Molecular and functional characterization of adenylate kinase 2He´ctor Villa1, Yolanda Pe´rez-Pertejo1, Carlos Garcı´a-Estrada1, Rosa M.. This study describes cloning and functional char
Trang 1Molecular and functional characterization of adenylate kinase 2
He´ctor Villa1, Yolanda Pe´rez-Pertejo1, Carlos Garcı´a-Estrada1, Rosa M Reguera1, Jose´ Marı´a Requena2, Babu L Tekwani3, Rafael Balan˜a-Fouce1and David Ordo´n˜ez1
1
Departamento de Farmacologı´a y Toxicologı´a (INTOXCAL), Facultad de Veterinaria, Universidad de Leo´n, Spain;2Centro de Biologı´a Molecular ‘Severo Ochoa’, Universidad Auto´noma de Madrid, Spain;3National Center for Natural Products Research, School of Pharmacy, University of Mississippi, USA
ATP-regenerating enzymes may have an important role in
maintaining ATP levels in mitochondria-like kinetoplast
organelle and glycosomes in parasitic protozoa Adenylate
kinase (AK) (ATP:AMP phosphotransferase) catalyses the
reversible transfer of the c-phosphate group from ATP to
AMP, releasing two molecules of ADP This study describes
cloning and functional characterization of the gene encoding
AK2 from a genomic library of Leishmania donovani and also
its expression in leishmania promastigote cultures AK2 was
localized on an 1.9-Mb chromosomal band as a single copy
gene L donovani AK2 gene is expressed as a single 1.9-kb
mRNA transcript that is developmentally regulated and
accumulated during the early log phase The overexpression
of L donovani AK gene in Escherichia coli yielded a 26-kDa
polypeptide that could be refolded to a functional protein
with AK activity The recombinant protein was purified to
apparent homogeneity Kinetic analysis of purified
L donovaniAK showed hyperbolic behaviour for both ATP and AMP, with Kmvalues of 104 and 74 lM, respectively The maximum enzyme activity (Vmax) was 0.18 lmolÆmin)1Æ
mg)1 protein P1,P5-(bis adenosine)-5¢-pentaphosphate (Ap5A), the specific inhibitor of AK, competitively inhibited activity of the recombinant enzymes with estimated Kivalues
of 190 nM and 160 nM for ATP and AMP, respectively
Ap5A also inhibited the growth of L donovani promastigotes
in vitrowhich could be only partially reversed by the addition
of ADP Thus, presence of a highly regulated AK2, which may have role in maintenance of ADP/ATP levels in
L donovani, has been demonstrated
Keywords: adenylate kinase; functional expression; leish-mania
Adenylate kinase (AK) (ATP:AMP phosphotransferase,
EC 2.7.4.3) catalyses the reversible transfer of the
c-phos-phate group from ATP to AMP, releasing two molecules of
ADP [1] Different isoenzymic forms of AK are involved in
maintenance of constant intracellular levels of adenine
nucleotides, necessary for energy metabolism and nucleic
acid synthesis [2] Three AK isoforms have been described in
mammals: AK1 in the cytosol, AK2 in the mitochondrial
intermembrane space [3] and AK3, a GTP:AMP
phospho-transferase that resides exclusively in the mitochondrial
matrix [4]
The role of AKs in the organisms of the order Kineto-plastida (that includes trypanosomes, leishmanias and other pathogenic parasites) has not been studied in detail yet As
in their mammalian hosts, AK in these parasites seems to be distributed in several intracellular compartments These eukaryotic microorganisms have some characteristic sub-cellular organelles, such as modified mitochondria called
kinetoplasts and several specific energy-producing micro-bodies called glycosomes [5] AK plays an important role in the ATP-regenerating system required for eukaryotic ciliary
or flagellar movements and has been found to be associated with Tetrahymena cilia [6], Paramecium caudatum [7] as well
as vertebrate spermatozoid flagella [8,9] AK activity in Leishmaniapromastigotes and Trypanosoma spp has been found to be associated with the membrane of glycosomes [10,11] A third form of AK located in the cytosol has been proposed as a virulence factor in bacteria, as it is secreted along with other ATP-related enzymes, contributing to modulation of ATP levels during macrophage death [12,13] Structural studies with AKs from different sources have revealed the presence of three distinct domains: the rigid CORE-domain and two smaller peripheral domains or mobile parts, the NMP-binding site and the LID-domain [4] The NMP-binding site has many intermolecular contacts with the nucleotide phosphoryl acceptor (NMP), whereas the LID-domain prevents the hydrolysis of the Mg-bound phosphoryl donor in the active site The relative movement of the NMP-binding site and LID-domain
Correspondence to D Ordo´n˜ez, Department Farmacologı´a y
Toxi-cologı´a (INTOXCAL), Ftad Veterinaria, Universidad de Leo´n,
Campus de Vegazana s/n 24071 Leo´n, Spain.
Fax: + 34 987 291 252, Tel.: + 34 987 291 590,
E-mail: dftrbf@isidoro.unileon.es
Abbreviations: AK, adenylate kinase; IPTG, isopropyl thio-b- D
-galactoside; Ap 5 A, P 1 ,P 5 -bis(adenosine)-5¢-pentaphosphate; NMP,
nucleoside monophosphate; IC 50 , 50% inhibitory concentration.
Enzymes: adenylate kinase (ATP:AMP phosphotransferase;
EC 2.7.4.3).
Note: The nucleotide sequence data reported has been submitted to
EMBL and GenBank Nucleotide Sequence Databases under the
accession number AF156853.
(Received 14 May 2003, revised 25 July 2003,
accepted 9 September 2003)
Trang 2induced by the substrates leads to two conformations called
closed and open states, which have been thoroughly
described by Schulz et al [14] Genes encoding AKs have
been characterized in many organisms, including bacteria,
fungi and mammals There are no reports about
character-ization of this enzyme in parasitic protozoa Leishmania
donovaniis the aetiological agent for visceral leishmaniasis, a
devastating disease which is mostly endemic in Asian and
Mediterranean countries [15] This paper describes the
cloning and functional characterization of an AK gene from
a genomic library of L donovani as well as its expression
and molecular characterization
Materials and methods
Materials
Plasmids pGEM-3Zf(+) and pQE30 were from Promega
and QIAGEN, respectively [32P]dCTP[aP] (3000 CiÆ
mmol)1) was from DuPont-NEN Restriction enzymes
and Taq DNA polymerase were from Boehringer
Mann-heim Isopropyl thio-b-D-galactoside (IPTG), pyruvate
kinase, lactate dehydrogenase, phosphoenolpyruvate,
pro-teinase K, antibiotics, ATP, AMP, dithiothreitol and P1,P5
-bis(adenosine)-5¢-pentaphosphate (Ap5A) were from Sigma
Lambda EMBL-3 genomic library was a gift from
J.C Meade (University of Mississippi, Medical Center,
MS, USA)
Parasite cell culture
Promastigote cultures of L donovani 1S2D strain (a
gener-ous gift from L Rivas, Centro de Investigaciones Biolo´gicas
CSIC, Madrid, Spain) were grown at 26.5C in M199
medium (Sigma) supplemented with sodium bicarbonate,
5 mMHepes, heat inactivated foetal bovine serum (10%)
(Boehringer Mannheim) and gentamicin (30 lgÆmL)1)
(Sigma)
Nucleic acid isolation
Genomic DNA from L donovani promastigotes was
isola-ted from 2.0 · 109cells in 10 mMEDTA, 150 mMNaCl,
0.4% SDS, 50 mgÆmL)1 proteinase K and incubated at
65C for 1 h followed by an overnight incubation at 37 C
The DNA was further purified by phenol/chloroform
extraction and ethanol precipitation RNA was isolated
with RNAeasy kit (QIAGEN) according to manufacture’s
protocol
Isolation of aL donovani AK gene fragment by PCR
To generate a DNA probe for the isolation of the
L donovani AKgene, PCR was carried out using degenerate
oligonucleotides based on conserved amino acid sequences
in human AK type 2A [16], Saccharomyces cerevisiae [17],
Trichomonas vaginalis[18], Oryza sativa [19] and Escherichia
coli [20] AK proteins The sense primer (5¢-GACGG
TTTTCCGCGCAC-3¢) corresponding to the amino acid
residues DGFPRT and an antisense primer (5¢-ACCAGG
GGCTCGCCGGT-3¢) corresponding to amino acid
resi-dues TGEPLV were used for isolation of AK gene fragment
by PCR with L donovani genomic DNA The PCR product was sequenced on both strands by an ALF automated sequencer (Sistemas Geno´micos, Valencia, Spain) according
to the dideoxy chain termination method using fluorescent primers and T7 DNA polymerase
Isolation ofL donovani AK gene The EMBL L donovani library was used for screening
50 000 bacteriophages were blotted onto positively charged nylon membranes (Amersham) and prehybridized for 4 h at
42C in 5 · Denhardt’s solution, 5 · NaCl/Cit, 50 mM sodium phosphate (pH 6.5), 100 lgÆmL)1 salmon sperm DNA, and 50% formamide Membranes were hybridized for 16–20 h at 42C in the same buffer containing
106)107c.p.m of 32P-labelled 215 bp-PCR product, that had been radiolabelled by the random primer method with [32P]dCTP[aP] (Amersham) Filters were washed twice with
2· NaCl/Cit plus 0.1% SDS at 42 C for 10 min each, followed by two washes in 1· NaCl/Cit, plus 0.1% SDS at
42C for 10 min Positive bacteriophages were carried through secondary and tertiary screenings, until all the plaques on the plate appeared positive The DNA was isolated from amplified phages by liquid culture method as described by Sambrook et al [21]
Subcloning and sequencing ofL donovani AK gene The isolated bacteriophage DNA was digested with restric-tion endonucleases, separated by electrophoresis on 0.7% agarose gels, and transferred to positively charged nylon membranes by the method of Southern [22] Southern blots were probed with the 215-bp PCR product under the same conditions described above for the screening of the genomic library A single 1.6-kb EcoRI–SphI fragment that hybrid-ized to the probe was ligated into pGEM-3Zf(+) and transformed into E coli DH5a Large-scale plasmid pre-paration of pGEM-3Zf(+) containing the 1.6-kb EcoRI– SphI fragment was processed using QIAGEN columns, and the insert was sequenced as above Analyses of nucleotide and amino acid sequences were performed using theBLAST algorithm from the database National Center for Biotech-nology Information
Southern and Northern analyses Genomic DNA prepared from L donovani promastigotes harvested at late log phase was digested with restriction enzymes, as described above, separated by electrophoresis in 0.8% agarose gels and transferred to nylon membranes Total RNA was extracted from L donovani promastigotes using RNeasy Mini kit (QIAGEN), following the manu-facturer’s instructions Approximately 20 lg total RNA were resolved onto Mops/formaldehyde 1.2% agarose gels, transferred to a nylon membrane, and fixed to the filters with an UV-crosslinker The filters were prehybridized for
2 h at 42C in 10 mL 10 · Denhardt’s, 6 · NaCl/Cit and 1% SDS solution containing 300 lgÆmL)1 herring sperm DNA Hybridization was performed overnight at 42C in
10 mL 50% formamide, 6· NaCl/Cit, 1% SDS,
150 lgÆmL)1 herring sperm DNA and the 32P-labelled probe The filters were washed stepwise in 6· NaCl/Cit 1%
Trang 3SDS for 30 min at room temperature, 1· NaCl/Cit, 0.5%
SDS for 45 min at 42C and 0.1 · NaCl/Cit 0.2% SDS
for 45 min at the same temperature Finally the filters
were exposed at)70 C for autoradiography
Chromosomal localization ofAK gene
Promastigotes (2· 108) were harvested by centrifugation,
washed twice in NaCl/Piand resuspended in NaCl/Pimixed
1 : 1 with 2% agarose Processing of the samples was
carried out at 50C for 48 h in 10 mL 0.5MEDTA pH 8.0,
1% Sarkosyl, and 150 lL of 2 mgÆmL)1freshly prepared
proteinase K Separation of the chromosomal bands was
achieved by electrophoresis at 14C in 1% agarose gels
with 0.5· TBE running buffer, using a Clamped
Homo-geneous Electrical Field apparatus (CHEF, BIO-RAD)
with a 35–120 s ramping pulse at 6 VÆcm)1 for 33 h
S cerevisiae (Amersham) chromosomes were used as
molecular weight markers After staining with ethidium
bromide, gels were blotted onto nylon filters (Sigma) by
alkaline transfer The membrane was probed to the 690-bp
probe as described above
Heterologous expression
The AK gene was amplified by PCR from L donovani
genomic DNA The sense primer 5¢-ACATGCATGCAT
GAAGATCGTGATGGAAGG-3¢ introduces an SphI
restriction site and the antisense one 5¢-AACTGCAG
GCTTTCACCAGAATTTCCACC-3¢ introduces a PstI
restriction site to subclone the AK gene in the
pGEM-3Zf(+) cloning vector (Promega), creating pGEM-AK To
subclone the AK gene into pQE-30 expression vector
(QIAGEN), pGEM-AK was digested with SphI and PstI
and the agarose purified AK gene insert was ligated into
SphI–PstI digested pQE30 plasmid with T4 DNA ligase
(Promega) This construct, called pQE30-AK, was
trans-formed into XL1-Blue E coli competent cells Overnight
cultures prepared from single colonies were used to
inoculate 100 mL of Luria–Bertani medium plus ampicilin
(50 lgÆmL)1) Cells were grown to D6000.5–0.8 and IPTG
was added to a final concentration of 0.1 mM After
induction with IPTG, growth was continued for 5 h under
the same conditions Cells were harvested by centrifugation,
washed in 1· NaCl/Piand resuspended into 50 mMTris/
HCl (pH 8), 10 mM MgCl2 before sonication Inclusion
bodies were obtained by centrifugation at 10 000 g for
10 min at 4C Pellets were washed twice with 4Murea,
20% Triton-X100, 50 mMTris/HCl pH 8, 10 mMMgCl2,
1 mMdithiothreitol and twice with 50 mMTris/HCl (pH 8),
10 mMMgCl2, 1 mMdithiothreitol After incubation with
50 lgÆmL)1DNAse I (Roche), cell debris were removed by
centrifugation and the insoluble fraction was dissolved in
equilibration buffer (10 mMTris/HCl, 300 mMNaCl
con-taining 8M urea) to be loaded onto a nickel column
equilibrated with the same buffer The column was washed
stepwise with equilibration buffer and equilibration buffer
plus 10 mM imidazole The elution of AK protein was
carried out with equilibration buffer containing 200 mM
imidazole, collecting 1-mL fractions which were analysed by
SDS/PAGE Fractions enriched in AK were dialysed
stepwise, first against 50 m Tris/HCl, 1 m EDTA,
10 mMdithiothreitol, containing 1Murea for 2 h and then against 50 mMTris/HCl, 1 mMEDTA, 10 mM dithiothre-itol containing 0.25Murea overnight [23]
SDS/PAGE and Western blotting
L donovanipromastigotes were harvested during exponen-tial growth phase (day 3) and washed twice with NaCl/Pi Once they were sonicated and centrifuged at 10 000 g for
20 min, the supernatant was removed Fifty lg protein were diluted in loading buffer (60 mM Tris/HCl pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 5% glycerol), heated in a boiling water bath for 5 min, and analysed by SDS/PAGE (12% acrylamide, 2.7% bisacrylamide) Proteins were electrotransferred to nylon membranes for 1 h at 25–30 VÆcm)1, blots were blocked by incubation in 10 mM Tris/HCl pH 7.5, 1MNaCl, 0.5% Tween 20, 5% nonfat milk powder (w/v) for 1 h at room temperature Primary, polyclonal antibodies (obtained from rabbit serum ino-culated with purified recombinant leishmanial AK) were added to this buffer and the blot incubated for an 2 h The blot was washed extensively in 10 mMTris/HCl pH 7.5, 1M NaCl, 0.5% Tween 20 and then incubated with an anti-rabbit antibody conjugated to alkaline phosphatase Anti-bodies were detected using Nitro-Blue-tetrazolium chloride
as substrate (Boehringer Mannheim)
AK assay Enzyme activity was assayed in the forward direction by the coupled reactions system [24] adding different concentra-tions of ATP and AMP as the substrates The rate of NADH disappearance was measured spectrophotometri-cally at 340 nm in a coupled enzyme assay to pyruvate kinase and lactate dehydrogenase at 30C The assay mixture contained, in a total volume of 1 mL, 0.1 mMTris/ HCl (pH 8.0), 120 mM KCl, 10 mM MgCl2, 1 mM phos-phoenolpyruvate, 0.2 mM NADH, 4.4 U pyruvate kinase (Sigma), 5 U lactate dehydrogenase (Sigma) and several concentrations of ATP (range 0.025–5 mM) and AM P (range 0.010–1.5 mM) Protein content was estimated by using the method of Bradford [25] For analysis of enzyme kinetics AK assay was done at varying concentrations of ATP and AMP and the results were analysed by double reciprocal Lineweaver–Burk plot by using SIGMA PLOT Kinetic parameters (Km and Vmax) were computed from these plots
In vitro anti-leishmania assay Anti-leishmanial activity of Ap5A the inhibitor of AK was tested on a transgenic cell line of L donovani promastigotes expressing firefly luciferase These cells show constant and stable expression of luciferase, which is directly proportional
to the number of live promastigotes The assay was performed in clear-bottomed, 96-well micro plates Promas-tigote culture (200 lL; 2· 106cells per well) was exposed to varying concentrations (5–250 lM) of Ap5A The plates were incubated at 26C for 72 h and growth of promastigotes was determined by luciferase assay with Steady Glo reagent (Promega) Fifty per cent inhibitory concentrations (IC50) were determined from the inhibition curves
Trang 4Cloning and characterization of theAK gene
inL donovani
An L donovani (1S2D strain) lambda EMBL3 genomic
library was screened with a [32P]dCTP[aP]-labelled AK
probe obtained by PCR from genomic DNA of L
dono-vanipromastigotes After three rounds of screening, three
different clones were isolated Analysis by digestion with
restriction enzymes indicated that all three clones were
the same, therefore only one was sequenced in both
strands (Sistemas Geno´micos, Valencia, Spain) The
analysis of the nucleotide sequence revealed the presence
of a 690-bp long ORF that encoded a protein of 230
amino acids, with an estimated molecular mass of
26 kDa This gene was designated the AK gene (see
GenBank accession number AF156853) The amino acid
sequence deduced from this ORF showed significant
homology with AK enzymes from different organisms
Multiple sequence alignment (Fig 1) of the L donovani
AK protein with other proteins from phylogenetically
diverse organisms revealed that the Leishmania AK gene
has the conserved motifs involved in NMP binding in a
sequence from 30 to 61 amino acids The amino acids
residues that possibly mediate the interactions with AMP
are T31, R36 and L59 In position 35 a valine residue
replaced the leucine residue, which is conserved in all
other sequences, while at position 60 an isoleucine residue
was present in place of a highly conserved valine The
decapeptide GPPQGGKTTV (in position 7–16) in
L donovaniAK resembles the P-loop related to the
ATP-binding site [26] The sequence containing four cysteine
residues C-X2-C-Xn-C-X2-C predicted to be a
zinc-binding motif was not found in L donovani LID
sequence, although it was present in P falciparum AK (AF308612), where it is located in positions 127–158 The LID domain, said to be related to the active site of the enzyme in most AK, starts at position 124 and extends to position 161) a total of 38 amino acids A number of phylogenetically conserved motifs (GFPRT in position 86–90) or amino acids (R121, G132, R133, R169, Y173, Q176, V201) with no defined functions are also found in
L donovani AK
Southern blot analysis (Fig 2A) was performed on genomic DNA to determine the gene copy number and genomic organization of AK genes in the L donovani genome Several enzymes with cleavage sites in the AK coding region were used to digest the DNA, and the blots were probed with the 690-bp coding region Complete digestion resulted in a single copy within the L donovani genome, as all enzymes showed only one band, except those which cut once into the gene sequence (BamHI, KpnI and SalI), which exhibited two hybridizing bands Chromosomal bands from L donovani 1S2D strain were resolved by pulsed-field gel electrophoresis as described in Materials and methods, transferred to a nylon membrane and hybridized to the random primer-labelled probe with
AKcoding region to determine the chromosomal location
of the AK gene A hybridization signal was observed in a chromosomal band of 1.9 Mb (Fig 2B) Expression of the AK gene during growth of leishmania promastigotes
in vitro was also investigated Total RNA was isolated from promastigotes harvested at different growth periods and analysed by Northern blotting (Fig 3) The promas-tigotes in these cultures entered the logarithmic phase of growth within 1–2 days after subculturing and reach the stationary phase by day 5 Fig 3B shows a single RNA transcript of 1.9 kb Transcription of the AK gene was higher during early period (days 1 and 2) of growth,
Fig 1 Multiple amino acid sequence align-ments of L donovani AK The predicted amino acid sequences for L donovani, Trypanosoma brucei, T vaginalis, P falciparum, S
cerevisi-ae, and human 2A AKs were aligned using the
CLUSTAL X multiple sequence alignment pro-gram Symbols: * identical or conserved residues in all sequences in the alignment; : conserved substitutions; . semiconserved substitutions Complete genomic sequences
of AK proteins are available from the GenBank, for L donovani accession number (AF156853), T brucei (AF047722),
T vaginalis (U07203), P falciparum (AF308612), S cerevisiae (Y00413) and human 2A (U39945).
Trang 5diminishing to very low levels in the logarithmic and
stationary phases (Fig 3C)
Overexpression and purification of AK2
The cultures of bacterial cells (XL1-Blue) transformed with
pQE30-AK were induced with IPTG for overexpression of
AK2 gene Marked overexpression of AK2 protein was
noticed but the recombinant AK protein accumulated
mainly in the inclusion bodies (Fig 4A) A 26-kDa
polypeptide was detected after induction of the culture with
IPTG, which matched the molecular mass predicted from
the amino acid sequence of AK2 protein The purification
and functional folding of L donovani AK from the E coli
cell lysates was carried out by washing and dissolving the
inclusion bodies in 8M urea Refolding was achieved by
equilibrium dialysis against 50 mM Tris/HCl pH 8.0,
con-taining either MgSO4, KCl or 10 mMdithiothreitol, extracts
were active and contained over 99% of the AK protein
Finally, the recombinant protein was purified to apparent
homogeneity by affinity chromatography on Ni–NTA
agarose columns The recombinant protein was analysed
for enzyme activity Western analysis of exponential phase
leishmanial extracts were performed with an anti-AK
polyclonal antibody obtained from previously immunized
rabbits A single band of 26 kDa was detected, as shown in
Fig 4B
Functional characterization of recombinant AK The recombinant protein purified from the inclusion bodies and refolded in the presence of dithiothreitol, MgSO4and KCl showed significant enzyme activity AK activity was linear in terms of both incubation time (up to
90 min) and protein concentration (0.5–5 lg protein; data not shown) Maximum enzyme activity (Vmax) at steady-state conditions and saturation concentrations of both substrates in the forward reaction, i.e 2.5 mM ATP and 1.5 mM AMP, was 0.18 lmol ADP formedÆmin)1Æmg)1 The enzyme exhibited hyperbolic behaviour with both ATP and AMP The Km value estimated for ATP was
104 ± 20 lM, while for AMP it was 74 ± 18 lM Stability of the recombinant leishmania AK was also checked The recombinant enzyme protein was incubated,
as described previously, at 4C, 13 C and 26.5 C for 10 consecutive days Activity was measured at different time
Fig 2 Southern blot analysis of wild-type AK loci (A) Genomic DNA
(20 lg) was isolated from L donovani promastigotes, digested with
PvuI, NotI, SalI, KpnI, BamHI, PstI, and resolved on a 0.7% agarose
gel, and blotted onto nylon membranes Blots were hybridized to the
690-bp PCR fragment under high stringency conditions (B)
Chromo-somal location of the AK gene in the L donovani 1S2D strain After
pulse-field gel electrophoresis of the L donovani 1S2D promastigotes
different sized groups were displayed, similar to other L donovani
strains The gel was blotted onto nylon membranes by alkaline transfer
and hybridized to random labelled 690-bp PCR product Under these
conditions only one band of 1.9 Mb was obtained.
Fig 3 Northern blots showing AK mRNA abundance during promas-tigote culture growth Total RNA was isolated from cultured pro-mastigotes in logarithmic phase (days 1–2), late logarithmic early stationary phase (days 3–5) and stationary phase (day 6) (A, B) The RNAs (20 lg per lane) were loaded onto agarose gel, separated by electrophoresis and transferred to a nylon membrane An ethidium bromide stain of the gel is shown (A) The membrane was probed with the coding region of the AK gene (B) (C) Relative abundance of AK mRNA during the growth curve of promastigotes.
Trang 6intervals as described in Fig 5 A time-dependent
reduc-tion in AK activity was noticed at all temperatures
studied, although it was more pronounced at 26.5C than
4C Semi-inactivation times were estimated to be 29,
24 and 12 h at temperatures of 4, 13 and 26.5C,
respectively
Inhibition of AK by the specific inhibitor Ap5A and its effect on growth ofL donovani promastigotes in vitro The effect of the nucleotide analogue Ap5A, was studied
on L donovani recombinant AK under standard assay conditions (Fig 6) Different concentrations (50–1000 nM)
of Ap5A were added to the assay buffer, which contained
6 lg refolded AK and three concentrations of one of the nucleotides (ATP: 0.62–2.5 mM; AMP: 0.5–1.5 mM) under forward assay conditions (Fig 6A,B) Dixon analyses of the inhibition of leishmania AK with Ap5A at saturating concentrations of the other substrate (ATP: 2.5 mM; AM P 1.5 mM), showed a competitive inhibitory pattern for both substrates, with Kivalues of 190 nMand 160 nMfor ATP and AMP, respectively Ap5A also inhibited growth of
L donovanipromastigotes in vitro (Fig 7) with IC50value
of 27 ± 5 lM The effect of Ap5A on leishmania growth could be partially reversed with ADP (IC50, 48 ± 9 lM) but addition of ATP to the culture medium did not show any reversal (IC50, 33 ± 6) The reversal with ADP was statistically significant (P < 0.01%) Ap5A did not show a direct effect on luciferase activity of the transgenic Leish-maniapromastigote extracts
Fig 4 Heterologous expression of recombinant AK from L donovani.
E coli cells (strain XL1-Blue) transformed with the plasmid
PQE30-AK were grown and the cultures were induced with 0.1 M IPTG for
5 h Samples of soluble and insoluble fractions were loaded onto SDS/
12% polyacrylamide gels under reducing conditions (A) Lane 1
solu-ble fraction; lane 2 insolusolu-ble fractions Lane 3 shows the inclusion
bodies after purification as outlined in Materials and methods The
arrow shows the 26-kDa expression product (B) Forty micrograms
exponential phase (day 3) promastigotes were loaded onto SDS/12%
PAGE The separated proteins were transblotted onto nylon
mem-branes and probed with an anti-AK polyclonal antibody MWM,
molecular mass markers; CNT, preimmune serum.
Fig 5 Half-life of recombinant L donovani AK at different
tempera-tures Inclusion bodies from E coli XL1-Blue, transformed with
PQE-30-AK, were washed, folded and purified as described in Materials and
methods Purified enzyme aliquots were maintained at 4 C (m), 13 C
(j) and 26.5 C (d) and AK activity was measured at different time
points Each point represents the mean of two different experiments.
Fig 6 Dixon plots of the inhibition of recombinant AK by the specific inhibitor Ap 5 A Six micrograms recombinant L donovani AK were incubated in presence of several concentrations of ATP (A) or AMP (B) and different concentrations of the inhibitor Each point is the mean of three separate trials.
Trang 7Amino acid sequence of AK2 cloned from L donovani
shows significant homology with the corresponding
enzyme from S cerevisiae (28%) [17], Homo sapiens
(29%) [16], P falciparum (15%) (AF308612), T vaginalis
(25%) [18] and T brucei rhodesiense (14%) (AF047722)
Analysis of leishmania AK sequence by BLAST and CD
search revealed the presence of a large (38 amino acids)
LID domain, which confirms its identity as a type 2 AK
A threonine residue specific to AMP binding is present at
position 31, and the P-loop attributed to the ATP binding
site is mostly conserved, although residues Q10 and G11
seem to be translocated Within the AMP binding
domain, some amino acid residues are well conserved
(L59, F87, R89) while two others, a leucine and a valine,
are replaced by V35 and I60, respectively A Mg2+
binding site, generally consisting of two aspartic acid
residues, contains two glutamic acid residues at positions
33 and 85 in AK2 of L donovani Some AKs are also
known to bind a Zn2+ cation, which plays a role in
protein stabilization and folding, although it is not
essential for catalysis The well-established C-X2-C-Xn
-C-X2-C motif that provides a Zn binding-site in some AKs
(i.e Chlamydia pneumoniae [27] or P falciparum
AF308612) is absent here, suggesting that L donovani
AK uses only Mg2+as a metal cofactor Three different
views of a theoretical three-dimensional ribbon model
diagram of the leishmania AK2, which were obtained using the SWISS-MODEL (Automated-Knowledge-Based-Protein-Modelling-Server; http://www.expasy.ch) (Fig 8), shows the structure of leishmania AK2 to be similar to
Fig 7 Effect of Ap5A on the growth of L donovani promastigotes
in vitro Effect of Ap5A at varying concentrations (5–250 l M ) was
tested alone (control) and also in combination with ATP or ADP
(1 m M ) Each point represents the mean ± SD of at least triplicate
observations.
Fig 8 Three-dimensional ribbon diagram of L donovani AK 2 The
SWISS-MODEL
(Automated-Knowledge-Based-Protein-Modelling-Ser-ver; http://www.expasy.ch) was used for prediction of theoretical
three-dimensional structure of the enzyme The three different views were
obtained by simple rotation of the same model The flexible LID
domain appears at top of the three figures, NMP-binding site and ATP
binding domain are displayed, NH 2 - and COOH-termini are also
indicated.
Trang 8that of E coli AK The flexible LID domain which
provides open and closed conformation to the protein
during binding of the substrate is shown in the top-most
portion of the protein ATP may bind to the glycine loop
on the left flap portion (Fig 8B) and AMP may bind to
the AMP flap shown on the right side (Fig 7B) AK
displays a closed configuration in the presence of bound
nucleotides and an open arrangement when products are
released to the medium, thus configuring a movable
structure [28] Reduction in the activity of recombinant
AK2 during storage at different temperatures indicate
inactivation of the proteins which seems to be faster at
26C than at 4 and 13 C
Heterologous expression in competent E coli strains
shows that the kinetic behaviour of L donovani
recombin-ant AK2 is hyperbolic for both AMP and ATP in the
forward reaction Like most AKs, the enzyme was strongly
inhibited by Ap5A, with Ki values of the order of those
found for other sources for both AMP and ATP [24,26,27]
Southern analysis of the L donovani AK gene showed that it
is present as a single-copy gene into the genome During the
progress of this work, six putative AK genes on
chromo-somes 4, 21, 25, 34(2) and 36 have been identified in the
L major genome (http://www.genedb.org/genedb/leish/
index.jsp) The L major AK gene CHR34_tmp.344,
cor-responds to the L donovani AK2 gene described in this
paper The amino acid sequence of L donovani AK2 gene
shows 96% homology to the CHR_tmp.344 while
homo-logy in nucleotide sequence was found to be 94% Other
putative AK genes were significantly different The results of
Southern blotting also indicated the AK2 as a single copy
gene in L donovani Investigation of transcription of the AK
gene by Northern blot analysis during the Leishmania life
cycle detects a single mRNA transcript of 1.9 kb
Leishmania cell seem to have strong regulation for
expres-sion of AK2 gene as the relative abundance of AK mRNA
during the early logarithmic phase was much higher than in
other phases of the promastigote cell cycle AK has been
detected in glycosomes in Leishmania [10] Recently, three
new putative AK gene sequences, one of them is incomplete
(AL 139794), have been identified by the L major Friedlin
Genome Project One of the L major AK genes seems to
encode for a short AK isoform (AQ 852692), with an LID
domain of 18 amino acids, while the other two genes
(AL354533) encode longer AKs Moreover, the AL354533
and AQ 852692 isoforms lack the threonine 31 residue,
which is replaced by a valine or a serine, respectively This
suggests possible involvement of AKs also in CMP/UMP
metabolism in leishmania The recombinant leishmanial
AK2 was also selectively recognized by the serum of
hamsters infected with L donovani (data not shown) AK2
may be a potential target antigen for a vaccine or diagnosis
of leishmaniasis The sequence of the AK genes identified in
the leishmania genome, including the present one, differ
significantly from each other Diverse AK isoforms may be
localized in different cellular compartments in leishmania
and may have important roles in energy metabolism and
adenine nucleotide equilibrium Inhibition of leishmania
growth with Ap5A, an inhibitor of leishmanial AK2 and its
partial reversal by ADP indicate the importance of this
enzyme in leishmania growth and proliferation The effect
of ApA on other metabolic functions of leishmania cell
may not be ruled out at this stage Detailed investigations of the distinct molecular characteristics of leishmania AK2 in comparison with its mammalian counterparts and its absolute need of the enzyme for growth and survival of the parasite within host macrophages are required to allow the development of selective inhibitors of the parasite enzyme as potential antileishmanial agents In leishmania promastigotes AK has been found to be associated with glycosomes [10,11] Beside the role of AK in maintenance and regeneration of intracellular ATP levels as shown in other organisms [7] partial reversal of antileishmanial action
of Ap5A by ADP indicate that the enzyme might also be important for regeneration of ADP in leishmania, which may be required for continuous synthesis of ATP through substrate level phophorylation AK in conjuction with an adenylate translocator has been shown to maintain the metabolic interconnection between malaria parasite P falci-parumand the host erythrocyte [29] High levels of ATP are generated in the malaria parasite by AK during intra-erythrocytic growth, and this is translocated to the eryth-rocytes AK may also play a role in the ATP regeneration system required for flagellar movements in leishmania promastigotes as shown earlier in the cilia of Paramecium caudatum[7] or may also be a virulence factor as shown in Pseudomonas[12] AK may thus be a potential drug target
in leishmania
Acknowledgements
This work was supported in part by Comisio´n Interministerial
de Ciencia y Tecnologı´a (CICYT, grants PM98/0036 and BMC2002 04107-C02-02) and Junta de Castilla y Leo´n (JCyL grants LE01/00B and LE54/03) We also thank to Dr J Zhou for his advice in
AK refolding experiments, Dr N Fasel, I Segura, P Bastien, and
F Fierro for their technical support in PFGE BLT is supported by Cooperative scientific agreement grant from Center for Disease Control, Atlanta USA (U50-CCU418839) and also by a USDA cooperative agreement no 58-6408-2-0009.
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