Characteristics of the Copper,Zinc Superoxide Dismutase of a Hadal Sea Cucumber (Paelopatides sp.) from the Mariana Trench (original) (raw)
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Molecular cloning of superoxide dismutase (Cu/Zn-SOD) from aquatic molluscs
Marine Environmental Research, 2004
The potential of the first line of the active oxygen-scavenging system, partial cDNA encoding Cu/Zn superoxide dismutase (SOD) was isolated in three aquatic mollusc species: Ruditapes decussatus (marine clam), Dreissena polymorpha (continental water mussel) and Bathymodiolus azoricus (hydrothermal vent mussel). These SOD cDNA fragments were amplified by PCR with degenerate oligonucleotide primers derived from the amino acid sequence conserved in the Cu/ Zn-SOD from several other organisms. A partial cDNA of CuZn-SOD was obtained for R. decussates (510 bp), D. polymorpha (510 bp) and B. azoricus (195 bp). The deduced amino acid sequence showed high similarity among the three mollusc species (57-63%) and among other species (50-65%). The residues involved in coordinating copper 49, 64, 121) and zinc 72,(81)(82)(83)(84) were well conserved among the three Cu/Zn-SOD sequences.
Fish & Shellfish Immunology, 2010
Superoxide dismutases (SODs, EC 1.15.1.1) are one family of important antioxidant metalloenzymes involved in scavenging the high level of reactive oxygen species (ROS) into molecular oxygen and hydrogen peroxide. In the present study, the intracellular CuZnSOD gene of Cristaria plicata (Cp-icCuZnSOD) was identified from hemocytes by homology cloning and the rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of Cp-icCuZnSOD consisted of 891 nucleotides with a canonical polyadenylation signal sequence ATTAAA, a poly (A) tail, and an open-reading frame of 468 bp encoding 155 amino acids. The deduced amino acids of CpSOD shared high similarity with the known icCuZnSODs from other species, and several highly conserved motifs including Cu/Zn ions binding sites (His-46, His-48, His-63, His-120 for Cu 2þ binding, and His-63, His-71, His-80, Asp-83 for Zn 2þ binding), intracellular disulfide bond and two CuZnSOD family signatures were also identified in CpSOD. Furthermore, the recombinant Cp-icCuZnSOD with high enzyme activity was induced to be expressed as a soluble form by IPTG supplemented with Cu/Zn ions at 20 C for 8 h, and then was purified by using the native Ni 2þ affinity chromatography. The specific activity of the purified rCp-icCuZnSOD enzyme was 5368 U/mg, which is 2.6-fold higher than that of zebrafish Danio rerio rZSOD and 5.3-fold higher than that of bay scallop Argopecten irradians rAi-icCuZnSOD. The enzyme stability assay showed that the purified rCp-icCuZnSOD enzyme maintained more than 80% activity at temperature up to 60 C, at pH 2.0e9.0, and was resistant to 8 mol/L urea or 8% SDS. In addition, the addition of active rCp-icCuZnSOD enzmye could protect hepatocyte L02 cells from oxidative damage as assessed using an alcohol-injured human liver cell model. Crown
Scientific Reports, 2022
Superoxide dismutases are important group of antioxidant metallozyme and play important role in ROS homeostasis in salinity stress. The present study reports the biochemical properties of a salt-tolerant Cu, Zn-superoxide from Avicennia marina (Am_SOD). Am_SOD was purified from the leaf and identified by mass-spectrometry. Recombinant Am_SOD cDNA was bacterially expressed as a homodimeric protein. Enzyme kinetics revealed a high substrate affinity and specific activity of Am_SOD as compared to many earlier reported SODs. An electronic transition in 360–400 nm spectra of Am_SOD is indicative of Cu2+-binding. Am_SOD activity was potentially inhibited by diethyldithiocarbamate and H2O2, a characteristic of Cu, Zn-SOD. Am_SOD exhibited conformational and functional stability at high NaCl concentration as well in alkaline pH. Introgression of Am_SOD in E. coli conferred tolerance to oxidative stress under highly saline condition. Am_SOD was moderately thermostable and retained functional...
Characterization of Fish Cu/Zn?Superoxide Dismutase and Its Protection from Oxidative Stress
Marine Biotechnology, 2003
Copper/zinc superoxide dismutase was cloned from the zebrafish (Danio rerio). The full coding region of the zebrafish superoxide dismutase (ZSOD) complementary DNA was ligated with pET-20b(+) and successfully expressed in Escherichia coli strain AD494(DE3)pLysS. The active enzyme was purified by His tagging. The ZSOD yield was 6 mg from 0.2 L of E. coli culture, and the specific activity was 2000 U/mg as assayed using a RANSOD kit. The enzyme stability was characterized by reaction to temperature, pH, and detergent treatment. The results showed enzyme activity was still active after heat treatment at 70°C for 10 minutes, resistant to pH treatment from 2.3 to 12, and resistant to treatment with sodium dodecyl sulfate (SDS) under 4%. In addition, the recombinant ZSOD was used to protect fish from 100 ppm of paraquatinduced oxidative injury by soaking fish larva in 55 lg/ml SOD enzyme. The results were significant.
Characterization of Cu,Zn Superoxide Dismutase from the Bathophile Fish, Lampanyctus crocodilus
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1997
The pathogenic yeast Cryptococcus neoformans ( Cn ) var. gattii causes meningoencephalitis in healthy individuals, unlike the better known Cn varieties grubii and neoformans , which are common in immunocompromised individuals. The virulence determinants and mechanisms of host predilection are poorly defined for var. gattii . The present study focused on the characterization of a Cu,Zn superoxide dismutase ( SOD1 ) gene knock-out mutant constructed by developing a DNA transformation system. The sod1 mutant was highly sensitive to the redox cycling agent menadione, and showed fragmentation of the large vacuole in the cytoplasm, but no other defects were seen in growth, capsule synthesis, mating, sporulation, stationary phase survival or auxotrophies for sulphurcontaining amino acids. The sod1 mutant was markedly attenuated in virulence in a mouse model, and it was significantly susceptible to in vitro killing by human neutrophils (PMNs). The deletion of SOD1 also resulted in defects in the expression of a number of virulence factors, i.e. laccase, urease and phospholipase. Complementation of the sod1 mutant with SOD1 resulted in recovery of virulence factor expression and menadione resistance, and in restoration of virulence. Overall, these results suggest that the antioxidant function of Cu,Zn SOD is critical for the pathogenesis of the fungus, but is dispensable in its saprobic life. This report constitutes the first instance in which superoxide dismutase has been directly implicated in the virulence of a fungal pathogen.
Fish & Shellfish Immunology, 2006
A copper/zinc superoxide dismutase (Cu,Zn-SOD) cDNA was cloned from the hepatopancreas of giant freshwater prawn Macrobrachium rosenbergii using reverse transcriptionepolymerase chain reaction (RTePCR) by degenerate primers. Both 3#and 5#-regions were isolated by the rapid amplification of cDNA ends method. Analysis of nucleotide sequence revealed that the Cu,Zn-SOD cDNA clone consists of 845 bp with an open reading frame of 603 bp encoding a protein of 201 amino acids with a 22 amino acid signal peptide. The calculated molecular mass of the mature proteins (179 amino acids) is 21 kDa with an estimated pI of 4.75. Two putative N-glycosylation sites, NXT and NXS, were observed in the Cu,Zn-SOD. Four conserved amino acids responsible for binding copper (H86, H89, H106 and H163) and four conserved amino acids responsible for binding zinc (H106, H114, H123 and D126) were observed. Sequence comparison showed that the Cu,Zn-SOD deduced amino acid sequence of M. rosenbergii has similarity of 60% and 64% to that of freshwater crayfish Pacifastacus leniusculus ecCu,Zn-SOD and blue crab Callinectes sapidus ecCu,Zn-SOD, respectively. Quantitative real-time RTePCR analysis showed that Cu,Zn-SOD transcripts in haemocytes of M. rosenbergii increased 3 h and 6 h after injection of Lactococcus garvieae, whereas Cu,Zn-SOD transcripts decreased in the hepatopancreas 3 h after L. garvieae injection.
Molecular Biology Reports, 2009
The full-length cDNA and genomic DNA of a cytoplasmic copper, zinc superoxide dismutase (CuZn-sod) were cloned from the hepatopancreas of small abalone Haliotis diversicolor supertexta by RT-PCR, RACE and TAIL PCR. The full-length cytoplasmic CuZn-sod cDNA (designated sasod) comprises 984 bp. Its ORF encodes a polypeptide of 154 amino acids with a predicted molecular mass of 15.7 kDa and theoretical isoelectric point of 6.30. The deduced amino acid (designated saSOD) shares a common consensus pattern with the SODs of vertebrate and invertebrate animals. The full-length sasod genomic DNA comprises 5,574 bp, containing five exons and four introns. The splice donor and acceptor sequence of the four introns is 5 0 GT-AG3 0 . Real time quantitative PCR analysis revealed that sasod expression level in hepatopancreas of small abalone was no significant difference at 2, 6, 48 and 192 h post TBT exposure (P [ 0.05). However, the sasod expression level at 12 and 24 h post TBT exposure was decreased significantly (P \ 0.05).
Superoxide dismutases (SODs) are dedicated to scavenge and dismutate the superoxide anions in order to protect the cells from oxidative stress by establishing the redox homeostasis. In this study, we describe a cytosolic Cu/ZnSOD, the second SOD member from rock bream Oplegnathus fasciatus (Of-cCu/ZnSOD) at molecular, geno-mic structural-, transcriptional-and functional-levels. The determination of genomic arrangement of Of-cCu/ ZnSOD by means of a BAC library revealed that its primary transcript is represented by five exons and encoded a peptide of 154 amino acids. In silico investigation of Of-cCu/ZnSOD indicated the presence of several family characteristics including two Cu/ZnSOD signatures, seven metal liganding residues and eight β-sheets forming a β-barrel topology. Alignment and modeling studies confirmed the conservation of Cu/ZnSOD at primary and tertiary levels. While invertebrate Cu/ZnSOD members mainly demonstrate a tetraexonic structure, the vertebrate members have acquired an additional intron in the third exon resulting in a quinquepartite arrangement with class-specific exon lengths. Although, teleost Cu/ZnSOD members resembled the mammalian orthologs in their genomic organization, they shared a proximal position with molluscan members in the phylogeny. The an-tioxidant (AO) activity of Of-cCu/ZnSOD was affirmed by a recombinant protein which was also used to examine the biophysical and biochemical properties. The pronounced activity was detected when the rOf-cCu/ZnSOD was expressed with the Cu 2+ and Zn 2+ supplementation. The optimum activities were observed at pH 10 and 25 °C, and KCN strongly inhibited the activity of the rOf-cCu/ZnSOD. Furthermore, a constitutive mRNA expression of Of-cCu/ZnSOD with higher levels in blood N liver N heart and brain was observed, which was consistent with the transcriptional profile of Of-mMnSOD, suggesting important physiological role(s). This idea was further strengthened by the temporal assessment of Of-cCu/ZnSOD transcripts in animals under pathological (bacteria-or viral-induced) and physiological (H 2 O 2-induced oxidative) stress conditions using qPCR, in which it exhibited significantly up-regulated levels. Screening of Of-cCu/ZnSOD 5'-flanking region revealed the presence of several important transcription factor binding sites that potentially govern the Cu/ZnSOD expression. These findings conjointly contribute to expand our understanding regarding the piscine Cu/ZnSODs and; in particular, the AO enzyme network of rock bream.
Background: Neocaridina denticulata sinensis possesses characters of rapid growth, tenacious vitality, short growth cycle, transparent, and easy feeding. Therefore, it is gradually being developed into an animal model for basic research on decapod crustaceans. Superoxide dismutase (SOD) family, the first line of defense to eliminate ROS, can catalyze ROS into O2 and H2O2. Particularly, Cu/Zn-SOD accounts for about 90% of total SOD, which play an important role in many creatures. Nevertheless, only a small number of related reports have cloned and characterized Cu/Zn-SOD in decapod crustaceans. Herein, a Cu/Zn superoxide dismutase (Cu/Zn-SOD), named as Nd-ecCu/Zn-SOD, was identified and characterized from N. denticulata sinensis. Results: The full-length cDNA sequence of Nd-ecCu/Zn-SOD is 829 bp containing a 684 bp open reading frame, which encoded a protein of 227 amino acid residues with a typical Sod_Cu domain. The quantitative real-time PCR analysis showed that Nd-ecCu/Zn-SOD mRN...