Molecular cloning and expression analysis of interferon-γ inducible lysosomal thiol reductase (GILT)-like cDNA from disk abalone (Haliotis discus discus)

Interferon Gamma (IFN-γ) Inducible Lysosomal Thiol reductase (GILT) has been described as a key enzyme in processing and presentation of major histocompatibility complex (MHC) class II restricted antigen (Ag) by catalyzing disulfide bond (S–S) reduction in mammals. Abalone GILT-like (AbGILT) full-length cDNA was isolated from the normalized disk abalone cDNA library. The 807-bp AbGILT cDNA consists of an open reading frame of 684-bp, encoding 228 amino acid residues. The predicted AbGILT protein has a molecular weight of 25 kDa and an isoelectric point of 7.8. The N-terminus of the AbGILT was found to have a putative signal peptide with a cleavage site amino acid position at 19–20. AbGILT contains two active site C-XX-C motifs, (²³CLDC²⁶ and ⁴⁶CPYC⁴⁹) which motif is highly conserved in GILT protein family. AbGILT exhibited a characteristic GILT signature sequence ⁹²CQHGX₂ECX₂NX₄C¹⁰⁷ and 12 cysteine residues representing 5% in the mature peptide. Phylogenetic analysis showed that AbGILT has been derived from a common ancestor with other GILT proteins. RT-PCR results showed that AbGILT expression was up-regulated in the gill, mantle and digestive tract 24 h post injection of phytohemagglutinin (PHA) while Vibrio alginolyticus up-regulation appeared in the gill and digestive tract after 48 h. In contrast, AbGILT expression was not up-regulated by poly inosinic–cytidylic acid (poly I:C) during the 48 h induction. However, AbGILT was constitutively expressed in gill, mantle, and digestive tract tissues suggesting that it may maintain first line of innate immune defense at basal level in disk abalone.     

Cloning, characterization and tissue expression of disk abalone (Haliotis discus discus) catalase

Catalase is an antioxidant enzyme that plays a significant role in protection against oxidative stress by detoxification of hydrogen peroxide (H2O2). A gene coding for a putative catalase was isolated from the disk abalone (Haliotis discus discus) cDNA library and denoted as Ab-catalase. The full-length (2864 bp) Ab-catalase cDNA contained 1,503 bp open reading frame (ORF), encoding 501 amino acid residues with 56 kDa predicted molecular weight. The deduced amino acid sequence of Ab-catalase has characteristic features of catalase family such as catalytic site motif (61FNRERIPERVVHAKGAG77), heme-ligand signature motif (351RLYSYSDT358), NADPH and heme binding residues. Phylogenetic and pairwise identity results indicated that Ab-catalase is more similar to scallop (Chlamys farreri) catalase with 80% amino acid identity except for other reported disk abalone catalase sequences. Constitutive Ab-catalase expression was detected in gill, mantle, gonad, hemocytes, abductor muscle and digestive tract in tissue specific manner. Ab-catalase mRNA was up-regulated in gill and digestive tract tissues for the first 3h post injection of H2O2, showing the inducible ability of abalone catalase against oxidative stress generated by H2O2. The purified recombinant catalase showed 30,000 U/mg enzymatic activity against H2O2 and biochemical properties of higher thermal stability and broad spectrum of pH. Our results suggest that abalone catalase may play an important role in regulating oxidative stress by scavenging H2O2.     

Mitochondrial thioredoxin-2 from disk abalone (Haliotis discus discus): molecular characterization, tissue expression and DNA protection activity of its recombinant protein

Thioredoxin-2 is a mitochondria-specific member of the thioredoxin (TRx) super-family that plays an important role as a component of the mitochondrial antioxidant system. The gene coding mitochondrial TRx-2 was isolated from the disk abalone (Haliotis discus discus) cDNA library, denoted as AbTRx-2. It contains 1214-bp full length with 519-bp open reading frame, encoding 173 amino acids. AbTRx-2 showed characteristic TRx active site at ⁹⁶WCGPC¹⁰⁰ and mitochondrial targeting peptide at the N-terminal amino acid sequence. The deduced amino acid comparison showed that AbTRx-2 shares 43 and 42% identity with Xenopus laevis and human TRx-2, respectively. Purified recombinant AbTRx-2 fusion protein was shown to catalyze insulin reduction and protect supercoiled plasmid DNA from damages induced by metal-catalyzed generation of reactive oxygen species. Constitutive AbTRx-2 mRNA was detected in gill, mantle, gonad, abductor muscle, digestive tract, and hemocytes, in a tissue specific manner. The AbTRx-2 mRNA was up-regulated in gill and digestive tract tissues initially at 3 h post-injection of H₂O₂ and maintained higher level at 6 h. Our results suggest that abalone TRx-2 may play an important role in regulating oxidative stress in mitochondria by catalyzing protein disulfide reduction, scavenging of ROS, and minimizing the DNA damage.     

First report of invertebrate Mx: cloning, characterization and expression analysis of Mx cDNA in disk abalone (Haliotis discus discus)

Myxovirus resistance (Mx) protein is one of the most studied antiviral proteins. It is induced by the type I interferon system (IFN alpha/beta) in various vertebrates, but its expression has not been identified or characterized in mollusks or other multi-cellular invertebrates to date. In this study, we isolated the Mx gene from a disk abalone (Haliotis discus discus) normalized cDNA library. Mx cDNA was sequenced, cloned and compared to other known Mx proteins. The full-length 1664 bp of abalone Mx cDNA contained a 1533-bp open reading frame that codes for 511 amino acids. Within the coding sequence of abalone Mx, characteristic features were found, such as a tripartite guanosine-5'-triphosphate (GTP)-binding motif and a dynamin family signature. In addition, leucine residues in the C-terminal region displayed a special leucine domain at L(468), L(475), L(489) and L(510), suggesting that abalone Mx may have a similar oligomerization function as other leucine zipper motifs. Abalone Mx protein exhibited 44% amino acid similarity with channel catfish Mx1, rainbow trout Mx2 and Atlantic halibut Mx. Abalones were injected intramuscularly with the known IFN inducer poly I:C and RT-PCR was performed for Mx mRNA analysis. The results showed enhanced Mx expression in abalone gill and digestive tissues 24h as well as 48 h after injection of poly I:C. Mx mRNA was expressed in gill, digestive gland, mantle and foot tissues in healthy abalone, suggesting that the basal level of Mx expressed is tissue-specific. There is no known Mx protein closely related to abalone Mx according to phylogenetic analysis. Abalone Mx may have diverged from a common gene ancestor of fish and mammalian Mx proteins, since abalone Mx showed high similarity in terms of conserved tripartite GTP-binding, dynamin family signature motifs and poly I:C enhancement of Mx mRNA expression.     

Comparative study of two thioredoxin peroxidases from disk abalone (Haliotis discus discus): cloning, recombinant protein purification, characterization of antioxidant activities and expression analysis

Thioredoxin peroxidase (TPx), also named peroxiredoxin (Prx), is an important peroxidase, which can protect organisms against various oxidative stresses. Two TPxs were isolated from a disk abalone (Haliotis discus discus) cDNA library, named as AbTPx1 and AbTPx2, respectively. AbTPx1 and AbTPx2 consist of 1315 and 1045 bp full-length cDNA with 753 and 597 bp open reading frames encoding 251 and 199 amino acids, respectively. The TPx signature motif 1 (FYPLDFTFVCPTEI) and motif 2 (GEVCPA) were conserved in both AbTPx1 and AbTPx2 amino acid sequences. Purified recombinant abalone TPx fusion proteins catalyzed the reduction of H2O2 and butyl hydroperoxide in peroxidase assays. Furthermore, both AbTPx fusion proteins were shown to protect super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Escherichia coli cells transformed with AbTPx1 and AbTPx2 coding sequences in pMAL-c2x showed resistance to H2O2 at 0.8 mM concentration by in vivo H2O2 tolerance assay. AbTPx1 and AbTPx2 mRNA were constitutively expressed in gill, mantle, abductor muscle and digestive tract in a tissue specific manner. Additionally, both TPxs mRNA were up-regulated in gill and digestive tract tissues against H2O2 at 3h post injection. The results indicate that AbTPx1 and AbTPx2 gene expressions are induced by oxidative stress and their respective proteins function in the detoxification of different ROS molecules to maintain efficient antioxidant defense in disk abalone.     

Two ferritin subunits from disk abalone (Haliotis discus discus): cloning, characterization and expression analysis

Ferritin plays a key role in cellular iron metabolism, which includes iron storage and detoxification. From disk abalone, Haliotis discus discus, the cDNA that encodes the two ferritin subunits abalone ferritin subunit 1 (Abf1) and abalone ferritin subunit 2 (Abf2) were cloned. The complete cDNA coding sequences for Abf1 and Abf2 contained 621 and 549 bp, encoding for 207 and 183 amino acid residues, respectively. The H. discus discus Abf2 subunit contained a highly conserved motif for the ferroxidase center, which consists of seven residues of a typical vertebrate heavy-chain ferritin with a typical stem-loop structure. Abf2 mRNA contains a 27 bp iron-responsive element (IRE) in the 5'UTR position. This IRE exhibited 96% similarity with pearl and Pacific oyster and 67% similarity with human H type IREs. However, the Abf1 subunit had neither ferroxidase center residues nor the IRE motif sequence; instead, it contained iron-binding region signature 2 (IBRS) residues. Recombinant Abf1 and Abf2 proteins were purified and the respective sizes were about 24 and 21 kDa. Abf1 and Abf2 exhibited iron-chelating activity 44.2% and 22.0%, respectively, at protein concentration of 6 microg/ml. Analysis of tissue-specific expression by RT-PCR revealed that Abf1 and Abf2 ferritin mRNAs were expressed in various abalone tissues, such as gill, mantle, gonad, foot and digestive tract in a wide distribution profile, but Abf2 expression was more prominent than Abf1.     

Molecular characterization and expression analysis of regucalcin in disk abalone (Haliotis discus discus): intramuscular calcium administration stimulates the regucalcin mRNA expression

Regucalcin is a novel calcium (Ca(2+)) binding protein and it has been demonstrated to play a multifunctional role in many organisms. Here, we report the molecular cloning of invertebrate regucalcin cDNA from disk abalone Haliotis discus discus. The full length cDNA showed 1321 bp of nucleotides with a polyadenylated sequence (AATAAA). Abalone regucalcin (HdReg) open reading frame (ORF) consists of 918 nucleotides encoding 305 amino acids (aa). Estimated molecular mass was 33 kDa and predicted isoelectric point (pI) was 4.9. The HdReg aa sequence did not contain the EF-hand motif as a Ca(2+) binding domain, suggesting a novel class of Ca(2+) binding protein. Moreover, it showed 45% identity to chicken and zebrafish, and 44% to rat and mouse regucalcin in deduced aa level. The tissue expression analysis of HdReg mRNA was investigated by RT-PCR and it was expressed in all the tissues tested such as gill, mantle, digestive tract, and abductor muscle. Semi-quantitative RT-PCR results showed that an intramuscular administration of calcium chloride (CaCl(2)) (0.5 mg CaCl(2)/g of abalone) could significantly induce regucalcin mRNA in abductor muscle after 30 min of administration and reached maximum after 1 h. Subsequently, the expression level was decreased after 2 h. This indicates that the expression of regucalcin mRNA is constitutive, and specifically up regulated in abalone abductor muscle by Ca(2+) administration.     

Molluskan fasciclin-1 domain-containing protein: Molecular characterizationand gene expression analysis of fasciclin 1-like protein from disk abalone (Haliotis discus discus)

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 909 bp open reading frame (ORF) encodes 303 amino acid (aa) residues with a predicted molecular mass of 33 kDa 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 48 h after the Vibrio parahaemolyticus infection, by 3.1-fold at 6 h after the Listeria monocytogenes infection and by ~ 9-fold at 6 h 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 12 h 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.     

Molluscan death effector domain (DED)-containing caspase-8 gene from disk abalone (Haliotis discus discus): molecular characterization and expression analysis

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.     

cDNA cloning of an alginate lyase from abalone, Haliotis discus hannai

An alginate lyase, termed HdAly in the present paper, was isolated from the hepatopancreas of abalone, Haliotis discus hannai, by ammonium sulfate fractionation, followed by TOYOPEARL CM-650M column chromatography. Enzymatic properties of HdAly were similar to those of previously reported Haliotis and Turbo poly(M) lyases, e.g., it preferentially degraded a poly(beta-D-mannuronate)-rich substrate with an optimal pH and temperature at pH 8.0 and 45 degrees C, respectively. In order to determine the primary structure of abalone lyase that is still poorly understood, cDNAs for HdAly were cloned by PCR from the abalone hepatopancreas cDNA library and sequenced. From the nucleotide sequences of the cDNAs, the sequence of 909 bp in total was determined, and the amino acid sequence of 273 residues was deduced from the translational region of 822 bp locating at nucleotide positions 27-848. The N-terminal region of 16 residues, except for the initiation Met in the deduced sequence, was regarded as the signal peptide since it was absent in the HdAly protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretary proteins. This suggests that HdAly is initially produced as a precursor possessing the signal peptide in hepatopancreatic cells and then secreted into digestive tract as the mature form. Thus, the mature HdAly was regarded to consist of 256 residues with the calculated molecular mass of 28895.5 Da. The amino acid sequence of HdAly showed 85 and 28% identity to those of Turbo cornutus alginate lyase SP2 and the C-terminal region of Chlorella virus lyase-like protein CL2, respectively, while it showed no significant identity to those of any bacterial alginate lyases. In order to provide the basis for the structure-function studies and various applications of the abalone lyase, a bacterial expression system was constructed by means of the HdAly-cDNA and pET-3a expression plasmid. Although the active recombinant HdAly was hardly produced at a cultivation temperature 37 degrees C in Escherichia coli BL21 (DE3), a small amount of soluble and active enzyme could be produced when the temperature was lowered to 19 degrees C.     

A novel oligoalginate lyase from abalone, Haliotis discus hannai, that releases disaccharide from alginate polymer in an exolytic manner

We previously reported the isolation and cDNA cloning of an endolytic alginate lyase, HdAly, from abalone Haliotis discus hannai [Carbohydr. Res.2003, 338, 2841-2852]. Although HdAly preferentially degraded mannuronate-rich substrates, it was incapable of degrading unsaturated oligomannuronates smaller than tetrasaccharide. In the present study, we used conventional chromatographic techniques to isolate a novel unsaturated-trisaccharide-degrading enzyme, named HdAlex, from the digestive fluid of the abalone. The HdAlex showed a molecular weight of 32,000 on SDS-PAGE and could degrade not only unsaturated trisaccharide but also alginate and mannuronate-rich polymers at an optimal pH and temperature of 7.1 and 42 degrees C, respectively. Upon digestion of alginate polymer, HdAlex decreased the viscosity of the alginate at a slower rate than did HdAly, producing only unsaturated disaccharide without any intermediate oligosaccharides. These results indicate that HdAlex degrades the alginate polymer in an exolytic manner. Because HdAlex split saturated trisaccharide producing unsaturated disaccharide, we considered that this enzyme cleaved the alginate at the second glycoside linkage from the reducing terminus. The primary structure of HdAlex was deduced with cDNAs amplified from an abalone hepatopancreas cDNA library by the polymerase chain reaction. The translational region of 822 bp in the total 887-bp sequence of HdAlex cDNA encoded an amino-acid sequence of 273 residues. The N-terminal sequence of 16 residues, excluding the initiation methionine, was regarded as the signal peptide of this enzyme. The amino-acid sequence of the remaining 256 residues shared 62-67% identities with those of the polysaccharide lyase family-14 (PL14) enzymes such as HdAly and turban-shell alginate lyase SP2. To our knowledge, HdAlex is the first exolytic oligoalginate lyase belonging to PL14.     

Molecular cloning and expression analysis of inhibitor of growth protein 3 (ING3) in the Manila clam,Ruditapes philippinarum

Inhibitor of growth protein 3 (ING3), a new member of ING family, is involved in the regulation of various processes. In this study, a full-length cDNA of ING3 (named as RpING3) was cloned from the gill of Ruditapes philippinarum by rapid amplification of cDNA ends method for the first time. The cDNA obtained was 1442 bp exclusive of poly (A) residues with a 1248 bp open reading frame encoding 415 amino acids. The RpING3 protein has a calculated molecular weight of 46.59 kDa and isoelectric point of 6.62. Two conserved motif and some functional sites were found. Tissue distribution analysis of the RpING3 mRNA revealed that the RpING3 expression level was much higher in gill and digestive gland while lower in mantle, foot and adductor muscle. The temporal expression of RpING3 in digestive gland after lead exposure was recorded by quantitative real-time PCR. The result showed that RpING3 was rapidly up-regulated at 6 h post-exposure and reached tenfold of the control group. These results suggest that RpING3 dependent signaling pathway is present in Manila clam and RpING3 may play important roles in protecting cells from heavy metal damage in R. philippinarum.