Molecular Cloning and Expression of Cu/Zn-Containing Superoxide Dismutase from Fasciola hepatica

Abstract

The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/ZnSODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.

Functional expression and characterization of a cytosolic copper/zinc-superoxide dismutase of Spirometra erinacei

“…Its activity was inhibited by H 2 O 2 and KCN, but not by NaN 3 . These results are comparable with those obtained with other helminth enzymes (Hong et al 1992;Kim et al 2000;Lee et al 2005;Li et al 2005a, b). SeCuZnSOD was determined to be functionally expressed in different developmental stages of S. erinacei, plerocercoid larvae and adult worms.…”

“…They play essential roles in protecting organisms from oxidation by the superoxide anion which is produced by enzymatic and non-enzymatic metabolic pathways (Fridovich 1998). SODs have been identified and characterized in various helminth parasites, and their essential roles for parasite survival in the hosts have been well addressed (Henkle et al 1991;Hong et al 1992;HenkleDührsen et al 1995;Kim et al 2000;Tawe et al 2000;Castellanos-González et al 2002;Lee et al 2005;Li et al 2005a, b;Wu et al 2008). Several studies have also suggested the potential of SODs of parasitic helminths as vaccine candidates (Shalaby et al 2003;Carvalho-Queiroz et al 2004;Cook et al 2004;Vaca-Paniagua et al 2008).…”

Biochemical assessment of oxidative status versus liver enzymes in patients with chronic fascioliasis

“…SODs have been demonstrated from various helminths of different species such as Schistosoma mansoni, Onchocerca volvulus, Dirofilaria immitis, Brugia pahangi, and F. hepatica (Callahan et al 1991;Hong et al 1992;James et al 1994;Kim et al 2000;Thorpe 1965). Infection with F. hepatica was accompanied by rising level of the superoxide radicals which opens a way to act as precursor of other ROS (Abo-Shousha et al 1999;Oberley and Oberley 1997).…”

Molecular cloning and characterization of the copper/zinc and manganese superoxide dismutase genes from the human parasiteClonorchis sinensis

“…Degenerate oligonucleotide primers (CZ-Df/CZ-Dr for Cu/ZnSOD and Mn-Df/Mn-Dr for MnSOD, Table 1) were designed based on two highlyconserved regions of Cu/ZnSOD or MnSOD cDNA sequences of other species Henkle-Duhrsen et al 1995 ;Kim et al 2000). PCR reactions were performed on C. sinensis cDNA library.…”