From the Divisions of Infection and Immunity and
¶ Biochemistry and Molecular Biology, Institute of Biomedical and
Life Sciences, University of Glasgow, Glasgow G12 8QQ and the
Wellcome Centre for Molecular Parasitology, University of
Glasgow, Anderson College,
Glasgow G11 6NU, Scotland, United Kingdom
Received for publication, September 13, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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Cytosolic 3-mercaptopyruvate sulfurtransferases
(EC 2.8.1.2) of Leishmania major and Leishmania
mexicana have been cloned, expressed as active enzymes in
Escherichia coli, and characterized. The leishmanial
single-copy genes predict a sulfurtransferase that is structurally
peculiar in possessing a C-terminal domain of some 70 amino acids.
Homologous genes of Trypanosoma cruzi and
Trypanosoma brucei encode enzymes with a
similar C-terminal domain, suggesting that this feature, not known in
any other sulfurtransferase, is a characteristic of trypanosomatid
parasites. Short truncations of the C-terminal domain resulted in
misfolded inactive proteins, demonstrating that the domain plays some
key role in facilitating correct folding of the enzymes. The
leishmanial recombinant enzymes exhibited high activity toward
3-mercaptopyruvate and catalyzed the transfer of sulfane sulfur to
cyanide to form thiocyanate. They also used thiosulfate as a substrate
and reduced thioredoxin as the accepting nucleophile, the latter being
oxidized. The enzymes were expressed in all life cycle stages, and the
expression level was increased under peroxide or hypo-sulfur stress.
The results are consistent with the enzymes having an involvement in
the synthesis of sulfur amino acids per se or iron-sulfur
centers of proteins and the parasite's management of oxidative stress.
Sulfurtransferases (EC 2.8.1.1-5) are widely distributed enzymes
of prokaryotes and eukaryotes (1, 2). The enzymes catalyze the transfer
of sulfane sulfur from a donor molecule, such as thiosulfate or
3-mercaptopyruvate, to a nucleophilic acceptor, such as cyanide or
mercaptoethanol. However, the natural sulfane donors and acceptors and
the physiological functions of most sulfurtransferases remain uncertain.
The rhodanese family sulfurtransferases are thought to occur in the
majority of organisms (1), with the mammalian enzymes being the most
extensively studied (3, 4). The first elucidated role of mitochondrial
bovine liver rhodanese was the detoxification of cyanide to form
thiocyanate, which is harmless and excreted by the kidney. This role
could be important, especially in the epithelial cells lining the gut
(5), but is thought not to account for the wide distribution of these
sulfurtransferases in different cell types (2). Another putative
function of at least some sulfurtransferases is the provision of
sulfane sulfur required for the formation of the iron-sulfur centers of
proteins, notably respiratory proteins (6-9).
Sulfurtransferases may also play a part in the management of the
cytotoxicity of reactive oxygen species in aerobic tissues (4). Bovine
rhodanese has a 1000-fold higher affinity for the reduced form of
thioredoxin than for cyanide and so may function in peroxide
detoxification (4, 10). A reaction analogous to that of sulfane-loaded
sulfurtransferase with thioredoxin is also thought to be a critical
step in the synthesis of thiouridine (7, 11, 12), and the formation of
thiocarboxylate during thiamine biosynthesis by the multidomain protein
ThiI of Escherichia coli (7, 12). Sulfurtransferases have
also been implicated in the synthesis of biotin (13) and molybdopterin
(14). Moreover, a role for sulfurtransferases in assimilatory sulfate
reduction by transferring a molecule of sulfide to
O-acetyl-L-serine in the synthesis of cysteine
has been postulated (15).
Most sulfurtransferases have an N-terminal "structural" domain and
a C-terminal domain containing the active site (1, 16, 17). The
vertebrate rhodaneses have been extensively studied in attempts to
understand the part played by the N-terminal structural domain in the
correct folding and stability of the enzymes. Current evidence
suggests, however, that correct protein folding also requires the
assistance of a chaperone molecule (18, 19).
Leishmania parasites are widespread and important parasites
of humans and dogs. The diseases they cause are most prevalent in the
tropics and subtropics, although leishmaniasis is also endemic in
Southern Europe and has been reported in the United States (20). There
is a pressing need to improve ways of treating the diseases; and in
particular, there is a requirement for better chemotherapy (21).
Leishmania is an excellent organism in which to investigate
the possible roles of sulfurtransferases in antioxidant and sulfur
amino acid metabolism. The parasites exist intracellularly in
macrophages while within their mammalian host and are thought to be
particularly well adapted to survive against oxidative stress arising
from the immune mechanisms of the host. Leishmania is unusual in possessing trypanothione, a conjugate of glutathione and
spermidine, as a major cellular thiol and apparently uses this and
associated enzymes as a prime means of protection against oxidative
damage (22, 23). However, the source of cysteine, essential for
trypanothione, is unknown. In this study, we describe the cloning,
expression, and characterization of a sulfurtransferase of the
protozoan parasites Leishmania major and Leishmania
mexicana and provide data on its unusual structure and possible roles.
Cultivation of L. major and L. mexicana--
Promastigotes of
L. major (MHOM/JL/80/Friedlin) and L. mexicana
(MNYC/BZ/62/M379) were normally grown in modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum
at 27 °C (24). Metacyclic promastigotes of L. major were purified from a stationary phase population of cells using the agglutination assay described by Sacks et al. (25).
Amastigotes of L. mexicana were purified from infected
BALB/c mice or grown axenically as described (26). The bloodstream form
of Trypanosoma brucei strain 427 was grown in rats and
isolated by DEAE ion-exchange chromatography as described (27). The
procyclic form of T. brucei strain 427 was grown
at 27 °C in semi-defined medium 79 containing 10% (v/v)
fetal bovine serum and 3.5 mg/ml hemin (28). Parasites were harvested
and used immediately or pelleted and stored at Identification, PCR Amplification, and Cloning of
3-Mercaptopyruvate Sulfurtransferase
(MST)1 of L. major (LmajMST)
and L. mexicana (LmexMST)--
Total RNAs of L. major and
L. mexicana promastigotes were isolated using TRIzol
(Invitrogen) and reverse-transcribed with Superscript II reverse
transcriptase (Invitrogen) into single-stranded cDNA as described
by the manufacturer. Genomic DNAs of L. major and L. mexicana were isolated using TELT buffer (50 mM
Tris-HCl, pH 8.0, 62.5 mM EDTA, 2.5 M LiCl,
49% (v/v) Triton X-100) as described (29). The 5'- and 3'-ends of the
genes from both L. major and L. mexicana were
amplified using 5'- and 3'-RACE kits (Invitrogen). The degenerate
reverse gene-specific primers NT22 (5'-ACGCGCACCTTCGATGTGGCC-3') and
NT41 (5'-ATTTCATCCACAAGGTAGTGATGC-3'), based on conserved motif
sequences from the alignment of the putative rhodanese of L. major (GenBankTM/EBI accession number AF163772)
and bacterial homologs in the NCBI Database, and the spliced leader
primers LMEXSLI (5'-TAACGCTATATAAGTATCAGTTTC-3') and
LMEXSLII (5'-AGTATCAGTTTCTGTACTTTATTG-3') were used for 5'-RACE. The perfect match primers NT21 (5'-CACCGCCCGGCATCCGCTACC-3') and NT54
(5'-ATCGACTGGTGCATGGCGAAC-3'), based on consensus sequences of
the genes detailed above, and oligo(T) primers with an adaptor attached, UAP (5'-CUACUACUACUAGGCCACGCGTGGACTAGTAC-3') and AUAP (5'-GGCCACGCGTGGACTAGTAC-3'), were used to amplify the 3'-ends of the
genes with Expand High Fidelity Pwo/Taq
polymerase. Both the 5'- and 3'-ends of the genes were cloned into the
pGEM-T vector (Promega) and sequenced.
PCR Amplification, Expression, and Purification of MSTs--
The
open reading frames (ORFs) of the genes encoding LmajMST and LmexMST
and four constructs, LmajMST
Plasmids were digested with NdeI and XhoI and
ligated into NdeI/XhoI-digested
pET21a+ vector (Invitrogen) to produce the respective pET
constructs (e.g. pETLmajMST). The plasmids were used to
transform E. coli BL21 Codon Plus (DE3)RP cells
(Stratagene). Expression was induced with 0.5 mM
isopropyl- Refolding of Denatured Enzyme--
Denaturation and reactivation
analyses of rLmajMST Genomic Southern Blot Analysis--
Genomic DNA was extracted
according to standard procedures (29). DNA (5 µg) was digested with
the appropriate enzymes, fractionated by agarose gel electrophoresis,
nicked, denatured, neutralized, and blotted onto a
HybondTM-N+ membrane (31) by capillary
transfer. Probes were prepared from 1110-bp
NdeI/XhoI ORF fragments from pETLmajMST and
pETLmexMST using a Prime-It kit (Stratagene) and purified on Microspin
S-200 HR columns (Amersham Biosciences). Filters were hybridized
overnight with [ Enzyme Activity Measurements--
Sulfurtransferase activity was
determined for recombinant sulfurtransferases and soluble extracts of
parasites by monitoring thiocyanate or sulfide formation as follows.
The "rhodanese" assay was based on the formation of thiocyanate
from the reaction between cyanide and either 3-mercaptopyruvate or
thiosulfate and the detection of the thiocyanate by reacting it with an
iron reagent to produce a red complex of FeSCN
The oxidation of reduced thioredoxin by recombinant sulfurtransferase
was measured by monitoring the reduction of oxidized thioredoxin by
NADPH-specific thioredoxin reductase (4). A reaction mixture (1 ml)
containing 20 µM NADPH, 2 µg Trichomonas vaginalis thioredoxin reductase, and 5 µg T. vaginalis thioredoxin2
in 0.1 M phosphate buffer, pH 7.0, was incubated at
37 °C for 5 min. A constant absorbance, as reported by Nandi
et al. (4), was not achieved, as the thioredoxin
reductase-thioredoxin system used has a low NADPH oxidase
activity.2 0.1 µg of recombinant sulfurtransferase was
added, and the reaction was started by the addition of 5 mM
3-mercaptopyruvate or thiosulfate. The reaction was incubated at
37 °C, and the consumption of NADPH was followed at 340 nm.
Western Blot Analysis and SDS-PAGE--
Rabbit polyclonal
anti-MST antibody was raised against rLmajMST by the Scottish Antibody
Production Unit (Carluke, UK) using standard protocols. Parasite
pellets were resuspended in an equal volume of lysis buffer, and
supernatant (15,000 × g for 10 min) samples (10 µg of protein)
were subjected to Western blot analysis as described previously (31)
with polyclonal immune rabbit serum diluted 1:500 in Tris-buffered
saline containing 1% (w/v) low fat dried milk and 0.1% Tween 20. Bound antibody was detected using horseradish peroxidase-coupled
secondary antibodies (Scottish Antibody Production Unit) and ECL
Western blotting detection reagents (Amersham Biosciences).
Effects of Oxidative and Hypo-sulfur Stress on the Expression of
LmajMST--
Promastigotes were cultured for 24 h under standard
conditions and from a starting density of 2.5 × 105
ml Analysis of LmajMST and Its Truncated Derivatives Using CD
Spectroscopy and Binding of Bis-ANS--
CD spectral measurements of
rLmajMST Cloning Genes Encoding Sulfurtransferases in L. major and L. mexicana--
Searches of L. major HTGS sequence in the
GenBankTM/EBI Data Bank revealed a gene (gi:13122208)
encoding a protein with similarity to sulfurtransferases. PCR cloning,
coupled with 5' and 3'-RACE, confirmed the identity of the gene, which
was subsequently annotated as a sulfurtransferase
(GenBankTM/EBI accession number AJ313201). The AG
dinucleotide spliced reader addition acceptor site was found to
be 354 nucleotides 5' of the ATG start codon, and the gene has a
1160-bp ORF and a 173-bp 3'-untranslated region. The same methodology
was used to clone the L. mexicana homolog (accession number
AJ313202), which has a 89-bp 5'-untranslated region, a 1160-bp ORF, and
a 255-bp 3'-untranslated region. The predicted amino acid sequences of
the sulfurtransferase of each Leishmania species comprise
370 residues, with a calculated molecular mass of 40.1 kDa.
Features of Predicted Amino Acid Sequences of the
Sulfurtransferases--
The predicted amino acid sequences of L. major and L. mexicana are 96.7% identical to each
other and 47% identical to a putative T. brucei
sulfurtransferase identified in the T. brucei genome data
base. Of the other sulfurtransferases known, the most similar are the
mammalian and plant mercaptopyruvate and thiosulfate
sulfurtransferases, which are ~23% identical within the main domains
(excluding the C-terminal extensions of the Leishmania
sequences). Sequence analysis of the sulfurtransferases from
vertebrates, plants, yeast, bacteria, and protozoa (Fig.
1) showed that the leishmanial proteins
are more similar to other MSTs (with an active-site motif of
CG(S/T)GVTA) than to eukaryotic thiosulfate sulfurtransferases and
rhodaneses (with an active-site motif of CRKGVTA). Interestingly,
prokaryotic genes thought to encode thiosulfate sulfurtransferases lack
the CRKGVTA motif, although, in most cases, the substrate specificity has not been fully analyzed. As the catalytic active-site CGSGVTA motif
of the leishmanial genes (underlined in Fig. 1) is
considered to be predictive of an MST rather than a rhodanese or
thiosulfate sulfurtransferase (49), the genes were designated LmajMST
and LmexMST.
Intriguingly, both of the leishmanial sulfurtransferases contain an
additional C-terminal domain compared with all other MSTs known. The
domain, which comprises 70 amino acids compared with the human
gene (GenBankTM/EBI accession number P25325), has no
sequence identity to any other known protein sequence. However, a
similar C-terminal domain is also present in a homologous protein of
T. brucei and also in the protein predicted from a contig
compiled from genomic sequence survey (GSS) gene fragments
identified in the Trypanosoma cruzi genome data base (Fig.
1). Thus, it appears that this domain of the protein is a feature
characteristic of, and perhaps unique to, the MSTs of trypanosomatids.
Genomic Organization of the MSTs--
To assess the copy number of
the leishmanial MSTs, genomic DNA was digested with five restriction
enzymes and analyzed by Southern blotting using the MST cDNAs as
probes (Fig. 2). With L. major, the enzymes used that did not cut the gene itself
(PstI, SalI, BsaI, and
ClaI) resulted in a single major hybridizing DNA fragment,
whereas Sau3A1, which cuts the ORF, resulted in multiple fragments (Fig. 2a). The hybridization patterns obtained for
L. major, together with analysis of the genome sequence in
the vicinity of the gene, indicate that the LmajMST gene is
single-copy. Analysis of the genome sequence data
base3 showed that the MST of
L. major is flanked at its 5'-end by NADP dehydrogenase (EC
1.6.99.3) and at its 3'-end by dipeptidyl peptidase III (EC 3.4.14.4).
The Southern data for the LmexMST gene (Fig. 2b) similarly
show that the enzymes PstI, SalI, and ClaI did not cut the gene itself and resulted in a single
major hybridizing DNA fragment, whereas Sau3A1 and
BsaI, both of which cut the ORF, resulted in multiple
fragments. These data suggest that the gene of L. mexicana
is also single copy.
Biochemical Characterization of Recombinant MSTs--
The LmajMST
and LmexMST genes were cloned into pET21a+ for E. coli expression of soluble recombinant enzyme (LmajMST and
LmexMST) with a C-terminal six-histidine tag (~25 mg/liter E. coli). The purified recombinant MST was highly pure as judged by
SDS-PAGE analysis and stable for at least 12 months without loss of
activity at
Recombinant LmajMST and LmexMST had activity toward both
3-mercaptopyruvate and thiosulfate in the assays for both
rhodanese-like and sulfurtransferase-like activities (Table
I). The enzyme was optimally active in
the pH range 6.9-7.6 toward both substrates. However, rLmajMST was
considerably more active toward 3-mercaptopyruvate than thiosulfate,
with a lower Km and higher
kcat. In contrast to the reported behavior of
bovine liver rhodanese (9, 42), cysteine and homocysteine were not used
by rLmajMST. The role of thiols such as mercaptoethanol, homocysteine,
cysteine, and glutathione was analyzed using the sulfurtransferase
assay involving sulfide trapping with lead acetate. In this
assay, the enzyme showed activity in the absence of added
2-mercaptoethanol, but the addition of 5 mM
2-mercaptoethanol increased the rate by 20-fold to 345 µmol/min/mg of
protein. Homocysteine (10 mM) and cysteine (50 µM), but not glutathione, also each increased the
production of sulfide from mercaptopyruvate, but only by
~10-fold.
Expression and Localization of Leishmanial
MSTs--
Sulfurtransferase activity was found in cell extracts
prepared from each developmental stage in the leishmanial life cycle (Table II). Polyclonal antibodies raised
against rLmajMST recognized a single 40-kDa protein in soluble extracts
of L. mexicana amastigotes and promastigotes and L. major log phase, stationary phase, and metacyclic promastigotes
(Fig. 3a).
The localization of the LmajMST gene was investigated using
immunofluorescence microscopy. Labeling was detected throughout the
cytoplasm of the cell (data not shown), suggestive of a cytosolic location for LmajMST. The subcellular distribution of MST in
promastigote fractions derived by differential centrifugation was also
analyzed by Western blot analysis. This analysis similarly showed that the protein was recovered primarily in the cytosolic fraction (data not
shown), whereas antibody raised against the lysosomal CPB
cysteine protease of L. mexicana (36) detected protein in the small organelle fraction as expected. Enzyme activity analyses revealed similar results. These data together suggest that the leishmanial MST is a cytosolic enzyme. This is consistent with analysis
using PSORT,4 which indicated
that the leishmanial sulfurtransferases lack any characteristic
mitochondrial or other targeting sequence.
LmajMST Is Up-regulated in Response to Oxidative and Hypo-sulfur
Stress--
Exposure of L. major promastigotes to the
oxidants hydrogen peroxide, cumene hydroperoxide, and
tert-butyl hydroperoxide (at 300, 10, and 10 µM, respectively) led to the inhibition of growth by
~50% during 24 h of incubation. Differing effects upon the expression of MST were noted. The sulfurtransferase activity of parasites exposed to cumene hydroperoxide was increased (to 0.98 ± 0.01 µmol/min/mg of protein) compared with the control (Table II).
In contrast, there were no significant changes in sulfurtransferase activity after exposure to tert-butyl hydroperoxide or
hydrogen peroxide. Western analyses of the same cells extracts (Fig.
3b) also showed an increase above the wild-type levels of
protein in the parasites exposed to cumene hydroperoxide stress.
Promastigotes of L. major also responded to hypo-sulfur
stress over 24 h by increased expression of MST (Fig.
3b), with the resultant sulfurtransferase activity being
0.85 ± 0.01 µmol/min/mg of protein.
LmajMST Oxidizes Reduced Thioredoxin--
It has been reported
that MSTs can both oxidize thioredoxin, a key intermediate in cellular
redox reactions, and react with peroxides and that this may be a
physiologically significant mechanism for combating oxidative
challenges (4). Addition of rLmajMST and 3-mercaptopyruvate to a
mixture of thioredoxin reductase, thioredoxin, and NADPH led to the
rapid oxidation of NADPH (Fig. 4).
Addition of thiosulfate rather than mercaptopyruvate resulted in
activity, albeit at a lower level (37 µmol/min/mg of protein compared
with 386 µmol/min/mg of protein for 3-mercaptopyruvate). The apparent
affinity for the reduced thioredoxin was high (Km= 300 nM). Similar oxidation did not occur if any substrate
was omitted or if 3-mercaptopyruvate was replaced by 10 µM cumene hydroperoxide.
The C-terminal Domain Is Required for LmajMST Activity--
Four
LmajMSTs were expressed with truncations in the C-terminal domain.
Although the truncated proteins were expressed in E. coli in
similar amounts compared with full-length rLmajMST, the removal of
C-terminal peptides substantially changed the solubility of the
protein. rLmajMST Denatured LmajMST Refolds Unassisted--
Mammalian rhodanese has
been studied extensively as a model for protein folding (34, 38).
However, refolding of rhodanese is relatively difficult because of the
presence of multiple disulfide bonds and the ability of folding
intermediates to form aggregates. Thus, rhodanese does not refold well
unassisted, and either a chaperone or a detergent is required for
success (38). LmajMST denatured using 6 M urea and then
either dialyzed or simply diluted into refolding buffer regained its
full enzyme activity without the need for assistance in the form of a
chaperone or detergent (Fig. 7). In
contrast, the truncated proteins similarly treated did not even regain
the low level of activity that the enzymes possessed when they were
initially purified from E. coli.
We have characterized at the molecular and biochemical levels an
MST that is expressed throughout the life cycle of L. major and L. mexicana. The leishmanial single-copy genes predict
an unusual sulfurtransferase that, in comparison with other known sulfurtransferases, has an additional C-terminal domain of some 70 amino acids. The discovery that this domain is also encoded in a gene
of T. cruzi and T. brucei suggests that the
feature is conserved among trypanosomatid parasites and so has some key function. Importantly, it distinguishes the parasites' enzymes from
their mammalian counterparts. A possible role of the unusual C-terminal
domain was highlighted by the finding that the leishmanial MST can
refold successfully without a chaperone, which is in contrast to the
results reported for sulfurtransferases from other sources (21,
39-41). We hypothesized that the unusual C-terminal domain may play
some part in this. The finding that the proteins with truncated C
termini were misfolded and showed very little activity is consistent
with this postulate.
Analysis of the recombinant enzyme has clearly shown that the enzyme
prefers 3-mercaptopyruvate to thiosulfate as the donor substrate. This
substrate preference correlates well with the active-site residues
being homologous to other eukaryotic MSTs rather than rhodaneses. The
Km and Vmax values obtained compare favorably with those reported for bacterial, plant, and vertebrate MSTs (1, 42). The very high activity toward
3-mercaptopyruvate suggests that this could function as a natural
substrate, although currently, there is nothing known about the levels
of this compound in Leishmania parasites or any roles that
it may have.
The finding that the leishmanial sulfurtransferases can use thioredoxin
as an acceptor with a Km of 300 nM
suggests that this could also be one natural substrate. It has been
demonstrated previously that the thioredoxin from E. coli
can serve as a sulfur acceptor substrate for the E. coli
sulfurtransferase when thiosulfate is near its Km
(10), and it has also been shown that mammalian sulfurtransferases can
utilize thioredoxin (4). At one time, thioredoxin was considered to be
absent in Leishmania (see Ref. 25); however, a
thioredoxin-like gene has been identified in L. major
(GenBankTM/EBI accession number AAG10802) and T. brucei (43). One possible function of the interaction between a
sulfurtransferase and thioredoxin could be the involvement of the
enzyme in reduction and detoxification of peroxides (4). The finding
that the leishmanial enzyme cannot catalyze this reaction suggests that
it must have some other role. The oxidation status of thioredoxin is
thought to be crucial in regulating a number of cellular reactions, and so the oxidation by the leishmanial sulfurtransferase of reduced thioredoxin implicates this enzyme in similar processes.
The antioxidant machinery of trypanosomatids has been considered to
rely almost exclusively on trypanothione and its associated enzymes
(23). However, other enzyme systems may well also be involved. The
up-regulation of LmajMST upon exposure of L. major promastigotes to the oxidant cumene hydroperoxide shows that MST may be
an important additional enzymatic mechanism for protection against
oxidative stress.
The up-regulation of MST upon culturing L. major
promastigotes without an exogenous source of sulfur suggests that the
enzyme might also have a physiological role in sulfur amino acid
metabolism. The expression of RhdA, a rhodanese-like protein of
Synechococcus sp. strain PCC7942, is also induced by sulfur
starvation (44). Furthermore, sulfurtransferases have been implicated
in the metabolism of sulfur as shown by the formation and regulation of
iron-sulfur centers of proteins (9). Another possible role of the
sulfurtransferase in Leishmania is that it functions to
provide sulfide for the cysteine synthesis pathway. We have shown that
LmajMST works in conjunction with both recombinant L. major
cysteine synthase (GenBankTM accession number AL499624) and
recombinant L. major cystathionine Eukaryotic MSTs can be cytosolic or mitochondrial (22, 42, 46). Both
the immunolocalization and fractionation data suggest that the MST of
L. major occurs within the cytosol. The only other report on
a sulfurtransferase of a protozoon (33) suggest that an enzyme in
Euglena gracilis is also distributed in the cytosol.
The results obtained provide compelling evidence that
Leishmania contains a high level of activity of a
structurally unusual sulfurtransferase that has a strong activity
toward 3-mercaptopyruvate and thioredoxin and that plays some role in
antioxidant and sulfur amino acid metabolism. The finding that the
enzyme itself differs structurally from mammalian counterparts and is
expressed in the mammalian form of the parasite suggests the
possibility that it may have a crucial role in the parasite and so
represent a possibly useful drug target. Very little is known about
sulfurtransferases in protozoa, but the findings reported here suggest
that study of them will yield interesting new insights into the roles
and structures of this widespread class of enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Parasite
lysates were produced by resuspension of parasite pellets in lysis
buffer comprising 0.25 M sucrose, 0.25% (v/v) Triton
X-100, 10 mM EDTA, and a mixture of protease inhibitors (10 µM E-64, 2 mM 1,10-phenanthroline, 4 µM pepstatin A, and 1 mM phenylmethylsulfonyl
fluoride). Lysates were centrifuged at 13,000 × g for
5 min at 4 °C, and the resulting supernatant (designated the soluble
fraction) was retained for analysis. Protein concentrations were
determined according to the Bradford procedure (Bio-Rad) using bovine
serum albumin as the protein standard.
300-370, LmajMST
320-370, LmajMST
338-370, and LmajMST
360-370, encoding truncated proteins lacking 70, 50, 32, and 10 amino acids of the C terminus
(rLmajMST
300-370, rLmajMST
320-370, rLmajMST
338-370,
and rLmajMST
360-370, respectively), were amplified from cDNA by
PCR with the Expand High Fidelity system. For this, the 5'-perfect
match primer NT42 (5'-CGCTGACATATGTCTGCTCCTGCTGCGCCGAA-3') was used for all constructs, together with the appropriate 3'-primer encoding the regions directly preceding the native stop codon and amino
acids 300, 320, 338 and 360, respectively: NT55
(5'-TTCCTCGAGTGGAGGGGCGGAAGAGGCCGCTGT-3') for
LmajMST, NT56 (5'-TTCCTCGAGCGGCAGCGGCGTCACCGC-3') for
LmexMST, NTMST1 (5'-TTCCTCGAGGCTGCGCATTATGG-3') for
LmajMST
300-370, NTMST2 (5'-TTCCTCGAGCTTCGGGTTGTCGCCGA-3') for LmajMST
320-370,
NTMST3 (5'-TTCTCGAGCTCCGCATCGGGTCTCTCG-3') for
LmajMST
338-370, and NTMST4 (5'-TTCCTCGAGGACGCGGCCGCTCTTGA-3')
for LmajMST
360-370. An NdeI restriction site
(underlined) was added to the 5'-end of NT42, and an XhoI
restriction site (underlined) was added to NT55, NT56, NTMST1, NTMST2,
NTMST3, and NTMST4 to facilitate cloning and purification. The PCR
products were cloned into pGEM-T to generate plasmids designated as
pLmajMST, pLmexMST, pLmajMST
300-370, pLmajMST
320-370, pLmajMST
338-370, and pLmajMST
360-370, respectively, and sequenced.
-D-thiogalactopyranoside for 5 h at
15 °C. Cells were pelleted and resuspended in 5 ml of buffer A (50 mM sodium phosphate and 3 M NaCl, pH 8.0) with
5 mM imidazole and disintegrated by sonication, and the
soluble fraction was recovered by centrifugation at 13,000 × g for 30 min at 4 °C. This was applied to a 13-ml
nickel-nitrilotriacetic acid column (bioCAD® 700E work
station) pre-equilibrated with buffer A. The column was washed
with 60 ml of buffer A containing 5 mM imidazole and then
with 300 ml of buffer A containing 60 mM imidazole. The His-tagged recombinant proteins were eluted with 250 mM
imidazole in buffer A. The eluant was dialyzed with excess 20 mM Tris-HCl, pH 7.9, and 1 mM
Na2S2O3 at 4 °C overnight and
stored at
20 °C. Approximately 25 mg of soluble recombinant
proteins (LmajMST and LmexMST) were obtained from 1-liter cultures of
E. coli.
338-370, rLmajMST
360-370, and rLmajMST with
urea were based on protocols detailed by Bhattacharyya and Horowitz
(30). Proteins were incubated at 1.0 mg/ml in 50 mM
potassium phosphate, pH 7.8, containing 50 mM
Na2S2O3 and 6 M urea
for 120 min at 20 °C. Attempts to reactivate the proteins involved
one of two procedures: (a) 25 µl of denatured protein at
1.0 mg/ml were diluted in 75 µl of refolding buffer containing 0.2 M 2-mercaptoethanol, 50 mM
Na2S2O3, 10 mM KCl, and
10 mM MgCl2; or (b) an aliquot of
the denatured sample was dialyzed at 4 °C against refolding buffer.
Successful refolding and hence reactivation were monitored by assaying
10-µl samples using the standard sulfurtransferase assay.
-32P]dATP-labeled LmajMST and LmexMST
probes in Church-Gilbert hybridization solution. Filters were washed
under high stringency and exposed to x-ray film (Konica Medical Film).
, which
absorbs at 460 nm. The optimized reaction mixture (1 ml) contained 0.1 M Tris-HCl, pH 7.3, 10 mM KCN, and enzyme
extract. The reaction was initiated by the addition of thiosulfate or
3-mercaptopyruvate (5 mM), and incubation was at 37 °C
for 10 min. The reaction was stopped by the addition of 500 µl of
formaldehyde, and 1.5 ml of iron reagent
(Fe(NO3)3, 50 g/liter; and 65%
HNO3, 200 ml/liter) were added for FeSCN
complex formation. The assay was calibrated using NaSCN. The "sulfurtransferase" assay was based on the formation of hydrogen sulfide from the reaction between 2-mercaptoethanol and
3-mercaptopyruvate or thiosulfate. This was detected using lead acetate
and measuring the production of lead sulfide. The reaction mixture (1 ml) contained 0.1 M Tris-HCl, pH 7.3, 0.2 mM
lead acetate, 5 mM mercaptoethanol, and enzyme extract. The
reaction was started by the addition of 5 mM sodium
thiosulfate or 3-mercaptopyruvate. The reaction at 37 °C was
monitored continuously by detecting the formation of lead sulfide
spectrophotometrically at 360 nm. The molar extinction coefficient was
taken as 5205 cm
1 M
1 (32).
1 in either (a) the presence of
concentrations of hydrogen peroxide, cumene hydroperoxide, and
tert-butyl hydroperoxide that inhibited growth by ~50%
(300, 10, and 10 µM, respectively) or in (b)
RPMI 1640 medium lacking cysteine and methionine (Labtech)
supplemented with 10% (v/v) dialyzed heat-inactivated fetal bovine
serum. Crude lysates of the parasites were obtained as described above
for further analyses.
338-370, rLmajMST
360-370, and rLmajMST were performed
on a Jasco J-600 spectropolarimeter at 20 °C. All samples were in 20 mM sodium phosphate buffer, pH 7.4. Protein concentrations
for far-UV and near-UV CD measurements were 0.26 and 1.1 mg/ml,
respectively, and samples were scanned in 0.05-cm and 0.5-mm path
length cells, respectively. CD data were calculated in terms of mean
molar residue ellipticity (
) using mean residue weights of 109 for
each enzyme (34, 35). The availability of hydrophobic binding sites on
each of the proteins was also assessed using bis-ANS. Protein samples
(50 µg/ml) were mixed with bis-ANS (to 30 µM) in 20 mM phosphate buffer, pH 7.8, containing 50 mM
Na2S2O3, and fluorescence was
monitored (excitation at 395 nm and emission at 500 nm) at 25 °C
using an LS55 luminescence spectrometer (PerkinElmer Life Sciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of deduced amino acid sequences of
sulfurtransferases from Leishmania and representative
organisms. The sequences are as follows: LmajMST and LmexMST (this
study); T. brucei (Tb) MST
(GenBankTM/EBI accession number AC091553); T. cruzi (Tc) MST (accession number AI667879);
Arabidopsis thaliana (At) MST (accession number
BAA85148; gi:6009981); Escherichia coli (Ec) SseA
protein (accession number P31142); Homo sapiens
(Hs) MST (accession number P25325); and H. sapiens thiosulfate sulfurtransferase (TST) (accession
number NM003312.2; gi:17402865). These sequences were aligned using
AlignX (VectorNTI). Black shading indicates identical amino
acids; gray shading indicates conserved amino acids. The
active-site sequence is underlined. The unique N-terminal
and hinge domains of A. thaliana MST have been omitted; the
number of amino acids that have been removed are indicated in
parentheses. The T. cruzi sequence is incomplete
and comprises only the C-terminal segment of the protein.
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Fig. 2.
Genomic organization of leishmanial
MSTs. 5 µg of genomic DNAs from L. major
(a) and L. mexicana (b) were digested with a
variety of restriction endonucleases; separated by agarose gel
electrophoresis; transferred to a nylon membrane; and
hybridized with [32P]dATP-labeled LmajMST and
LmexMST cDNA probes corresponding to the entire ORFs, respectively.
The restriction endonucleases used were PstI (lane
1), BsaI (lane 2), SalI
(lane 3), and EcoRI (lane 4), and
Sau3A1 (lane 5) for L. major
(a) and PstI (lane 1),
Sau3A1 (lane 2), BsaI (lane
3), ClaI (lane 4), and SalI
(lane 5) for L. mexicana (b). The
sizes of DNA standards are indicated in kilobase pairs.
20 °C when stored in 20 mM Tris-HCl, pH
7.9, 1 mM Na2S2O3, and
4 mg/ml bovine serum albumin.
Activities of recombinant LmajMST and LmexMST
Sulfurtransferase activities in L. major and L. mexicana
View larger version (27K):
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Fig. 3.
Western blot analysis of the expression of
leishmanial MST during the life cycle and in cells subjected to
hypo-sulfur and oxidative stress. 80 µg of soluble cell extract
were subjected to Western blot analysis using rabbit anti-recombinant
MST antiserum. a, expression during the life cycle.
Lane 1, L. mexicana amastigotes; lane 2,
L. mexicana stationary phase promastigotes; lane
3, L. major mid-log phase promastigotes; lane
4, L. major stationary phase promastigotes; lane
5, L. major metacyclic promastigotes. b,
expression in L. major promastigotes subjected to
hypo-sulfur and oxidative stress. Lane 1, control;
lane 2, hypo-sulfur stress; lane 3, 10 µM cumene hydroperoxide; lane 4, 300 µM hydrogen peroxide; lane 5, 10 µM
tert-butyl hydroperoxide.
View larger version (11K):
[in a new window]
Fig. 4.
rLmajMST reacts with thioredoxin.
rLmajMST (enzyme (E)) and 3-mercaptopyruvate (substrate
(S)) were added at the times indicated by the
arrows to the reduced thioredoxin system, which was at
equilibrium, and the subsequent NADPH oxidation was followed
spectrophotometrically at 340 nm. Abs, absorbance.
300-370, the protein lacking the entire C-terminal domain, and rLmajMST
320-370 were expressed entirely in
inclusion bodies irrespective of the induction and growth conditions used. Solubilization of the protein using 8 M urea and
subsequent attempts to refold the enzyme failed to result in active
enzyme. Shorter truncations of 24 or 10 amino acids yielded some
soluble recombinant protein (~50 and 70%, respectively). However,
these recombinant MSTs had greatly diminished enzyme activity (0.3 and 3 µmol/min/mg of protein, respectively) compared with that of the
full-length protein (343 µmol/min/mg of protein). CD analyses of the
full-length and two soluble truncated proteins showed that all three
had similar secondary structure contents (18%
-helix, 26%
-sheet, 23% turn, 33% other) when analyzed by the SELCON method
(37). However, there was a marked difference in the near-UV spectra,
especially in the region between 270 and 290 nm, between the wild-type
protein and the truncated derivatives (Fig.
5). These results suggested that the
consequence of the truncations was a less well folded protein with
significant alterations to the environments of the tyrosine side
chains. Bis-ANS fluorescence studies (Fig.
6) further supported the conclusions from
the CD analyses in that the truncated proteins exhibited greater
fluorescence, which is consistent with hydrophobic residues normally
buried in the native enzyme being exposed in the truncated
proteins.
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Fig. 5.
CD spectra of rLmajMST proteins.
The near-UV CD spectrum of full-length rLmajMST (trace
1) differed considerably from those of rLmajMST 360-370
(trace 2) and rLmajMST
338-370 (trace 3).
Mol. Ellip., molecular ellipticity in
degrees/cm2/dmol.
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Fig. 6.
Bis-ANS fluorescence of rLmajMST
proteins. Full-length rLmajMST ( ) bound less bis-ANS than did
rLmajMST
360-370 (
) and rLmajMST
338-370 (
). The
fluorescence intensity (FI) is given in arbitrary
units.
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Fig. 7.
LmajMST refolds in the absence of a
chaperone. rLmajMST refolded to give active enzyme most
efficiently when simply diluted into refolding buffer ( ), with 97%
of the original activity being recovered by 2 h. Dialysis (
)
also yielded active enzyme, but more slowly.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-synthase (accession
number AL499619) to synthesize cysteine in vitro. T. cruzi cystathionine
-synthase was also recently reported to
possess cysteine synthase activity (45).
![]() |
ACKNOWLEDGEMENT |
---|
We thank N. C. Price for helpful advice with the CD analyses.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ313201 and AJ313202.
§ Supported by a Commonwealth scholarship.
** Medical Research Council (United Kingdom) Senior Research Fellow.
To whom correspondence should be addressed: Div. of Infection
and Immunity, University of Glasgow, Joseph Black Bldg., Glasgow G12
8QQ, Scotland, UK. Tel.: 44-141-330-4777; Fax: 44-141-330-3516; E-mail:
g.coombs@bio.gla.ac.uk.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M209395200
2 G. H. Coombs, G. D. Westrop, P. Suchan, G. Puzova, T. Hirt, T. M. Embley, J. C. Mottram, and S. Muller, submitted for publication.
3 Available at www.sanger.ac.uk.
4 Available at psort.nibb.ac.jp.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MST, 3-mercaptopyruvate sulfurtransferase; LmajMST, L. major MST; rLmajMST, recombinant LmajMST; LmexMST, L. mexicana MST; RACE, rapid amplification of cDNA ends; ORF, open reading frame; bis-ANS, 1,1'-bis(4-anilino)naphthalene-5,5'disulfonic acid; contig, group of overlapping clones.
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REFERENCES |
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