Protein sequence analysis of this parasite product showed lower identity to known GSTs.. Like other omega class GSTs, SmGSTO showed very low activity toward classical GSTs substrates as
Trang 1Characterization of an omega-class glutathione S -transferase
dehydroascorbate reductase and thiol transferase activities
Javier Girardini1,*, Alejandro Amirante2,†, Khalid Zemzoumi1and Esteban Serra1
1
Instituto de Biologı´a Molecular y Celular de Rosario, IBR-CONICET, Facultad de Ciencias Bioquı´micas y Farmace´uticas, UNR; and 2 Facultad de Odontologı´a, UNR, Rorario, Argentina
Glutathione S-transferases (EC 2.5.1.18) (GSTs), are a
family of multifunctional enzymes present in all living
organisms whose main function is the detoxification of
electrophilic compounds GSTs are considered the most
prominent detoxifying class II enzymes in helminths We
describe here the characterization of novel dehydroascorbate
reductase and thiol transferase activities that reside in the
human parasite Schistosoma mansoni GSTx Protein
sequence analysis of this parasite product showed lower
identity to known GSTs However, phylogenic analysis
placed SmGSTx among the recently described omega class
GSTs (GSTO1-1) We report here that SmGSTO protein is a
28-kDa polypeptide, detected in all life stages of the parasite,
being highly expressed in adult worms Like other omega class GSTs, SmGSTO showed very low activity toward classical GSTs substrates as 1-chloro-2,4-dinitrobenzene, and no binding affinity to glutathione–agarose matrix but showed some biochemical characteristics related with thio-redoxins/glutaredoxins Interestingly, SmGSTO was able to bind S-hexyl glutathione matrix and displayed significant glutathione-dependent dehydroascorbate reductase and thiol transferase enzymatic activities
Keywords: glutathione S-transferase; dehydroascorbate reductase; thiol transferase; Schistosoma
Glutathione S-transferases (GSTs, EC 2.5.1.18) constitute a
family of multifunctional enzymes that mainly catalyze the
nucleophilic attack of reduced glutathione (GSH) to a wide
variety of electrophilic endogenous and exogenous
com-pounds GSTs are found in all living organisms tested to
date, present as unique enzymes in lower organisms and as a
large number of tissue-specific isoforms in more complex
species like mammals [1,2]
The expression level of GSTs is modulated by many
compounds including carcinogens, drugs and
oxidative-stress metabolites [1] Several additional functions were
attributed to GSTs including the transport of hydrophobic
ligands, binding to bilirubin and carcinogens [3,4], the isomerization of maleylacetoacetate and the regulation of stress kinases and apoptosis [5,6]
Based on their sequence structure, catalytic activitiy, immunogenicity, substrate specificity and sensitivity to inhibitors, the mammalian GSTs form six evolutionary distinct classes termed alpha, kappa, mu, pi, sigma, and zeta [1,7,8] A new class of the GST superfamily, designated GST omega (GSTO) in accordance with the established human GST nomenclature convention [9], has been recently characterized in humans on the basis of structural data [10] This new enzyme (GSTO1-1) has similar functional characteristics with previously described proteins in rats [11] and mouse [12] Although the mammalian GSTO has low sequence similarity to other known GSTs, its crystallo-graphic structure showed a GST fold composed of an N-terminal glutathione-binding domain and a C-terminal domain composed entirely of a-helices In contrast, unlike other GSTs, GSTO has an active site cysteine that is able to form a disulfide bond with GSH and exhibits glutathione-dependent thiol transferase (TTase) and dehydroascorbate reductase (DHA) activities, reminiscent of thioredoxin (Trx) and glutaredoxin (Grx) enzymes [10] Recent studies have shown new additional functions for human GSTO including monomethylarsonic acid (MMA) reductase activity and the modulation of ryanodine calcium channels [13,14] A particular member of the GST superfamily, designated Tc52, exhibiting GSH-dependent TTase activity, has been characterized in the human causative agent of Chagas’ disease, Trypanosoma cruzi [15,16] Further studies have shown that Tc52 is essential for the parasite development and is involved in the immunomodulatory processes asso-ciated with Chagas’ disease [17–20]
Correspondence to E Serra, Instituto de Biologı´a Molecular y
Celular de Rosario, IBR-CONICET, Facultad de Ciencias
Bioquı´micas y Farmace´uticas, UNR, Suipacha 531 CP 2000,
Rorario, Argentina Fax: 54 3414390465, Tel.: 54 3414370008,
E-mail: eserra@arnet.com.ar
Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; DHA,
dehydro-ascorbate reductase; DCNB, 1,2-dichloro-4-nitrobenzene; Grx,
glutaredoxin; GSH, glutathione; GST, glutathione S-transferase;
GSTO, GST omega; HEDS, hydroxyethyl disulfide; MMA,
mono-methylarsonic acid; Trx, thioredoxin; TTase, thiol transferase.
Enzyme: glutathione S-transferase (EC 2.5.1.18).
Note: nucleotide sequence data are available in the GeneBank database
under the accession number AF484940.
*Note: These authors contributed equally to this work.
Present address: Hoˆpital N-Dame, Allergy, M4211-K, 1560 rue
sherbrooke Est, Montre´al, QC, H2L 4M1, Canada
(Received 8 July 2002, revised 3 September 2002,
accepted 11 September 2002)
Trang 2At least five GST activities have been described in the
human parasite Schistosoma mansoni (SmGST-1 to
SmGST-5) SmGST-5 has been characterized as an
unstable enzyme that may be involved in the conjugation
of epoxide substrates and dichlorovos, the active form of
the anti-schistosomal drug metrifonate, but no
corres-ponding parasite gene has been cloned to date [21–23]
Two members of SmGSTs, 28-kDa Sm28GST and
26-kDa Sm26GST, have been cloned and were found to
correspond to the previously reported SmGST-1, -2, -3
and -4 isoenzymes, respectively [22,24] Although DHA
activity was first described in plants several years ago, and
then in mammals, insects and protozoans, little is known
about nonvertebrate GSH-dependent DHA proteins at
the molecular level
We report here the characterization of a new member
of the GST superfamily from S mansoni When compared
with other GSTs, S mansoni protein showed a limited
sequence identity with omega-class GSTs Additional
phylogenic analysis, including known GSTs classes and
S mansoni GSTs, allowed us to place the new parasite
product among the newly identified GSTO class, and the
previously characterized Sm28GST and Sm26GST as
mu- and sigma-class, respectively Additional evidence
placed the S mansoni protein among the omega class of
GSTs, as the recombinant parasite protein (a) did not
have significant affinity for glutathione, but bound
strongly to S-hexyl glutathione matrix; (b) exhibited low
activity towards the classical GST substrate
1-chloro-2,4-dinitrobenzene (CDNB); and (c) showed significant
GSH-dependent TTase and DHA activities The data presented
here provide the first evidence for a potential new ascorbic
pathway within S mansoni
E X P E R I M E N T A L P R O C E D U R E S
Materials
Reduced glutathione, oxidized glutathione, S-hexyl
gluta-thione, 1-chloro-2,4-dinitrobenzene, 1,2
dichloro-4-nitro-benzene, cibacron blue 3GA, and t-butyl hydroquinone,
were obtained from Sigma;
7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, ethacrynic acid, p-nitrobenzyl chloride,
dehydroascorbate, bromosulfophthalein, Evan’s blue,
hem-atine, and p-chloranil were obtained from ICN;
hydroxy-ethyl disulfide (HEDS) and vinylene trithiocarbonate was
obtained from Aldrich
EST identification and cDNA isolation
The S mansoni EST data base search was performed using
the tBLASTn version of blast program [25] with the T cruzi
Tc52 amino acid sequence as query An EST encoding an
unknown protein was obtained (GeneBank accession
num-ber AI975843) A 549-bp fragment was amplified by PCR
using oligonucleotides GSTX1: 5¢- GTTGTCGACAAA
CATCTCAACTAG-3 and GSTX3: 5¢-GTAAGTGTGG
GAATAAGATCAAATC from adult S mansoni
reverse-transcribed RNA The product of the PCR was sequenced
and used as probe to screen an adult worm S mansoni
lambda gt10 cDNA library by conventional methods [26]
Southern blot analysis was performed using S mansoni
cercariae purified DNA as described [26]
Alignments and phylogenetic analysis
ofS mansoni GSTs Sm26GST, Sm28GST and SmGSTO amino acid sequences were aligned manually on the alignment provided by
L Jermiin (see [10]) From the original alignment used by Board et al [10] eight not clearly class-defined sequences were avoided A phylogenetic tree was obtained by maximum likelihood analysis of all the sites in the above-mentioned alignment The data was analyzed using the
JTT-F substitution model [27], and local bootstrap probabilities were estimated for the internal branches using thePROTML program [28] More than one analysis was performed by using different input order of the sequences Each analysis involved two steps: stepwise addition and nearest neighbor interchanges The most likely tree was obtained by using the test of Kishino and Hasegawa [29] All calculations were performed using theMOLPHY 2.3 molecular phylogenetics programs package [28]
Expression and purification of recombinant SmGSTO Recombinant SmGSTO was expressed in Escherichia coli and purified by two separate methods using either the pQE30 vector (Qiagen) and nickel agarose affinity chroma-tography, or the pT7-7 vector [30] and S-hexyl glutathione affinity chromatography To produce N-terminal 6· His tag fused SmGST, the SmGSTO cDNA was amplified
by polymerase chain reaction (sense primer GSTX2: 5¢-AAGGATCCATGCACCTTAAACGAAATGACC-3¢; antisense primer odTSalI: 5¢-AAGTCGACTTTTTTTTTT TTTTTTTTTS-3¢) and was inserted between the BamHI and SalI sites of the bacterial expression vector pQE30 (Qiagen), the cloned vector was transformed into BL21 [DE3] cells (Novagen, Milwaukee, WI, USA) Briefly, a seed culture of the transformed cells was grown to D600 of 0.4–0.6, scaled up, grown again to the same density, induced with IPTG (0.5 lM), and grown for a further 3 h at 30C His-tagged SmGSTO product was purified on nickel agarose as described by manufacturers (Qiagen) The enzyme was eluted with 250 mM imidazole, 50 mM potas-sium phosphate, pH 7.6, and exhaustively dialyzed against the same buffer to remove imidazole before storage at
80C in 50% glycerol Recombinant SmGSTO was also expressed from it’s own methionine initiation codon Briefly, SmGSTO cDNA was amplified by polymerase chain reaction (sense primer GSTX4: 5¢-AAACATATGAT GCACCTTAAACGAAATGACC-3¢; antisense primer odTSalI) and cloned between the NdeI and SalI sites of the expression vector pT7-7 Protein was purified from soluble extracts on S-hexyl glutathione agarose (Sigma) as previously described [31] The enzyme was eluted with 5 mM S-hexyl glutathione, 50 mM Hepes, pH 8, and dialyzed against 50 mMHepes, pH 8.0, before storage Purification yield approximately 500 lg of protein per milliliter of S-hexyl glutathione agarose In all cases, protein purity was determined by SDS/PAGE and protein concentration was measured by bicinchoninic acid method following manu-facturers indications (Sigma) Antiserum against the puri-fied protein was raised in rabbits using standard immunization protocols
Glutathione and S-hexyl glutathione affinity assays were performed in batch Briefly, 20 lL of 50% resuspended
Trang 3resin was centrifuged and the supernatant was eliminated.
Ten microliters of 1 mgÆmL)1purified enzyme was added to
the same tube and incubated in ice with gentle agitation
After 30 min the supernatant was recovered by
centrifuga-tion The resin was washed four times with 250 lL of
50 mMHepes, pH 8.0, and eluted by adding 10 lL of the
same buffer containing 5 mMGSH, GSSG or S-hexyl GSH
Ten microliters of the original protein, binding assay
supernatant and eluate were analyzed by SDS/PAGE
Expression of SmGSTO along the parasite life cycle
Total proteins were prepared as follows: S mansoni
schist-osomule or sporocysts were resuspended in extraction buffer
(100 mM Tris, 3 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, 2.5 lgÆmL)1 leupeptin, 4 lgÆmL)1 pepstatin A);
adults or cercariae were first homogenized with liquid
nitrogen and then resuspended in extraction buffer The
extracts were homogenized by pulses of 1 min at 25%
amplitude and centrifuged at 10 000 g for 20 min at 4C
The soluble fraction was ultracentrifuged at 105 000 g for
30 min at 4C and the supernatants were used for assays
One hundred micrograms protein from each extract were
separated by SDS/PAGE, electroblotted to a nitrocellulose
membrane (Amersham Pharmacia) Western blot
experi-ments were carried out according to standard techniques
Ten micrograms of DNAse I-treated total RNA from
S mansonimiracidia, sporocyst, cercariae and adult worm
were reverse transcribed by using 100 U of SuperScriptTM
reverse transcriptase (Life Technologies) in 50 lL of
supplied reaction buffer PCRs were performed on 1 lL
of each reverse transcription reaction and resolved by
agarose gel electrophoresis Primers used were: GSTX1/
GSTX3 for SmGSTO and TUB3 (5¢-GAAGTGGAT
ACGAGGATAAGGTACCAG-3¢)/TUB4 (TGGAACTT
ATCGTCAACTTTTCCATCC-3¢) for S mansoni
a-tubu-lin SmGSTO amplification bands were quantified by using
GelPro and normalized by comparing to a-tubulin
ampli-fication products
Enzyme assays
Enzymatic activity towards a range of substrates and
inhibitors was determined as described [32] Thiol
trans-ferase activity was measured according to Axelsson et al
[33] using HEDS as substrate The reaction mixture
contained 0.2 mM NADPH, 0.5 mM GSH, 50 mM
phos-phate buffer, 0.5 units of glutathione reductase and an
aliquot of the protein solution The reaction was initiated by
the addition of 2 mMof HEDS at 30C and followed by
340 nm absorption decrease Absorption coefficient used
for NADPH oxidation at 340 nm was 6.22 mM )1Æcm)1
Glutathione-dependent dehydroascorbate reductase activity
was determined by following the dehydroascorbate (DHA)
reduction spectrophotometrically at 265 nm The standard
reaction mixture contained 50 mMphosphate buffer, pH 8,
1 mMGSH, and was started with the addition of 0.25 mM
DHA after a 1 min preincubation [34]
Construction of a homology model of SmGSTO
SmGSTO structure was built using the human GSTO 1–1
structure as template in the SWISS-MODEL modeling
environment and structure accuracy was assayed byWHAT CHECKtool in the same environment [35] Briefly, structurally driven alignments were performed using SmGSTO sequence Best alignment, obtained based on GSTO1-1, was refined and used to obtain an optimized model The WhatCheck tool (from theWHAT IFpackage program) was used to estimate accuracy of the structure obtained Parameters taken into account were: Ramachandran plot appearance Z-score, chi-1/chi-2/Z-score, packing quality Z-score and RMS Z-scores (http://www.cmbi.kun.nl/gv/pdbreport/checkhelp/)
R E S U L T S
SmGSTO DNA and protein sequence BLAST search of the S mansoni EST database with the complete sequence of Tc52 revealed a clone (EST AI975843) with around 25% sequence identity with the GST-like domain of the T cruzi protein A fragment of 549 bp was amplified by PCR using specific primers, designed based on the EST sequence, and cDNA from S mansoni adult worms The amplified fragment was sequenced and used as probe to screen an adult worm cDNA library After three rounds of hybridization, two independent clones were purified and sequenced The sequences obtained were identical in both clones and corresponded to a cDNA of
934 bp including an open reading frame encoding for a 241 amino acid polypeptide, with a predicted molecular mass of 27.6 kDa (Fig 1) Sequence identity ranging from 18 to 25% was obtained with human GST-theta, mouse p28, rat DHA, human GSTO 1-1, as well as with several plant DHAs and non characterized GST-like proteins In all cases, the most conserved amino acids were localized in the N-terminal domain of SmGSTO The best hit obtained (E¼ 8.1e)11) when searched at HMM motifs databases was the pfam glutathione S-transferase N-terminal domain (GST-Nter, PF02996)
In order to determine whether our sequence belongs to the GST omega class, a phylogenetic analysis using the maximum-likelihood approach was carried out To achieve this, our sequence, as well as Sm28GST and Sm26GST sequences, were included in a multiple-sequence alignment similar to that previously used to characterize human omega class GST [10] The tree in Fig 2 is the most likely tree obtained by neighbor-interchange analysis of 2000 likeli-hood trees, and shows the eight families previously described
by Board et al [10] The tree grouped the new SmGST with human, rat, mouse and Caenorhabditis elegans omega GSTs
as well as two plant GST-like sequences already proposed to belong to the omega class by Board et al [10] leading us to name it SmGSTO Concerning the two other S mansoni GSTs, Sm26GST grouped within mu-class GSTs with a high local bootstrap value and Sm28GST remains in a nondefined position between sigma and pi classes
A Southern blot analysis was performed in order to ascertain that the cloned sequence belonged to the parasite and was not due to an artifact, and also to examine the copy number of SmGSTO genes in the parasite genome Cerca-riae genomic DNA was digested with several restriction enzymes and hybridized with radiolabeled SmGSTO probe (Fig 3) The pattern of hybridization obtained suggested that one copy of SmGSTO exists per haploid genome in
S mansoni
Trang 4Fig 1 Nucleotide and deduced amino acid
sequences of SmGSTO 1 The codon
corres-ponding to the initial methionine is
underlined.
Fig 2 Unrooted phylogeny showing the most likely relationship between class representative GSTs and S mansoni GST amino acid sequences Branch lengths are proportional to estimates of evolutionary change The number associated with each internal branch is the local bootstrap probability that is an indicator of confidence The sequences are (species name; GenBankTMaccession number): Schistosoma omega (Schistosoma mansoni, AF484940), nematode omega (Caenorhabditis elegans, L23651), mouse omega (Mus musculus, U80819), rat omega (Rattus rattus, AB008807), human omega (Homo sapiens, AF212303), soybean heat-shock protein (HsPr) (Glycine max, M20363), potato GST (Solanum tuberosum, J03679), nematode zeta (Caenorhabditis elegans, Z66560), human zeta (Homo sapiens, NM_001513), carnation zeta (Dianthus caryophyllus, M64268), mouse theta (Mus musculus, U48419), human theta (Homo sapiens, NM_000854), blowfly delta (L cuprina, L23126), house fly delta (Musca domestica, X61302), fruit fly Delta (Drosophila melanogaster, X14233), Arabidopsis phi (Arabidopsis thaliana, D17672), Petunia phi (Petunia hybrida, Y07721), mouse mu (Mus musculus, J03952), human mu (Homo sapiens, NM_000848), chicken mu (Gallus gallus, X58248), rat Pi (Rattus norvegicus, L29427), human pi (Homo sapiens, NM_000852), rat sigma (Rattus norvegicus, D82071), human sigma (Homo sapiens, D82073), squid2 sigma (Ommastrephens sloanei, M36938), squid1 sigma (O sloanei, M36937), Schistosoma 28 kDa (Schistosoma mansoni, S71584), human alpha (Homo sapiens, NM_000846), mouse alpha (Mus musculus, M73483), and chicken alpha (Gallus gallus, L15386), Schistosoma 26 kDa (Schistosoma mansoni, M31106).
Trang 5GSH binding affinity of recombinant SmGSTO
Recombinant SmGSTO was first produced as a fusion to a
histidine-tag and purified in one step using an Ni2+
nitrilotriacetic acid resin (Fig 4A) There are some
discrep-ancies in the literature about the ability of omega class GSTs
to bind different glutathione-coupled matrixes To
deter-mine whether SmGSTO was able to bind glutathione
agarose, we have used the purified enzyme in batch assays
As showed in Fig 4B,C, His-tagged SmGSTO was not able
to bind to agarose-coupled glutathione, but was able to bind
to S-hexyl glutathione agarose and was recovered after
elution with S-hexyl glutathione, as was already described
for Tc52 [16] In order to ascertain our observation and to
rule out the (possibility that the lack of binding of SmGSTO
to glutathione was neither due to the presence of the His-tag
sequence nor to a deletion in the parasite protein sequence,
we constructed a second plasmid which directed the
production of SmGSTO from its own methionine initiation
codon, lacking the His-tag In this way, the recombinant
SmGSTO could be purified using an S-hexyl glutathione–
agarose matrix (Fig 4D) Furthermore, batch experiments
performed with this protein determined that S-hexyl
glutathione agarose-bound SmGSTO was not eluted by
reduced nor by oxidized glutathione (Fig 4E) These results
suggested that SmGSTO has a low affinity to glutathione,
which coincides with the Kmvalue obtained for GSH in
kinetic experiments (see below)
Expression of SmGSTO
The recombinant SmGSTO was used to produce polyclonal
antibodies in immunized rabbits Expression of SmGSTO
along the parasite life cycle was analyzed by Western blot
In cercariae, sporocysts, schistosomule and adult worms, a
immunoreactive band was observed with a calculated molecular weight of nearly 28 kDa (Fig 5A) However, higher SmGSTO levels were observed in sporocysts (para-sitic stage of the intermediate host Biomphalaria glabrata) and adult worms (parasitic stage of human) than in others stages Expression of SmGSTO was also studied at the transcriptional level by RT-PCR using cDNA prepared from miracidia, sporocysts, cercariae and adult worms (Fig 5B) A unique amplification product of the 549 bp expected size was observed in all reactions The intensity of the amplified products was compared after normalization using a-tubulin cDNA as internal control Relative values showed as a bar graphic determine that SmGSTO tran-scription is higher in sporocysts and adult worms in comparison with cercariae and miracidia This results are
in agreement with those obtained by Western blot Taken together, these results suggested that SmGSTO is expressed more in parasitic stages than in free living stages during the
S mansonilife cycle
Characterization of recombinant SmGSTO enzymatic activity
Recombinant GSTO was used to assay its enzymatic activity Results of substrate specificity are shown in Table 1 SmGSTO showed a negligible activity against CDNB and ethacrynic acid and no measurable activity against other GST substrates like 1,2-dichloro-4-nitroben-zene (DCNB), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; p-nitrobenzyl chloride, vinylene thiocarbonate, t-butyl hydroquinone and p-chloranil In contrast, SmGSTO showed DHA and HEDS-measured TTase activities Kinetic parameters were obtained for DHA and TTase activities SmGSTO showed a Km¼ 0.23 mM for HEDS and a Km¼ 0.32 mMfor GSH in the TTase reaction These values were similar to those obtained for the same reaction for glutaredoxins from several origins [36] When kinetic parameters were calculated for DHA activity, a
Km¼ 2.3 mMfor DHA was obtained Graphical analysis
of the data obtained for GSH concentrations between
300 lM and 4 mM in a Lineweaver–Burk plot resulted reproducibly in a Kmvalue of 6.5 mM As GSH concentra-tions >4 mMcould not be tested, we prefer to give a Kmof
>4 mMfor GSH in this reaction Even though differences
in Kmvalues for GSH in the two assayed reactions were obtained, it was clear that a Kmlower than 0.32 mMcould not be reached Finally, SmGSTO showed differential sensitivity to several GST inhibitors tested (Table 2) Among them, it is interesting to note the inhibitory activity
of CDNB which is considered as a classical GST substrate Specific activity was first measured for both reactions in standard conditions using a phosphate buffer pH 7.6 [33] However, when the pH profile for SmGSTO activity was carried out, optimal activities were recorded at pH 8.0 and
pH 8.6 when phosphate buffer or Tris/HCl buffer were used, respectively (Fig 6) A similar optimal pH profile was recently reported for Plasmodium falciparum glutaredoxin 1 activity [37]
Construction of a homology model of SmGSTO SmGSTO structure was built using the human GSTO 1–1 structure as template in the SWISS-MODEL modeling
Fig 3 Southern blot analysis of SmGST DNA from S mansoni
cercaria (10 lg) digested with different restriction enzymes was
hybridized with the coding region of SmGST-O cDNA radiolabeled by
a random primer.
Trang 6environment [35] Structure accuracy was assayed by WhatCheck tool in the same environment As can be observed in Fig 7(A), only N-terminal SmGSTO domain structure could be predicted with accuracy The program was unable to model the C-terminal domain of SmGSTO due to the low rate of identity between the two proteins in this region However, as the active sites of the GSTO1-1 are located at the N-terminal domain, several shared structural features with SmGSTO could be pointed out Residues S(86), E(85), V(72), K(59) and C(32) which contact GSH in GSTO 1–1 correspond to S(79), E(78), V(65), K(52) and C(25) in SmGSTO, and were predicted to be placed in the same spatial orientation (Fig 7B)
D I S C U S S I O N
We report here the characterization of a new member of the GST superfamily from the human blood fluke S mansoni BLAST searches for sequence similarities showed that the cloned parasite gene has sequence homology to members of the recently discovered omega class of GSTs [10], and was
Fig 4 Expression and purification of His-tagged SmGSTO and native SmGSTO (A) SDS/PAGE analysis of His-tagged SmGST purified on nickel-agarose Lane 1, 50 lg soluble extract of BL21 (ED3) expressing His-tagged SmGSTO Lane 2, 5 lg purified His-tagged SmGSTO (B) Batch analysis of His-tagged SmGSTO to GSH-agarose Lane 1, protein used in the binding step Lane 2, supernatant of the binding-step Lane 3, GSH eluted fraction (C) Batch analysis of His-tagged SmGSTO to S-hexyl glutathione-agarose Lane 1, protein used in the binding step Lane 2, supernatant of the binding-step Lane 3, S-hexyl glutathione eluted fraction (D) SDS/PAGE analysis of recombinant native GST purified on S-hexyl glutathione-agarose Lane 1, 50 lg soluble extract of BL21 (ED3) expressing native SmGSTO Lane 2, 5 lg purified SmGSTO (E) Elution
of S-hexyl glutathione agarose-bound SmGSTO by GSH, GSSG or S-hexyl glutathione In all cases: Lane 1, protein used in the binding step Lane
2, supernatant of the binding-step Lane 3, eluted fraction Compound used to elute in each case are indicated in the figure.
Fig 5 Expression of SmGSTO in S mansoni (A) Immunoblotting
analysis of parasite extracts Each lane contains total protein (50 lg)
from cercariae (lane 1), schistosomula (lane 2), sporocysts (lane 3), and
adult worms (lane 4) electroblotted and immunodetected by
a-SmG-STO serum (B) RT-PCR analysis of different S mansoni stages.
Reverse-transcribed RNA from miracidia (lane 1), sporocysts (lane 2),
cercariae (lane 3) and adult worms (lane 4) were amplified using
SmGSTO and a-tubulin specific primers as indicated in experimental
section.
Trang 7referred here as SmGST omega (SmGSTO) In addition, sequence alignment of SmGSTO with representative sequences from the recently reported GSTO class or from the EST data base highlights similarities, but also significant differences In all cases, the most conserved amino acids were localized in the N-terminal domain of the SmGSTO protein However, the remaining regions of SmGSTO presented significant divergences from the known GSTO,
at the primary structure level Indeed, the GSTO class represents a particular class of the GST superfamily which possesses specific structural features, such as an active site cysteine that is able to form a disulfide bond with GSH, a novel domain formed by the proximity of a specific N-terminal extension to the C-terminus and a large H-site,
as well as the ability to catalyze the GSH-dependent reduction of dehydroascorbate [10]
We further undertook a phylogenetic protein sequence analysis to ascertain whether the SmGSTO was a divergent member of the GSTO class The deduced SmGSTO protein sequence was used in a phylogenic analysis using the maximum-likelihood approach Two other previously char-acterized S mansoni GSTs, Sm26GST and Sm28GST, were amongst the GST proteins included in this study The most likely tree obtained showed the eight proposed GST families [7,8] The tree grouped the new cloned schistosome protein
as a divergent member of the GSTO class We could also place the Sm26GST among the Mu-class Even if position
of Sm28GST was not well defined in the tree, the fact that Sm28GST has a prostaglandin D2 synthetase activity, typical of the sigma class of GSTs, strongly suggest that it belongs to this group (J F Trottein, Institut Pasteur de Lille, France, personal communication) All S mansoni GSTs showed particularly long branches when compared with those obtained for free living invertebrates GSTs This high rate of evolution for Schistosoma GSTs was already reported [38] The same abnormality was also observed when the phylogenetic analysis of S mansoni nuclear receptors was performed [39] The proposed explanation for this observation is that the human parasites of Schisto-soma genus were subjected during evolution to a biased selection pressure due to host biochemical characteristics, including the immune and endocrine systems The host pressure resulted in abnormally divergent parasite sequences
as shown for GSTs and nuclear receptors of the parasite, or
by abnormally host-parasite converged sequences as was reported for the tropomyosins of S mansoni and its intermediate host Biomphalaria glabrata [40]
SmGSTO expression in the different life forms of
S mansoniwas performed by RT-PCR and Western blot The results obtained support the higher expression of SmGSTO observed in S mansoni parasitic life stages rather than in free-living life stages, suggesting that this protein may play a role in the survival of the parasite within the host An increased expression during cercariae transforma-tion to mature adults in mammalian host was already described as a general feature for detoxifying enzymes in Schistosoma [41–43] Immunohistochemical analysis of human tissues confirmed a widespread expression of GSTO1-1, suggesting that it has important biological functions Specific expression of GSTO1-1 was localized in the nuclei and in nuclear membranes of many cell types [44] However, no putative nuclear localization signals could be found within GSTO1-1 or SmGSTO Nuclear localization
Table 1 Substrate specificities of recombinant SmGSTO Activity for
each substrate was determined in standard conditions ND, not
detected.
Substrate
Specific activity (lmolÆmin)1Æmg)1) 1,2-Dichloro-4-nitrobenzene ND
1-Chloro-2,4-dinitrobenzene 0.02
7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole
ND
p-Nitrobenzyl chloride ND
Vinylene trithiocarbonate ND
t-Butyl hydroquinone ND
Hydroxyethyl disulfide 0.11
Table 2 Inhibitor sensitivities of recombinant SmGSTO Inhibitor
sensitivities are presented as I 50 and were calculated in standard
reac-tion condireac-tions Values in the table represent the mean of three
determinations.
Dehydroascorbate activity inhibitor
Hydroxyethyl disulfide activity
I 50 (l M ) I 50 (l M )
Bromosulfophthalein 0.3 8.3
Cibacron blue 3GA < 0.001 < 0.001
S-Hexyl glutathione < 0.001 < 0.001
Fig 6 pH optimum of SmGSTO The SmGSTO pH profile was
car-ried out using the glutathione:dehydroascorbate reductase assay.
Grafic indicates relative DHA activity measured in 100 m M phosphate
buffer and 100 m Tris/HCl, using standard substrate concentrations.
Trang 8of S mansoni 28 kDa GST, which has no detectable nuclear
localization signal, was already described [45] A
cytolocali-zation study of SmGSTO is being undertaken in our
laboratory
Some contrasting findings were reported concerning the
ability of omega GSTs to bind matrix-linked glutathione
Mouse p28 was reported to bind glutathione-agarose, but
human GSTO1-1 was unable to bind S-linked
glutathione-sepharose Here, we report that SmGSTO binds S-hexyl
glutathione-agarose but not glutathione-agarose Moreover,
S-hexyl glutathione-agarose-bound SmGSTO was not
dis-placed neither by reduced nor oxidized glutathione These
results are in line with the high GSH Kmvalue obtained for
SmGSTO and the strong inhibitory effect of S-hexyl
glutathione on the enzyme activity The preference of
SmGSTO for more hydrophobic alkyl-bound S-hexyl
glutathione rather than glutathione could reflect some
particular characteristics of the active site of this enzyme
As other GSTs from the omega class the parasite protein
was unable to use known GST family substrates like CDNB
and other GST substrates assayed (Table 1) The most
significant enzymatic activities observed for SmGSTO were
the ability to act as GSH-dependent DHA and TTase
SmGSTO activity was inhibited to different extents by
classical GSTs inhibitors In addition, GSTs’ substrate,
CDNB, which inhibits thioredoxin activity, also inhibited
GSTO activity Our phylogenic analysis showed that
omega, zeta and theta GST classes, which demonstrated
low activities towards CDNB substrate, branch together in
the left side of the tree whereas the remaining classes, which use CDNB as substrate, branch in the right side of the tree This observation may reflect an evolutionary relationship among this group of proteins To confirm this assumption, the inhibitory activity of CDNB over zeta- and theta-class GSTs should be tested
Finally, the SmGSTO structure was built based on homology modeling Even if sequence divergence makes it impossible to produce a model of the whole protein, a prediction of the N-terminal domain was obtained This GSTO domain shares structural features with the recently described glutaredoxin-2 from E coli [46] Glutaredoxins can catalyze the reduction of mixed disulfides between GSH and proteins or low molecular mass disulfides, in a reaction that only requires N-terminal active-site cysteine residue, and the reverse reaction called glutathionylation [47] Xia et al recently proposed a three subfamilies classification for glutaredoxins The first group, which includes E coli Gsx1 and human Grx1 amongst others, corresponds to small
two-cysteine (dithiol) classical glutaredoxins which contain the consensus sequence C-P-Y-C and have a high activity with HEDS The second group corresponds to variable molecular mass one-cysteine (monothiol) glutaredoxins with a conserved sequence C-G-F-S, such as yeast gluta-redoxins Grx3–5 E coli Grx2 and GSTO 1–1 were proposed
to be grouped into a third subfamily, having an N-terminal Grx-like domain and the helical C-terminal domain and the general structure reminiscent to the GST superfamily of proteins Sequence similarity and predicted structure show
Fig 7 Model showing SmGSTO structure.
Structure was built based on homology
modeling using human GSTO1-1 as template
in the SWISS-MODEL modeling
environ-ment (A) General chain fold view of human
GSTO 1–1 and SmGSTO (B) Scheme
illus-trating position of GSH contacting residues
determined for human GST 1–1 and modeled
for SmGSTO.
Trang 9that SmGSTO belongs to this last group At this point, it
should be noted that the questions as to whether E coli
Grx-2 is a GST or if GST-O are glutaredoxins is not completely
solved When active cysteine-containing tetramers were
sought in these proteins, a striking sequence divergence was
observed E coli Grx2 contains a Trx1-like two-cysteine
sequence, C-P-Y-C; GSTO 1–1 has a one-cysteine C-P-F-A
sequence; and SmGSTO contains C-P-Y-V, similar to the
Trx1-like sequence but with only one cysteine Sequence
comparison at the active site level and HEDS-measured
SmGSTO TTase activity strongly suggest that SmGSTO
could participate in glutathionylation and reduction of
mixed disulfides, a typical glutaredoxin activity Sensitivity to
CDNB and alkaline optimal pH, which stabilize the GSH
thiolate at the active cysteine, are considered as GSTO
biochemical characteristics that support the idea of a
functional relationship between omega-class GSTs and
glutaredoxins/thioredoxins superfamily In this way, Caccuri
et al [48] recently proposed that Proteus mirabilis low
molecular weight GST could be an intermediary enzyme
somewhere between thioldisulfide oxidoreductases and the
GST superfamily P mirabilis GST differs from SmGST-O
because of it shows both TTase and CDNB-measured
transferase activities Keeping in mind that GSTO contains
an additional domain, new activities can not be ruled out for
GSTO
To resume, SmGSTO can be considered as a
multifunc-tional enzyme displaying thioredoxin/glutaredoxin features
The additional C-terminal domain could allow this enzyme
to react with a large substrate spectrum in comparison with
low molecular weight thioredoxins and glutaredoxins To
date, very little is known about thioredoxin or glutaredoxin
metabolism in S mansoni Recently a
thioredoxin/glutathi-one reductase containing a thioredoxin/glutaredoxin-like
motif at the N-terminal was described in this parasite [49]
Structural and functional relationships between these two
multifuctional enzymes should be explored in the future
Finally, this work is the first evidence that S mansoni may
take advantage of host ascorbic acid The physiological
significance of all these findings will need much investigation
in order to be understood
A C K N O W L E D G M E N T S
The authors wish to thank Dr Lars Jermiin for GST sequences
alignments and for His help with Molphy 2.3 utilization, Dr Luis
Esteban for His help with Linux operative system installation and
utilization and Dr Eleonora Garcı´a Ve´scovi for her critical reading
of the manuscript This research was supported by Fundacion
Antorchas, Third World Academy of Sciences and the Research
Program of the UNR ECS is member of the National Research
Council (CONICET, Argentina) and JEG is Fellow of the same
institution.
R E F E R E N C E S
1 Hayes, J.D & Pulford, D.J (1995) The glutathione S-transferase
supergene family: regulation of GST and the contribution of the
isoenzymes to cancer chemoprotection and drug resistance CRC
Crit Rev Biochem Mol Biol 30, 445–600.
2 Vuilleumier, S (1997) Bacterial glutathione S-transferases: What
are they good for? J Bacteriol 179, 1431–1441.
3 Tipping, E & Ketterer, B (1978) The role of intracellular proteins
in the transport and metabolism of lipophilic compounds In
Transport by Proteins (Blauer, G & Sund, H., eds), pp 369–382 Walter de Gruyter, Berlin.
4 Litwack, G., Ketterer, B & Arias, I.M (1971) Ligandin: a hepatic protein which binds steroids, bilirubin, carcinogens and a number
of exogenous organic anions Nature 234, 466–467.
5 Fernandez-Canon, J.M & Penalva, M.A (1998) Characterization
of a fungal maleylacetoacetate isomerase gene and identification of its human homologue J Biol Chem 273, 329–337.
6 Adler, V., Yin, Z., Fuchs, S.Y., Benezra, M., Rosario, L., Tew, K.D., Pincus, M.R., Sardana, M., Henderson, C.J., Wolf, C.R., Davis, R.J & Ronai, Z (1999) Regulation of JNK signaling by GSTp EMBO J 18, 1321–1334.
7 Pemble, S.E., Wardle, A.F & Taylor, J.B (1996) Glutathione S-transferase class kappa: characterization by the cloning of rat mitochondrial GST and identification of a human homologue Biochem J 319, 749–754.
8 Board, P.G., Baker, R.T., Chelvanayagan, G & Jermiin, L (1997) Zeta, a novel class of glutathione transferases in a range of species from plants to humans Biochem J 328, 929–935.
9 Mannervik, B., Awasthi, Y.C., Board, P.G., Hayes, J.D., Di Ilio, C., Ketterer, B., Listowsky, I., Morgenstern, R., Muramatsu, M., Pearson, W.R., Pickett, C.B., Sato, K., Widersten, M & Wolf, C.R (1992) Nomenclature for human glutathione transferases Biochem J 282, 305–306.
10 Board, P.G., Coggan, M., Chelvanayagam, G., Easteal, S., Jermiin, L.S., Schulte, G.K., Danley, D.E., Hoth, L.R., Griffor, M.C., Kamath, A.V., Rosner, M.H., Chrunyk, B.A., Perregaux, D.E., Gabel, C.A., Geoghegan, K.F & Pandit, J (2000) Identi-fication, characterization, and crystal structure of the omega class glutathione transferases J Biol Chem 275, 24798–247806.
11 Ishikawa, T., Casini, A & Nishikimi, M (1998) Molecular cloning and functional expression of rat liver glutathione-dependent dehydroascorbate reductase J Biol Chem 273, 28708–28712.
12 Kodym, R., Calkins, P & Story, M (1999) The cloning and characterization of a new stress response protein J Biol Chem.
274, 5135–5137.
13 Zakharyan, R.A., Sampayo-Reyes, A., Healy, S.M., Tsaprailis, G., Board, P.G., Liebler, D.C & Aposhian, H.V (2001) Human monomethylarsonic acid (MMA(V)) reductase is a member of the glutathione-S-transferase superfamily Chem Res Toxicol 14, 1051–1057.
14 Dulhunty, A., Gage, P., Curtis, S., Chelvanayagam, G & Board,
P (2001) The glutathione transferase structural family includes a nuclear chloride channel and a ryanodine receptor calcium release channel modulator J Biol Chem 276, 3319–3323.
15 Schoneck, R., Plumas-Marty, B., Taibi, A., Billaut-Mulot, O., Loyens, M., Gras-Masse, H., Capron, A & Ouaissi, A (1994) Trypanosoma cruzi cDNA encodes a tandemly repeated domain structure characteristic of small stress proteins and glutathione S-transferases Biol Cell 80, 1–10.
16 Moutiez, M., Quemeneur, E., Sergheraert, C., Lucas, V., Tartar,
A & Davioud-Charvet, E (1997) Glutathione-dependent acti-vities of Trypanosoma cruzi p52 makes it a new member of the thiol: disulphide oxidoreductase family Biochem J 322, 43–48.
17 Ouaissi, A., Guevara-Espinoza, A., Chabe, F., Gomez-Corvera,
R & Taibi, A (1995) A novel and basic mechanism of immunosuppression in Chagas’ disease: Trypanosoma cruzi releases in vitro and in vivo a protein which induces T cell unre-sponsiveness through specific interaction with cysteine and gluta-thione Immunol Lett 48, 221–224.
18 Fernandez-Gomez, R., Serra, E., Gomez-Corvera, R., Kerrou-che, Z & Ouaissi, A (1998) Trypanosoma cruzi: Tc52 released protein-induced increased expression of nitric oxide synthase and nitric oxide production by macrophages J Immunol 160, 3471– 3479.
19 Allaoui, A., Francois, C., Zemzoumi, K., Guilvard, E & Ouaissi,
A (1999) Intracellular growth and metacyclogenesis defects in
Trang 10Trypanosoma cruzi carrying a targeted deletion of a Tc52
protein-encoding allele Mol Microbiol 32, 1273–1286.
20 Borges, M., Guilvard, E., Cordeiro da Silva, A., Vergnes, B.,
Zemzoumi, K & Ouaissi, A (2001) Endogenous Trypanosoma
cruzi Tc52 protein expression upregulates the growth of murine
macrophages and fibroblasts and cytokine gene expression.
Immunol Lett 78, 127–134.
21 O’Leary, K.A & Tracy, J.W (1988) Purification of three cytosolic
glutathione S-transferases from adult Schistosoma mansoni Arch.
Biochem Biophys 264, 1–12.
22 O’Leary, K.A & Tracy, J.W (1991) Schistosoma mansoni:
glu-tathione S-transferase-catalysed detoxication of dichlorvos Exp.
Parasitol 72, 355–361.
23 O’Leary, K.A., Hathaway, K.M & Tracy, J.W (1992)
Schisto-soma mansoni: single step purification and characterization of
glutathione S-transferase )4 Exp Parasitol 75, 47–55.
24 Pierce, R.J., Khalife, J., Williams, D.L., Kanno, R., Trottein, F.,
LePresle, T., Sabatier, J., Achstetter, T & Capron, A (1994)
Schistosoma mansoni: characterization of sequence variants of the
28-kDa glutathione S-transferase Exp Parasitol 79, 81–84.
25 Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang,
Z., Miller, W & Lipman, D.J (1997) Gapped BLAST and
PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res 25, 3389–3402.
26 Sambrook, J., Fritsch, E.F & Maniatis, T (1989) Molecular
Cloning: a Laboratory Manual, 2nd edn Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY.
27 Jones, D.T., Taylor, W.R & Thornton, J.M (1992) The rapid
generation of mutation data matrices from protein sequences.
Bioinformatics 8, 275–282.
28 Adachi, J & Hasegawa, M (1996) MOLPHY , Version 2.3 The
Institute of Mathematical Statistics, Tokyo.
29 Kishino, H & Hasegawa, M (1989) Evaluation of the maximum
likelihood estimate of the evolutionary tree topologies from DNA
sequence data, and the branching order in hominoidea J Mol.
Evol 29, 170–179.
30 Studier, F.W., Rosenberg, A.H., Dunn, J.J & Dubendorf, J.W.
(1990) Use of T7 RNA polymerase to direct expression of cloned
genes Methods Enzymol 185, 60–89.
31 Moutiez, M., Aumercier, M., Schoneck, R., Meziane-Cheri, F.D.,
Lucas, V., Aumercier P., Ouaissi A., Sergheraert, C & Tartar, A.
(1995) Glutathione-dependent activities of Trypanosoma cruzi p52
makes it a new member of the thiol: disulphide oxidoreductase
family Biochem J 310, 433–437.
32 Mannervik, B & Danielson, U.H (1988) Glutathione reductases –
Structure and catalitic activity CRC Crit Rev Biochem 23, 283–337.
33 Axelsson, K., Eriksson, S & Mannervik, B (1978) Purification
and characterization of cytoplasmic thioltransferase (glutathione:
disulfide oxidoreductase) from rat liver Biochem 17, 2978–2984.
34 Wells, W.W., Xu, D.P & Washburn, M.P (1995) Glutathione:
dehydroascorbate oxidoreductases Methods Enzymol 252, 30–38.
35 Guex, N & Peitsch, M.C (1997) SWISS-MODEL and the
Swiss-PdbViewer: an environment for comparative protein modelin.
Electrophoresis 18, 498–501.
36 Holmgren, A & Aslund, F (1995) Glutaredoxins Methods Enzymol 252, 283–292.
37 Rahlfs, S., Fischer, M & Becker, K (2001) Plasmodium falci-parum possesses a classical glutaredoxin and a second, gluta-redoxin-like protein with PICOT homology domain J Biol Chem 276, 37133–37140.
38 Hughes, A.L (1993) Rates of amino acids evolution in the 26- and 28-kDa glutathione S-transferases of Schistosoma Mol Biochem Parasitol 58, 43–52.
39 de Mendonca, R.L., Escriva, H., Bouton, D., Zelus, D., Vanacker, J.M., Bonnelye, E., Cornette, J., Pierce, R.J & Laudet, V (2000) Structural and functional divergence of a nuclear receptor of the RXR family from the trematode parasite Schistosoma mansoni Eur J Biochem 267, 3208–3219.
40 Dissous, C., Torpier, G., Duvaux-Miret, O & Capron, A (1990) Structural homology of tropomyosins from the human trematode Schistosoma mansoni and its intermediate host Biomphalaria glabrata Mol Biochem Parasitol 43, 245–255.
41 LoVerde, P (1998) Do antioxidants play a role in schistosome host–parasite interactions? Parasitol Today 14, 284–289.
42 Serra, E., Zemzoumi, K & Dissous, C (1997) Deletion analysis of the Schistosoma mansoni 28 kDa glutathione S-transferase gene promoter Functionality of a proximal AP-1 site Eur J Biochem.
248, 113–119.
43 Serra, E., Lardans, V & Dissous, C (1999) Identification of a NF-AT-like transcription factor in Schistosoma mansoni: its possible involvement in the antiparasitic action of cyclosporin A Mol Biochem Parasitol 101, 33–41.
44 Yin, Z.L., Dahlstrom, J.E., Le Couteur, D.G & Board, P.G (2001) Immunohistochemistry of omega class glutathione S-transferase in human tissues J Histochem Cytochem 49, 983–987.
45 Liu, J.L., Fontaine, J., Capron, A & Grzych, J.M (1996) Ultrastructural localization of Sm28 GST protective antigen
in Schistosoma mansoni adult worms Parasitology 113, 377–391.
46 Xia, B., Vlamis-Gardikas, A., Holmgren, A., Wright, P.E & Dyson, J (2001) Solution structure of Escherichia coli gluta-redoxin-2 shows similarity to mammalian glutathione S-trans-ferases J Mol Biol 310, 907–918.
47 Cotgreave, I.A & Gerdes, R.G (1998) Recent trends in glu-tathione biochemistry – gluglu-tathione–protein interactions: a molecular link between oxidative stress and cell proliferation? Biochem Biophys Res Com 242, 1–9.
48 Caccuri, A.M., Antonini, G., Allocati, N., Di Ilio, C., De Maria, F., Innocenti, F., Parker, M.W., Masulli, M., Lo Bello, M., Turella, P., Federici, G & Ricci, G (2002) GSTB1-1 from Proteus mirabilis: a snapshot of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme J Biol Chem 277, 18777–18784.
49 Alger, H.M & Williams, D.L (2002) The disulfide redox system
of Schistosoma mansoni and the importance of a multifunctional enzyme, thioredoxin glutathione reductase Mol Biochem Para-sitol 121, 129–139.