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INTRODUCTION |
Methionine adenosyltransferase
(MAT1;
S-adenosyl-L-methionine synthetase, EC 2.5.1.6)
catalyzes the synthesis of S-adenosylmethionine (AdoMet)
from L-methionine and ATP (1, 2) in all living organisms.
AdoMet is a critical metabolite in transmethylation and transulfuration
reactions and a precursor in polyamine metabolism. AdoMet is the major
donor of methyl groups for transmethylation reactions in eukaryotic
systems (3, 4). S-Adenosylhomocysteine (AdoHcy), generated
by the transfer of a methyl group from AdoMet, is converted to
adenosine and homocysteine by AdoHcy hydrolase (5). Because cells try
to maintain low AdoHcy levels, the AdoMet/AdoHcy ratio is indicative of
the relative potential for transmethylation reactions, once the
S-methyl moiety has been transferred and the AdoHcy is
cleaved by AdoHcy hydrolase (6). Homocysteine can be readily
transformed into cysteine, thereby participating in the metabolism of
glutathione, the scavenger tripeptide involved in the sulfur redox
status of eukaryotes (7). Finally, 2-5% of total intracellular AdoMet
is used as a precursor for higher polyamines such as spermidine and
spermine in all living cells (8). After AdoMet is decarboxylated, the
released aminopropyl moiety can be captured by putrescine and
spermidine to synthesize spermidine and spermine, respectively (9).
MAT enzymes are involved in two enzymatic activities. In the first
reaction AdoMet and tripolyphosphate (PPPi) are
synthesized from ATP and L-methionine. In the second, the
tripolyphosphate is hydrolyzed to orthophosphate (Pi) and
pyrophosphate (PPi) before the products are released. MAT-I
and MAT-III, products of the same MAT1A gene, are
predominantly expressed in adult liver (10). MAT-II, a product of the
MAT2A gene, is present in most tissues of the fetus and
newborn, and in the kidney, brain, lymphocytes, testis, lens, as well
as the early regenerating liver of adults (11, 12). The tetramer MAT-I
(Mr 200,000) and dimer MAT-III (Mr 100,000) are oligomeric forms of the 
1
subunit (13), whereas MAT-II (Mr 185,000) is a
heterooligomeric complex comprised of 2/2' (11) and 
subunits
(14). The 2' subunit of the MAT-II isozyme is considered to be a
post-translational modification of the 2 subunit that occurs as the
organism matures.
There are few reports on AdoMet metabolism in Trypanosomatids. Yarlett
et al. (15) described two different MAT isoforms in
Trypanosoma brucei, which were evident from Eadie-Hofstee
and Hanes-Woolf plots, that had different affinities for
L-methionine and ATP. The depletion of polyamine metabolism
with the irreversible enzyme-activated inhibitor of ornithine
decarboxylase, -DL-difluoromethylornithine (DFMO),
caused massive accumulation of AdoMet and decarboxylated AdoMet
(dcAdoMet) in trypanosomes. The reason for this buildup is due to the
unregulated nature of the trypanosome MAT (16). In mammalian tissues
this enzyme is closely regulated by its product, whereas the MAT
activity in trypanosomes is not. After DFMO treatment and increases in
AdoMet concentration, the methylation index (the ratio of AdoMet to
AdoHcy) in trypanosomes raises to >100, whereas in mammalian cells
this ratio is on the order of 5-10 (17). This striking difference
between host and parasite may be the grounds for establishing MAT as a
drug target in polyamine-based chemotherapy (3).
In this report we describe the cloning and organization of the
MAT2 gene from Leishmania infantum, as well as
the heterologous expression, protein purification, and kinetic analysis
of the recombinant enzyme.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
L. infantum LEM 75 zymodeme
1 was a generous gift from Dr. Requena (Centro de Biología
Molecular Severo Ochoa, Consejo Superior de Investigaciones
Científicas Madrid). The cells were grown as promastigotes
(flagellated, insect form) in Schneider's insect medium (Sigma
Chemical Co., St. Louis, MO), supplemented with 10% heat-inactivated
fetal calf serum (Roche Molecular Biochemicals, Germany), penicillin
(50 units/ml), and streptomycin (50 µg/ml).
Nucleic Acid Isolation--
Genomic DNA from L. infantum promastigotes was isolated from 2.0 × 109 cells in 10 mM EDTA, 150 mM
NaCl, 0.4% SDS, and 50 µg/ml proteinase K and incubated at 65 °C
for 1 h followed by 37 °C overnight incubation. DNA was further
purified by phenol and phenol/chloroform extraction and ethanol
precipitation. Qiagen columns were used for RNA isolation following the
manufacturer's instructions.
Isolation of an L. infantum MAT2 Fragment by PCR--
To
generate a DNA probe for isolation of the L. infantum MAT2
gene, PCR was employed using degenerated oligonucleotides based on
conserved amino acid sequences in mouse (18), rat liver (19), human
liver (20), Saccharomyces cerevisiae (21, 22), and Escherichia coli (23) MAT proteins. A sense primer, 5'-GAG
GGC CA(C/T) CC(C/G) GA(C/T) AAG-3', was designed encoding EGHPDK
amino acid residues. The antisense primer 5'-(C/G)GG GCC GCC (G/A)AT (G/C)AC GAA-3' was designed for amino acid residues PGGIVF. 500 ng of
L. infantum genomic DNA was added to a reaction mixture containing 50 mM KCl, 10 mM Tris-HCl, pH 9.0, 1% Triton X-100, 2.5 mM MgCl, 200 µM of each
deoxynucleotide triphosphate, 50 pmol of each oligonucleotide primer,
and 2.5 units of Taq DNA polymerase in a final volume of 50 µl. A 636-bp product was generated after 30 cycles of denaturation at
94 °C for 1 min, annealing at 50 °C for 2 min, and extension at
72 °C for 1 min. PCR products were isolated from 2% low melting
point agarose gels, and the purified DNA fragments were ligated to
pGEM-T vector (Promega) and transformed into a competent E. coli, DH5 strain. Plasmids were isolated on Wizard columns
(Promega), according to the manufacturer's recommendations. The
amplified product was sequenced on both strands using the dideoxy chain
termination method (24) with fluorescent primers and T7 polymerase.
Sequencing was performed in an ALF automated sequencer (Amersham
Biosciences, Inc.) placed at the core facility of Universidad de
León.
Isolation of the L. infantum MAT2 Gene--
The EMBL L. infantum library (25) was kindly supplied by Dr. Requena (Centro
de Biología Molecular Severo Ochoa, Consejo Superior de
Investigaciones Científicas, Madrid). A total of 50,000 bacteriophages was blotted onto positively charged nylon membranes
(Amersham Biosciences, Inc.) and prehybridized for 4 h at 42 °C
in 5× Denhardt's solution, 5× SSC, 50 mM sodium
phosphate, pH 6.5, 100 µg/ml salmon sperm DNA, and 50% formamide for
screening with DNA probes. The membranes were hybridized for 16-20 h
at 42 °C in the same buffer containing
106-107 cpm of the 636-PCR product radiolabeled
with [-32P]dCTP (Amersham Biosciences, Inc.), using
the random primer method. Filters were washed twice with 2× SSC plus
0.1% SDS at 42 °C for 10 min each, followed by two washes in 1×
SSC, plus 0.1% SDS at 42 °C for 10 min. Positive bacteriophages
were subjected to a second and third purification, and DNA was isolated
from amplified phages using the liquid culture method as described by
Sambrook et al. (26).
Subcloning and Sequencing of the L. infantum MAT2 Gene--
The
isolated bacteriophage DNA was digested with restriction endonucleases,
electrophoresed on 0.7% agarose gels, and transferred by Southern's
method (27) to positively charged nylon membranes. Southern blots were
probed with the 636-bp PCR product under the conditions described above
for screening the genomic library. The 3.2- and 1.8-kb
PstI-fragments that hybridized to the probe were ligated
into pGEM-3Zf(+) and transformed into E. coli, DH5 strain. Large scale preparation of the pGEM-3Zf(+) plasmid containing the 3.2- and 1.8-kb PstI fragments was done using Qiagen
columns, and the inserts were sequenced as mentioned above. Analyses of nucleotide and amino acid sequences were performed using the BLAST algorithm from the National Center for Biotechnology Information database.
Southern and Northern Hybridizations--
Genomic DNA from
L. infantum was digested with the appropriate restriction
enzymes under the conditions recommended by the suppliers. Digested DNA
was electrophoresed on 0.7% agarose gels and transferred to positively
charged nylon membranes as described above. For identification of
specific MAT2 transcripts, total RNA was electrophoresed on
1.2% agarose (formaldehyde gels) and transferred to positively charged
nylon membranes.
Mapping the 5'-End Using RT-PCR--
To amplify the 5'-end of
MAT2, total RNA was used as a template for RT-PCR. Reverse
transcription steps were performed with avian myeloblastosis
virus-reverse transcriptase (Promega) following the manufacturer's
protocol (10 µg of total RNA/reaction). PCR was performed using
Taq polymerase (Amersham Biosciences, Inc.) with 30 cycles
of denaturation at 95 °C for 30 s, annealing at 55 °C for 2 min, and extension at 72 °C for 2 min. The primers used for RT were:
5'-TCG GGA TCC CAA CGC TAT ATA AGT ATC AGT TTC TGT ACT TTA TTG-3', a
sense primer containing the sequence that codifies the spliced leader
region, and 5'-CTT GTC TGG ATG GCC CTC CGT-3', an antisense primer
located at the end of the MAT2 gene.
Cloning and Expression of the MAT2 Gene--
The genomic DNA
prepared from L. infantum cultures was amplified by PCR
using the proofreading DNA polymerase Pfu (Stratagene) with
the following specific primers, 5' sense primer,
5'-CGGGATCCATGTCTGTCCACAGCATTCTCTTC-3', and the 3' antisense primer,
5'-AACTGCAGTTACTCGACCATCTTCTTTGGCAC-3'. A BamHI restriction
site was created upstream of the initiation ATG codon in the sense
primer, and a PstI restriction site was introduced into the
antisense primer downstream of the termination codon. Subcloning of
this product into the pQE-30 (Qiagen) expression vector yields
pQE30-MAT2. Competent E. coli, JM109 cells were transformed with pQE30-MAT2. Transformed bacterial clones
were grown at 37 °C in LB medium containing 100 µg
ml
1 ampicillin to an absorbance at 600 nm
(A600) of 0.5 unit. Protein expression was
induced by adding isopropyl--D-thiogalactopyranoside to
a final concentration of 0.5 mM. The cells were grown for
an additional 3 h at 37 °C, and then separated by
centrifugation (8000 × g for 15 min) at 4 °C. The
pellets were resuspended in ice-cold phosphate-buffered saline (PBS)
solution, washed three times in PBS, and stored at 
80 °C until use.
Purification of Recombinant MAT-II--
The protein was
partially purified using the method described by Lopez-Vara et
al. (28). The bacterial pellet was resuspended in water at 0 °C
for 10 min to facilitate cell wall lysis and centrifuged at 8000 × g for 10 min. The pellet was resuspended in 10 ml of
solution A: 50 mM Tris-HCl, pH 8.0, 10 mM DTT,
10 mM MgSO4, and protease inhibitors (2 µg/ml
aprotinin, 1 µg/ml pepstatin A, 0.5 µg/ml leupeptin, 2.5 µg/ml
antipain, 0.1 mM benzamidine, and 0.1 M
phenylmethylsulfonyl fluoride). Cell extracts were prepared by
sonication with ten 30-s pulses at 50 watts and 30-s intervals with a
U50-control sonicator (Kika Labortechnick). Soluble and insoluble
fractions (including inclusion bodies) were separated by centrifugation
for 15 min at 10,000 × g. Pellets from the insoluble fraction were resuspended in 10 ml of buffer A. Nucleic acids were
digested by incubating the cell debris with 40 µg ml1
DNase I and 130 units ml1 T1 RNase at room temperature
for 20 min. The inclusion bodies were diluted in solution A (w/v 1:4),
centrifuged at 10,000 × g for 10 min, and washed three
times by resuspension in 10 ml of solution A supplemented with 5%
Triton X-100 and 4 M urea and collected by centrifugation
at 4 °C (10,000 × g for 10 min). A final wash with
the same buffer without urea and Triton X-100 was performed. The washed
pellet was solubilized in 10 ml of solution B (50 mM
Tris-HCl, pH 8.0, 10 mM MgSO4), supplemented
with 8 M urea, and subjected to mild agitation for 2 h
at 10 °C. Protein refolding was achieved by a 20-fold dilution
(protein concentration: 0.3-18 mg/ml) in solution B containing 2 M urea. The suspension was left overnight at 4 °C under
gentle agitation and centrifuged at 13 000 × g for 30 min, after which the pellet was discarded. The resulting supernatant
was dialyzed three times for 4 h each against buffer C (50 mM Tris-HCl, pH 8.0, 10 mM MgSO4,
and 10 mM DTT) at 4 °C to remove the urea. Dialyzed
samples were clarified by centrifugation at 10,000 × g
for 30 min before any determination was carried out.
Purification by Nickel-Agarose Bead Affinity Capture--
The
recombinant MAT-II protein was purified from inclusion bodies by
affinity on nickel-agarose beads (Qiagen). In a 15-ml tube, 2 ml of the
50% nickel-agarose slurry was spun down at 1000 × g
for 2 min, and then the pellet was equilibrated with a buffer containing 300 mM NaCl, 50 mM sodium phosphate,
pH 8.0, and 8 M urea. Inclusion bodies resuspended in
solution B, supplemented with 8 M urea (containing about 10 mg of total proteins) were added and incubated for 30 min at 4 °C
with gentle mixing. The equilibrated gel was poured into the column and
allowed to settle. The column was washed several times with 5 ml of
equilibrated buffer until the absorbance at 280 nm of the flow-through
material was undetectable. Bound proteins were eluted with 10 ml of 300 mM imidazole and dialyzed against 50 mM
Tris-HCl, 10 mM MgSO4, and 10 mM
DTT as described above.
Generation of MAT-II Antisera--
Polyclonal antisera against
L. infantum MAT-II was generated by inoculating the purified
protein in New Zealand White male rabbits (Criffa, Barcelona, Spain).
Preimmune blood was drawn prior to the initial immunizations, and the
sera were used as a negative control. The recombinant protein was
combined and emulsified in an equal volume of Freund's complete
adjuvant (Sigma Chemical Co.) and injected into the rabbits. The
initial injections were made in three sites: each hindquarter and the
back of the neck. Boosters were made every 2 weeks following the
removal of 10 ml of blood from the ear vein to test the immune
response. The final rabbit serum was collected after the titers had
reached a plateau (12-14 weeks). Antisera titers were assessed by
immunoblotting after preparation of L. infantum crude
extracts on a reducing 15% SDS slab gel and transfer to a nylon membrane.
SDS-PAGE and Western Blotting--
L. infantum
promastigotes were harvested at different times during the growth and
washed twice with PBS. After sonication and centrifugation at
10,000 × g for 20 min, the supernatant was removed. 50 µg of protein from each time point were diluted in loading buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, and
5% glycerol), heated in a boiling water bath for 5 min, and analyzed
by SDS-PAGE (10% acrylamide, 2.7% bisacrylamide). Proteins were
electrotransferred onto nylon membranes for 1 h at 25-30 V/cm,
and the blots were blocked by incubation in 10 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.5% Tween 20, 5% nonfat milk powder
(w/v) for 1 h at room temperature. Primary antibody was added to
this buffer, and the blot was incubated for 2 h. The blot was
washed thoroughly in 10 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.5% Tween 20 and then incubated with an
anti-rabbit antibody conjugated to alkaline phosphatase. Antibodies
were detected using nitro blue tetrazolium chloride as substrate (Roche
Molecular Biochemicals).
MAT Assay--
MAT activity was assayed as described previously
(29). The assay included, in a total volume of 250 µl, 5 mM L-methionine, 1 mM ATP
(containing [2,8-3H]adenosine 5'-triphosphate, 46 Ci/mmol, Amersham Biosciences, Inc.) in 100 mM Tris-HCl, pH
8.0, 240 mM KCl, 12 mM MgCl2, and 10 mM DTT. The reaction was stopped with 4 ml of ice-cold
water. Reaction mixtures were loaded onto AG 50W-X4 cationic exchanger columns, washed twice with 10 ml water, and eluted with 4 ml of 3 M NH4OH. Samples were measured in a
scintillation counter using 10 ml of an Optiphase-Hisafe 3 (Wallac)
mixture for aqueous mixtures. One unit of MAT activity is defined as
the amount of enzyme that catalyzes the formation of 1 nmol of AdoMet
per hour and per mg of protein. Protein was determined using the
Bradford method (30). Each data point was measured by triplicate and
presented as the mean. The kinetic parameters for
L-methionine and ATP were calculated under steady-state
conditions. Kinetic constants for L-amino acid were
determined in the presence of 0.05 to 5 mM ATP and 0.025 to
2.5 mM L-methionine, whereas nucleoside
constants were ascertained in the presence of 0.05 to 1 mM
L-methionine and 0.05-5 mM ATP.
Native Molecular Weight Determination--
Gel-filtration
chromatography was used to determine both native and recombinant
L. infantum MAT-II molecular weight. 2 ml of freshly
prepared recombinant MAT-II was loaded onto a Sephacryl S-300 column
(75 × 1.5 cm) (Amersham Biosciences, Inc.), equilibrated with 50 mM Tris-HCl, pH 8.0, 10 mM MgSO4,
and 10 mM DTT. Chromatography was performed at 4 °C and
a flow rate of 10 ml/h with the same elution buffer, containing
protease inhibitors, and 2.5-ml fractions were collected. The column
was previously calibrated with S-300 molecular weight markers;
chymotrypsinogen A (23,000), ovoalbumin (43,000), bovine serum
albumin (67,000), and aldolase (158,000) (Sigma), whose
A280 profile for standards were
spectrophotometrically determined. The eluted fractions were
used to assay either for MAT activity, with the method described
above, or for immunoblotted detection against the MAT-II antibody.
One liter of promastigotes were grown as described previously for gel
filtration analysis of native MAT-II. The cells were harvested after 3 days and washed twice by centrifugation in PBS. Pellet was resuspended
in 2 ml of 50 mM Tris-HCl, pH 8.0, 10 mM MgSO4, and 10 mM DTT in the presence of
protease inhibitors, and disrupted by sonication. The cytosolic
fraction, obtained after centrifugation for 60 min at 100,000 × g at 4 °C, was loaded onto the same column and
eluted under similar conditions.
L. infantum Promastigotes Transient Transfections--
Cells
were grown in Schneider's insect medium until the log late phase was
reached. 4 × 107 cells were electroporated with 10 µg of DNA, 20 µg of sheared salmon sperm DNA as carrier, and 2 µg
of pX63NEO-gal plasmid as control for transfection
efficiency. Transfections were performed in a Gene Pulser II unit
(Bio-Rad) using 2250 V/cm and a capacitance of 500 microfarads.
Following electroporation, cells were inoculated into 10 ml of
Schneider's insect medium and grown for 20 h at 26.5 °C
(31).
Plasmid Constructions--
Three fragments of different lengths
belonging to the upstream region of the MAT2 gene were
generated by PCR, using an MAT2 genomic clone as template.
The resulting fragments were 415-, 355-, and 136-bp long and contained
new BamHI and NotI restrictions sites at the
ends. The sense primers for the fragments extended from 415 to 398,
from 355 to 336, and from 136 to 116, respectively. The
antisense primer was the same for all fragments and extended from
position 21 to 1. After cleavage with BamHI and
NotI, these fragments were cloned into the BamHI
and NotI sites just upstream of the luciferase coding region
in the promoterless pGEM-luc vector (Promega). The identity
of the constructions was confirmed by sequencing. At the same time,
another fragment, containing the 3'-UTR and IR between both
MAT2 genes, was amplified by PCR with specific primers
containing SacI restriction sites. After cleavage, it was
cloned into SacI site, located downstream of the luciferase coding region in the promoterless pGEM-luc vector. Insert
orientation was confirmed by sequencing.
Luciferase and Galactosidase Assays--
Cells were collected by
centrifugation, resuspended in 1.4 ml of Hanks' balanced salt
solution, and pelleted. The pellets were suspended in 160 µl of 50 mM Tris-HCl, pH 7.5, containing protease inhibitors (TPI
buffer). The cells were lysed by sonication and centrifuged at 4 °C
for 15 min. 80 µl of the supernatant was assayed for luciferase
activity using the Luciferase Assay System (Promega). Luminescence was
measured in a Labsystems Luminoskan luminometer. The rest of the
supernatant (80 µl) was assayed for -galactosidase activity to
adjust the transfection efficiency by adding 320 µl of assay buffer
(23 mM Tris-HCl, pH 7.5, 125 mM NaCl, 2 mM MgCl2, 12 mM
-mercaptoethanol) and 0.3 mM 4-methyl-umbelliferyl -D-galactoside (Sigma). After incubation for 5 h at
37 °C, the reaction was stopped by adding 2 ml of 133 mM
glycine/83 mM Na2CO3, pH 10.7 (32).
4-Methylumbelliferone fluorescence was measured in a spectrofluorometer
(Hitachi F2500). Luciferase activity values were adjusted to the
-galactosidase activity results to verify transfection efficiency,
taking the pX63NEO-luc-transfected cells values as 100%.
The results are shown as the mean ± S.D. from five independent measurements.
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RESULTS |
Molecular Cloning and Sequencing of the L. infantum MAT2
Gene--
The cloning strategy employed was based on the design of
degenerate primers representing conserved internal protein sequences from phylogenetically unrelated organisms (20-25) and PCR
amplification using purified L. infantum DNA as template. A
636-bp PCR product that encodes a portion of internal
Leishmania MAT2 gene was amplified from genomic
DNA and used as a probe to isolate the entire genomic locus. The
suitability of this 636-bp PCR fragment was initially evaluated by
sequencing and comparison to the NCBI sequence database (Basic Blast)
(33). 50,000 bacteriophages from a Lambda-EMBL3 Leishmania
library were screened with the random primer-labeled 636-bp PCR probe
as described under "Experimental Procedures." One positive plaque
was identified and isolated. This clone contained ~20 kb of
Leishmania DNA. More than one band was identified with this
probe in Southern blot analysis of bacteriophage DNA digested with
restriction endonucleases (Fig.
1A).
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Fig. 1.
Southern blot analysis of MAT2
locus in L. infantum. A, bacteriophage DNA
(2 µg) containing L. infantum MAT2 was digested with the
restriction endonucleases indicated (ApaI, EcoRV,
HindIII, PstI, SacII, SalI,
SmaI, and XhoI) or combined with EcoRI
to excise the bacteriophage arms and subjected to Southern blot
analysis using the 636-bp PCR probe. The three faint bands
(approximately 2.0 kb) located between the
PstI-SacII and SalI-SmaI
lanes correspond to a band hybridizing with the molecular
weight markers. The 3.2- and 1.8-kb fragments obtained by
PstI digestion were used for cloning and are indicated with
an asterisk. B, Southern blot analysis of the
wild type MAT2 loci. Genomic DNA (20 µg) was isolated from
L. infantum promastigotes, digested with ApaI,
EcoRV, KpnI, NcoI, SalI,
XhoI, BamHI, EcoRI, and
HindIII, resolved on a 0.7% agarose gel, and blotted onto
nylon membranes. Blots were hybridized to a 636-bp PCR fragment under
high stringency conditions. The KpnI lane did not
show any hybridization band due to sample degradation.
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Two fragments, 3.2 and 1.8 kb from the isolated bacteriophage obtained
by PstI digestion (marked in Fig. 1A with an
asterisk), were chosen for subcloning into pGEM-3Zf(+) and
completely sequenced on both strands. Sequence analysis identified the
presence of two identical 1179-nucleotide ORFs, which could each be
expected to encode a protein consisting of 394 amino acids with an
estimated molecular mass of 43 kDa. We have termed these ORFs
MAT2 (see GenBankTM accession number AF031902).
In addition to the MAT2 coding region, the 3.2-kb fragment
contained 421 bp upstream of the initial MAT2 codon and ~2
kb downstream of the stop codon. In these genes, which have no introns
(confirmed by 
-gt11 cDNA L. infantum library screening), G + C accounted for 62% of the overall base composition. Multiple sequence alignment (Fig. 2) of
the L. infantum MAT-II protein with other proteins from
phylogenetically diverse organisms, revealed that MAT-II contained all
the sequence motifs related to ATP binding (the nonapeptide
GGGAFSGKD (34)), metal binding (Asp-19, Asp-282, and Glu-45
(35)), and the MAT active site (GAGDQG at position 118-123)
found in mammalian, yeast, Plasmodium falciparum, and
E. coli MAT isozymes.
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Fig. 2.
Multiple amino acid sequence alignments of
L. infantum MAT-II. The predicted amino acid
sequences predicted for the L. infantum, P. falciparum, E. coli, S. cerevisiae, human
liver, and human kidney MAT were aligned using the ClustalX multiple
sequence alignment program. Symbols: "*" represents
identical or conserved residues in all sequences in the alignment,
":" conserved substitutions and "." semi-conserved
substitutions. Complete genomic sequences of MAT proteins are available
in GenBankTM, for L. infantum accession number
(AF031902), P. falciparum (AF097923), E. coli
(K02129), S. cerevisiae (M23368), human liver (X69078), and
human kidney (X68836).
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Genomic Organization of the MAT2 Locus--
To determine the
number and organization of MAT2 genes in L. infantum genome, a Southern blot analysis was performed. Several enzymes whose cleavage sites are not present in the MAT2
coding region were used to digest DNA, and blots were probed with the 636-bp PCR-labeled product. Complete digestion resulted in two copies
in the L. infantum genome, because ApaI,
EcoRV, SalI, and XhoI (which do not
cut inside the MAT2 coding region), each excised two
hybridizing bands of unequal size (Fig. 1B).
These two copies may be organized either head to head or head to tail
as Fig. 3A shows. We designed
oligonucleotides based on the sequences inferred from 3.2- and 1.8-kb
fragments (see Fig. 1A) to obtain a more detailed picture on
the genomic organization of this gene and determine the intergenic
region (IR). The oligonucleotides were designed to be able to amplify
the region between these genes via PCR. Primer 1, 5'-CCAAAGAAGATGGTCGAC-3', is a sense primer whose sequence corresponded
to the 3'-end coding region; primer 2, 5'-AATGCTGTGGACAGACAT-3', is an
antisense primer with a sequence corresponding to the 5'-end of the
coding region; and primer 3, 5'-GACTGACCGGCTTTCTAG-3', is also an
antisense primer corresponding to the 3'-end of the coding region.
After PCR amplification under the conditions described under
"Experimental Procedures," in which genomic L. infantum
DNA was used as a template, a single 4-kb band was originated with
primers 1 and 2, whereas with primers 1 and 3 no fragments were
amplified (Fig. 3B, lanes 2 and 3). The 4-kb band was subcloned into pGEM-3Zf(+) and sequenced on both
strands, in which the sequence of the last 421 bp was identical to the
sequence found upstream of the MAT2 gene in the 3.2-kb fragment (Fig. 3D). To ascertain whether this homology
extends beyond the 421-bp fragment, bacteriophage DNA isolated from the L. infantum EMBL3 genomic library was digested with
KpnI, an enzyme that cleaves the region between both copies
of MAT2 gene at a single site, generating two fragments,
with 5.5 and 4.5 kb, respectively, both hybridizing with the 636-bp PCR
probe (Fig. 3C). The two fragments were separated in low
melting point agarose gels and subcloned into pGEM-3Zf(+) for
sequencing. Sequence analysis showed that they contained the
MAT2 gene, included in the previously isolated 3.2-kb
fragment, as well as a second 2.0-kb fragment located upstream of the
ATG codon. This 2.0-kb piece was identical to the sequence found at the
end of the 4-kb IR and contained an ORF located upstream of both
MAT2 genes. These ORFs showed a 37% amino acid homology
with an unknown protein named L2259.4 (see GenBankTM
accession number AC009602), ascertained by PSORT algorithm analysis to
be located in the nucleus.
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Fig. 3.
Organization of MAT2 gene
copies. A, schematic representation showing possible
organization of MAT2 gene copies (gray boxes) in
the L. infantum genome; the small arrows indicate
the position of the primers used in the amplification of the intergenic
region. B, agarose gel showing the PCR product obtained
under the conditions described under "Experimental Procedures" and
the negative control. C, bacteriophage DNA digested with
KpnI and Southern blot analysis with the 636-bp PCR
fragment. D, schematic representation showing the L. infantum MAT2 locus. Gray boxes symbolize
MAT2 gene copies separated by a 4-kb IR, white
boxes represent an ORF found upstream of both MAT2 gene
copies, dotted boxes indicate the 2.0-kb conserved fragment
located upstream of each MAT2 genes, and stripped
boxes contain the spliced leader junction site. A thick
black line indicates the location of the probe. Capital
letters denote several restriction sites: EcoRV
(E), KpnI (K), PstI
(P), SmaI (S), and XhoI
(X).
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Attempts were also made to identify the location of the spliced leader
junction site. The 5'-end of the mature mRNAs was mapped with
reverse transcriptase, and PCR was carried out as described under
"Experimental Procedures." Electrophoresis of the PCR products on
agarose gels yielded only one 120-bp product. Nucleotide sequence analysis demonstrated that the MAT2 transcript spliced
leader junction site was located at nucleotide 42 (boldface AG
dinucleotide in Fig. 4) from the
predicted translation initiation site.
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Fig. 4.
RT-PCR mapping of the 5'-UTR of the
MAT2 transcript. A, ethidium bromide
stained agarose gel of the RT-PCR product. Total RNA was amplified
using nondegenerate primers as described under "Experimental
Procedures." The spliced leader sequence was used as the sense
primer, and as antisense primer, an internal gene sequence was used.
Lane 1 shows the amplified product, whereas lane
2 exhibits the negative control, in which no antisense primer was
used. B, 421 nucleotides located upstream of the ATG codon,
containing the polypyrimidine tracts (underlined) and the
spliced leader junction site (boldface AG dinucleotide)
predicted by mapping of the 5' terminus of the MAT2
transcript.
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Expression of the MAT2 Genes--
Total RNA was isolated from
promastigotes at various times during culture growth and probed in
Northern blots with MAT2 coding region (Fig.
5A). Promastigotes in these
cultures entered the logarithmic phase of growth 1-2 days after
passage and reached the stationary phase by day 5. Fig. 5B
shows a single 2.5-kb RNA species that appears to show constitutive
expression during the experimental interval, whereas panel A
shows variations in RNA loading. In this particular experiment more RNA
was added to lane 4 and less to lanes 5 and
6. When differences in gel loading are taken into account,
the increases in late logarithmic phase MAT2 RNA production
are not consistent with the relative band intensities in panel
A. Antibodies against MAT-II fusion protein were used to determine
polypeptide expression at different stages of culture growth.
Panel C shows the expression of the MAT-II protein in L. infantum. Only one 49-kDa peptide was detected on any of
the growth days tested, and the relative intensity of the band was the
same throughout the experiment.
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Fig. 5.
Northern blots showing MAT2
mRNA abundance during promastigote culture growth.
A, ethidium bromide staining. Total RNA was isolated from
cultured promastigotes in the logarithmic (days 2-3), late
logarithmic-early stationary (days 4-5), and stationary (day 6)
phases. RNAs (20 µg per lane) were loaded onto agarose
gel-formaldehyde, separated by electrophoresis, and transferred to a
nylon membrane. B, the membrane was probed with the
MAT2 gene coding region. The arrow indicates the
2.5-kb transcript. C, Western blot analysis using polyclonal
antiserum raised against His6-MAT-II. Cells were harvested
at different growth times, proteins were extracted, and 50 µg of
L. infantum whole cell lysate was applied to a 10%
SDS-PAGE. Only one 49-kDa immunoreactive band was detected in L. infantum promastigotes.
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Functional Analysis of Recombinant MAT-II and Molecular Weight
Determination--
The expression of the L. infantum MAT-II
recombinant protein in E. coli JM109 resulted in an abundant
protein with an Mr of ~49,000 (Fig.
6A, lanes 7-9).
Analysis of the soluble and insoluble fractions of the bacterial
culture at different induction times indicated that the expression
product was concentrated in the insoluble inclusion bodies. MAT-II
recombinant protein was expressed as an His6-tag fusion for
more efficient nickel-agarose resin purification. The MAT-II
recombinant protein obtained with this method was used to produce
specific antiserum. The enzyme activity of the preparation was very
low, however. An alternative method for enzyme purification and
activation, based on a previous study (28), was highly successful.
Triton X-100 and 8 M urea-buffered extraction of the
inclusion body phase resulted in a significant increase in purity (Fig.
6B, lane 3). After equilibrium dialysis against
50 mM Tris-HCl, pH 8.0, containing 10 mM
MgSO4 and 10 mM DTT, extracts were active and
contained over 99% of the MAT protein. MAT activity was linear in
terms of both time (up to 90 min) and protein concentration (data not
shown). The steady-state activity at saturation of both substrates,
i.e. 5 mM ATP and 5 mM
L-methionine, was 12 µmol/mg/h
(kcat = 0.32 s1). The enzyme
exhibited slightly sigmoidal behavior with both L-methionine and ATP. Fig.
7A shows that there is
positive cooperativity between MAT and L-methionine at
different ATP concentrations. Cooperativity, estimated to be
n = 2.3 (ATP = 0.5 mM) declined with
increasing ATP levels to close 1 (ATP = 5 mM). The
K' values for L-methionine were not
significantly affected by ATP and were estimated to be 250 ± 25 µM. Conversely, when assessed as a function of ATP at
different L-methionine levels (Fig. 7B), MAT
activity was sigmoidal (n = 1.8). The K'
values for ATP were estimated to be 370 ± 80 µM,
with the sigmoidal shape of the curve persisting at rising
concentrations of the L-amino acid. The tripolyphosphatase activity of L. infantum recombinant MAT-II was measured
under the standard assay conditions described under "Experimental
Procedures." Tripolyphosphatase activity (Fig.
8A) was linear in terms of
time and protein concentration, and no Pi was released in
the absence of recombinant MAT-II. Under steady-state conditions,
behavior was found to be sigmoidal, showing a
kcat of 0.04 s1 and a
K' value of 30 µM. The
kcat values determined at saturating concentrations of tripolyphosphate, ATP, and L-methionine
showed that the rate of AdoMet synthesis is higher than the rate of
tripolyphosphate cleavage. Because the final process should be
dependent on the slowest reaction, tripolyphosphatase activity might be
responsible for the AdoMet synthesis rate in Leishmania.
However, tripolyphosphate affinity and the presence of the AdoMet
synthesized in the first step of the reaction, in the active site,
stimulate tripolyphosphatase activity at values severalfold higher
than required.
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Fig. 6.
SDS-PAGE analysis of the expression and
purification of recombinant MAT-II protein from E. coli.
A, expression of MAT-II recombinant protein. Coomassie
Blue-stained SDS-PAGE gel. Lane 1, molecular weight markers;
lane 2, E. coli lysate prior to
-D-thiogalactoside induction; lanes 3-5,
soluble fractions of E. coli lysate 30 min, 1 h, and
3 h after induction; lane 6, insoluble fraction of
E. coli lysate prior to -D-thiogalactoside
induction; lanes 7-9, insoluble fractions of E. coli lysate 30 min, 1 h, and 3 h after induction. The
arrow shows the expression product. B,
purification from inclusion bodies. Lane 1, molecular weight
markers; lane 2, E. coli whole cell lysate;
lane 3, inclusion body fraction, washed, solubilized in
urea, and post-dialyzed.
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Fig. 7.
Kinetic analysis of recombinant L. infantum MAT activity. A,
concentration-dependent MAT activity at different ATP
concentrations and four L-methionine levels. B,
concentration-dependent MAT activity at different
L-methionine concentrations and four ATP levels. Kinetic
constants are provided in the text (K',
kcat, and n). C, effect of
some potential inhibitors on MAT activity. Each point is the mean of
three separate trials.
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Fig. 8.
Kinetic analysis of recombinant L. infantum tripolyphosphatase activity. A,
concentration-dependent tripolyphosphatase activity at
different PPPi concentrations, and the effect of increasing
levels of AdoMet. Kinetic constants are provided in the text
(K', and kcat). B, effect
of some potential activators on tripolyphosphatase activity. Each point
is the mean of three separate trials.
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The molecular weight of recombinant MAT-II was determined by gel
filtration on a Sephacryl S-300 column (75 × 1.5 cm, flowing at
10 ml/h). L. infantum promastigotes were suspended in 50 mM Tris-HCl, pH 8.0, 10 mM DTT, and 10 mM MgSO4, cell suspension was treated as
described (33), and the enzymatic extract was loaded onto the Sephacryl
S-300 column equilibrated in the same buffer. Recombinant L. infantum MAT-II was prepared as described under "Experimental
Procedures." After the fractions collected were separated on
SDS-PAGE, the proteins were transferred to a nylon membrane (Sigma) and
immunoblotted against the MAT-II antibody. Most of the native protein
appeared as a dimer with a molecular mass of 96 kDa, whereas a
small monomer fraction with a molecular mass of 49 kDa was also
detected. Assays were conducted for MAT activity (33), although only
the dimeric fractions were observed to be active.
Effects of Substrate, Products, and Products Analogues on
Recombinant L. infantum MAT and Tripolyphosphatase Activity--
The
effect of L-amino acid analogues, products, and products
analogues on both MAT and tripolyphosphatase activities were assessed.
The L-methionine analogues, cycloleucine,
L-ethionine, seleno-DL-ethionine, and
seleno-L-methionine, showed no appreciable inhibitory
effect on MAT activity at any of the concentrations studied (Fig.
7C). However, AdoMet, the natural product of MAT, and other
nucleotide analogues such as AdoHcy and
S-adenosyl-L-ethionine were found to inhibit the
enzyme to a certain extent. AdoMet was the most effective of these
compounds reducing MAT activity more than 50% at 1 mM,
under standard assay conditions. A Dixon analysis of this compound, at
L-methionine concentrations of 0.5-5.0 mM, resulted in a noncompetitive inhibitory pattern with a
Ki value of 4 mM. By contrast, in a
similar analysis with ATP concentrations of 0.5 to 5.0 mM,
a competitive inhibitory effect was observed with a
Ki value for the nucleobase of 0.8 mM
(data not shown). The presence of the AdoMet S-methyl
substituent seems to be an absolutely prerequisite to AdoMet
inhibition. AdoMet analogues lacking this methyl moiety such as
S-adenosyl-L-cysteine and AdoHcy had little
effect on MAT activity. Moreover, replacing the AdoMet methyl group
with an ethyl moiety, S-adenosyl-L-ethionine, significantly reduced the inhibitory effect of this analogue. The MAT
reaction involves the full dephosphorylation of ATP yielding tripolyphosphate (PPPi) as an intermediate, followed by the
release of pyrophosphate (PPi) and orthophosphate
(Pi). All these by-products of MAT activity were assayed as
potential feedback inhibitors of the enzyme. PPPi was found
to be a competitive inhibitor with respect to ATP and
L-methionine, with Ki values determined using 0.25 and 0.2 mM Dixon plots, respectively.
PPi, one of the final by-products of MAT activity,
effectively reduced AdoMet synthesis, with estimated
Ki values of 0.3 and 0.35 mM for ATP and
L-methionine, respectively. Pi had no
inhibitory effect on MAT activity, however. Tripolyphosphatase activity
was assessed in the presence of AdoMet and nucleotide analogues (Fig.
8B). Like AdoMet,
S-adenosyl-L-ethionine increased
tripolyphosphatase activity more than 3-fold at a concentration of 50 µM, and, as in the case of MAT activity, the presence of
S-methyl derivative seems to be absolutely requisite to this
effect, because neither S-AdoHcy nor
S-adenosyl-L-cysteine were found to stimulate
enzyme activity.
Analysis of Upstream and Downstream MAT2 Sequences--
A series
of luciferase reporter constructs were derived from the
pGEM-luc vector (Promega) to analyze the upstream sequences required for expression of a foreign gene in Leishmania.
Three different fragments from the region upstream of the
MAT2 gene were inserted upstream of the luciferase gene
(luc) (Fig. 9). One feature
that these fragments have in common is the presence of polypyrimidine
tracts upstream of the splice acceptor site. 415-, 355-, and 136-bp
fragments were cloned into the pGEM-luc vector, giving rise
to p415/luc, p355/luc, and p136/luc
constructs used to directly test the effect of polypyrimidine tracts on
luciferase expression.
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Fig. 9.
Luciferase activity assays in transiently
transfected promastigotes. A, plasmid construction used
in L. infantum transfections. B, luciferase
activity values adjusted to the results of -galactosidase assays.
Values for pX63NEO-luc transfected cells were set to 100%.
Results are shown as mean ± S.D. from five independent
experiments.
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Parasites were simultaneously transfected with each construction.
pGEM-luc vector was used as the negative control.
pX63NEO-luc was constructed and used as the positive control
by cloning the luciferase gene (derived from pGEM-luc
plasmid) into XbaI-NdeI sites in the
pX63NEO-gal plasmid (kindly provided by Dr. S. M. Beverley, University of Washington, St. Louis, MO). This cleavage released the -galactosidase gene making possible its change by the
luciferase gene. All the transient transfections included the
pX63NEO-gal to control experimental variability. The
simultaneous presence of plasmids containing reporter luciferase and
-galactosidase did not interfere with respective enzymatic
activities (data not shown).
Parasites transiently transfected with p415/luc, which
contains several polypyrimidine tracts, exhibited high levels of
luciferase activity, about 40-fold above pGEM-luc activity
and 200-fold above the background level. Similar results were obtained
when p355/luc and p136/luc were transfected.
Therefore, deletions at upstream sequences and polypyrimidine tracts
did not affect luciferase activity. To study the role of IR on MAT-II
expression, the 4-kb IR was cloned on the p415/luc plasmid,
downstream of the luciferase gene. This new construction was called
p415/luc/IR. As shown in Fig. 9, parasites transiently
transfected with this construction yielded levels of luciferase
activity similar to the levels obtained with p415/luc. When
the 415-bp fragment was removed, the new construct (called
pluc/IR) reduced the activity of transfected parasites to
pGEM-luc levels.
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DISCUSSION |
The existence of multiple MAT genes and isozymes in
different species is well established. Previous kinetic studies (15) reported two isoforms of the enzyme in T. brucei. The
sequence described in this report conserved homologies with S. cerevisiae (60%), Mus musculus (59%), Homo
sapiens (59%), Rattus novergicus (58%), P. falciparum (53%), and E. coli (52%) MAT-II (encoded by the MAT2 gene). Sequence homology analysis showed
similarities with S. cerevisiae (59%), R. novergicus (57%), H. sapiens (56%), and E. coli (52%) MAT-I/III (encoded by the MAT1 gene).
MAT1 and MAT2 sequences described so far have
shown high homology, and the BLAST algorithm sequence analysis
indicated a higher degree of conserved homology with MAT-II. The reason
for the existence of two MAT isozymes is still obscure. Only AdoMet
generated by the reaction catalyzed by the metK enzyme would
be used to repress the methionine biosynthetic enzyme (36). Because
none of the E. coli strains showed size differences in the
metK region, it is tempting to speculate that
metX arose from a duplication of the metK locus
(38). The occurrence of duplicated genes encoding proteins is frequent
in Leishmania sp. (38-42). In our study we found two
identical copies for the gene that encodes MAT-II. We were not able
to determine whether these copies in the L. infantum genome meet cellular needs for AdoMet, i.e. needs that are
accommodated for in other source cell MAT-I/III and MAT-II isoforms.
These two copies, arranged in a head-to-tail tandem, are separated by a
4-kb IR. There is no evidence of introns. Most tandemly arranged genes
in Trypanosomatids are transcribed into large polycistronic precursor
RNAs, which are subsequently processed into monocistronic mRNAs by
trans-splicing, in turned followed by addition of a 39-nucleotide spliced leader to the 5'-ends and a poly(A)-tail to the 3'-ends (32).
This "cut and paste" mechanism is interrelated, whereby the poly(A)
addition is governed by the location of the splice acceptor site on the
downstream gene (32). Because no poly(A) signals have been described in
Trypanosomatids, the explanation generally accepted for this phenomenon
is that the distance between splice acceptor site and the
polyadenylation site is conserved (43). The 3'-UTR of L. infantum
MAT2 transcript was not analyzed in detail in this study, because
no difference in luciferase activity was observed between L. infantum promastigotes transfected with plasmids containing or
lacking IR sequences. The minimal vector for transient gene expression
in Leishmania consists of a circular plasmid containing a
signal for trans-splicing followed by a reporter gene (36). Because
polyadenylation and trans-splicing are coupled, a second trans-splicing
signal should exist downstream of the reporter gene, a signal which, in
our case, seems to be provided by the vector sequence itself. In
addition to the suitable distance between the spliced leader acceptor
site and the polyadenylation site, the presence of several
polypyrimidine-rich motifs is also crucial, because only AG
dinucleotides located downstream of a polypyrimidine tract are used as
splice acceptor sites. The presence of a 2-kb conserved region upstream
of each MAT2 gene, containing both the polypyrimidine tracts
and the splice acceptor site, not only suggests the duplication of the
ORF but also the surrounding regions, and is indicative of similar
control elements for transcription of these two MAT2 genes.
Because the sequences located upstream of the coding regions in both
copies of the MAT2 gene are exactly the same, it is
impossible for us to ascertain, by 5' mapping analysis, whether both
ORFs are able to originate mature mRNAs. It has been shown here
that expression of the firefly luciferase gene transfected in L. infantum is dependent on the presence of Leishmania
5'-DNA sequences. These DNA sequences can be provided by truncated
415-, 355-, or 136-bp fragments from the same region that contains the
AG trans-splicing acceptor site. All these fragments gave rise to high
levels of the luciferase activity. We have shown that a polypyrimidine
tract and an AG site located upstream of the luc gene start
codon were sufficient to yield luciferase activity without the presence
of a promoter region.
The 42-kDa polypeptide predicted on the grounds of theoretical ORF for
MAT-II is considerably smaller than 49-kDa estimated by SDS-PAGE in
this study. Such discrepancies have been reported for MAT isozymes from
different species (3). Anomalously slow migration in SDS-PAGE has been
attributed to excess charge density, or more specifically to an
unusually high acidic amino acids content. Although the entire MAT-II
peptide has a net charge of 7, because many of the basic residues are
located in the carboxyl-terminal portion, the first 210 amino acids
have a net charge of 19.
L. infantum recombinant MAT-II shows significant sigmoidal
kinetic behavior for both L-methionine and ATP. Positive
cooperativity for L-methionine was found using
Lineweaver-Burk plots but was observed to disappear in the presence of
saturating concentrations of ATP (5 mM). Nevertheless,
sigmoidal behavior was not ruled out for ATP in the presence of
saturating concentration of the L-amino acid. The
K' values calculated for L-methionine and ATP correspond to a low affinity enzyme, resembling the dimeric form (MAT-III) of hepatic MAT-I/III isoforms. The tripolyphosphatase activity observed in recombinant L. infantum MAT-II has a
sigmoidal behavior and is extremely sensitive to the presence of AdoMet in the incubation medium. The presence of AdoMet in the assay buffer
has a paradoxical effect on both MAT and tripolyphosphatase activity.
MAT-II is negatively regulated by its product AdoMet in the micromolar
range in most of mammalians sources, as well as in S. cerevisiae and E. coli, whereas the simultaneous
presence of AdoMet enhances tripolyphosphatase activity several times. The Ki for AdoMet inhibition of
Leishmania MAT-II is very high in comparison with its host
counterparts and T. brucei. The estimated
Ki of 1.5 mM suggests that, unlike the mammalian enzyme, Leishmania MAT activity is weakly
regulated by its product. In addition, Northern blot analysis shows
that MAT2 is only synthesized by promastigotes in a low
range. These characteristics may explain the anomalous accumulation of
intracellular AdoMet and dcAdoMet after treatment with DFMO in
Trypanosomatids. Other substrate and product analogues showed a weak
inhibitory effect on recombinant MAT activity. The final products of
dephosphorylation, PPi and Pi, inhibit MAT-II
more efficiently than AdoMet. AdoMet analogues, such as AdoHcy,
S-adenosylcysteine, or S-adenosylethionine, moreover, only proved to have an inhibitory effect at the millimolar level.
All these results suggest poor post-translational MAT-II regulation in
Leishmania parasites. Further experiments will be necessary to assess the MAT2 gene regulation, either at the
transcriptional or post-transcriptional level.
§,
TOP
ABSTRACT
INTRODUCTION
