Molecular and Structural Basis of Receptor Binding and Signaling of a Fish Type I IFN with Three Disulfide Bonds.

In mammals, type I IFNs, which commonly contain one or two disulfide bonds, activate the JAK-STAT signaling pathway through binding to the common cell surface receptor formed by IFN-α/ß receptor (IFNAR)1 and IFNAR2 subunits. Although type I IFNs are also known to be essential for antiviral defense in teleost fish, very little is known about mechanisms underlying the recognition of fish type I IFNs by associated receptors. In this study, we demonstrate that a type I IFN of large yellow croaker Larimichthys crocea (LcIFNi), belonging to a new subgroup of fish type I IFNs, triggers antiviral response via the conserved JAK-STAT pathway through stable binding with a heterodimeric receptor comprising subunits LcCRFB5 and LcCRFB2. LcIFNi binds to LcCRFB5 with a much higher affinity than to LcCRFB2. Furthermore, we determined the crystal structure of LcIFNi at a 1.39 Å resolution. The high-resolution structure is, to our knowledge, the first reported structure of a type I IFN with three disulfide bonds, all of which were found to be indispensable for folding and stability of LcIFNi. Using structural analysis, mutagenesis, and biochemical assays, we identified key LcIFNi residues involved in receptor interaction and proposed a structural model of LcIFNi bound to the LcCRFB2-LcCRFB5 receptor. The results show that LcIFNi-LcCRFB2 exhibits a similar binding pattern to human IFN-ω-IFNAR2, whereas the binding pattern of LcIFNi-LcCRFB5 is quite different from that of IFN-ω-IFNAR1. Altogether, our findings reveal the structural basis for receptor interaction and signaling of a type I IFN with three disulfide bonds and provide new insights into the mechanisms underlying type I IFN recognition in teleosts.

Whole genome sequencing of a snailfish from the Yap Trench (~7,000 m) clarifies the molecular mechanisms underlying adaptation to the deep sea.

Hadal environments (depths below 6,000 m) are characterized by extremely high hydrostatic pressures, low temperatures, a scarce food supply, and little light. The evolutionary adaptations that allow vertebrates to survive in this extreme environment are poorly understood. Here, we constructed a high-quality reference genome for Yap hadal snailfish (YHS), which was captured at a depth of ~7,000 m in the Yap Trench. The final YHS genome assembly was 731.75 Mb, with a contig N50 of 0.75 Mb and a scaffold N50 of 1.26 Mb. We predicted 24,329 protein-coding genes in the YHS genome, and 24,265 of these genes were successfully functionally annotated. Phylogenetic analyses suggested that YHS diverged from a Mariana Trench snailfish approximately 0.92 million years ago. Many genes associated with DNA repair show evidence of positive selection and have expanded copy numbers in the YHS genome, possibly helping to maintain the integrity of DNA under increased hydrostatic pressure. The levels of trimethylamine N-oxide (TMAO), a potent protein stabilizer, are much higher in the muscles of YHS than in those of shallow-water fish. This difference is perhaps due to the five copies of the TMAO-generating enzyme flavin-containing monooxygenase-3 gene (fmo3) in the YHS genome and the abundance of trimethylamine (TMA)-generating bacteria in the YHS gut. Thus, the high TMAO content might help YHS adapt to high hydrostatic pressure by improving protein stability. Additionally, the evolutionary features of the YHS genes encoding sensory-related proteins are consistent with the scarce food supply and darkness in the hadal environments. These results clarify the molecular mechanisms underlying the adaptation of hadal organisms to the deep-sea environment and provide valuable genomic resources for in-depth investigations of hadal biology.

Molecular characterization, expression analysis and cellular location of IL-4/13 receptors in large yellow croaker (Larimichthys crocea).

Interleukin (IL)-4 and IL-13 are closely related class I cytokines that play key roles in the T helper (Th)-2 immune response via heterodimeric receptors. IL-4 signals via both the type I (IL-4Rα/γc) and type II (IL-4Rα/IL-13Rα1) receptor complexes, while IL-13 signals only via the type II receptor complex. IL-13Rα2 is traditionally considered a "decoy" receptor for IL-13. However, the IL-4/13 system and its response to pathogenic infection are still not fully understood in fish. In this study, we identified four IL-4/13 receptor subunit genes in the large yellow croaker (Larimichthys crocea): LcIL-4Rα1, LcIL-4Rα2, LcIL-13Rα1, and LcIL-13Rα2. Sequence analysis showed that these receptors possessed typical characteristic domains, including a signal peptide, two fibronectin type III (FN III)-like domains, and a transmembrane domain, but their cytoplasmic regions were not well conserved. The mRNA and protein of the four IL-4/13 receptors were constitutively expressed in all examined tissues of large yellow croaker. Their mRNAs were also detected in primary head kidney macrophages (PKMs), primary head kidney granulocytes (PKGs), and primary head kidney lymphocytes (PKLs). Immunofluorescence assay further showed that LcIL-4Rα and LcIL-13Rα1 were expressed on the membrane of IgM + B cells. After stimulation by Vibrio alginolyticus and poly (I:C) (a viral dsRNA mimic), the mRNA levels of LcIL-4/13 receptors were significantly upregulated in the head kidney and spleen. Their mRNA levels were also upregulated in head kidney leukocytes in response to poly (I:C) and lipopolysaccharide (LPS) treatment. Moreover, both recombinant LcIL-4/13A and LcIL-4/13B upregulated LcIL-4Rα1 and LcIL-4Rα2 in primary leukocytes, but only recombinant LcIL-4/13A upregulated LcIL-13Rα1 and LcIL-13Rα2. These results indicated that LcIL-4/13 receptors, containing conserved functional domains, may be involved in the IL-4/13-mediated immune response to pathogenic infections in the large yellow croaker.

Molecular characterization and role in virus infection of Beclin-1 in large yellow croaker (Larimichthys crocea).

Beclin-1, the ortholog of yeast autophagy-related gene 6 (Atg6), has a central role in autophagy, which has been linked to diverse biological processes including immunity, development, tumor suppression, and lifespan extension. However, understanding of function of fish Beclin-1 is limited now. In this study, the complete Beclin-1 cDNA of large yellow croaker Larimichthys crocea (LcBeclin-1) was cloned, whose open reading frame (ORF) is 1344 bp long and encodes a protein of 447 amino acids (aa). The deduced LcBeclin-1 possesses a typical Bcl-2 homology domain 3(BH3) and an APG6 domain that contains a central coiled-coil domain (CCD, residues 174 to 231) and a C-terminal evolutionarily conserved domain (ECD, residues 241 to 334). LcBeclin-1 shared a high amino acid identity of 81.66-98.66% with reported Beclin-1 molecules from other vertebrate species. LcBeclin-1 gene was constitutively expressed in all tissues tested, with the highest levels in heart. LcBeclin-1 transcripts were also detected in primary head kidney granulocytes (PKGs), primary head kidney macrophages (PKMs), primary head kidney leukocytes (PKLs), and large yellow croaker head kidney cell line (LYCK), and were significantly upregulated by poly (I:C) in PKMs and LYCK cells. Subcellular localization showed that LcBeclin-1 was evenly distributed in the cytoplasm and nucleus of LYCK cells. Overexpression of LcBeclin-1 significantly increased the replication of SVCV, as evidenced by increased severity of the cytopathic effects, enhanced viral titre, and upregulated transcriptional levels of viral genes. Further studies showed that LcBeclin-1 induced the occurrence of autophagy in LYCK cells. Additionally, LcBeclin-1 also decreased the expression levels of large yellow croaker interferons (IFNs; IFNc, IFNd, and IFNh), interferon regulatory factor 3 (IRF3) and IRF7, IFN-stimulated genes (ISGs; Mx, PKR, and Viperin) in LYCK cells. All these data suggest that LcBeclin-1 promoted the viral replication possibly by inducing autophagy or negatively modulating IFN response, which will help us to further understand the function of fish Beclin-1.

Molecular and functional identification of a ß-defensin homolog in large yellow croaker (Larimichthys crocea).

ß-defensin (BD) is a cysteine-rich cationic antibacterial peptide that is active against a wide range of bacteria. Here, a ß-defensin homolog (LcBD2) was identified in large yellow croaker (Larimichthys crocea). The open reading frame of LcBD2 contains 195 nucleotides, encoding a protein of 64 amino acids that possesses a typical arrangement of six conserved cysteine residues (C31 , C37 , C41 , C53 , C59 and C60 ). LcBD2 transcripts were constitutively expressed in all examined tissues and significantly increased in head kidney, spleen and gills by Vibrio alginolyticus. The synthetic LcBD2 peptide imparted antimicrobial effects on both Gram-negative bacteria (V. campbellii, V. parahaemolyticus, V. alginolyticus, V. harveyi and Pseudomonas plecoglossicida) and Gram-positive bacteria (Bacillus subtilis). We also observed that after treatment with synthetic LcBD2 peptide, numerous blisters appeared on the membrane of P. plecoglossicida, which in turn may result in cell membrane breakage and bacterial death. Moreover, the synthetic LcBD2 peptide significantly upregulated the expression levels of TNF-α2, IL-1ß and CXCL8_L1 in monocytes/macrophages, while downregulated expression level of IL-10. The LcBD2 peptide also remarkedly enhanced the phagocytosis of monocytes/macrophages. These results indicate that LcBD2 not only protects large yellow croaker against multiple bacterial pathogens but also plays a role in activation of monocytes/macrophages.

Transcriptome analysis reveals molecular strategies in gills and heart of large yellow croaker (Larimichthys crocea) under hypoxia stress.

The gills and heart are two major targets of hypoxia in fish. However, the molecular responses in fish gills and heart to hypoxia challenge remain unclear. Here, RNA-Seq technology was used to study the gene expression profiles in gills and heart of large yellow croaker (Larimichthys crocea) at 6, 24, and 48 h after hypoxia stress. A total of 1,546 and 2,746 differentially expressed genes (DEGs) were identified in gills and heart, respectively. Expression changes of nine genes in each tissue were further validated by the qPCR. Based on KEGG and Gene ontology enrichments, we found that various innate immunity-related genes, such as complement components (C1qs, C2, C3, C6, and C7), chemokines (CCL3, CCL17, CCL19, CCL25, and CXCL8_L3), chemokine receptors (CCR9, CXCR1, and CXCR3), and nitric oxide synthase (NOS), were significantly down-regulated in gills and/or heart, suggesting that innate immune processes mediated by these genes may be inhibited by hypoxia. The genes involved in both glycolysis pathway (LDHA) and tricarboxylic acid cycle (IDH2 and OGDH) were up-regulated in gills and heart of hypoxic large yellow croakers, possibly because gill and heart tissues need enough energy to accelerate gas exchange and blood circulation. Hypoxia also affected the ion transport in gills of large yellow croaker, through down-regulating the expression levels of numerous classical ion transporters, including HVCN1, SLC20A2, SLC4A4, RHBG, RHCG, and SCN4A, suggesting an energy conservation strategy to hypoxia stress. All these results indicate that the immune processes, glycolytic pathways, and ion transport were significantly altered in gills and/or heart of large yellow croaker under hypoxia, possibly contributing to maintain cellular energy balance during hypoxia. Our data, therefore, afford new information to understand the tissue-specific molecular responses of bony fish to hypoxia stress.

Transcriptome analysis reveals new insights into immune response to hypoxia challenge of large yellow croaker (Larimichthys crocea).

Fish live in direct contact with aquatic environment, which exhibits much wider temporal and spatial variations in oxygen content. The molecular mechanisms underlying fish response to hypoxia have become a subject of great concern in recent years. In the present study, we performed transcriptome analysis of spleen and head kidney tissues from large yellow croaker (Larimichthys crocea) at 6 h, 24 h and 48 h after hypoxia challenge. A total of 2,499 and 3,685 differentially expressed genes (DEGs) were obtained in spleen and head kidney, respectively. The expression changes of 10 selected genes in each tissue were further validated by quantitative real-time PCR. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichments revealed that numerous DEGs were immune genes, involved in multiple immune-relevant pathways. In spleen, several pattern recognition receptors (PRRs), including Toll-like receptors (TLR1, TLR2-1, TLR2-2, TLR5 and TLR8), Fucolectins (FUCL1, FUCL4 and FUCL5) and macrophage mannose receptor (MRC1), were significantly down-regulated, suggesting that the immune processes mediated by these PRRs may be suppressed by hypoxia stress. However, some PRRs (FUCL4, FUCL5 and MRC1) and other innate immunity genes, such as C-type lectin domain gene family members, chemokines, chemokine receptors and complement components were up-regulated in head kidney, which may be due to the increases in phagocytosis and cytokine secretion by macrophages after hypoxic stimulus. The expression of genes involved in B cell receptor signaling pathway, Natural killer cell-mediated cytotoxicity and NF-κB signaling pathway decreased rapidly, but regained normal or increased over time, suggesting an early adjustment pattern of fish immune response to cope with hypoxia stress. Moreover, the anaerobic ATP-generating pathway was activated and energy consumption processes were repressed concurrently in both spleen and head kidney. These data provide valuable information for understanding the tissue-specific and temporal changes of immune gene expression in hypoxic large yellow croakers.

Molecular characterization of a new fish specific chemokine CXCL_F6 in large yellow croaker (Larimichthys crocea) and its role in inflammatory response.

Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in regulating cell migration and activation. Currently, five subgroups of fish specific CXC chemokines, named CXCL_F1-CXCL_F5, have been identified in teleost fish. However, understanding of the functions of these fish specific CXC chemokines is still limited. Here, a new member of fish specific CXC chemokines, LcCXCL_F6, was cloned from large yellow croaker Larimichthys crocea. Its open reading frame (ORF) is 369 nucleotides long, encoding a peptide of 122 amino acids (aa). The deduced LcCXCL_F6 protein contains a 19-aa signal peptide and a 103-aa mature polypeptide, which has four conserved cysteine residues (C28, C30, C56, and C72), as found in other known CXC chemokines. Phylogenetic analysis showed LcCXCL_F6 formed a separate clade with sequences from other fish species, tentatively named CXCL_F6, distinct from the clades formed by fish CXCL_F1-5 and mammalian CXC chemokines. The LcCXCL_F6 transcripts were constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney tissues by poly (I:C) and Vibrio alginolyticus. Its transcripts were also detected in primary head kidney leukocytes (HKLs), peripheral blood leucocytes (PBLs), and large yellow croaker head kidney (LYCK) cell line, and significantly up-regulated by poly(I:C), lipopolysaccharide (LPS), and peptidoglycan (PGN) in HKLs. Recombinant LcCXCL_F6 protein (rLcCXCL_F6) could not only chemotactically attract monocytes/macrophages and lymphocytes from PBLs, but also enhance NO release and expression of proinflammatory cytokines (TNF-α, IL-1ß, and CXCL8) in monocytes/macrophages. These results indicate that LcCXCL_F6 plays a role in mediating the inflammatory response.

Molecular characterization and functional activity of CXCL8_L3 in large yellow croaker Larimichthys crocea.

CXCL8, also called interleukin-8, is a typical CXC chemokine that plays a key role in promoting inflammation. Phylogenetically, fish CXCL8 chemokines can be divided into three subgroups, CXCL8_L1, CXCL8_L2, and CXCL8_L3, of which CXCL8_L3 is a new subgroup. The CXCL8_L3 gene sequences have been reported in many fish species, but their function remains unknown. Here, a CXCL8_L3 (LycCXCL8_L3) gene was cloned from large yellow croaker Larimichthys crocea. Its open reading frame (ORF) was 309 nucleotides long, encoding a peptide of 102 amino acids. The deduced LycCXCL8_L3 protein contains an 18-aa signal peptide and an 84-aa mature polypeptide, which has four conserved cysteine residues (C30, C32, C57, and C73) as found in other known CXCL8 chemokines. Phylogenetic analysis showed LycCXCL8_L3 formed a major clade with CXCL8_L3 sequences from other fish species. The LycCXCL8_L3 transcript was constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney tissues by inactivated trivalent bacterial vaccine. The LycCXCL8_L3 transcript was also detected in peripheral blood leukocytes (PBLs), primary head kidney macrophages (PKM), and large yellow croaker head kidney cell line (LYCK), with the highest levels in PKM. Recombinant LycCXCL8_L3 (rLycCXCL8_L3) protein could not only chemotactically attract lymphocytes and eosinophils in PBLs, but also enhance the respiratory burst activity of PKM. These results indicate that LycCXCL8_L3 may play an important role in the inflammatory response of large yellow croaker. To our knowledge, this is the first report on functional study of the CXCL8_L3 in fish.

Identification of two IL-4/13 homologues in large yellow croaker (Larimichthys crocea) revealed their similar roles in inducing alternative activation of monocytes/macrophages.

Mammalian interleukin-4 (IL-4) and -13 (IL-13), two anti-inflammatory T helper cell type 2 (Th2) cytokines, play the central roles in mediating the alternative activation of monocytes/macrophages (MO/Mφs). However, exact functions in MO/Mφs polarization of IL-4/13 homologues in teleost fish remain largely unknown. In this study, we identified two IL-4/13 homologues from large yellow croaker Larimichthys crocea, LcIL-4/13A and LcIL-4/13B, which share low amino acid sequence identities to the known fish IL-4/13 molecules. Phylogenetic analysis showed that LcIL-4/13A is evolutionarily closely related to Dicentrarchus labrax IL-4/13A, and LcIL-4/13B to Takifugu rubripes IL-4/13B. The two LcIL-4/13 genes were constitutively expressed in all examined tissues, but with different expression levels. Both LcIL-4/13A and LcIL-4/13B were up-regulated by inactivated trivalent bacterial vaccine in the head kidney, and LcIL-4/13B appeared more responsive to bacterial vaccine than LcIL-4/13A. Recombinant LcIL-4/13A and LcIL-4/13B proteins (rLcIL-4/13A and rLcIL-4/13B) produced in Escherichia coli could significantly decrease production of reactive oxygen species (ROS) and nitrogen oxide (NO) in the head kidney MO/Mφs from large yellow croaker. Furthermore, rLcIL-4/13A and rLcIL-4/13B obviously down-regulated expression of pro-inflammatory cytokine (IL-1ß and TNF-α) and inducible NO synthase (iNOS) genes in MO/Mφs, while they increased mRNA expression of anti-inflammatory cytokines (TGF-ß and VEGF) and arginase-2. Additionally, the phagocytic activity of MO/Mφs was also inhibited by rLcIL-4/13A or rLcIL-4/13B. All these results therefore indicated that both LcIL-4/13A and LcIL-4/13B, although exhibiting a lower degree of sequence identity of 15.6% and differential expression pattern, have the similar roles in promoting alternative activation of head kidney MO/Mφs.

Genome sequencing of the perciform fish Larimichthys crocea provides insights into molecular and genetic mechanisms of stress adaptation.

The large yellow croaker Larimichthys crocea (L. crocea) is one of the most economically important marine fish in China and East Asian countries. It also exhibits peculiar behavioral and physiological characteristics, especially sensitive to various environmental stresses, such as hypoxia and air exposure. These traits may render L. crocea a good model for investigating the response mechanisms to environmental stress. To understand the molecular and genetic mechanisms underlying the adaptation and response of L. crocea to environmental stress, we sequenced and assembled the genome of L. crocea using a bacterial artificial chromosome and whole-genome shotgun hierarchical strategy. The final genome assembly was 679 Mb, with a contig N50 of 63.11 kb and a scaffold N50 of 1.03 Mb, containing 25,401 protein-coding genes. Gene families underlying adaptive behaviours, such as vision-related crystallins, olfactory receptors, and auditory sense-related genes, were significantly expanded in the genome of L. crocea relative to those of other vertebrates. Transcriptome analyses of the hypoxia-exposed L. crocea brain revealed new aspects of neuro-endocrine-immune/metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia. Proteomics data demonstrate that skin mucus of the air-exposed L. crocea had a complex composition, with an unexpectedly high number of proteins (3,209), suggesting its multiple protective mechanisms involved in antioxidant functions, oxygen transport, immune defence, and osmotic and ionic regulation. Our results reveal the molecular and genetic basis of fish adaptation and response to hypoxia and air exposure. The data generated by this study will provide valuable resources for the genetic improvement of stress resistance and yield potential in L. crocea.

Molecular characterization and expression analysis during embryo development of CD4-1 homologue in large yellow croaker Larimichthys crocea.

CD4+ helper T (Th) cells are a master component of the adaptive immune response. CD4 is one of the most effective surface markers for identifying Th cells. In the present study, we cloned and characterized a CD4-1 homologue, LycCD4-1, from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCD4-1 is 1695 bp long, encoding a protein of 462 amino acids. The deduced LycCD4-1 protein has a typical domain architecture as found in mammalian CD4 molecules, including a signal peptide, four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane region, and a CXC signaling motif in the cytoplasmic tail. Four N-glycosylation sites and 10 cysteine residues were also found in LycCD4-1, which may be essential for its tertiary structure and succeeding function. Homology comparison showed that LycCD4-1 has 27.9-58.4% identity to other teleost fish CD4-1 molecules, and 16.4-20% identity to those of higher vertebrates. Genomic analysis revealed that the LycCD4-1 gene consisted of nine exons and eight introns and exhibited a similar exon-intron organization to other species CD4 genes except for a different intron length. Phylogenetic analysis showed that LycCD4-1 form a cluster with CD4-1 molecules in other fish species. The LycCD4-1 was constitutively expressed in all tissues tested, with a higher expression in gills and spleen. LycCD4-1 mRNA expression in the spleen and head kidney tissue was increased by poly (I:C) at 48 h, whereas its expression levels were somewhat down-regulated at 6 h and 72 h after bacterial vaccine induction in spleen. Unexpectedly, LycCD4-1 mRNA could be detected in each stage of early embryo development since fertilized eggs, with a higher level before mid-gastrula and the highest level in high blastocysts. These results will be helpful for better understanding molecular characteristics of CD4-1 and tracing origin of CD4-1+ cell precursors in fish.

Functional activities of interferon gamma in large yellow croaker Larimichthys crocea.

Interferon gamma (IFN-γ) is a T helper cell type 1 (Th1) cytokine that plays important roles in almost all phases of immune and inflammatory responses. Although IFN-γ gene in large yellow croaker Larimichthys crocea has been reported, little is known about its bioactivity. In this study, large yellow croaker IFN-γ (LycIFN-γ) gene was found to be constitutively expressed in all tissues tested, with the highest levels in blood and heart. Based on stimulation with polyinosinic-polycytidylic acid [poly (I:C)] or inactivated trivalent bacterial vaccine, LycIFN-γ mRNA was significantly increased in spleen and head kidney tissues. LycIFN-γ transcripts were also detected in head kidney granulocytes, primary head kidney macrophages (PKM), head kidney leukocytes, and large yellow croaker head kidney cell line (LYCK), and were significantly up-regulated by poly(I:C) or lipopolysaccharide (LPS) in head kidney leukocytes. Recombinant LycIFN-γ protein (rLycIFN-γ) produced in Escherichia coli could enhance respiratory burst responses in PKM. Furthermore, rLycIFN-γ not only induced the expression of iNOS gene and release of NO, but also up-regulated the expression of proinflammatory cytokines TNF-α and IL-1ß in PKM. These findings therefore indicated that LycIFN-γ has a role in mediating inflammatory response. In addition, rLycIFN-γ could significantly up-regulate expression of IFN-γ receptor CRFB13, signal transduction factor STAT1, transcription factors IRF1 and T-bet, and Th1-related cytokines IFN-γ and IL-2 in head kidney leukocytes, suggesting that LycIFN-γ may have the potential to promote Th1 immune response in large yellow croaker. Taken together, our results show that LycIFN-γ may be involved in inflammatory response and promote Th1 immune response as its mammalian counterpart.

Identification of a fish specific chemokine CXCL_F2 in large yellow croaker (Larimichthys crocea) reveals its primitive chemotactic function.

Chemokines are a superfamily of cytokines regulating immune cell migration under both inflammatory and normal physiological conditions. Currently, a number of fish specific CXC chemokines, named as CXCL_F1-5, have been identified in several species. However, understanding of their functional characteristics is still limited. In this study, we identified a fish specific chemokine CXCL_F2 (LycCXCL_F2) from large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of LycCXCL_F2 is 348 nucleotides long, encoding a protein of 115 amino acids (aa). The deduced LycCXCL_F2 protein contains a 20-aa signal peptide and a 95-aa mature polypeptide. Phylogenetic analysis showed that LycCXCL_F2 fell into a major clade formed by CXCL_F2 sequences and was separated from CXCL_F1 and CXCL_F3-5 subgroups. LycCXCL_F2 mRNA transcript was constitutively expressed in various tissues, with the highest levels in spleen and head kidney. After stimulation with inactivated trivalent bacterial vaccines, LycCXCL_F2 mRNA transcription was significantly increased in both spleen and head kidney. Moreover, recombinant LycCXCL_F2 protein exhibited obvious chemotaxis to monocytes, lymphocytes and eosnophils of PBLs isolated from large yellow croaker, but could not induce the respiratory burst of macrophages. These results indicate that this fish specific CXC chemokine LycCXCL_F2 possesses primitive chemotactic activity and may play a role in immune response in large yellow croaker.

A cystatin F homologue from large yellow croaker (Larimichthys crocea) inhibits activity of multiple cysteine proteinases and Ii chain processing in vitro.

Cystatin F, a member of the family II cystatins, plays important roles in immune response-related processes through inhibiting specific enzyme targets. In this study, a cystatin F homologue, LycCysF, was identified and characterized from large yellow croaker (Larimichthys crocea). The deduced LycCysF protein exhibits a typical structural feature of type II cystatins, including three evolutionally conserved motifs, Gly(35), QVVRG(79-83) and PW(130-131). Tissue expression analysis showed that LycCysF mRNA was expressed in all tissues examined, albeit at different levels. Recombinant LycCysF (rLycCysF) produced in Pichia pastoris could inhibit the activity of multiple cysteine proteases, including papain, legumain and recombinant large yellow croaker cathepsin B, L and S. Moreover, rLycCysF could inhibit the Ii chain processing by recombinant cathepsin S in vitro. These data suggest that LycCysF may participate in regulation of cathepsins and MHC-II associated Ii chain processing. In addition, mammalian cystatin F is produced as an inactive dimer, becoming activated by proteolysis in the endo/lysosome of immune cells and then exerts its function of regulating downstream proteases activity. However, the N-terminal extension and two additional cysteine residues responsible for dimer formation are absent in LycCysF and cystatin F from other fish species, reptiles and Aves, indicating that these proteins can not form dimer and may regulate the proteases activity via an alternate pathway distinct from mammalian cystatin F. To our knowledge, this is the first report on molecular characteristics of a teleost cystatin F and its role in Ii chain processing.

Molecular and functional characterization of Toll-like receptor 21 in large yellow croaker (Larimichthys crocea).

Toll-like receptor 21 (TLR21) is a non-mammalian TLR that functions similar to mammalian TLR9 in recognizing CpG DNA. In the present study, we identified a TLR21 homologue, LycTLR21, from large yellow croaker (Larimichthys crocea). The complete coding sequence of LycTLR21 is 2946 nucleotides long, encoding a protein of 981 amino acids. The deduced LycTLR21 protein has typical TLR domain architecture, including a signal peptide, 13 leucine-rich repeats (LRRs) in the extracellular region, a transmembrane region, and a cytoplasmic Toll-Interleukin-1 receptor (TIR) domain. Phylogenetic analysis showed that LycTLR21 falls into a major clade formed by all fish TLR21 sequences and is closely related to TLR21 in Epinephelus coioides and Oplegnathus fasciatus. LycTLR21 mRNA was constitutively expressed in all tissues tested, with higher levels in immune-related tissues, such as spleen, head kidney, and gills. Upon stimulation with inactivated trivalent bacterial vaccine, LycTLR21 mRNA was significantly increased in these three tissues. Overexpression of a chimeric plasmid containing the extracellular domain of human cluster of differentiation 4 (CD4) and the transmembrane and cytoplasmic domains of LycTLR21 could activate NF-κB, but not IFN-ß in Chinese hamster ovary (CHO) cells, suggesting that LycTLR21 could mediate activation of NF-κB. LycTLR21 could specifically recognize three CpG-oligodeoxynucleotides (CpG-ODNs), CpG-ODN 1826, 2006, and 2007, but not other CpG-ODNs detected, poly(I:C), lipopolysaccharide (LPS), and lipoteichoic acid (LTA-SA). These three CpG-ODNs were found to significantly up-regulate the expression of LycTLR21 and downstream proinflammatory cytokines IL-1ß and IL-6 of NF-κB pathway in large yellow croaker head kidney (LYCK) cells. In addition, the expression levels of LycTLR21, c-Rel subunit of NF-κB, IL-1ß and IL-6 genes were quickly increased in the spleen and head kidney by bacterial infection, suggesting that LycTLR21 signaling pathway may play a role in immune response to bacterial infection.

Molecular characterization and evolution analysis of five interleukin-17 receptor genes in large yellow croaker Larimichthys crocea.

Interleukin-17s (IL-17s) play critical roles in inflammatory response and host defense against extracellular pathogens. IL-17s induce the immune response signaling through the specific IL-17 receptors (IL-17Rs) that consist of five members (IL-17RA to E). In the present work, we have identified the five IL-17R orthologs (LycIL-17Rs) from large yellow croaker Larimichthys crocea. The deduced protein of each LycIL-17R exhibits a typical IL-17R domain architecture, including a signal peptide, the extracellular FNIII domain (IL-17RA/RB/RD) or IL-17_R_N domain (IL-17RC/RE), a transmembrane domain, and a SEFIR domain in cytoplasmic region. In particular, the extracellular regions of teleost IL-17RB are much shorter than those in mammals and lack an FNIII domain (FN2). Phylogenetic tree shows that IL-17Rs are classified into two main groups: IL-17RA/RB/RD group and IL-17RC/RE group, which is distinct from previous proposal that grouped IL-17RB into IL-17RC/RE. The surrounding genes of IL-17Rs are conservatively aligned in genomes between teleosts and mammals. The five LycIL-17Rs were constitutively expressed in all tissues examined, but with different expression patterns. Aeromonas hydrophila infection significantly upregulated LycIL-17RA, RC, RD and RE in both mucosal tissue (gills) and systemic immune tissues (head kidney and spleen), while the increase of LycIL-17RB expression could be detected in gills, indicating that LycIL-17Rs may be involved in host defense against bacterial infection. Thus, these results suggest that teleost IL-17Rs may function in mediating immune response as their mammalian orthologs. To our knowledge, this is the first report of molecular characterization of the five IL-17Rs (IL-17RA/RB/RD and IL-17RC/RE) in teleost fish.

Identification and characterization of a novel Toll-like receptor 2 homologue in the large yellow croaker Larimichthys crocea.

Toll-like receptors (TLRs) are key components of innate immunity that play significant roles in immune defence against pathogen invasion. In the present study, we identified a novel TLR2 homologue (LycTLR2b) in large yellow croaker (Larimichthys crocea) that shared low sequence identity with the previously reported large yellow croaker TLR2 (tentatively named LycTLR2a). The full-length cDNA of LycTLR2b was 2926 nucleotides (nt) long and encoded a protein consisting of 797 amino acids (aa). The deduced LycTLR2b protein exhibited a typical TLR domain architecture including a signal peptide, seven leucine-rich repeats (LRRs) in the extracellular region, a transmembrane domain, and a Toll-Interleukin 1 receptor (TIR) domain in the cytoplasmic region. Phylogenetic analysis showed that both LycTLR2a and LycTLR2b fall into a major clade formed by all TLR2 sequences, and are divided into two distinct branches. Genomic organization revealed that the LycTLR2b gene lacks intron, which is similar to zebrafish and human TLR2 genes, whereas the LycTLR2a gene contains multiple introns, as found in damselfish TLR2a and Fugu TLR2 genes. Syntenic analysis suggested that the occurrence of LycTLR2a and LycTLR2b may result from a relatively recent genome duplication event. LycTLR2b mRNA was constitutively expressed in all tissues examined although at different levels. Following bacterial vaccine challenge, LycTLR2b expression levels were significantly up-regulated in both spleen and head kidney tissues. Taken together, these results indicated that two different TLR2 homologues, which may play roles in antibacterial immunity, exist in large yellow croaker.

Molecular characterization and biological effects of a CXCL8 homologue in large yellow croaker (Larimichthys crocea).

CXCL8 also called interleukin-8, is a CXC-type chemokine that plays a key role in promoting inflammation. Three subgroups of CXCL8 homologues have been reported in teleost fish, including CXCL8_L1, CXCL8_L2 and CXCL8_L3. In the present study, we identified a CXCL8 homologue belonging to CXCL8_L1 subgroup (LycCXCL8_L1) in large yellow croaker (Larimichthys crocea) that shares low identity to the previously reported large yellow croaker CXCL8 (LycCXCL8). The full-length cDNA of LycCXCL8_L1 is 716 nucleotides (nt) long and encodes a protein consisting of 99 amino acids (aa) with a putative molecular weight of 11.2 kDa. The deduced LycCXCL8_L1 protein contains a 22-aa signal peptide and a 77-aa mature polypeptide, which possesses an arrangement of four cysteines typical of other known CXC chemokines (C(34), C(36), C(60), and C(77)). Genomic analysis revealed that the LycCXCL8_L1 gene consisted of four exons and three introns and exhibited a similar exon-intron organization to LycCXCL8 and other species CXCL8 genes except for a different intron length. Phylogenetic analysis showed that both LycCXCL8_L1 and LycCXCL8 belong to CXCL8_L1 subgroup. LycCXCL8_L1 mRNA was constitutively expressed in all tissues examined although at different levels. Upon bacterial vaccine induction, LycCXCL8_L1 mRNA expression was rapidly increased in the spleen and head kidney tissues. Recombinant LycCXCL8_L1 and LycCXCL8 proteins produced in Escherichia coli both induced chemotaxis and superoxide production in peripheral blood leucocytes from large yellow croaker. These results indicate that two CXCL8_L1 molecules exist in large yellow croaker and play roles in inflammatory response.

Cathepsin S, but not cathepsin L, participates in the MHC class II-associated invariant chain processing in large yellow croaker (Larimichthys crocea).

Two cysteine proteases, cathepsin S (CatS) and cathepsin L (CatL), have been identified as the key enzymes involved in the processing of invariant chain (Ii chain) in mammals. However, little is known about the roles of fish cathepsins in the Ii chain processing. In this study, large yellow croaker cathepsin S (LycCatS) and L (LycCatL) were identified and characterized. Based on the sequence comparison and phylogenetic analysis, both LycCatS and LycCatL are highly conserved to their counterparts in teleost. These two cathepsins were constitutively expressed in all tissues and immune-related cells tested, although at different levels. Both recombinant LycCatS (rLycCatS) and LycCatL (rLycCatL) possess the typical cysteine protease activity. Like other mammalian endopeptidase cathepsins, rLycCatS and rLycCatL could be autocatalytically activated to remove propeptides and release active mature peptides. On the other hand, the autocatalytic activation of rLycCatL could be inhibited by recombinant large yellow croaker Ii chain (rLyc-TR-Ii), but the autocatalytic activation of rLycCatS was not affected by rLyc-TR-Ii. Furthermore, the activated rLycCatS can efficiently process rLyc-TR-Ii in a stepwise manner in vitro, while the activated rLycCatL can not. These data indicate that cathepsin S may be the main cathepsin involved in the Ii chain processing in bony fish.