Structural and functional characterization of Schistosoma mansoni Thioredoxin

Abstract

Schistosomiasis, the human parasitosis caused by various species of the blood-fluke Schistosoma, is a debilitating disease affecting 200 million people in tropical areas. The massive administration of the only effective drug, praziquantel, leads to the appearance of less sensitive parasite strains, thus, making urgent the search for new therapeutic approaches and new suitable targets. The thiol-mediated detoxification pathway has been identified as a promising target, being essential during all the parasite developmental stages and sufficiently different from the host counterpart. As a part of a project aimed at the structural characterization of all the proteins involved in this pathway, we describe hereby the high-resolution crystal structure of Schistosoma mansoni Thioredoxin (SmTrx) in three states, namely: the wild-type oxidized adult enzyme and the oxidized and reduced forms of a juvenile isoform, carrying an N-terminal extension. SmTrx shows a typical thioredoxin fold, highly similar to the other components of the superfamily. Although probably unlikely to be a reasonable drug target given its high similarity with the human counterpart, SmTrx completes the characterization of the whole set of thiol-mediated detoxification pathway components. Moreover, it can reduce oxidized glutathione and is one of the few defence proteins expressed in mature eggs and in the hatch fluid, thus confirming an important role in the parasite. We believe its crystal structure may provide clues for the formation of granulomas and the pathogenesis of the chronic disease.

Introduction

Neglected tropical diseases are a major threat to human health. Under WHO auspices some academic research groups and governmental agencies have undergone systematic biological and epidemiological studies of the causative agents of these diseases. The research group at the “Sapienza” University of Rome has chosen a focussed structural genomics approach to find suitable drug targets against Schistosoma mansoni, a blood fluke, which is endemic in 75 tropical and subtropical countries. The pathway we decided to tackle is the thiol-mediated detoxification one, in which electrons flow from NADPH through several redox proteins to finally reduce hydrogen and organic peroxides. In schistosomes, this metabolic pathway is peculiar because the two parallel pathways present in mammals, based on Thioredoxin (Trx) and on glutathione (GSH) are condensed in one single enzyme called thioredoxin glutathione reductase (TGR).¹ This feature combined with the fact that all proteins belonging to this pathway are expressed throughout all life stages of schistosomes² makes them appealing structural targets that our group has been characterizing in the last years.^3–8

Thioredoxins (Trx) are a family of small (about 12 kDa) proteins sharing a conserved catalytic site (WCGPC), which undergoes reversible oxidation to cystine disulfide (Trx-S₂) through the transfer of reducing equivalents from the two catalytic Cys usually to a disulfide substrate (X-S₂). In mammals, oxidized Trx is then reduced back to the Cys form [Trx-(SH)₂] by the NADPH-dependent flavoprotein thioredoxin reductase,⁹ whereas in schistosomes, the same reaction is undertaken by the peculiar TGR enzyme.¹

In addition to their antioxidant properties, Trxs are involved in a variety of cellular redox reactions, ranging from protein folding to transcription regulation, as well as being growth factors for a variety of cells (reviewed in Ref. 9).

Here, we present the work carried out on two isoforms of SmTrx, which we have cloned in E. coli, expressed in high yield, purified, and functionally and structurally characterized.

Results

Cloning, expression, and purification of SmTrx

According to its latest release, the complete sequence of S. mansoni genome contains one gene for Trx, whose construct is processed differently in the juvenile and in the adult stages. In the former, an extra N-terminal sequence of four amino acids, namely QLVI, is present before the first Met residue. These amino acids are absent in the adult form.

Herein, we shall refer to the adult isoform as wt-SmTrx, and to the juvenile one as K3E-SmTrx, because the gene originally fished out from a cDNA library contained the above-mentioned residues and a mutation leading to a substitution in position +3 from Lys to Glu. Both proteins, expressed in good yield in E. coli (15 mg/L culture), were purified to homogeneity (SDS-PAGE not shown), tested for in vitro activity and crystallized at high resolution.

Structural characterization

The structure of oxidized wt-SmTrx was solved at 1.6 Å. It crystallized in space group P2₁2₁2₁, with one molecule per asymmetric unit. Crystals of K3E-SmTrx were obtained both in the oxidized and reduced forms. K3E-SmTrx^ox structure was solved at 1.56 Å resolution; it crystallized in space group P2₁2₁2₁, with one molecule per asymmetric unit. K3E-SmTrx^red crystals diffracted at 1.67 Å in space group P3₂; this time, two molecules were found in the asymmetric unit. The statistics of the diffraction and refinement data for the three structures are summarized in Table I. The coordinates and the structure factors of wt-SmTrx, K3E-SmTrx^ox, and K3E-SmTrx^red have been deposited in the Protein Data Bank and assigned the following accession numbers: 2xbi, 2xc2, 2xbq.

Table I.

Summary of Data Collection and Refinement Statistics

	wt-SmTrx (2xbi)	K3E-SmTrxox (2xc2)	K3E-SmTrxred (2xbq)
Data Collection statistics
Space Group	P212121	P212121	P32
Cell dimensions
a, b, c (Å)	39.2, 46.8, 56.4	33.9, 52.0, 59.3	62.1, 62.1, 58.3
α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 120
Resolution in Å (last shell range)	36.00–1.60 (1.64–1.60)	39.00–1.56 (1.60–1.56)	53.80–1.67 (1.71–1.67)
Rmerge (last shell value)	0.11 (0.49)	0.06 (0.18)	0.10 (0.54)
I/σI (last shell)	14.6 (4.7)	26.9 (5.1)	15.2 (2.2)
% Completeness (last shell)	99.8 (99.5)	100 (96.5)	99.9 (99.3)
Redundancy (last shell)	6.9 (6.9)	10.1 (9.2)	5.7 (5.7)
Refinement statistics
Resolution in Å	20.00–1.60	20.00–1.56	25.0–1.67
Number of reflections	13487	14281	27704
Rwork	0.19	0.17	0.18
Rfree	0.24	0.22	0.21
No. molecules in asymmetric unit	1	1	2
Total number of atoms	959	997	1852
Protein	853	888	1713
Ions	6	2	2
Water	100	107	137
B factor analysis (Å2)
B Wilson	9.4	18.6	19.9
Overall	10.2	18.6	17.1
Protein	8.9	17.2	15.4
Ions	23.4	26.0	17.5
Water	21.4	29.7	28.5
R.M.S. deviations
bond lengths (Å)	0.016	0.012	0.009
bond angles (°)	1.41	1.46	1.32
Ramachandran plot % of residues in
Most favored regions	96.7	97.9	96.3
Additionally allowed	3.3	2.1	3.7
Generously allowed	0	0	0
Disallowed	0	0	0

The structure of the oxidized wt-SmTrx is shown in Figure 1. The map gave the possibility to fit residues from −1 to 106. The two additional residues before M1 (G-1 and S0) belong to the thrombin cleavage site of the expression vector. wt-SmTrx conserved the typical thioredoxin fold, made up of a five-stranded β sheet (β1-5) capped on each side by two α helices (α1 and 3 and α2 and 4). The sequence identity between SmTrx and those from other organisms ranges between 45% and 65%.² In particular, the identity between SmTrx and human Trx (HsTrx) is 47% [Fig. 2(B)]. The conserved active site amino acids W33–C34–G35–P36–C37 link the strand β2 to helix α2 and are arranged in a β-turn shape. The redox site, formed by C34 and C37, is oxidized, the distance between the two sulphurs being 2.1 Å. This cystine pair is shielded from the solvent by the C-terminal portion of helix α2, by the loop between α3 and β4, and by the active site loop itself. As a result of this architecture, C34 acts as the nucleophilic Cys toward its substrates, being more exposed to the solvent, whereas C37 is buried and could act as the resolving one. A conserved Asp residue (D28) with a H-bonded water molecule (D28(OD1)–O = 2.7 Å) responsible for the deprotonation of the resolving Cys during the redox cycle¹⁰ is present (Fig. 1).

Figure 1.

Stereo ribbon representation of the overall fold of oxidized wt-SmTrx. Residues of the active site (Asp28, Trp33, Cys34, and Cys37) are in stick representation, together with the water molecule putatively involved in catalysis (see text). The electron density 2F_o – F_c contoured at 1.5σ is also shown for the same residues. An interactive view is available in the electronic version of the article.

Figure 2.

Structure comparison. Panel A: Electrostatic surface of wt-SmTrx (left) and HsTrx (1ERT) (right). The view is rotated 90° on x-axis from Fig. 1, looking at the molecule through the active site. Side chains of the active site are shown as green sticks, residues of the second sphere are in grey. Panel B: Sequence alignment between wt-SmTrx (SCHMA) and HsTrx (HUMAN). The residues shown in Panel A are highlighted with the same colour scheme. Stars represent identity, colons represent high similarity; dots represent low similarity. Panel C: Superposition of oxidized wt-SmTrx (blue) and oxidized K3E-SmTrx (cyan). Panel D: Enlarged view of the active site of superposed oxidized (cyan) and reduced (magenta) K3E-SmTrx. An interactive view is available in the electronic version of the article.

Unlike many other eukaryotic Trxs (such as human, Plasmodium falciparum and Drosophila melanogaster), SmTrx has no Cys residues other than those of the active site.² Side chains around the active site, including their rotamers and electrostatic potential, are conserved between wt-SmTrx and HsTrx [highlighted in green in Fig. 2(A,B)]. A few differences can be located in the second sphere residues, including the additional C69 and C73 in HsTrx, replaced by Y70 and A74 in wt-SmTrx, and a few charged residues [highlighted in grey in Fig. 2(A,B)]. We cannot assess whether these differences might be enough to account for any selectivity between the two proteins within their multiple functions. Nevertheless, we have proven that SmTrx is indeed a substrate of human Trx-Reductase (see later).

The two oxidized structures, wt-SmTrx and K3E-SmTrx^ox, are completely superimposable [r.m.s.d. overall 0.33 Å calculated with SSM¹¹; Fig. 2(C)], the main differences being confined to the N-terminal, encompassing the extra residues and the mutation K3E. This region is floating into the solvent, on the opposite side of the molecule with respect to the active site. Moreover, K3E-SmTrx was found to have the same enzymatic properties as wt-SmTrx (see later). We have no obvious explanation for the presence of the acidic mutation in the cDNA prepared from worm extract, nor for its in vivo role, if any.

The Trx fold is very rigid and the only detectable movements on reduction (overall rmsd = 0.61 Å between K3E-SmTrx^ox and K3E-SmTrx^red) are limited to a rotamer change in the Cys couple and to a slight opening of the active site stretch W33-K38 [Fig. 2(D)], besides the differences in the C-terminal stretch due to crystallographic packing.

Functional characterization: Insulin reduction

SmTrx can reduce insulin if maintained in the reduced state by the addition of DTT. The activity of wt-SmTrx and K3E-SmTrx was assessed by recording the start of insulin precipitation and its rate (see Methods). In the presence of SmTrx, the start time was 100 s and the rate (ΔA₆₅₀ min⁻¹) was 0.35. The activity of SmTrx is thus comparable with that of HsTrx (80 s and 0.41 ΔA₆₅₀ min⁻¹) and slightly higher than that of EcTrx (240 sec and 0.28 ΔA₆₅₀ min⁻¹) assayed under the same conditions.

When GSH was used as reducing agent, insulin reduction activity was lower than in the presence of DTT. Also in this case, both schistosome enzymes are slightly more efficient than HsTrx and EcTrx (starting times 1100, 1080, 1020, and 1440 s and rates 0.09, 0.10, 0.06, and 0.07 ΔA₆₅₀ min⁻¹ for wt-SmTrx, K3E-SmTrx, HsTrx, and EcTrx, respectively).

Functional characterization: GSSG reduction

In the presence of NADPH and human Trx Reductase, wt-SmTrx, but not EcTrx, is able to reduce oxidized glutathione. The steady state parameters for the reduction of GSSG were measured as: k_cat = 0.085 s⁻¹ and K_m = 253.5 μM for SmTrx. These values are comparable with those obtained for HsTrx (k_cat = 0.115 s⁻¹ and K_m = 202.1 μM).

Functional characterization: Interaction with SmTGR

By the effective reduction of insulin in the presence of NADPH and SmTGR, we were able to assess the functional interaction between the two natural partners. Both wt-SmTrx and K3E-SmTrx proved to be fully functional.

Functional characterization: pKa determination of the active site Cysteines

The pKa values of the nucleophilic C34 and the buried C37 of wt-SmTrx were determined by pH titration at 240 nm. Generally, the pKa of Cys is about 8.7. The pH titration yielded a first pKa = 6.5, which was attributed to the solvent-exposed C34, by analogy with other known Trx. A second, less evident, pKa ≈ 9 was attributed to C37, which is buried in the structure.

Discussion

In the quest for novel drugs to combat schistosomiasis as well as other parasitic diseases, the biochemical approach is to identify a suitable macromolecular target. This implies a deep knowledge of the biology of the schistosome parasite, which is able of digenesis, because it enters the human body as monomorphic nonfeeding cercaria and then develops into a sex-differentiated blood-feeding worm. To be effective against the juvenile and the adult forms, a drug needs to target proteins, which are expressed by all life cycles and are crucial to the survival in the host blood stream. In this respect, undertaking a systematic study of the thiol-mediated detoxifying metabolic pathway makes sense, because all the proteins involved are expressed in high yield from cercaria to the adult stage. This is a necessary but not sufficient condition to succeed, because the parasite target(s) ought to be significantly different from the human ortholog(s), to avoid cross-inhibition leading to severe side effects.

The thiol-mediated detoxifying system under study has been validated as a good target, but in refining the search to a few candidates, we had to exclude SmTrx, because its physical–chemical properties do not significantly differ from HsTrx. This result is not surprising given that this small protein is multitasking, acting as a potent antioxidant, as redox regulator in signal transduction, and as electron donor during DNA synthesis. Moreover, it is present and conserved in all species from Archebacteria to mammals. Not only the structural features are conserved but also the structure and stereochemistry around the active site, which is designed to fit different proteins, do not change during the redox cycle.

Despite its limited usefulness in drug targeting, SmTrx structural and functional studies are important to complete the knowledge of Schistosoma redox metabolism. We have also performed in vitro experiments of SmTrx reduction by the endogenous reductase SmTGR, confirming the full functionality of the recombinant enzyme not only toward small molecules (DTT, insulin, and GSSG) but also toward macromolecular ligands. We used two SmTGR isoforms, a truncated one lacking the two C-terminal Sec-Gly residues,⁶ and a full-length Sec597Cys mutant.⁷ The electron transfer from NADPH to insulin through TGR and Trx was efficient with the full-length reductase but not with the truncated one, thus demonstrating the role of TGR C-terminus in Trx reduction.

The observed ability of SmTrx to reduce GSSG may have a great importance in the worm survival under high oxidative stress, also in view of the relatively low efficiency versus glutathione of the worm's TGR. This additional way to lower the GSSG/GSH ratio is active in other parasitic platyhelminths¹² and protozoa such as Plasmodium falciparum¹³ that have to fight the host immune system attack but has also a fundamental role in insects, such as Drosophila melanogaster, Anopheles gambiae, and Apis mellifera,^13–15 that lack glutathione reductase and have a tracheal respiratory system highly exposed to ROS. Interestingly, free living, not parasitic platyhelminths maintain the two typical separate pathways for thioredoxin and glutathione reductions.¹²

A further interesting aspect of SmTrx lies in its presence in the mature egg secretory products.² It is well known that eggs are mainly responsible for the pathology correlated to schistosomiasis, because they elicit the formation of granulomas in liver and bladder. Moreover, anti-Trx antibodies are able to elicit circumoval precipitin reaction,² which is still considered a useful diagnostic test for schistosomiasis, being highly specific and sensitive. In summary, even though SmTrx cannot be exploited as a drug target, its functional and structural characterizations may help to design more specific antibodies to develop new diagnostic tests and to better understand the interplay of the enzymes belonging to the peculiar redox pathway of the parasite.

Materials and Methods

Cloning, expression, and purification of SmTrx

SmTrx gene (Accession number AF473536) was initially amplified from S. mansoni cDNA prepared from a 3′ RACE (by courtesy of Dr Cristiana Valle, CNR-IBC Monterotondo). The gene was then cloned into pGEX-4T-1 (GE Healthcare) expression vector via EcoRI and XhoI restriction sites. After sequencing, we found that the amplified protein contained four extra amino acids at the N-terminus (QLVI), which belong to the deposited preprotein, typical of schistosomula and cercaria larval stages (EST from GenBank AM043494). Moreover, a single-base mutation occurred in the DNA, and Lys3 was replaced by Glu. Hence, the N-terminal sequence of this preprotein isoform of SmTrx is QLVIMSE. In addition, the expressed protein has seven extra residues, due to the restriction sites used and the thrombin cleavage site. The resulting N-terminal arm (from −10 to +3) is the following: GSPEFTSQLVIMSE, and we shall refer to this long construct as K3E-SmTrx. The protein was expressed fused to GST-tag in BL21(DE3) bacterial cells on induction with 0.3 mM IPTG, incubating overnight at 20°C. Successful expression of soluble K3E-SmTrx was confirmed by SDS-PAGE, highlighting a band at ∼40 kDa. Cells were lysed by sonication in 20 mM Tris/HCl pH 7.4, 0.2M NaCl buffer, on addition of 10 mM beta-mercaptoethanol, 0.1% Triton-X100, 3U DNase, 1 mM PMSF, and 1 mM EDTA. Protein was purified from the soluble fraction by affinity chromatography on glutathione sepharose (GE Healthcare); the GST tag was cleaved by thrombin (Sigma-Aldrich), which was finally removed by elution on a 1 mL benzamidine FF (HS) column (GE Healthcare). K3E-SmTrx was exchanged into crystallization buffer [20 mM Tris/HCl (pH 7.4), 5 mM β-ME, and 50 mM NaCl], concentrated to 15 mg/mL by ultrafiltration (Amicon, Millipore), aliquoted, and stored at −20°C.

To avoid the extra amino acids due to the plasmid and to the preprotein and to eliminate the mutation, we decided to clone wt-SmTrx between BamHI and XhoI sites of pGEX-4T-1, using proper PCR primers. The protein was successfully expressed and purified using the same protocol as for the mutant, and it was used for functional and structural experiments.

Crystallization of SmTrx

Crystals of K3E-SmTrx and wt-SmTrx were grown by vapour diffusion according to standard hanging drop methods. Crystals of reduced K3E preprotein (K3E-Trx^red) grew over 3 days in a drop composed by 1 μL protein (15 mg/mL) and 1 μL well solution (24% (w/v) PEG 8000, 5 mM zinc acetate, 5 mM TCEP, and 0.1M sodium cacodylate, pH 6.5). The oxidized form (K3E-Trx^ox) was obtained in the same manner, with the addition of 10 mM zinc acetate to the well solution in the absence of TCEP.

wt-SmTrx crystals grew in 0.1M BisTris, pH 6.5, 45% polypropylene glycol P400. We were unable to obtain crystals of wt-SmTrx more that 20% reduced, despite the use of TCEP, β-ME, and N-ethymaleimide (NEM).

Data collection, processing, and refinement

Diffraction data of SmTrx were collected at ESRF (Grenoble, France) and ELETTRA (Trieste, Italy) synchrotrons. Data were indexed with Mosflm and processed with programs of the CCP4 Suite.¹⁶ Crystals of K3E-SmTrx^ox diffracted to 1.56 Å, those of K3E-SmTrx^red diffracted to 1.67 Å, and those of oxidized wt-SmTrx to 1.6 Å. All the structures were solved by molecular replacement. In the case of K3E-SmTrx^ox, the structure of D. melanogaster Trx (52% identity—PDB entry: 1XWA¹⁰) was taken as a model. In the other two cases, we used K3E-SmTrx^ox (without the extra N-terminal residues and the mutation) as a starting model. The structures were refined using REFMAC5¹⁷ and fitted to generated electron density maps by Coot.¹⁸ The quality of the model was assessed with MolProbity¹⁹ and ProCheck.²⁰ Data collection and refinement statistics are summarized in Table I. Figures were prepared with PyMOL (http://www.pymol.org) and CCP4MG.²¹ The alignment in the figure was made with ClustalW2.²²

Insulin reduction assay

A standard turbidimetric assay was performed. Insulin solution was prepared according to Holmgren²³ by dissolution of insulin in Tris/HCl buffer pH 8, acidification to pH 2–3 and rapid titration of the solution back to pH 8. This procedure leads to the cleavage of the polypeptide into two chains, linked by a disulfide. The Trx-catalyzed reduction of the S–S bond causes the separation of the two insulin chains and their precipitation, with consequent increasing of turbidity that can be followed spectrophotometrically at 650 nm. The assay mixture contained 0.1M potassium phosphate buffer pH 7.0, 1 mM EDTA, 130 μM insulin, 500 μM dithiothreitol (DTT) and varying amounts of each protein: wt-SmTrx, K3E-SmTrx, EcTrx (SIGMA-Aldrich) and HsTrx (SIGMA-Aldrich). The baseline control reaction contained insulin and DTT but lacked Trx. The assay was performed also with GSH instead of DTT as reducing agent.

GSSG reduction assay

Rate constants were determined using an enzymatic Trx^ox reducing system and subsequently transforming it to a GSSG reducing system.¹³ In a cuvette containing 2 mM EDTA, 100 μM NADPH, and 100 nM human Trx Reductase (SIGMA-Aldrich), GSSG was added at various concentrations (100 μM to 1 mM). Reaction was started by the addition of 6 μM SmTrx^ox, and NADPH consumption was followed at 340 nm. The rate represents a reducing flux from NADPH to Trx reductase to Trx, leading to GSSG reduction.

Functional interaction with SmTGR

The functional interaction of SmTrx with its endogenous reductase, SmTGR, was assessed by a coupled reaction between NADPH, TGR, Trx, and insulin. The mixture contained 0.1M potassium phosphate pH 7.0, 1 mM EDTA, 130 mM insulin, 300 μM NADPH, and 8 μM SmTrx^ox. The reaction was started by the addition of 1.6 μM SmTGR and was followed both at 340 and 650 nm, to monitor NADPH consumption and insulin turbidity, respectively.

Determination of active site Cys pKa

The pKa values of the nucleophilic Cys34 and the buried Cys37 were determined by pH titration at 240 nm, because the thiolate ion has a higher absorption at this wavelength than the thiol group.²⁴ Spectra of oxidized and reduced Trx were recorded between 200 and 400 nm at 25°C in 1.3 mL of 1 mM citrate, 1 mM borate, and 1 mM phosphate, 0.2M KCl, pH 5.1, purged with nitrogen. The pH was adjusted from pH 5.1 to 10.5 by adding known volumes of either 0.2M or 0.1M KOH. The spectra were measured against air in a sealed quartz cuvette in a spectrophotometer. The spectrum of the buffer recorded in the same cuvette was subtracted from the spectrum of the respective protein solution. The absorbance was converted into molar extinction coefficients (ɛ = 5200 M⁻¹ cm⁻¹) and corrected for the dilution due to pH adjustment. The protein concentration was calculated from absorbance at 280 nm.

Glossary

Abbreviations:

DNase

deoxyribonuclease

DTT

dithiothreitol

EcTrx

Escherichia coli thioredoxin

GSH

glutathione

GSSG

oxidized glutathione

HsTrx

Homo sapiens thioedoxin

IPTG

isopropyl β-d-1-thiogalactopyranoside

K3E-SmTrx

Lys3Glu-mutated isoform of juvenile thioredoxin from Schistosoma mansoni

NEM

N-ethylmaleimide

PEG

polyethylene glycol

PMSF

phenyl-methane-sulfonylfluoride

ROS

reactive oxygen species

TCEP

tris(2-carboxyethyl)phosphine

Trx

thioredoxin

WHO

World Health Organization

wt-SmTrx

wild-type thioredoxin from Schistosoma mansoni.

β-ME

β-MercaptoEthanol