some bony fish. Here, a TLR22 homolog, EcTLR22, was characterized in the orange-spotted grouper (Epinephelus coioides) via homology cloning. The 3321 bp full-length cDNA sequence of EcTLR22 was obtained, which included an open reading frame of 2880 bp encoding a putative peptide of 960 amino acids containing three highly typical domains with the characteristics of TLR family members. The deduced amino acid sequence of EcTLR22 showed a relatively high similarity to flounder TLR22. Phylogenetic analysis showed that the orange-spotted grouper TLR22 sequence was clustered with those of Perciforme, such as flounder and croaker. Real-time quantitative PCR analysis revealed broad expression of EcTLR22, with relatively high expression detected in the head kidney, trunk kidney, spleen, peripheral blood leukocytes (PBLs) and heart of orange-spotted grouper. After injection with Vibrio alginolyticus, there was significant up-regulation of the expression of EcTLR22 in the spleen. In evaluating unstimulated/stimulated head kidney leukocytes and spleen leukocytes, a significant increase in EcTLR22 mRNA expression was detected, which implied a sensitive immune response. Furthermore, four important molecules for signal transduction, MyD88, TRIF, TNF-α and IRF3, were chosen to analyze the role of the EcTLR22 signaling pathway in anti-pathogen responses. Upon LPS or Poly I:C challenge, expression of the four genes was induced, with an increasing tendency detected in head kidney leukocytes, suggesting that the four genes might work with EcTLR22 in host defense against pathogenic microbes.     

Myostatin gene organization and nodavirus-influenced expression in orange-spotted grouper (Epinephelus coioides)

The relationship(s) between nodavirus infection and myostatin expression in the skeletal muscle tissue of grouper is unclear. To investigate, the grouper (Epinephelus coioides) myostatin gene was cloned and cDNA was utilized to examine the expression of the gene in skeletal muscle and serum of healthy (uninfected) grouper and fish naturally infected with nodavirus. The myostatin gene comprises three exons and two introns and is transcribed as a 2778-bp mRNA length that encodes a 376-aa precursor protein. All exon–intron boundaries conformed to the consensus sequences. Alignment of the upstream sequences indicated that the grouper myostatin promoter has been highly conserved during evolution. Sequence analyses of 1936 bp of the upstream region revealed ten E-box motifs. The protein was consistent with the predicted molecular weight (approximately 42 kDa) of the unprocessed monomeric precursor protein and the processed myostatin form of the protein secreted into the plasma. Transient transfection studies revealed that the activity of the myostatin promoter decreased in a subset of viral titers. Grouper naturally infected with nodavirus displayed downregulation of the myostatin protein.     

GRIM-19 inhibits the STAT3 signaling pathway and sensitizes gastric cancer cells to radiation

Gastric cancer is one of the most common malignancies, and radiation resistance is one of the key obstacles in gastric cancer treatment. In this study, we demonstrate that “genes associated retinoid–IFN induced mortality-19” (GRIM-19) expression was lower in patients with radiotherapy-resistant tumors compared to patients with radiotherapy-sensitive tumors. In order to further investigate the effects of GRIM-19 expression on the radiation response in gastric cancer cells, we established BGC-803 clones stably expressing exogenous GRIM-19. We found that the percentage of apoptotic cells was higher in cells expressing GRIM-19 than untransfected cells post-radiation treatment. Furthermore, caspase-3, -8, and -9 activity was significantly increased in GRIM-19-expressing cells compared to untransfected cells after radiation. Finally, we demonstrate that expression of GRIM-19 in BGC-803 cells suppresses accumulation of STAT3. Collectively, these data show that GRIM-19 expression sensitizes BGC-803 cells to radiation, and this is likely due to suppression of STAT3 accumulation. In summary, our results indicate that GRIM-19 expression might be a useful therapy to enhance apoptosis in gastric cancer cells in response to radiation treatment.     

Orange-spotted grouper (Epinephelus coioides) orexin: molecular cloning, tissue expression, ontogeny, daily rhythm and regulation of NPY gene expression

Orexin-A and -B, collectively called orexins, are hypothalamic neuropeptides involved in the regulation of food intake, sleep and energy balance. In this study, the full-length cDNA of prepro-orexin was isolated from the hypothalamus of orange-spotted grouper (Epinephelus coioides) using RT-PCR and RACE. The grouper prepro-orexin cDNA is 711 bp in length and encodes a 149-amino acid precursor protein that contains a 46-amino acid signal peptide, a 43-amino acid mature orexin-A peptide, a 27-amino acid mature orexin-B peptide and a 33-amino acid C terminus of unknown function. The tissue distribution and ontogeny of prepro-orexin were examined by quantitative real-time PCR. We found that the prepro-orexin mRNA is widely expressed in brain and peripheral tissues, with abundant expression in the hypothalamus. During the embryonic development, prepro-orexin mRNA was first detected in neurula stage embryos, and its expression gradually increased during the remainder of embryogenesis. Our analysis of grouper hypothalamic prepro-orexin expression showed that prepro-orexin mRNA levels were greater in the light phase than in the dark phase and increased significantly at meal-time. Intraperitoneal injection of orexin-A caused a dose-related increase in hypothalamus NPY mRNA expression level after 4 h. Orexin-A also increased NPY mRNA expression level from static hypothalamic fragments incubation. Our results imply that orexin may be involved in feeding in the orange-spotted grouper and orexin-A is a stimulator of NPY mRNA expression in vivo and in vitro.