Cloning,production and characterisation of a recombinant Cu/Zn superoxide dismutase from Taenia solium

A full-length complementary DNA clone encoding a cytosolic Cu/Zn superoxide dismutase with a M(r) of 15,588 Da was isolated from a Taenia solium larvae complementary DNA library. Comparison analysis of its deduced amino acid sequence revealed a 71% identity with Schistosoma mansoni, 57.2-59.8% with mammalian and less than 54% with other helminth cytosolic Cu/Zn superoxide dismutase. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc enzymatic function are conserved. The T. solium Cu/Zn superoxide dismutase was expressed in the pRSET vector. Enzymatic and filtration chromatographic analysis showed a recombinant enzyme with an activity of 2,941 U/mg protein and a native M(r) of 37 kDa. Inhibition assays using KCN, H(2)O(2), NaN(3) and SDS indicated that Cu/Zn is the metallic cofactor in the enzyme. Thiabendazole (500 microM) and albendazole (300 microM) completely inhibited the activity of T. solium Cu/Zn superoxide dismutase. Thiabendazole had no effect on bovine Cu/Zn superoxide dismutase; in contrast, albendazole had a moderate effect on it at same concentrations. Antibodies against T. solium Cu/Zn superoxide dismutase did not affect the enzymatic function; nevertheless, it cross reacts with several Taenia species, but not with trematodes, nematodes, pig, human and bovine Cu/Zn superoxide dismutase enzymes. Western blot analysis indicated the enzyme was expressed in all stages. These results indicate that T. solium possesses a Cu/Zn superoxide dismutase enzyme that can protect him from oxidant-damage caused by the superoxide anion.     

cDNA cloning,high-level expression,purification, and characterization of an avian Cu,Zn superoxide dismutase from Peking duck

As a special species of avian, Peking duck is often used as a model for exploring effective factors against cardio-cerebrovascular diseases, and therefore investigations of antioxidant enzymes including superoxide dismutase are intriguing. By using 3(')-RACE with a gene-specific primer, a cDNA encoding duck Cu,Zn SOD was amplified from the total RNA extracted from Peking duck liver. Three free cysteine residues are found in the deduced amino acid sequence of duck SOD, among which Cys153 at the carbonyl-terminal is a distinctive feature. Production with a high yield of recombinant duck Cu,Zn SOD was achieved in Escherichia coli after the reconstituted expression vector pET-3a-dSOD was transformed into the bacterial strain BL21(DE3)pLysS. After two steps of anion exchange chromatography, a great quantity of the purified enzyme (100mg/L fermented culture) with an enzymatic activity comparable to that of native duck and bovine SOD was finally obtained. Duck SOD is a homodimer with 153 residues for each subunit. The molecular mass of the recombinant enzyme is 15,540.0Da measured by mass spectrum, which well coincides with the estimated size of the sequence but significantly differs from that of the native counterpart. Five charge isomers were observed on isoelectricfocusing (IEF). The most interesting observation is that the thermal stability of duck SOD is much lower than that of the bovine enzyme as revealed by irreversible heat inactivation at 70 degrees C. These properties are discussed in relation to the distinctive free Cys residues in duck Cu,Zn SOD.     

Metabolism of steroid hormones by Taenia solium and Taenia crassiceps cysticerci

Previous in vitro experiments showed that both, Taenia crassiceps and Taenia solium cysticerci have the ability to metabolize exogenous androstenedione to testosterone. Here we evaluate on the capacity of both cysticerci to synthesize several sex steroid hormones, using different hormonal precursors. Experiments using thin layer chromatography (TLC) showed that both cysticerci were able to produce ³H-hydroxyprogesterone, ³H-androstenedione and ³H-testosterone when ³H-progesterone was used as the precursor. They also synthesized ³H-androstenediol and ³H-testosterone when ³H-dehydroepiandrosterone was the precursor. In addition, both cysticerci interconverted ³H-estradiol and ³H-estrone. These results, strongly suggest the presence and activity of the Δ4 and Δ5 steroid pathway enzymes, 3β-hydroxysteroid dehydrogenase/Δ^5-4 isomerase-like enzyme (3β-HSD), that converts androstenediol into testosterone; and the 17β-hydroxysteroid dehydrogenase that interconverts estradiol and estrone, in both types of cysticerci.     

Steroid hormone production by parasites: the case of Taenia crassiceps and Taenia solium cysticerci

Many examples of reciprocal endocrine interactions between parasites and hosts have been found in insects, arthropods and mammals. Cysticercosis produced by Taenia solium metacestodes is a widely distributed parasite infection that affects the human and the pig. Taenia crassiceps experimental murine cysticercosis has been used to explore the role of biological factors involved in host–parasite interactions. We had shown that T. crassiceps cysticercosis affects the serum concentration of steroid hormones and the reproduction behavior of the male mice host. In an effort to understand the biology of the parasite, we had investigated the parasite capacity to produce sex steroids. For this purpose, T. crassiceps cysticerci were incubated in the presence of different steroid precursors. TLC and recrystallization procedures showed that testosterone is produced from ³H-androstenedione in cysticerci. The conversion of ³H-testosterone to androstenedione, although present is much less significant. In addition, we had studied the production of testosterone by T. solium cysticerci. For this purpose, cysticerci were dissected from pork meat and incubated as above described. The results showed that T. solium cysticerci also produce testosterone. We have speculated about the importance of androgens in the growth of T. crassiceps cysticerci and found that the addition of the antiandrogen flutamide to the culture media of the parasites significantly decreased ³H-thymidine incorporation. We therefore hypothesized, that the ability of cysticerci to produce testosterone from steroid precursors might be important for the parasite growth and development.     

Divalent-metal-dependent nucleolytic activity of Cu, Zn superoxide dismutase

The known action of Cu, Zn superoxide dismutase (holo SOD) that converts O₂ ⁻ to O₂ and H₂O₂ plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of holo SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much holo SOD may be injurious to the cells. In the in vitro study, we report a finding that the holo SOD from bovine erythrocytes and its apo form possess a divalent-metal-dependent nucleolytic activity, which was confirmed by UV–vis absorption titration of calf thymus DNA (ctDNA) with the holo SOD, quenching of holo SOD intrinsic fluorescence by ctDNA, and by gel electrophoresis monitoring conversion of DNA from the supercoiled DNA to nicked and linear forms, and fragmentation of a linear λDNA. Moreover, the DNA cleavage activity was examined in detail under certain reaction conditions. The steady-state study indicates that DNA cleavage supported by both forms of SOD obeys Michaelis–Menten kinetics. On the other hand, the assays with some other proteins indicate that this new function is specific to some proteins including the holo SOD. Therefore, this study reveals that the divalent-metal-dependent DNA cleavage activity is an intrinsic property of the holo SOD, which is independent of its natural metal (copper and zinc) sites, and may provide an alternative insight into the link between SOD enzymes and neurodegenerative disorders.     

Intracellular Cu/Zn superoxide dismutase (Cu/Zn-SOD) from hard clam Meretrix meretrix: its cDNA cloning, mRNA expression and enzyme activity

Hard clam (Meretrix meretrix) is an economically important bivalve in China. In the present study, a gene coding for an intracellular Cu/Zn-SOD was cloned and characterized from hard clam. The full-length cDNA of this Cu/Zn-SOD (designated as Mm-icCuZn-SOD) consisted of 1,383?bp, with a 462-bp of open reading frame (ORF) encoding 153 amino acids. Several highly conserved motifs, including the Cu/Zn binding sites [H(46), H(48), H(63), and H(119) for Cu binding; H(63), H(71), H(80), and D(83) for Zn binding], an intracellular disulfide bond and two Cu/Zn-SOD signatures were identified in Mm-icCu/Zn-SOD. The deduced amino acid sequence of Mm-icCu/Zn-SOD has a high degree of homology with the Cu/Zn-dependent SODs from other species, indicating that Mm-icCu/Zn-SOD should be a member of the intracellular Cu/Zn-dependent SOD family. Real-time PCR analysis showed that the highest level of Mm-icCu/Zn-SOD expression was in the hepatopancreas, while the lowest level occurred in the hemocytes. Hard clam challenged with Vibrio anguillarum showed a time-dependent increase in Mm-icCu/Zn-SOD expression that reached a maximum level after 6?h. Mm-icCu/Zn-SOD purified as a recombinant protein expressed in E. coli retained a high level of biological activity, 83?% after 10?min incubation at 10–50?°C, and more than 87?% after incubation in buffers with pH values between 2.2 and 10.2. These results indicated that Mm-icCu/Zn-SOD may play an important role in the innate immune system of hard clam.     

Cu,Zn superoxide dismutase from chicken erythrocytes.

1. Copper, zinc superoxide dismutase (Cu,Zn SOD) has been purified to homogeneity from chicken erythrocytes by anion-exchange, immobilized metal affinity and size exclusion chromatography. 2. Molecular properties (amino acid composition, molecular mass, subunit composition and spec. act.) of the chicken enzyme are similar to those of a bovine erythrocyte Cu,Zn SOD. 3. The chicken and bovine enzymes are immunologically similar since antisera raised against each enzyme are cross-reactive.     

Structure,chromosomal location,and analysis of the canine Cu/Zn superoxide dismutase (SOD1) gene

Mutations in Cu/Zn superoxide dismutase (SOD1), a major cytosolic antioxidant enzyme in eukaryotic cells, have been reported in approximately 20% of familial amyotrophic lateral sclerosis (FALS) patients. Hereditary canine spinal muscular atrophy (HCSMA), a fatal inherited motor neuron disease in Brittany spaniels, shares many clinical and pathological features with human motor neuron disease, including FALS. The SOD1 coding region has been sequenced and cloned from several animal species, but not from the dog. We have mapped the chromosomal location, sequenced, and characterized the canine SOD1 gene. Extending this analysis, we have evaluated SOD1 as a candidate for HCSMA. The 462 bp SOD1 coding region in the dog encodes 153 amino acid residues and exhibits more than 83% and 79% sequence identity to other mammalian homologues at both the nucleotide and amino acid levels, respectively. The canine SOD1 gene maps to CFA31 close to syntenic group 13 on the radiation hybrid (RH) map in the vicinity of sodium myo/inositol transporter (SMIT) gene. The human orthologous SOD1 and SMIT genes have been localized on HSA 21q22.1 and HSA 21q21, respectively, confirming the conservation of synteny between dog syntenic group 13 and HSA 21. Direct sequencing of SOD1 cDNA from six dogs with HCSMA revealed no mutations. Northern analysis indicated no differences in steady-state levels of SOD1 mRNA.     

Mechanism of the peroxidase activity of Cu,Zn superoxide dismutase

In addition to its very efficient catalysis of the dismutation of superoxide ( O₂^- ) into O₂ plus H₂O₂, Cu, Zn SOD acts less efficiently as a non-specific peroxidase. This peroxidase activity is CO₂ dependent although very slow peroxidation of some substrates occurs in the absence of CO2. The mechanism of that CO₂ dependence is explained by the generation of a strong oxidant at the copper site by two sequential reactions with H₂O₂, followed by the oxidation of CO₂ to the carbonate radical that then diffuses into the bulk solution. This diffusible carbonate radical is then responsible for the diverse oxidations that have been reported. A different mechanism that involves the reduction of peroxymonocarbonate by the reduced superoxide dismutase to yield carbonate radical has been proposed. We will demonstrate that this mechanism is not supported by the available data. It seems likely that generation of the carbonate radical has relevance to the oxidative stress faced by aerobic organisms.     

Preparation of reduced bovine Cu,Zn superoxide dismutase.

N.m.r. and e.p.r. were used to measure the oxidation state of copper in Cu,Zn superoxide dismutase treated with reducing agents such as NaBH4, K4Fe(CN)6, Na2S2O4 and H2O2. The activity and the electrophoretic pattern of the treated enzyme were also studied. On the basis of the reducing ability and of the absence of inactivating effects, NaBH4 was the most suitable reducer of those tested. Some characteristics of the reduction of superoxide dismutase by NaBH4 were further investigated. The results obtained indicate that NaBH4 can be used to prepare, in a few minutes, solutions of completely reduced enzyme without any apparent change of the activity and of the structure.     

Sequence and structural determinants of Cu, Zn superoxide dismutase aggregation

Diverse point mutations in the enzyme Cu, Zn superoxide dismutase (SOD1) are linked to its aggregation in the familial form of the disease amyotrophic lateral sclerosis. The disease-associated mutations are known to destabilize the protein, but the structural basis of the aggregation of the destabilized protein and the structure of aggregates are not well understood. Here, we investigate in silico the sequence and structural determinants of SOD1 aggregation: (1) We identify sequence fragments in SOD1 that have a high aggregation propensity, using only the sequence of SOD1, and (2) we perform molecular dynamics simulations of the SOD1 dimer folding and misfolding. In both cases, we identify identical regions of the protein as having high propensity to form intermolecular interactions. These regions correspond to the N- and C-termini, and two crossover loops and two beta-strands in the Greek-key native fold of SOD1. Our results suggest that the high aggregation propensity of mutant SOD1 may result from a synergy of two factors: the presence of highly amyloidogenic sequence fragments ("hot spots"), and the presence of these fragments in regions of the protein that are structurally most likely to form intermolecular contacts under destabilizing conditions. Therefore, we postulate that the balance between the self-association of aggregation-prone sequences and the specific structural context of these sequences in the native state determines the aggregation propensity of proteins.     

Prion-like activity of Cu/Zn superoxide dismutase

Neurodegenerative diseases belong to a larger group of protein misfolding disorders, known as proteinopathies. There is increasing experimental evidence implicating prion-like mechanisms in many common neurodegenerative disorders, including Alzheimer disease, Parkinson disease, the tauopathies, and amyotrophic lateral sclerosis (ALS), all of which feature the aberrant misfolding and aggregation of specific proteins. The prion paradigm provides a mechanism by which a mutant or wild-type protein can dominate pathogenesis through the initiation of self-propagating protein misfolding. ALS, a lethal disease characterized by progressive degeneration of motor neurons is understood as a classical proteinopathy; the disease is typified by the formation of inclusions consisting of aggregated protein within and around motor neurons that can contribute to neurotoxicity. It is well established that misfolded/oxidized SOD1 protein is highly toxic to motor neurons and plays a prominent role in the pathology of ALS. Recent work has identified propagated protein misfolding properties in both mutant and wild-type SOD1, which may provide the molecular basis for the clinically observed contiguous spread of the disease through the neuroaxis. In this review we examine the current state of knowledge regarding the prion-like properties of SOD1 and comment on its proposed mechanisms of intercellular transmission.     

Molecular cloning, expression, and characterization of the Cu,Zn superoxide dismutase (SOD1) gene from the entomopathogenic fungus Cordyceps militaris

We describe the molecular characterization of the Cu,Zn superoxide dismutase (SOD1) gene of Cordyceps militaris, which is one of the entomopathogenic fungi called a vegetable wasp and plant worm. The SOD1 gene of C. militaris spans 922 bp and consisted of three introns and four exons coding for 154 amino acid residues. The deduced amino acid sequence of the C. militaris SOD1 cDNA showed 88% identity to Claviceps purpurea SOD1, 82% to Neurospora crassa SOD1, and 75-64% to SOD1 sequences from other fungi. The C. militaris SOD1 possesses the typical metal binding ligands of six histidines and one aspartic acid common to fungal SOD1s. The cDNA encoding C. militaris SOD1 was expressed as a 17-kDa polypeptide in the baculovirus-infected insect Sf9 cells. The enzyme activity of the purified recombinant C. militaris SOD1 was approximately 568 U per mg(-1) . Southern blot analysis of the genomic DNA suggested the C. militaris SOD1 was a single gene. Northern and Western blot analysis and enzyme activity assays indicated SOD1 was expressed constitutively. This is the first report of an SOD1 gene from any entomopathogenic fungus.