Differential expression of disulfide reductase enzymes in a free-living platyhelminth (<i>Dugesia dorotocephala</i>)

<div><p>A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth <i>Dugesia dorotocephala</i> was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the <i>D</i>. <i>dorotocephala</i> extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a <i>D</i>. <i>dorotocephala</i> extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.</p></div

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (<i>Dugesia dorotocephala</i>)

<div><p>A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth <i>Dugesia dorotocephala</i> was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the <i>D</i>. <i>dorotocephala</i> extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a <i>D</i>. <i>dorotocephala</i> extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.</p></div

Effect of GSSG and protein concentration on the full progress curves of NADPH consumption with GSSG as substrate.

<p>The GSSG reductase activity was measured at 25°C and pH 7.8 as described in Materials and Methods. A) Effect of GSSG. The enzyme assays were carried out at the following micromolar concentrations of the disulfide: (○) 67; (□) 150; (●) 350; (■) 500; (▲) 700; (▼) 1000. An enzyme concentration of 15 nM was used. B) Effect of protein concentration. The following nanomolar concentrations of protein were used: (●) 1.9; (○) 2.9; (▼) 3.8; (Δ) 7.7; (■) 15.3. Inset at panels A and B shows the dependence of the lag time on either GSSG or protein, respectively.</p

Electrophoretic analysis of the disulfide reductase activities purified from <i>D</i>. <i>dorotocephala</i>.

<p>A) Protein band pattern of the purified sample. Lane 1: An aliquot from the last purification step was incubated for 10 min at 80°C in the presence of 1% SDS and 5 mM β-mercaptoethanol; then, it was loaded on a top of a 12% polyacrylamide gel and ran during 3 h. Lane 2: molecular weight markers. B) Densitometric analysis of the 65 kDa and 55 kDa protein bands. The relative intensity of the latter was estimated with the software IMAGEJ.</p

Effect of GSSG and protein concentration on the full progress curves of NADPH consumption with GSSG as substrate.

<p>The GSSG reductase activity was measured at 25°C and pH 7.8 as described in Materials and Methods. A) Effect of GSSG. The enzyme assays were carried out at the following micromolar concentrations of the disulfide: (○) 67; (□) 150; (●) 350; (■) 500; (▲) 700; (▼) 1000. An enzyme concentration of 15 nM was used. B) Effect of protein concentration. The following nanomolar concentrations of protein were used: (●) 1.9; (○) 2.9; (▼) 3.8; (Δ) 7.7; (■) 15.3. Inset at panels A and B shows the dependence of the lag time on either GSSG or protein, respectively.</p

Effect of auranofin on the disulfide reductase activities of <i>D</i>. <i>dorotocephala</i>.

<p>An enzyme aliquot was incubated during 3 min in the presence of the corresponding concentration of auranofin and 100 μM NADPH; then, reaction was started by addition of either GSSG (○) or DTNB (▲) at final concentrations of 67 μM or 490 μM, respectively. The initial velocity obtained in the absence of auranofin was taken as 100% relative activity.</p

Summary of the purification procedure of the GSSG reductase activity from <i>Dugesia dorotocephala</i>.

<p>Summary of the purification procedure of the GSSG reductase activity from <i>Dugesia dorotocephala</i>.</p

Amino acid sequence of proteolytic fragments obtained from the electrophoretic bands of the purified sample of <i>D</i>. <i>dorotocephala</i>.

<p>Amino acid sequence of proteolytic fragments obtained from the electrophoretic bands of the purified sample of <i>D</i>. <i>dorotocephala</i>.</p

Saturation kinetics of the GR activities of <i>D</i>. <i>dorotocephala</i>.

<p>Enzyme assays were carried out as described in Materials and Methods. In all cases a NADPH concentration of 100 μM was used. (■) Total GSSG reductase activity (TGR + GR); (▲) GSSG reductase activity due to GR; (▼) GSSG reductase activity due to TGR. An enzyme concentration of 6 nM was used in the assays. The upper abscissa represents the range of GSSG concentrations used for the kinetic analysis of TGR. The continuous line was obtained through non-linear regression analysis of the corresponding data to the Michaelis-Menten equation. In all cases, error bars were omitted for clarity.</p

Chromatofocusing separation of the TrxR activities of <i>D</i>. <i>dorotocephala</i>.

<p>An active fraction was loaded on a DEAE-cellulose column (1.6 x 17 cm) previously equilibrated in 25 mM Tris/HCl buffer (pH 8.5). After washing, the adsorbed proteins were eluted by application of a polybuffer solution supplemented with 25 mM Hepes and 25 mM Tris (final pH of 6). Fractions containing DTNB reductase activities were pooled separately and concentrated. The major active peak (Pool 1) contain activities associated with TGR, GR and TrxR, while in the minor active peak (Pool 2) TGR is essentially absent. Insert: electrophoretic profile of both Pool 1 (line 1) and Pool 2 (line 2) after silver staining.</p