Structural and functional analysis of glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn-superoxide dismutase (Cu/ZnSOD) (HLSOD). To improve its yield, the effect of increased concentration of Cu2+ (from 1 to 750 microg/ml) on growth and enzyme biosynthesis was studied. The primary structure of this fungal enzyme has been determined by Edman degradation of peptide fragments derived from proteolytic digest. A single chain of the protein, consisting of 152 amino acid residues, reveals a very high degree (74-85%) of structural homology in comparison to the amino acid sequences of other fungal Cu/ZnSODs. The difference of the molecular masses of H. lutea Cu/ZnSOD, measured by MALDI-MS (15,935 Da) and calculated by its amino acid sequence (15,716 Da), is attributed to the carbohydrate chain of one mole of N-acetylglucosamine, attached to the N-glycosylation site Asn23-Glu-Ser. HLSOD protected mice from mortality after experimental influenza A/Aichi/2/68 (H3N2) virus infection. Using the glycosylated HLSOD, the survival rate is increased by 66% (protective index=86.1%) and the survival time prolonged by 5.2 days, similar to the application of ribavarin, while non-glycosylated bovine SOD conferred lower protection.     

Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method

The coding region of copper/zinc-superoxide dismutase (Cu/Zn-SOD) cDNA from sweet potato, Ipomoea batatas (L.) Lam. cv. Tainong 57, was introduced into an expression vector, pET-20b(+). The Cu/Zn-SOD purified by His-tagged technique showed two active forms (dimer and monomer). The amount of proteins of dimer and monomer appeared to be equal, but the activity of dimeric form was seven times higher than that of monomeric form. The enzyme was dissociated into monomer by imidazole buffer above 1.0 M, acidic pH (below 3.0), or SDS (above 1%). The enzyme is quite stable. The enzyme activity is not affected at 85 °C for 20 min, in alkali pH 11.2, or in 0.1 M EDTA and also quite resistant to proteolytic attack. Dimer is more stable than monomer. The thermal inactivation rate constant k _dcalculated for the monomer at 85 °C was 0.029 min^-1 and the half-life for inactivation was about 28 min. In contrast, there is no significant change of dimer activity after 40 min at 85 °C. The enzyme dimer and monomer retained 83% and 58% of original activity, respectively, after 3 h incubation with trypsin at 37 °C, while those retained 100% and 31% of original activity with chymotrypsin under the same condition. These results suggest subunit interaction might change the enzyme conformation and greatly improve the catalytic activity and stability of the enzyme. It is also possible that the intersubunit contacts stabilize a particular optimal conformation of the protein or the dimeric structure enhances catalytic activity by increasing the electrostatic steering of substrate into the active site.     

Lipid molecules induce the cytotoxic aggregation of Cu/Zn superoxide dismutase with structurally disordered regions

Cu/Zn-superoxide dismutase (SOD1) is present in the cytosol, nucleus, peroxisomes and mitochondrial intermembrane space of human cells. More than 114 variants of human SOD1 have been linked to familial amyotrophic lateral sclerosis (ALS), which is also known as Lou Gehrig's disease. Although the ultimate mechanisms underlying SOD1-mediated cytotoxicity are largely unknown, SOD1 aggregates have been strongly implicated as a common feature in ALS. This study examined the mechanism for the formation of SOD1 aggregates in vitro as well as the nature of its cytotoxicity. The aggregation propensity of SOD1 species was investigated using techniques ranging from circular dichroism spectroscopy to fluorescence dye binding methods, as well as electron microscopic imaging. The aggregation of SOD1 appears to be related to its structural instability. The demetallated (apo)-SOD1 and aggregated SOD1 species, with structurally disordered regions, readily undergo aggregation in the presence of lipid molecules, whereas metallated (holo)-SOD1 does not. The majority of aggregated SOD1s that are induced by lipid molecules have an amorphous morphology and exhibit significant cytotoxicity. The lipid binding propensity of SOD1 was found to be closely related to the changes in surface hydrophobicity of the proteins, even at very low levels, which induced further binding and assembly with lipid molecules. These findings suggest that lipid molecules induce SOD1 aggregation under physiological conditions and exert cytotoxicity, and might provide a possible mechanism for the pathogenesis of ALS.     

Isolation and characterization of a novel superoxide dismutase from fungal strain Humicola lutea 110.

A novel thermostable MnSOD was purified to electrophoretic homogeneity from the fungal strain Humicola lutea 110. The preparation of the pure metalloenzyme was performed using treatment with acetone followed by ion exchange and gel permeation chromatography. We found that the activity of this enzyme comprises about 80% of the total superoxide dismutase activity in the crude extract, containing two proteins: MnSOD and Cu/ZnSOD. The MnSOD has a molecular mass of approximately 76 kDa and 7200 U/mg protein specific activity. It is a tetrameric enzyme with four identical subunits of 18 860 Da each as indicated by SDS-PAGE, amino acid analysis and mass spectrometry. N-terminal sequence analysis of MnSOD from the fungal strain revealed a high degree of structural homology with enzymes from other eukaryotic sources. Physicochemical properties were determined by absorption spectroscopy and circular dichroism measurements. The UV absorption spectrum was typical for an MnSOD enzyme, but displayed an increased absorption in the 280 nm region (epsilon280 = 10.4 mM(-1). cm(-1)), attributed to aromatic amino acid residues. The CD data show that MnSOD has two negative Cotton effects at 208 and 222 nm allowing the calculation of its helical content. The ellipticity at 222 nm is 6800 deg. x m(2) x dmol(-1) and thus similar to the values reported for other MnSODs. The MnSOD from H. lutea 110 is stable over a wide range of pH (4.5-8), even in the presence of EDTA. The enzyme is thermostable at 70-75 degrees C, and more stable than MnSODs from other sources.     

Cloning of a putative extracellular Cu/Zn superoxide dismutase and functional differences of superoxide dismutases in invasive and indigenous whiteflies

Superoxide dismutases (SODs) are a group of important antioxidant defense enzymes. In this study, a putative extracellular Cu/Zn superoxide dismutase (ecCuZnSOD) complementary DNA was cloned and characterized from the whitefly, Bemisia tabaci. Quantitative polymerase chain reaction analysis showed that the expression level of Bt‐ecCuZnSOD was more than 10‐fold higher in the invasive Middle East Asia Minor 1 (MEAM1) than in the native Asia II 3 species of the B. tabaci species complex. After exposure to low temperature (4 °C), the expression of Bt‐ecCuZnSOD gene was significantly up‐regulated in MEAM1 but not in Asia II 3. Furthermore, the expression level of B. tabaci intracellular CuZnSOD (Bt‐icCuZnSOD), Bt‐ecCuZnSOD and mitochondrial MnSOD (Bt‐mMnSOD) was compared after transferring MEAM1 and Asia II 3 whiteflies from favorable (cotton) to unfavorable host plants (tobacco). On cotton, both CuZnSOD genes were expressed at a higher level in MEAM1 compared with Asia II 3. Interestingly, after transferring onto tobacco, the expression of Bt‐ecCuZnSOD was significantly induced in Asia II 3 but not in MEAM1. On the other hand, while Bt‐mMnSOD was expressed equally in both species on cotton, Bt‐mMnSOD messenger RNA was up‐regulated in MEAM1 on tobacco. Consistently, enzymatic activity assays of CuZnSOD and MnSOD demonstrated that CuZnSOD might play an important protective role against oxidative stress in Asia II 3, whereas MnSOD activation was critical for MEAM1 whiteflies during host adaptation. Taken together, our results suggest that the successful invasion of MEAM1 is correlated with its constitutive high activity of CuZnSOD and inducible expression of MnSOD under stress conditions.     

Structure of Cu/Zn superoxide dismutase from the heavy-metal-tolerant yeast Cryptococcus liquefaciens strain N6

The deep-sea yeast Cryptococcus liquefaciens strain N6 shows high tolerance towards heavy metals, and can grow in the presence of high concentrations of copper ions. Enzymatic analysis indicated that copper ions induced the Cu/Zn superoxide dismutase activity of strain N6 (Cl-SOD1). In this study, the 1.2 Å resolution crystal structure of Cl-SOD1 has revealed several significant residue substitutions compared to the other Cu/Zn SODs. In the electrostatic loop, notably, His135 and Pro136 replace the well-conserved linear residues, while Thr133 substitutes a highly conserved glycine. The electrostatic loop has been shown to be involved in the copper uptake process, and these substitutions have caused an inward dragging of the turn region of the loop. As the introduction of proline and abolishment of glycine decrease loop flexibility, this structural reorganization may have helped stabilize the loop conformation, possibly resulting in more efficient copper uptake and a more stabilized copper-bound form.     

蜡样芽胞杆菌M22铜锌超氧化物歧化酶基因的克隆及表达

采用PCR技术 ,从蜡样芽胞杆菌M2 2基因组DNA扩增到长 132 0bp的基因片段 .该片段含编码 179个氨基酸残基的开放阅读框 ,推定蛋白序列与报道的BacillusanthracisCu ,Zn SOD序列有96 %同源性 ,其中N端 16个氨基酸残基推定为信号肽序列 .将Cu ,Zn SOD编码区插入载体pET 2 2b(+ )构建表达载体pET 2 2b sodC ,转化E .coliBL2 1(DE3) ,IPTG诱导融合蛋白表达 .SDS PAGE显示 ,融合蛋白表观分子质量约 2 4kD ,占菌体裂解液中总蛋白的 2 1 3% .将此表达载体转入SOD缺陷型大肠杆菌PN132 ,赋予了该菌株对paraquat的抗性 .NBT光抑制法测定SOD活性显示 ,与PN132无SOD活性相比 ,重组子在IPTG诱导前SOD活性极低 (1 79U/mg) ,诱导后活性高达 6 9 76U/mg .非变性电泳结果显示 ,该酶活性不同程度地受到 5mmol LH2 O2 和 5mmol LKCN的抑制     

Molecular characterization and oxidative stress response of an intracellular Cu/Zn superoxide dismutase (CuZnSOD) of the whitefly, Bemisia tabaci

Superoxide dismutases (SODs) are important for the survival of insects under environmental and biological stresses; however, little attention has been devoted to the functional characterization of SODs in whitefly. In this study, an intracellular copper/zinc superoxide dismutase of whitefly (Bemisia tabaci) (Bt-CuZnSOD) was cloned. Sequence analysis indicated that the full length cDNA of Bt-CuZnSOD is of 907 bp with a 471 bp open reading frame encoding 157 amino acids. The deduced amino acid sequence shares common consensus patterns with the CuZnSODs of various vertebrate and invertebrate animals. Phylogenetic analysis revealed that Bt-CuZnSOD is grouped together with intracellular CuZnSODs. Bt-CuZnSOD was then over-expressed in E. coli and purified using GST purification system. The enzymatic activity of purified Bt-CuZnSOD was assayed under various temperatures. When whiteflies were exposed to low (4°C) and high (40°C) temperatures, the in vivo activity of Bt-CuZnSOD was significantly increased. Furthermore, we measured the activities of several antioxidant enzymes, including SOD, catalase and peroxidase, in the whitefly after transferring the whitefly from cotton to tobacco (an unfavorable host plant). We found that the activity of SOD increased rapidly on tobacco plant. Taken together, these results suggest that the Bt-CuZnSOD plays a major role in protecting the whitefly against various stress conditions.     

Modification and inactivation of Cu,Zn-superoxide dismutase by the lipid peroxidation product,acrolein

Acrolein is the most reactive aldehydic product of lipid peroxidation and is found to be elevated in the brain when oxidative stress is high. The effects of acrolein on the structure and function of human Cu,Zn-superoxide dismutase (SOD) were examined. When Cu,Zn-SOD was incubated with acrolein, the covalent crosslinking of the protein was increased, and the loss of enzymatic activity was increased in a dose-dependent manner. Reactive oxygen species (ROS) scavengers and copper chelators inhibited the acrolein-mediated Cu,Zn-SOD modification and the formation of carbonyl compound. The present study shows that ROS may play a critical role in acrolein-induced Cu,Zn-SOD modification and inactivation. When Cu,Zn-SOD that has been exposed to acrolein was subsequently analyzed by amino acid analysis, serine, histidine, arginine, threonine and lysine residues were particularly sensitive. It is suggested that the modification and inactivation of Cu,Zn-SOD by acrolein could be produced by more oxidative cell environments. [BMB Reports 2013; 46(11): 555-560]     

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODs; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 °C for 1 h at pH from 8 to 11.5, while maintaining its biological activity.     

Cloning and DNA sequencing of cytosolic Cu/Zn superoxide dismutase gene from chinese cabbage

A cDNA clone for the cytosolic Cu/Zn superoxide dismutase (Cu/Zn SOD) from Chinese cabbage (Brassica campestris ssp.pekinensis) was isolated and its DNA sequence was determined. The cDNA clone contains a complete coding sequence which encodes a protein of 152 amino acids and a 3-untranslated region including a poly A signal. The deduced amino acid sequence shows that it is highly homologous to the Cu/Zn SODs from other plants (60–90%). The lack of a putative chloroplast targeting transit peptide indicates that the clone represents a cytosolic form of Cu/Zn SOD. Genomic Southern hybridization suggests that cytosolic Cu/Zn SOD genes are present in 1 or 2 copies per genome.     

Dominant role of copper in the kinetic stability of Cu/Zn superoxide dismutase

Mutations in Cu/Zn superoxide dismutase (SOD) are involved in some cases of familial amyotrophic lateral sclerosis, and it appears that misfolding and aggregation, perhaps mediated by abnormal binding or loss of copper (Cu) and/or zinc (Zn), may play a pathological role. It is known that the absence of both metals kinetically destabilizes wild type and mutant SOD leading to a 60-fold increase in their rate of unfolding. Here, the individual contributions of Cu and Zn to the kinetic stability of SOD were investigated, and the results show that Cu plays a greater role. Thus, the deficiency of Cu or Zn, especially the former, will compromise the kinetic stability of SOD, thereby increasing the probability that pathogenic mutants and even the WT protein may misfold and self-assemble into toxic species.     

Modification of cysteine 111 in Cu/Zn superoxide dismutase results in altered spectroscopic and biophysical properties

Cu/Zn superoxide dismutase (SOD) mutations are involved in about 20% of all cases of familial amyotrophic lateral sclerosis (FALS). Recently, it has been proposed that aberrant copper activity may be occurring within SOD at an alternative binding, and cysteine 111 has been identified as a potential copper ligand. Using a commercial source of human SOD isolated from erythrocytes, an anomalous absorbance at 325 nm was identified. This unusual property, which does not compromise SOD activity, had previously been shown to be consistent with a sulfhydryl modification at a cysteine residue. Here, we utilized limited trypsin proteolysis and mass spectrometry to show that the modification has a mass of 32 daltons and is located at cysteine 111. The reaction of SOD with sodium sulfide, which can react with cysteine to form a persulfide group, and with potassium cyanide, which can selectively remove persulfide bonds, confirmed the addition of a persulfide group at cysteine 111. Gel electrophoresis and glutaraldehyde cross-linking revealed that this modification makes the acid-induced denaturation of SOD fully irreversible. Furthermore, the modified protein exhibits a slower acid-induced unfolding, and is more resistant to oxidation-induced aggregation caused by copper and hydrogen peroxide. Thus, these results suggest that cysteine 111 can have a biochemical and biophysical impact on SOD, and suggest that it can interact with copper, potentially mediating the copper-induced oxidative damage of SOD. It will be of interest to study the role of cysteine 111 in the oxidative damage and aggregation of toxic SOD mutants.     

Cu/Zn superoxide dismutase is differentially regulated in period gene-mutant mice

The circadian clock in the brain coordinates the phase of peripheral oscillators that regulate tissue-specific physiological outputs. Here we report that circadian variations in the expression and activity of Cu/Zn superoxide dismutase (SOD1; EC 1.15.1.1) are present in liver homogenates from mice. The SOD1 mRNA expression from wild-type (WT) mice peaked at Zeitgeber Time 9 (ZT9; 9 h after lights-on time). While there was no rhythmicity in that from period2 (per2) gene knockout (P2K) mice, the level of SOD1 from per1/per2 double knockout (DKO) mice was significantly elevated at ZT5. The enzyme activity of SOD1 was also rhythmic in the mouse liver. Moreover, the total amount of the SOD1 exhibited a rhythmic oscillation with a peak at ZT9 in the liver from WT mice. We also found that tert-butylhydroperoxide (t-BHP)-induced oxidative damage in both WT and P2K mouse embryonic fibroblast (MEF) cells resulted in the up-regulation of SOD1 levels. Our data suggest that the expression of an important antioxidant enzyme, SOD1, is under circadian clock control and that mice are more susceptible to oxidative stress depending on the time of day.     

Heat-shock protein 105 interacts with and suppresses aggregation of mutant Cu/Zn superoxide dismutase: clues to a possible strategy for treating ALS

A dominant mutation in the gene for copper-zinc superoxide dismutase (SOD1) is the most frequent cause of the inherited form of amyotrophic lateral sclerosis. Mutant SOD1 provokes progressive degeneration of motor neurons by an unidentified acquired toxicity. Exploiting both affinity purification and mass spectrometry, we identified a novel interaction between heat-shock protein 105 (Hsp105) and mutant SOD1. We detected this interaction both in spinal cord extracts of mutant SOD1(G93A) transgenic mice and in cultured neuroblastoma cells. Expression of Hsp105, which is found in mouse motor neurons, was depressed in the spinal cords of SOD1(G93A) mice as disease progressed, while levels of expression of two other heat-shock proteins, Hsp70 and Hsp27, were elevated. Moreover, Hsp105 suppressed the formation of mutant SOD1-containing aggregates in cultured cells. These results suggest that techniques that raise levels of Hsp105 might be promising tools for alleviation of the mutant SOD1 toxicity.     

Purification and partial characterization of Cu, Zn containing superoxide dismutase from entomogenous fungal species Cordyceps militaris

Cordyceps militaris mycelium produced mainly Cu, Zn containing superoxide dismutase (Cu, Zn-SOD). Cu, Zn-SOD activity was detectable in the culture filtrates, and intracellular Cu, Zn-SOD activity as a proportion protein was highest in early log phase culture. The effects of Cu²⁺, Zn²⁺, Mn²⁺ and Fe²⁺ on enzyme biosynthesis were studied. The Cu, Zn-SOD was isolated and purified to homogeneity from C. militaris mycelium and partially characterized. The purification was performed through four steps: (NH₄)₂SO₄ precipitation, DEAE-sepharose™ fast flow anion-exchange chromatography, CM-650 cation-exchange chromatography, and Sephadex G-100 gel filtration chromatography. The purified enzyme had a molecular weight of 35070 ± 400 Da and consisted of two equal-sized subunits each having a Cu and Zn element. Isoelectric point value of 7.0 was obtained for the purified enzyme. The N-terminal amino acid sequence of the purified enzyme was determined for 12 amino acid residues and the sequences was compared with other Cu, Zn-SODs. The optimum pH of the purified enzyme was obtained to be 8.2–8.8. The purified enzyme remained stable at pH 5.8–9.8, 25 °C and up to 50 °C at pH 7.8 for 1.5 h incubation. The purified enzyme was sensitive to H₂O₂, KCN. 2.5 mM NaN₃, PMSF, Triton X-100, β-mercaptoethanol and DTT showed no significant inhibition effect on the purified enzyme within 5 h incubation period.