Molecular cloning and characterization of Mn-superoxide dismutase from disk abalone (Haliotis discus discus)

The mitochondrial enzyme manganese superoxide dismutase (mitMn-SOD) is one of the antioxidant enzymes involved in cellular defense against oxidative stress and catalyzes the conversion of O₂⁻ into the stabler H₂O₂. In this study, a putative gene encoding Mn-SOD from disk abalone (Haliotis discus discus, aMn-SOD) was cloned, sequenced, expressed in Escherichia coli K12(TB1) and the protein was purified using pMAL protein purification system. Sequencing resulted ORF of 681 bp, which corresponded to 226 amino acids. The protein was expressed in soluble form with molecular weight of 68 kDa including maltose binding protein and pI value of 6.5. The fusion protein had 2781 U/mg activity. The optimum temperature of the enzyme was 37 °C and it was active in a range of acidic pH (from 3.5 to 6.5). The enzyme activity was reduced to 50% at 50 °C and completely heat inactivated at 80 °C. The alignment of aMn-SOD amino acid sequence with Mn-SODs available in NCBI revealed that the enzyme is conserved among animals with higher than 30% identity. In comparison with human mitMn-SOD, all manganese-binding sites are also conserved in aMn-SOD (H28, H100, D185 and H189). aMn-SOD amino acid sequence was closer to that of Biomphalaria glabrata in phylogenetic analysis.     

Molecular cloning and characterization of three sigma glutathione S-transferases from disk abalone (Haliotis discus discus)

Three novel glutathione S-transferase (GSTs) cDNAs were cloned from a disk abalone (Haliotis dicus discus) cDNA library. Multiple alignment and phylogenetic analysis of three GSTs revealed that their closest relationship is with insect sigma GSTs. Recombinant GSTs were over-expressed in Escherichia coli as soluble fusion proteins. HdGSTS1 and HdGSTS2 were active towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid, whereas HdGSTS3 appeared to be a non-enzymatic GST. Two active GSTs had similar optimum conditions for enzymatic reaction at pH 8.0 and temperature of approximately 30 degrees C. Molecular modeling analysis of three GSTs implicates their diverse active sites as being responsible for their different enzymatic features. Three sigma GSTs had significantly different expression patterns and levels of expression in abalone tissues, indicating their different functions. After 48 h-exposure to three model marine pollutants, only HdGSTS1 exhibited a proper inducibility, exhibiting its good biomarker potential for organic contaminants in marine environment. In contrast, the other two sigma GSTs revealed a minor role in the response of pollutants exposure.     

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.     

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.     

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.     

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

Interferon Gamma (IFN-gamma) 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 25kDa 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, ((23)CLDC(26) and (46)CPYC(49)) which motif is highly conserved in GILT protein family. AbGILT exhibited a characteristic GILT signature sequence (92)CQHGX(2)ECX(2)NX(4)C(107) 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 24h post injection of phytohemagglutinin (PHA) while Vibrio alginolyticus up-regulation appeared in the gill and digestive tract after 48h. In contrast, AbGILT expression was not up-regulated by poly inosinic-cytidylic acid (poly I:C) during the 48h 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.     

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.     

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.     

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.