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Molecular cloning of an Onchocerca volvulus extracellular Cu-Zn superoxide dismutase.

ABSTRACT: Onchocerca volvulus, a human parasitic nematode, is the third leading cause of preventable blindness worldwide. This study describes the molecular cloning of a novel superoxide dismutase (SOD) from the parasite. This putative O. volvulus extracellular SOD (OvEcSOD) is 628 nucleotides (nt) long, including a 22-nt 5' spliced leader (SL1) and a portion encoding an N-terminal hydrophobic 42-amino-acid signal peptide. The remainder of the cDNA shares 71% identity with an O. volvulus cytosolic SOD sequence and is 3 nt longer. All residues involved in metal ion binding, active site formation, folding, and dimer formation in SODs are conserved. Data indicate the OvEcSOD and O. volvulus cytosolic SOD are separate gene products and that the OvEcSOD appears to possess the characteristics of a membrane-bound or secreted enzyme which may be involved in the parasite defense against phagocyte-generated reactive oxygen species.

SUBMITTER: James ER

PROVIDER: S-EPMC186164 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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Molecular cloning of an Onchocerca volvulus extracellular Cu-Zn superoxide dismutase.

James E R ER McLean D C DC Perler F F

Infection and immunity 19940201 2

Onchocerca volvulus, a human parasitic nematode, is the third leading cause of preventable blindness worldwide. This study describes the molecular cloning of a novel superoxide dismutase (SOD) from the parasite. This putative O. volvulus extracellular SOD (OvEcSOD) is 628 nucleotides (nt) long, including a 22-nt 5' spliced leader (SL1) and a portion encoding an N-terminal hydrophobic 42-amino-acid signal peptide. The remainder of the cDNA shares 71% identity with an O. volvulus cytosolic SOD seq ...[more]

PMID: 8300230

Similar Datasets

Characterization and molecular cloning of a Cu/Zn superoxide dismutase from the human parasite Onchocerca volvulus.

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| S-EPMC257966 | biostudies-other

Cloning and expression analysis of Drosophila extracellular Cu Zn superoxide dismutase.

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| S-EPMC174485 | biostudies-other

Crystal structure of an extracellular superoxide dismutase from Onchocerca volvulus and implications for parasite-specific drug development.

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Steered molecular dynamic simulations of conformational lock of Cu, Zn-superoxide dismutase.

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Cu,Zn-superoxide dismutase without Zn is folded but catalytically inactive.

Project description:Amyotrophic lateral sclerosis has been linked to the gain of aberrant function of superoxide dismutase, Cu,Zn-SOD1 upon protein misfolding. The mechanism of SOD1 misfolding is thought to involve mutations leading to the loss of Zn, followed by protein unfolding and aggregation. We show that the removal of Zn from SOD1 may not lead to an immediate unfolding but immediately deactivates the enzyme through a combination of subtle structural and electronic effects. Using quantum mechanics/discrete molecular dynamics, we showed that both Zn-less wild-type (WT)-SOD1 and its D124N mutant that does not bind Zn have at least metastable folded states. In those states, the reduction potential of Cu increases, leading to the presence of detectable amounts of Cu(I) instead of Cu(II) in the active site, as confirmed experimentally. The Cu(I) protein cannot participate in the catalytic Cu(I)-Cu(II) cycle. However, even without the full reduction to Cu(I), the Cu site in the Zn-less variants of SOD1 is shown to be catalytically incompetent: unable to bind superoxide in a way comparable to the WT-SOD1. The changes are more radical and different in the D124N Zn-less mutant than in the Zn-less WT-SOD1, suggesting D124N being perhaps not the most adequate model for Zn-less SOD1. Overall, Zn in SOD1 appears to be influencing the Cu site directly by adjusting its reduction potential and geometry. Thus, the role of Zn in SOD1 is not just structural, as was previously thought; it is a vital part of the catalytic machinery.

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Structural consequences of cysteinylation of Cu/Zn-superoxide dismutase.

Project description:The metalloenzyme Cu/Zn-superoxide dismutase (SOD1) catalyzes the reduction of superoxide anions into molecular oxygen and hydrogen peroxide. Hydrogen peroxide can oxidize SOD1, resulting in aberrant protein conformational changes, disruption of SOD1 function, and DNA damage. Cells may have evolved mechanisms of regulation that prevent such oxidation. We observed that cysteinylation of cysteine 111 (Cys111) of SOD1 prevents oxidation by peroxide (DOI 10.1021/bi4006122 ). In this article, we characterize cysteinylated SOD1 using differential scanning fluorometry and X-ray crystallography. The stoichiometry of binding was one cysteine per SOD1 dimer, and there does not appear to be free volume for a second cysteine without disrupting the dimer interface. Much of the three-dimensional structure of SOD1 is unaffected by cysteinylation. However, local conformational changes are observed in the cysteinylated monomer that include changes in conformation of the electrostatic loop (loop VII; residues 133-144) and the dimer interface (loop VI; residues 102-115). In addition, our data shows how cysteinylation precludes oxidation of cysteine 111 and suggests possible cross-talk between the dimer interface and the electrostatic loop.

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Cu,Zn-superoxide dismutase increases toxicity of mutant and zinc-deficient superoxide dismutase by enhancing protein stability.

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| S-EPMC2962488 | biostudies-literature

Preparation of reduced bovine Cu,Zn superoxide dismutase.

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