Molecular characterization and expression of Onchocerca volvulus glutathione reductase

Abstract

Glutathione metabolism represents a potential target for anti-parasite drug design. The central role of glutathione reductase (GR) in maintenance of the thiol redox state and in anti-oxidative defence has to be evaluated in more detail in order to establish the essential function of this enzyme for the survival of the filarial parasite Onchocerca volvulus. The O. volvulus GR (OvGR) gene was cloned and sequenced. The gene is composed of 13 exons and 12 introns and spans 4065 bp. The first intron is located within the 5′-untranslated region of the gene, 16 nucleotides upstream of the translation initiation codon. Southern-blot analysis and structural characterization of the genomic sequence indicate that OvGR is encoded by a single-copy gene. Isolation of various cDNA clones revealed a polymorphism of polyadenylation initiation with no consensus polyadenylation sites in any of the cDNAs analysed. The entire cDNA is 1977 bp long and carries the nematode-specific spliced leader sequence SL1 at its 5′ end, 236 nucleotides upstream of the first in-frame methionine. The cDNA codes for a polypeptide of 462 amino acids with 53.5% sequence identity with human GR (HsGR). A total of 18 out of 19 residues contributing to glutathione binding are identical in OvGR and HsGR. However, one of the arginine residues (Arg-224 in HsGR) involved in discrimination between NADPH and NADH in all known GRs is substituted by tryptophan (Trp-207 in OvGR). The coding region of OvGR was expressed in Escherichia coli as a histidine-fusion protein, and it was established that the parasite protein still favours the binding of NADPH (Km 10.9 μM) over NADH (Km 108 μM). The histidine-fusion protein has a subunit size of 54 kDa and is active as a homodimer of 110 kDa.

Low-Molecular-Mass Antioxidants in Parasites

“…The main lowmolecular-mass thiol is GSH ( Table 1). The GSH1 and GR from O. volvulus exert structural and kinetic properties comparable to those of the enzymes from other origins (151,170).…”

The Glutathione Reductase GSR-1 Determines Stress Tolerance and Longevity in Caenorhabditis elegans

“…The activity of the GSR-1 was determined spectrophotometrically at 25°C (UVIKON-932 spectrophotometer, Goebel, Germany) as previously described by Müller et al [38]. The decrease of absorbance at 340 nm due to the oxidation of NADPH was recorded.…”

Laser‐assisted microdissection for the study of the ecology of parasites in their hosts

“…1995; Liebau et al . 1996; Müller et al . 1997), and they were selected because their intron/exon structure was known and so the size of PCR fragments could be predicted.…”

“…Six sets of primers were designed from three different single-copy genes from Onchocerca volvulus (glutathione reductase, GD; manganese superoxide dismutase, MnSOD; and Glutathione S-transferase, GST2). All three genes were Glutathione reductase (GD) 5%: 9/12 5%: 5/12 GD7F + GD7R (exon only) 1.7%: 4/11 (expected size: 135 bp) 1%: 5/12 1%: 1/12 0.5%: 2/8 0.5%: 0/8 0.5%: 0/8 Glutathione reductase (GD) 5%: 0/12 5%: 0/12 GD5F + GD7R (exon + intron) 1.7%: 1/11 (expected size: 556 bp) 1%: 0/12 1%: 0/12 0.5%: 3/8 0.5%: 4/8 0.5%: 0/8 Manganese superoxide dismutase 5%: 0/12 5%: 0/12 (MnSOD) 1.7%: 0/11 MnSOD4F + MnSOD4R (exon only) 1%: 0/12 1%: 0/12 (expected size: 138 bp) 0.5%: 0/8 0.5%: 3/8 0.5%: 0/8 Manganese superoxide dismutase 5%: 0/12 5%: 0/12 (MnSOD) 1.7%: 1/11 MnSOD2F + MnSOD4R 1%: 0/12 1%: 0/12 (exon + intron) 0.5%: 0/8 0.5%: 1/8 0.5%: 0/8 (expected size: 1454 bp) Glutathione S-transferase (GST2) 5%: 12/12 5%: 11/12 GST26F + GST26R (exon only) 1.7%: 2/11 (expected size: 120 bp) 1%: 9/20 1%: 1/12 0.5%: 8/8 0.5%: 5/8 0.5%: 3/8 Glutathione S-transferase (GST2) 5%: 9/12 5%: 6/12 GST24F + GST26R (exon + intron) 1.7%: 1/11 (expected size: 343 bp) 1%: 0/12 1%:0/12 0.5%: 3/8 0.5%: 0/8 0.5%: 0/8 previously reported as single copy on the basis of Southern blot hybridizations (Henkle-Dührsen et al 1995;Liebau et al 1996;Müller et al 1997), and they were selected because their intron/exon structure was known and so the size of PCR fragments could be predicted. For reactions that were designed to amplify short fragments spanning only one exon, the PCR conditions used were: 2 min at 94°C followed by 40 cycles of 94°C for 45 s, 53°C for 45 s and 72°C 1 min 30 s, followed by a final 10 min round of synthesis at 72°C.…”