[show abstract][hide abstract] ABSTRACT: Apoptosis based immune responses are important component of host defense in mollusks. In this study, we have identified two novel molluscan BCL-2 cDNAs from Manila clam, Ruditapes philippinarum and named as RpBCL-2A and RpBCL-2B. There were four and three highly conserved BCL-2 homology (BH) regions in RpBCL-2A and RpBCL-2B, respectively suggesting these two genes could be different isoforms of anti-apoptotic BCL-2 family. Phylogenetic results revealed that Manila clam BCL-2 genes were clustered closely with invertebrate BCL-2 members. It gives evidence of their common origin and conserved features of invertebrate BCL-2 family. RpBCL-2A and 2B were expressed in tissue specific manner showing the highest and lowest level of expression in gills and hemocytes, respectively. However there was no clear expression profile difference between two genes. After Vibrio tapetis challenge, transcriptional responses of RpBCL-2A and RpBCL-2B were induced in gills and hemocytes with high variation that could be due to effects of immune reactions of other host defense molecules.
Fish & Shellfish Immunology 04/2013; · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ferritins are iron chelating proteins, which involve in iron metabolism and sequestration, contributing to the iron homeostasis in living organisms. In the present study, one ferritin subunit which was identified as H-like subunit was completely characterized in cDNA and protein levels from Manila clam (Ruditapes philippinarum); (RpFeH). The full length cDNA of RpFeH was 776 bp and it consisted of open reading frame of 513 bp, encoding a 171 amino acid peptide with a calculated molecular mass of 19.6 kDa and isoelectric point of 5.23. The amino acid sequence resembled the characteristic features of typical ferritin H subunits, including seven metal ligands in ferroxidase center, two iron binding region signatures and potential bio-mineralization residue (Thy(27)). Moreover, it was possible to identify an iron response element in 5' untranslated region, showing an agreement with previously reported ferritin H like subunits. The generated 3 dimensional tertiary structure of RpFeH showed a substantial consistency with the human ferritin H subunit, reinforcing its validity. Recombinant RpFeH was overexpressed in Escherichia coli BL21 (DE3) and purified. The recombinant protein showed a detectable iron binding activity according to the results obtained in our iron chelating activity assay. Furthermore it showed a noticeable anti-bacterial activity through suppressing the growth of Vibrio tapetis bacteria. The quantitative real time PCR detected ubiquitous RpFeH expression in tissues examined. However expression was found to be elevated in hemocyte and gill tissues. Transcriptional profile of Manila clam gill tissue, challenged with V. tapetis demonstrated a prolonged significant (P < 0.05) transcriptional upregulations from 12 h post injection to 48 h post injection. Our findings suggest that RpFeH functions as an iron chelating protein subunit in Manila clam while contributing to the innate immune responses against bacterial infections, via its iron withholding function.
Fish & Shellfish Immunology 10/2012; 33(4):926-36. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: a b s t r a c t Ferritins are iron chelating proteins, which involve in iron metabolism and sequestration, contributing to the iron homeostasis in living organisms. In the present study, one ferritin subunit which was identified as H-like subunit was completely characterized in cDNA and protein levels from Manila clam (Ruditapes philippinarum); (RpFeH). The full length cDNA of RpFeH was 776 bp and it consisted of open reading frame of 513 bp, encoding a 171 amino acid peptide with a calculated molecular mass of 19.6 kDa and isoelectric point of 5.23. The amino acid sequence resembled the characteristic features of typical ferritin H subunits, including seven metal ligands in ferroxidase center, two iron binding region signatures and potential bio-mineralization residue (Thy 27). Moreover, it was possible to identify an iron response element in 5 0 untranslated region, showing an agreement with previously reported ferritin H like subunits. The generated 3 dimensional tertiary structure of RpFeH showed a substantial consistency with the human ferritin H subunit, reinforcing its validity. Recombinant RpFeH was overexpressed in Escherichia coli BL21 (DE3) and purified. The recombinant protein showed a detectable iron binding activity according to the results obtained in our iron chelating activity assay. Furthermore it showed a noticeable anti-bacterial activity through suppressing the growth of Vibrio tapetis bacteria. The quantitative real time PCR detected ubiquitous RpFeH expression in tissues examined. However expression was found to be elevated in hemocyte and gill tissues. Transcriptional profile of Manila clam gill tissue, challenged with V. tapetis demonstrated a prolonged significant (P < 0.05) transcriptional up-regulations from 12 h post injection to 48 h post injection. Our findings suggest that RpFeH functions as an iron chelating protein subunit in Manila clam while contributing to the innate immune responses against bacterial infections, via its iron withholding function.
Fish & Shellfish Immunology 09/2012; · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cysteine proteases are present in all living organisms and, in animals, function in a vast array of physiological and pathological processes. Cysteine protease inhibitors act upon the cysteine proteases to regulate their activity. The cystatin superfamily of cysteine protease inhibitors has members represented in all living organisms studied to date. Here, we report the identification of a new member of the family 1 cystatin in Oplegnathus fasciatus rock bream (denoted as RbCyt B) and the characterization at the molecular level. The complete genomic sequence of RbCyt B consists of three exons and a promoter region. The open reading frame (ORF) encodes for a 100 amino acids length polypeptide with a single cystatin-like domain and a cysteine protease inhibitor signature motif. The conserved N-terminal glycine, glutamine-valine-glycine motif, QxVxG, and a variant of the proline-tryptophan, PW, motif were identified. RbCyt B showed closest phylogenetic distance to Dicentrarchus labrax cystatin B, and shared up to 73% amino acid identity and 90% amino acid similarity with known cystatin B genes. RbCyt B mRNA expression was detected in nine different tissues and was highly expressed in liver, spleen, gill, brain, intestine, kidney, head kidney, and blood, as compared with muscle. In vivo immune stimulation with Edwardsiella tarda bacteria caused significant up-regulation of RbCyt B mRNA in head kidney and spleen at 24h post-infection (P<0.05). Recombinant RbCyt B was expressed in Escherichia coli, and the purified protein demonstrated 82% papain inhibitory activity at 500 × 10(-3) μg μL(-1) in a concentration-dependent manner. These results suggest that RbCyt B is a member of family 1 cystatin with high homology to cystatin B, and is a biologically active protein possessing papain inhibitory activity and potentially involved in immune responses against invading Gram-negative bacteria in rock bream.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 05/2012; 163(1):138-46. · 1.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Acute phase response is a significant component of innate immunity, playing a vital role in the signaling processes and elimination of invading pathogens. Acute phase proteins are synthesized in liver and secreted into the blood for transportation to an infection site, where the defense function is exerted. Serum amyloid A (SAA) and C-reactive proteins are the major positive acute phase proteins. In this study, we have identified and characterized a novel SAA related gene from rock bream (Oplegnathus fasciatus), designated OfSAAL1. Genomic characterization revealed the presence of 13 exons and 12 introns, similar to SAAL1 in zebrafish. Multiple protein sequence alignment revealed high conservation with other SAAL1 homologues. Phylogenetic analysis showed that OfSAAL1 clustered with another fish homologue, and pairwise alignment revealed highest identity and similarity at the amino acid level with zebrafish SAAL1. Promoter region analysis revealed the presence of immunologically significant transcription factor binding sites. Tissue distribution profiling to indicate physiological relevance showed the highest levels occur in blood, followed by liver, suggesting a positive immune role in rock bream. Transcriptional analysis by reverse transcription polymerase chain reaction to understand OfSAAL1 responsiveness to immune challenge with poly I:C, Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus, revealed a significant level of elevation from 12h to 48 h post-infection in blood, spleen, head kidney, and liver. To our knowledge, OfSAAL1 is the first characterized SAAL1 homologue from teleosts. We anticipate that its identification will prove inspiring for further studies of SAAL1 homologues as biomarkers of the acute phase response.
Developmental and comparative immunology 04/2012; 37(2):294-305. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine widely expressed by both immune and non-immune cells. MIFs have been largely identified in a variety of species, playing a central role as a regulator of innate immune and inflammatory responses. In this present study, we reported the cloning and functional characterization of two MIFs of disk abalone H. discus discus. The full-length cDNA of two MIFs, HdMIF1 and HdMIF2, were identified and sequenced from a disk abalone cDNA library. Two MIF cDNAs contained two open reading frames (ORF) of 357 and 351 bp that encoded two proteins with 119 and 117 amino acid (aa) residues, respectively. In addition, a 14 aa signal peptide was predicted in the N-terminus of both two MIF proteins. The protein sequences of HdMIF1 and HdMIF2 shared a similarity of 82%, and were over 60% identical to another MIF from small abalone (H. diversicolor). Two MIF genes were expected to locate closely on the abalone chromosome DNA through the screening of disk abalone BAC-library. Two MIFs exhibited similar expression patterns in different tissues of disk abalone, where digestive tract and hepatopancreas possessed the highest expression levels. The immune responses of two MIFs were investigated under the microbial challenge and tissue injury by qRT-PCR. The expression of HdMIF1 and HdMIF2 in hemocyte were rapidly induced within 3 hours post bacterial (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) and viral hemorrhagic septicemia virus (VHSV) challenges, indicating as an acute phase response. Furthermore, their expression levels in mantle were also significantly up-regulated in response to mantle tissue injury. Taken together, these findings suggest that abalone MIFs may involve in immune responses of pathogen invasion and the wound healing after tissue injury.
GIA2011, Institute of Marine Biology and Genetics- Hellenic Centre for Marine Research, Heraklion, Crete; 09/2011
[show abstract][hide abstract] ABSTRACT: Lysozyme (muramidase) represents an important defense molecule of the fish innate immune system. Known for its bactericidal properties, lysozyme catalyzes the hydrolysis of β-(1,4)-glycosidic bonds between the N-acetyl glucosamine and N-acetyl muramic acid in the peptidoglycan layer of bacterial cell walls. In this study, the complete coding sequence of a g-type lysozyme (RBgLyz) was identified in the Oplegnathus fasciatus rock bream fish genome by means of multi-tissue normalized cDNA pyrosequencing using Roche 454 GS-FLX™ technology. RBgLyz is composed of 669 bp, with a 567 bp open reading frame that encodes 188 amino acids. Protein motif searches indicated that RBgLyz contains the soluble lytic transglycosylase domain involved in maintaining cell wall integrity. Furthermore, RBgLyz shares significant identity (81.4%) with Chinese perch Siniperca chuatsi. Quantitative real-time RT-PCR analysis results showed that RBgLyz transcripts are constitutively expressed in various tissues from healthy rock breams. In order to determine RBgLyz function in immunity, its expression was analyzed in head kidney following exposure to known immune stimulants or pathogens. RBgLyz transcripts were significantly up-regulated in response to challenge with lipopolysaccharide (LPS) and Edwardsiella tarda, as compared to non-injected control fish. Polyinosinic:polycytidylic acid (poly I:C) dsRNA stimulated a moderate expression of RBgLyz, as did Streptococcus iniae but to a lesser extent. There were no specific time-dependent effects on RBgLyz mRNA expression observed in response to rock bream iridovirus (RBIV) infection. Taken together, the gene expression results indicated that g-type lysozyme plays a role in the innate immune response to LPS, poly I:C, E. tarda and S. iniae in rock bream. Thus, we generated recombinant RBgLyz in an Escherichia coli expression system and characterized its antimicrobial activity. Our results indicated that recombinant RBgLyz had lytic activity against Gram-negative Vibrio salmonicida, Gram-positive Listeria monocytogenes, S. iniae and Micrococcus lysodeikticus. In addition, observations by scanning electron microscope (SEM) confirmed that the cell morphology of M. lysodeikticus was altered in the presence of recombinant RBgLyz.
Fish & Shellfish Immunology 02/2011; 30(2):532-42. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: The caspase family represents aspartate-specific cysteine proteases that play key roles in apoptosis and immune signaling. In this study, we cloned the first death effector domain (DED)-containing molluscan caspase-8 gene from disk abalone (Haliotis discus discus), which is named as hdCaspase-8. The full-length hdCaspase was 2855 bp, with a 1908 bp open reading frame encoding 636 amino acids. The hdCaspase-8 had 72 kDa predicted molecular mass with an estimated isoelectric point (PI) of 6.0. The hdCaspase-8 amino acid sequence contained the characteristic feature of an N-terminal two DED, a C-terminal catalytic domain and the caspase family cysteine active site ⁵¹³KPKLFFLQACQG⁵²⁴. Phylogenetic analysis results showed that hdCaspase-8 is more similar to the invertebrate Tubifex tubifex (sludge worm) caspase-8. Real-time RT-PCR results showed that hdCaspase-8 constitutively and ubiquitously expressed in all tested tissue of unchallenged disk abalone. The basal expression level of hdCaspase-8 in gill tissue was higher than all other tested tissues. The hdCaspase-8 mRNA expression in gill and hemocytes was significantly up-regulated by exposure to bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Listeria monocytogenes) and VHSV (viral hemorrhagic septicemia virus), as compared to control animals. These results suggest that hdCaspase-8 may be involved in immune response reactions in disk abalone.
Fish & Shellfish Immunology 02/2011; 30(2):480-7. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cathepsins are lysosomal cysteine proteases of the papain family that play an important role in intracellular protein degradation and turn over within the lysosomal system. In the present study, full-length sequences of cathepsin B (RbCathepsin B) and L (RbCathepsin L) were identified after transcriptome sequencing of rock bream Oplegnathus fasciatus mixed tissue cDNA. Cathepsin B was composed of 330 amino acid residues with 36 kDa predicted molecular mass. RbCathepsin L contained 336 amino acid residues encoding for a 38 kDa predicted molecular mass protein. The sequencing analysis results showed that both cathepsin B and L contain the characteristic papain family cysteine protease signature and active sites for the eukaryotic thiol proteases of cysteine, asparagine and histidine. In addition, RbCathepsin L contained EF hand Ca(2+) binding and cathepsin propeptide inhibitor domains. The rock bream cathepsin B and L showed the highest amino acid identity of 90 and 95% to Lutjanus argentimaculatus cathepsin B and Lates calcarifer cathepsin L, respectively. By phylogenetic analysis, cathepsin B and L exhibited a high degree of evolutionary relationship to respective cathepsin family members of the papain superfamily. Quantitative real-time RT-PCR analysis results confirmed that the expression of cathepsin B and L genes was constitutive in all examined tissues isolated from un-induced rock bream. Moreover, activation of RbCathepsin B and L mRNA was observed in both lipopolysaccharide (LPS) and Edwardsiella tarda challenged liver and blood cells, indicating a role of immune response in rock bream.
Fish & Shellfish Immunology 12/2010; 30(3):763-72. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: The regulation of transcriptional activation is an essential and critical point in gene expression. In this study, we describe a novel transcription factor activator protein-1 (Ap-1) gene from disk abalone Haliotis discus discus (AbAp-1) for the first time in mollusk. It was identified by homology screening of an abalone normalized cDNA library. The cloned AbAp-1 consists of a 945 bp coding region that encodes a putative protein containing 315 amino acids. The AbAp-1 gene is composed of a characteristic Jun transcription factor domain and a highly conserved basic leucine zipper (bZIP) signature similar to known Ap-1 genes. The AbAp-1 shares 46, 43 and, 40% amino acid identities with fish (Takifugu rubripes), human and insect (Ixodes scapularis) Ap-1, respectively. Quantitative real time RT-PCR analysis confirmed that AbAp-1 gene expression is constitutive in all selected tissues. AbAp-1 was upregulated in gills after bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) challenge; and, upregulated in hemocytes and gills by viral hemorrhagic septicemia virus (VHSV) challenge. Shell damage and tissue injury also increased the transcriptional level of Ap-1 in mantle together with other transcription factors (NF-kB, LITAF) and pro-inflammatory TNF-α. All results considered, identification and gene expression data demonstrate that abalone Ap-1 is an important regulator in innate immune response against bacteria and virus, as well as in the inflammatory response during tissue injury. In addition, stimulation of Ap-1 under different external stimuli could be useful to understand the Ap-1 biology and its downstream target genes, especially in abalone-like mollusks.
Fish & Shellfish Immunology 12/2010; 29(6):1028-36. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Heparin cofactor (HCII) is a serine protease inhibitor (SPI), and plays important physiological roles in various biological events including hemostasis. The gene encoding the HCII was isolated from GS-FLX™ genomic data of rock bream (Oplegnathus fasciatus), designated as RbHCII. The RbHCII (1950 bp) consists of a 1512 bp open reading frame (ORF) encoding 504 amino acids (aa), with a signal peptide of 19 aa residues. The predicted molecular mass and the estimated isoelectric point of RbHCII were 58 kDa and 5.9, respectively. The deduced aa sequence of RbHCII displayed a characteristic serpin domain and a serpin signature motif (FTVDQPFLFLI). RbHCII demonstrated homology with vertebrate HCIIs and the greatest degree of similarity (90.1%) was observed with Gasterosteus aculeatus HCII. Various functional domains including the reactive center loop (RCL), glycosaminoglycan (GAG) and thrombin binding sites and acidic repeats of human and RbHCII were found to be orthologs through the molecular modeling studies. Phylogenetic analysis revealed that RbHCII belongs to the clade D serpins, and is closely related to the clade A members. Constitutive expression of RbHCII mRNA was detected at different levels in various tissues in a tissue-specific manner. Interestingly, RbHCII transcription was significantly downregulated (p < 0.05) in liver after challenge with lipopolysaccharide (LPS), Edwardsiella tarda and rock bream iridovirus (RBIV). However, after the immune challenges, RbHCII showed a significant downregulation in blood tissue only at the late-phase of investigation. The recombinant RbHCII (rRbHCII) was overexpressed in Rosetta-gami (DE3) cells and purified using the pMAL™ system. The rRbHCII inhibited thrombin and chymotrypsin in a dose-dependent manner. Remarkably, heparin was found to be an enhancer of RbHCII's thrombin-inhibitory activity. Correlating the heparin-dependent thrombin-inhibition activity of RbHCII with its temporal downregulation against immune stimulants, it could be suggested that it is not only involved in the blood coagulation cascade, but also plays an incognito role in immune modulation.
Fish & Shellfish Immunology 10/2010; 30(1):194-208. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gene-encoded antimicrobial peptides (AMPs) serve a major role in host defense systems against pathogens. In this study, cDNA of a new mollusk defensin was identified from a normalized cDNA library constructed from whole tissues of disk abalone. Abalone defensin peptide (pro-defensin) has a 198-bp coding sequence comprised of a putative 66 amino acids with a mature defensin consisting of 48 amino acid residues. The presence of an invertebrate defensin family domain, an arrangement of six cysteine residues and their disulfide linkage in C(1)-C(4), C(2)-C(5) and C(3)-C(6) form, an alpha helix in three-dimensional structure and a phylogenetic relationship suggests that abalone defensin could be a new member of the invertebrate defensin family, and related to arthropod defensins. In non-stimulated abalone, defensin transcripts were constitutively expressed in all examined tissues including hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas. It was observed that abalone defensin transcripts were significantly induced in hemocytes, gills and digestive tract at different time intervals after infection by pathogenic bacteria mixture containing Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes. Our overall results suggest that disk abalone defensin could be involved in the immune response reactions as a host defense against pathogenic bacteria.
Fish & Shellfish Immunology 11/2009; 28(2):261-6. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: This study describes the transcriptional analysis of antioxidant and immune defense genes in gills tissue of disk abalone exposed to thermal, salinity and hypoxia-related stress, using quantitative real-time PCR. Results showed that manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD), catalase (CAT), thioredoxin peroxidase (TPx), selenium dependant glutathione peroxidase (SeGPx), and thioredoxin-2 (TRx-2) transcripts were expressed differently in gills, and that they respond collectively as a classical enzymatic antioxidant defense system in abalone. Comparative analysis of expression profiles indicated that CAT, TPx and SeGPx transcripts were significantly up-regulated (p<0.05) by all three physical stress conditions - thermal (28 degrees C), salinity (25 per thousand) and hypoxia - relative to levels in respective controls. In contrast, CuZnSOD and TRx-2 transcription were down-regulated in response to thermal stress. Interestingly, all the antioxidant transcripts exhibited significant up-regulation in response to salinity-related stress. Meanwhile, hypoxia caused up-regulation of the MnSOD, CAT, TPx and SeGPx, but not the CuZnSOD and TRx-2 transcripts. One of the most significant outcomes was the more than 10-fold induction of SeGPx, suggesting that SeGPx is a potential biomarker gene among antioxidant enzymes, under conditions of physical stress. The effects of physical stress on the transcriptional responses of immune functional genes namely suppressor of cytokine signaling-2 (SOCS-2) and myxovirus resistance (Mx) were investigated. We observed that salinity and hypoxia increase both the SOCS-2 and Mx transcripts, while thermal stress at 28 degrees C exerts less of an effect, producing mixed transcriptional responses (both up and down regulation). Based upon these results, we postulate that abalones utilize antioxidant and immune defense mechanisms together to overcome physical stresses.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 08/2009; 154(4):387-95. · 1.61 Impact Factor