Purification and characterization of Cu,Zn-superoxide dismutase from common carp liver

1. Common carp (Cyprinus carpio L.) liver Cu,Zn-superoxide dismutase (Cu,Zn-SOD) was purified and characterized. 2. Its molecular weight, isoelectric point, electrophoretic mobility, amino acid pattern and some other characteristics were determined.     

Molecular cloning and biochemical characterization of a Cu,Zn-superoxide dismutase from Scedosporium apiospermum

A Cu,Zn-superoxide dismutase has been characterized from Scedosporium apiospermum, a fungus which often colonizes the respiratory tract of patients with cystic fibrosis. Enzyme production was stimulated by iron starvation. Purification was achieved from mycelial extract from 7-day-old cultures on Amberlite XAD-16. The purified enzyme presented a relative molecular mass of 16.4 kDa under reducing conditions and was inhibited by potassium cyanide and diethyldithiocarbamate, which are two known inhibitors of Cu,Zn-SODs. Its optimum pH was 7.0 and the enzyme retained full activity after pretreatment at temperatures up to 50 degrees C. Moreover, a 450-bp fragment of the gene encoding the enzyme was amplified by PCR using degenerate primers designed from sequence alignment of four fungal Cu,Zn-SODs. Sequence data from this fragment allowed us to design primers which were used to amplify by walking-PCR the flanking regions of the known fragment. SaSODC gene (890 bp) corresponded to a 154 amino acid polypeptide with a predicted molecular mass of 15.9 kDa. A database search for sequence homology revealed for the deduced amino acid sequence 72 and 83% identity rate with Cu,Zn-SODs from Aspergillus fumigatus and Neurospora crassa, respectively. To our knowledge, this enzyme is the first putative virulence factor of S. apiospermum to be characterized.     

Molecular cloning and characterization of copper/zinc-superoxide dismutase of Paragonimus westermani

Superoxide dismutases (SODs; EC 1.15.1.1) play important roles in the protection of the parasites against cellular oxygen-mediated killing of the hosts. A copper/zinc-containing SOD (Cu/Zn-SOD) was identified previously from lung fluke, Paragonimus westermani. To expand our understanding of P. westermani SOD, we isolated a complementary DNA encoding a Cu/Zn-SOD, expressed the active enzyme in Escherichia coli, and characterized its biochemical properties. The deduced amino acid (aa) sequence of the gene shared up to 73.7% identities with Cu/Zn-SODs of other helminths and shared well-conserved characteristic motifs and essential aa residues involved in coordinating copper and zinc enzymatic functions. Recombinant Cu/ Zn-SOD exhibited comparable biochemical properties with that of the native enzyme, including pH optima and potassium cyanide-and hydrogen peroxide-sensitive inhibition profiles. The active enzyme consisted of 2 identical subunits covalently linked by disulfide bonds. The enzyme was constitutively expressed throughout various developmental stages of the parasite. The levels increased as P. westermani matured and plateaued in adult stage. Our result suggests the enzyme might play an important role for parasites to survive in the hosts through its superoxide anion-detoxifying function.     

Identification and characterization of Paragonimus westermani leucine aminopeptidase

Paragonimus westermani is a tissue-invading trematode parasite that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals. While aminopeptidases play important roles in trematodes in the catabolism of host hemoglobin, an essential source of nutrient for the parasite, little is known about aminopeptidase in Paragonimus. Presently, we isolated a cDNA encoding a 58 kDa P. westermani leucine aminopeptidase (PwLAP). Deduced amino acid sequence of PwLAP exhibited significant sequence homology with LAP from Schistosoma spp. and Fasciola hepatica. Biochemical analysis of the recombinant PwLAP protein demonstrated preferential substrate specificity for Leu-NHMec and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of leucine aminopeptidase. PwLAP exhibited relatively higher enzyme activity in the presence of Mn2+ compared to Schistosoma mansoni LAP. Based on the biochemical properties and immunohistochemical analysis, PwLAP is concluded to represent a leucine aminopeptidase. The enzyme is most likely responsible for the catabolism of host hemoglobin, and, hence, represents a potential target of Paragonimus chemotherapy.     

Developmental regulation of rat lung Cu,Zn-superoxide dismutase.

In the present investigation we found that lung Cu,Zn-superoxide dismutase (SOD) activity (units/mg of DNA) increases steadily in the rat from birth to adulthood. The specific activity (units/micrograms of enzyme) of Cu,Zn-SOD was unchanged from birth to adulthood, excluding enzyme activation as a mechanism responsible for the increase in enzyme activity. Lung synthesis of Cu,Zn-SOD peaked at 1 day before birth and decreased thereafter to adult values. Calculations, based on rates of Cu,Zn-SOD synthesis and the tissue content of the enzyme, indicated that lung Cu,Zn-SOD activity increased during development owing to the rate of enzyme synthesis exceeding its rate of degradation by 5-10%. These calculations were supported by measurements of enzyme degradation in the neonatal (half-life, t1/2, = 12 h) and adult lung (t1/2 = greater than 100 h); the difference in half-life did not reflect the rates of overall protein degradation in the lung, since these rates were not different in lungs from neonatal and adult rats. We did not detect differences in the Mr or pI of Cu,Zn-SOD during development, but the susceptibility of the enzyme to inactivation by heat or copper chelation decreased with increasing age of the rats. We conclude that the progressive increase in activity of Cu,Zn-SOD is due to a rate of synthesis that exceeds degradation of the enzyme. The data also suggest that increased stabilization of enzyme conformation accounts for the greater half-life of the enzyme in lungs of adult compared with neonatal rats.     

Putative denitrosylase activity of Cu,Zn-superoxide dismutase

The Cu,Zn-superoxide dismutase (SOD1) has been reported to exert an S-nitrosylated glutathione (GSNO) denitrosylase activity that was augmented by a familial amyotrophic lateral sclerosis (FALS)-associated mutation in this enzyme. This putative enzymatic activity as well as the spontaneous decomposition of GSNO has been reexamined. The spontaneous decomposition of GSNO exhibited several peculiarities, such as a lag phase followed by an accelerating rate plus a marked dependence on GSNO concentration, suggestive of autocatalysis, and a greater rate in polypropylene than in glass vessels. Dimedone caused a rapid increase in absorbance likely due to reaction with GSNO, followed by a slower increase possibly due to reaction with an intermediate such as glutathione sulfenic acid. SOD1 weakly increased the rate of decomposition of GSNO, but did so only when GSH was present; and FALS-associated mutant forms of SOD1 were not more active in this regard than was the wild type. Decomposed GSNO, when added to fresh GSNO, hastened its decomposition, in accord with autocatalysis, and when added to GSH, generated GSNO in accord with the presence of nitrite. A mechanism is proposed that is in accord with these observations.     

Nitric oxide protects Cu,Zn-superoxide dismutase from hydrogen peroxide-induced inactivation

Reaction of Cu,Zn-superoxide dismutase (SOD1) and hydrogen peroxide generates a putative oxidant SOD-Cu2+-.OH that can inactivate the enzyme and oxidize 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to DMPO-.OH. In the presence of nitric oxide (.NO), the SOD1/H2O2 system is known to produce peroxynitrite (ONOO-). In contrast to the proposed cytotoxicity of .NO conferred by ONOO-, we report here a protective role of .NO in the H2O2-induced inactivation of SODI. In a dose-dependent manner, .NO suppressed formation of DMPO-.OH and inactivation of the enzyme. Fragmentation of the enzyme was not affected by .NO. Bicarbonate retarded formation of ONOO-, suggesting that .NO competes with bicarbonate for the oxidant SOD-Cu2+-.OH. We propose that .NO protects SOD1 from H2O2-induced inactivation by reducing SOD-Cu2+.OH to the active SOD-Cu2+ with concomitant production of NO+ which reacts with H2O2 to give ONOO-.     

Preparation and characterization of Cu,Zn-superoxide dismutase covalently modified by polyunsaturated fatty acids

To improve its stability and lipophilicity, Cu,Zn-superoxide dismutase (SOD) was chemically modified with linoleic and α-linolenic acids using two different methods. Higher retained enzymatic activity has been observed compared with SOD modified by macromolecular substances. Enhanced heat stability, acid and alkali resistance, and anti-pepsin/trypsin ability of the modified SOD were observed compared with those of the natural enzyme, the apparent oil-water partition coefficient being especially increased. The results characterize SOD modified with polyunsaturated fatty acids as a promising pharmacological tool.     

Purification and properties of a neutral thiol protease from larval trematode parasite Paragonimus westermani metacercariae

1. A neutral thiol protease was isolated from the extract of larvae of the mammalian trematode parasite, Paragonimus westermani metacercariae, by arginine-Sepharose, Ultrogel AcA-54 and DEAE-toyopearl column chromatography, measuring its activity by the hydrolysis of Boc-Val-Leu-Lys-MCA as a substrate. 2. The molecular weight of the purified enzyme was estimated to be 22,000 as a single polypeptide by SDS-polyacrylamide gel electrophoresis and was estimated to be 20,000 by size exclusion high-performance liquid chromatography. 3. The activity was suppressed by antipain, E-64, leupeptin, chymostatin, N-tosyl-L-lysine chloromethyl ketone, but was not affected by metallo protease inhibitors or serine protease inhibitors. 4. Studies on the substrate specificity showed that the enzyme hydrolyzed Boc-Val-Leu-Lys-MCA, Z-Phe-Arg-MCA, fluorescein isothiocyanate-labeled collagen, azocoll and casein. 5. The enzyme was found to hydrolyze peptide bonds of oxidized insulin B chain preferentially at the carboxy side of hydrophobic and basic amino acids.     

Molecular and biochemical characterization of hemoglobinase, a cysteine proteinase, in Paragonimus westermani

The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.     

Molecular characterization of two myoglobins of Paragonimus westermani

Myoglobins (Mbs), globin proteins, are present in high concentrations in trematodes. In Paragonimus westermani, 2 cDNAs were found to encode Mbs. The first clone, Pwmyo1, codes a total of 149 amino acids with a calculated mass of 16.6 kDa. The second, Pwmyo2, encodes a 146-amino acid protein with a calculated mass of 16.2 kDa. The predicted secondary structures showed the presence of 8 helices, which is the basic characteristic of Mbs. Sequence alignment revealed a high homology with the other trematode Mbs. The 2 clones contained the characteristic tyrosyl residues at helical positions B10 and distal E7, which are substitutions that have been previously shown to contribute to the high oxygen affinity of Mbs. Polyclonal antibodies against the recombinant Mbs were raised with no cross-reactivity observed. Immunolocalization revealed the proteins to be distributed generally throughout the parenchymal tissues, but absent from the tegument and reproductive organs. The cell mass of the eggs of the worm stained positive to Pwmyo2 but not Pwmyo1, suggesting the stage-specific expression of these Mbs.     

Modification of Cu,Zn-superoxide dismutase by oxidized catecholamines

Oxidation of catecholamines may contribute to the pathogenesis of Parkinson's disease (PD). The effect of the oxidized products of catecholamines on the modification of Cu,Zn-superoxide dismutase (SOD) was investigated. When Cu,Zn-SOD was incubated with the oxidized 3,4-dihydroxyphenylalanine (DOPA) or dopamine, the protein was induced to be aggregated. The deoxyribose assay showed that hydroxyl radicals were generated during the oxidation of catecholamines in the presence of copper ion. Radical scavengers, azide, N-acetylcysteine, and catalase inhibited the oxidized catecholamine-mediated Cu,Zn-SOD aggregation. Therefore, the results indicate that free radicals may play a role in the aggregation of Cu,Zn-SOD. When Cu,Zn-SOD that had been exposed to catecholamines was subsequently analyzed by an amino acid analysis, the glycine and histidine residues were particularly sensitive. These results suggest that the modification of Cu,Zn-SOD by oxidized catecholamines might induce the perturbation of cellular antioxidant systems and led to a deleterious cell condition.     

A novel heme protein, the Cu,Zn-superoxide dismutase from Haemophilus ducreyi

Haemophilus ducreyi, the causative agent of the genital ulcerative disease known as chancroid, is unable to synthesize heme, which it acquires from humans, its only known host. Here we provide evidence that the periplasmic Cu,Zn-superoxide dismutase from this organism is a heme-binding protein, unlike all the other known Cu,Zn-superoxide dismutases from bacterial and eukaryotic species. When the H. ducreyi enzyme was expressed in Escherichia coli cells grown in standard LB medium, it contained only limited amounts of heme covalently bound to the polypeptide but was able efficiently to bind exogenously added hemin. Resonance Raman and electronic spectra at neutral pH indicate that H. ducreyi Cu,Zn-superoxide dismutase contains a 6-coordinated low spin heme, with two histidines as the most likely axial ligands. By site-directed mutagenesis and analysis of a structural model of the enzyme, we identified as a putative axial ligand a histidine residue (His-64) that is present only in the H. ducreyi enzyme and that was located at the bottom of the dimer interface. The introduction of a histidine residue in the corresponding position of the Cu,Zn-superoxide dismutase from Haemophilus parainfluenzae was not sufficient to confer the ability to bind heme, indicating that other residues neighboring His-64 are involved in the formation of the heme-binding pocket. Our results suggest that periplasmic Cu,Zn-superoxide dismutase plays a role in heme metabolism of H. ducreyi and provide further evidence for the structural flexibility of bacterial enzymes of this class.     

Paragonimus westermani: molecular cloning,expression, and characterization of a recombinant yolk ferritin

Ferritin is an intracellular protein involved in iron metabolism. A cDNA PwYF-1 cloned from the adult Paragonimus westermani cDNA library encoded a putative polypeptide of 216 amino acids homologous with ferritins of vertebrates and invertebrates. Febinding motifs identified in PwYF-1 polypeptide were conserved and predicted to form a ferroxidase center. PwYF-1 polypeptide contained an extended peptide of 45 amino acids at its C-terminus. Recombinant PwYF-1 protein, expressed and purified from Escherichia coli, showed iron-uptake ability and ferroxidase activity. Ferroxidase activity of recombinant PwYF-1 protein was reactivated by secondary addition of apotransferrin to assay mixture. Mouse immune serum raised against the recombinant PwYF-1 protein recognized specifically 24 kDa protein from adult P. westermani lysate. PwYF-1 protein was localized to vitelline follicles and the eggs of P. westermani. Collectively, PwYF-1 protein was identified as a P. westermani yolk ferritin.     

Modification of Cysteine 111 in human Cu,Zn-superoxide dismutase

Human Cu,Zn-superoxide dismutase (hSOD1) has 4 cysteines per subunit. Cys57 and Cys148 are involved in an intrasubunit disulfide bond, while Cys6 and Cys111 are free. Cys6 is buried within the protein while Cys111 is on the surface, near the dimer interface. We examined by liquid chromatography-mass spectrometry the commercially purchased hSOD1 isolated from erythrocytes as well as hSOD1s isolated from human erythrocytes, brain, and hSOD1 expressed in Sf9, yeast, and E. coli. Our goal was to ascertain whether the Cys111 modification occurred naturally in vivo. Only the Sigma erythrocyte hSOD1 appeared to contain a trisulfide crosslink between the Cys111 residues. Thus it failed to react with N-ethylmaleimide, showed absorbtion at 325 nm that was eliminated by 2-mercaptoethanol, and had a mass 30 units more than expected for the native dimer. We examined the possibility that different purification methods might cause this modification in erythrocyte hSOD1. None of the procedures examined for hSOD1 purification produced such a trisulfide. In disagreement with Liu et al. [Biochemistry, 2000, 39, 8125-8132], complete derivitization of both Cys111s of hSOD1 from Sf9 cells with N-ethylmaleimide, 4-vinylpyridine, and by 5,5′-dithiobis(2-nitrobenzoic acid) were readily achieved; indicating that steric hindrance was not a problem.     

Paragonimus westermani: a cytosolic glutathione S-transferase of a sigma-class in adult stage

We purified cytosolic glutathione S-transferase (GST) of adult Paragonimus westermani monitoring its activity with 1-chloro-2,4-dinitrobenzene (CDNB). The enzyme was purified 18.4-fold to electrophoretic homogeneity with 21% recovery rate through a three-step procedure. The purified enzyme (Pw28GST) has a subunit molecular weight of 28 kDa with an isoelectric point at 4.6. Monoclonal antibody (anti-Pw28GST) against Pw28GST did not cross-react with GSTs from other helminths. cDNA library was constructed in lambdaZAP II bacteriophage and screened with anti-Pw28GST. The corresponding gene containing a single open reading frame of 804 bp encoded 211 amino acids. The predicted amino acid sequence exhibited a higher homology with catalytic domain near N-terminus of class sigma GSTs (58%) than with schistosome 28-kDa GSTs (45-41%) or with class sigma GSTs themselves (33-31%). The sequence contained both Tyr-6 and Tyr-10 that are highly conserved in mammalian and helminth GSTs. The apparent K(m) value of a recombinant enzyme was 0.78 mM. Both native and recombinant enzymes showed the highest activity against CDNB, relatively weak activity against ethacrynic acid and reactive carbonyls, and no activity against epoxy-3-(p-nitrophenoxy)-propane. The activities were inhibited by bromosulfophthalein, cibacron blue, and albendazole, but not by praziquantel. These findings indicate that adult P. westermani has a class sigma GST.