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Eur. J. Biochem. 271, 4845–4854 (2004) FEBS 2004
doi:10.1111/j.1432-1033.2004.04450.x
Cloning, over-expression, purification and characterization of
Plasmodium falciparum enolase
Ipsita Pal-Bhowmick, K. Sadagopan, Hardeep K. Vora, Alfica Sehgal*, Shobhona Sharma and
Gotam K. Jarori
Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
We have cloned, over-expressed and purified enolase from
Plasmodium falciparum strain NF54 in Escherichia coli in
active form, as an N-terminal His6-tagged protein. The
sequence of the cloned enolase from the NF54 strain is
identical to that of strain 3D7 used in full genome sequencing. The recombinant enolase (r-Pfen) could be obtained in
large quantities ( 50 mg per litre of culture) in a highly
purified form (> 95%). The purified protein gave a single
band at 50 kDa on SDS/PAGE. MALDI-TOF analysis
gave a mean ± SD mass of 51396 ± 16 Da, which is in
good agreement with the mass calculated from the sequence.
The molecular mass of r-Pfen determined in gel-filtration
experiments was 100 kDa, indicating that P. falciparum
enolase is a homodimer. Kinetic measurements using
2-phosphoglycerate as substrate gave a specific activity
of 30 UÆmg)1 and Km2PGA ¼ 0.041 ± 0.004 mM. The
Michaelis constant for the reverse reaction (KmPEP) is
0.25 ± 0.03 mM. pH-dependent activity measurements
gave a maximum at pH 7.4–7.6 irrespective of the direction
of catalysis. The activity of this enzyme is inhibited by Na+,
whereas K+ has a slight activating effect. The cofactor
Mg2+ has an apparent activation constant of 0.18 ±
0.02 mM. However, at higher concentrations, it has an
inhibitory effect. Polyclonal antibody raised against pure
recombinant P. falciparum enolase in rabbit showed high
specificity towards recombinant protein and is also able
to recognize enolase from the murine malarial parasite,
Plasmodium yoelii, which shares 90% identity with the
P. falciparum protein.
Malaria remains one of the most infectious diseases in the
third world with about 500 million infections and over one
million deaths per year [1]. In the face of increasing threats
by resurgent infections and an expanding array of drugresistant phenotypes, the requirement of alternative preventive therapeutics is evident, especially for the most severe
form of human malaria parasite Plasmodium falciparum.
The first step in rational drug development involves
identification of macromolecular targets, which are unique
and essential for the survival of the parasite. Glycolytic
enzymes seem to be promising candidates from this
perspective, as energy production in P. falciparum depends
entirely on the glycolytic pathway as the parasite and its
mammalian host (red cells) lack a complete Krebs cycle
and active mitochondria [2,3]. The level of glycolytic flux
in parasite-infected cells is 100-fold greater than that
observed in uninfected cells, and the activity of many of the
glycolytic enzymes is higher in the infected cells than in
uninfected ones [4]. Therefore an antimalarial that selectively inhibits the parasite ATP-generating machinery
would be expected to arrest parasite development and
growth. Extensive work has already been carried out with
many P. falciparum glycolytic enzymes, with aldolase,
lactate dehydrogenase and triose phosphate isomerase
showing quite promising behavior as detection tools, drug
targets and vaccine candidates [5–8]. P. falciparum enolase
(Pfen) (EC 4.2.1.11), the dehydrating glycolytic metalloenzyme that catalyzes the inter conversion of 2-phosphoglyceric acid (2-PGA) and phosphoenolpyruvate (PEP), has
not yet been characterized. Enolases are highly conserved
across species [9]. In most species, it exists as a symmetric
homodimer [10]. However, in several bacterial species,
octameric enolases have been reported [11,12]. Conservation
is particularly pronounced for the active-site residues,
leading to similar kinetic properties among enolases from
diverse sources. For activity, enolase requires the binding of
2 mol bivalent cations (in vivo this is usually Mg2+) per
subunit. Binding at site I leads to changes in the tertiary
structure of the enzyme (conformational site) whereas
binding to site II is essential for catalysis (catalytic site)
[13]. At higher concentrations, bivalent cations inhibit
activity, suggesting the existence of a third inhibitory site.
Univalent cations also influence the activity of enolases.
Most of the enolases are inhibited by Na+, whereas the
effect of K+ depends on the source of the enzyme. K+ has
no effect on yeast enolase whereas it activates rabbit
enolases [14].
Correspondence to G. K. Jarori, Department of Biological Sciences,
Tata Institute of Fundamental Research, Homi Bhabha Road,
Colaba, Mumbai 400 005, India. Fax: +91 22 2280 4610,
Tel.: +91 22 2280 4545, E-mail: [email protected]
Abbreviations: DAPI, 4¢,6¢-diamidinophenylindole; PEP, phosphoenolpyruvate; 2-PGA, 2-phosphoglyceric acid; r-Pfen, recombinant
Plasmodium falciparum enolase.
Enzyme: enolase (EC 4.2.1.11).
*Present address: Section of Infectious Diseases/Internal Medicine,
Yale University, New Haven, CT 06511, USA.
(Received 4 September 2004, accepted 22 October 2004)
Keywords: enolase; homodimer; localization; Plasmodium
falciparum; purification.
FEBS 2004
4846 I. Pal-Bhowmick et al. (Eur. J. Biochem. 271)
There have been reports of antibodies to enolase detected
in high titers in Japanese and Thai P. falciparum patient sera
and use of yeast enolase for immunodiagnostic purposes
[15]. The activity of enolase in parasite-infected red blood
cells increases 15-fold [16]. The gene for P. falciparum
(strain K1) enolase (Pfen) has been cloned and characterized
[17]. However, Pfen protein has not yet been characterized.
The deduced sequence of Pfen exhibits high homology with
mammalian enolases (68–69%), but differs in containing
a plant-like pentapeptide (EWGWS), a dipeptide insertion,
and some different residues [17]. These include Cys157. The
analogous residue in Trypanosoma brucei enolase (Cys147)
has recently been shown to be modified with iodoacetamide
[18,19]. Reaction with iodoacetamide also leads to partial
inactivation of the enzyme. It will be interesting to examine
whether modification of Cys157 and other P. falciparumspecific residues in the vicinity of the active site leads to
irreversible inactivation of Pfen. Comparative studies on the
structural and kinetic properties of parasitic and mammalian enolases may provide clues for obtaining specific
inhibitors that can be developed as chemotherapeutic
reagents. To address questions related to the detailed
characterization of the molecular structure and kinetic
properties and to develop immunological reagents for
subcellular localization, we cloned Pfen and over-expressed
it in Escherichia coli to obtain adequate quantities of pure
recombinant P. falciparum enolase (r-Pfen). The results of
these experiments are presented in this paper.
Materials and methods
Materials
Taq DNA polymerase, T4 DNA ligase, endonucleases
(KpnI and PstI), 4¢,6¢-diamidinophenylindole (DAPI) and
2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid) powder were purchased from Roche Diagnostics Corp.
(Indianapolis, IN, USA). Mouse anti-His sera were from
Qiagen, Hilden, Germany. Horseradish peroxidase-conjugated anti-mouse secondary IgG was obtained from Santa
Cruz Biotech (Santa Cruz, CA, USA), and Coomassie
Brilliant Blue R-250 was acquired from USB (Cleveland,
OH, USA). Nitrocellulose membrane, dithiothreitol,
molecular mass markers used for gel filtration and Superdex-75 HiLoad 16/60 (Prep grade) column were from
Amersham Pharmacia. Oligonucleotide primers, dianilinobenzene, sodium salt of 2-PGA, rabbit muscle enolase
(b-isoform), yeast enolase, iodoacetamide, N-ethylmaleimide
and unstained high molecular mass protein markers for gel
electrophoresis were purchased from Sigma, St Louis, MO,
USA. Freund’s complete and incomplete adjuvants were
from Gibco-BRL, Alexa Fluor 488-conjugated anti-rabbit
IgG was from Molecular Probes, Inc. (Eugene, OR, USA),
and vectashield-mounting medium was from Vector Laboratories, Inc. (Burlingame, CA, USA). Maxisorp plates for
ELISA were from Nunc, Roskilde, Denmark. All other
chemicals used in this study were of analytical grade.
PCR amplification
Sense and antisense primers were designed according to the
multiple cloning sites present in the pQE30 expression
vector and the published sequence of the P. falciparum
enolase gene [17]. The two primers were: PfenoEcoRIKpnI
(32-mer) 5¢-CCGGAATTCGGTACCATGGCTCATGT
AATAAC-3¢ and PfenoPstIXhoI (30-mer) 5¢-CATTCT
CGAGCTGCAGATTTAATTGTAATC-3¢.
A gametocytic cDNA library constructed from the
NF54 strain was used for the amplification of the enolase
gene (cDNA library used here was a gift from N. Kumar,
Johns Hopkins University, Baltimore, MD, USA).
Amplification was carried out in the standard Robocycler
Gradient Stratagene machine (Stratagene, La Jolla, CA,
USA) in a reaction consisting of 400 ng of each of the
primers, 100 lM dNTP mix, pH 8.8 buffer, 2 mM MgCl2,
50 mM KCl, 0.01% gelatin, 2 U Taq polymerase and
2 lL of the template library in a final volume of 20 lL.
The amplified enolase PCR product and the pQE30
plasmid vector were digested with KpnI and PstI
restriction enzymes, and these were ligated using T4
ligase. Competent XL1Blue E. coli cells were transformed
with the ligation mixture to obtain the required recombinants, which were screened by PCR and plasmid DNA
preparation, and finally sequencing was performed (Macrogen Inc., Seoul, South Korea) using standard protocols
[20].
Expression in E. coli and preparation of crude cellular
extracts
Expression was carried out in E. coli strain XL1Blue.
Cultures transformed with recombinant plasmid were
grown in Luria–Bertani medium containing 100 lgÆmL)1
ampicillin. Cultures were induced with 0.5 mM isopropyl
thio-b-D-galactoside. Before induction, cultures were grown
at 37 C to an A600 of 0.6–0.8. For analytical studies, culture
aliquots were taken at different time intervals (0, 3, 4, 5, 6 h)
after the induction and analyzed for protein production.
The cells were pelleted by centrifugation at 5000 g for
10 min and stored at )80 C. The cells were lysed by
incubation in 50 mM sodium phosphate (10 mL per g wet
weight), pH 8.0, containing 300 mM NaCl, 1 mgÆmL)1
lysozyme and 1 mM phenylmethanesulfonyl fluoride for
30 min on ice and sonicated for six cycles, 15 s each with
15 s cooling between successive bursts at 5 output in a
Branson sonifier 450. The lysate was centrifuged at 45 000 g
for 30 min in a Beckman Ultracentrifuge (model LE-80K,
70 Ti rotor).
Affinity chromatography
His6-tagged r-Pfen was purified from soluble cell extract
using Ni-nitrilotriacetic acid affinity chromatography.
The binding was carried out by the batch method.
Soluble cell extract was mixed with Ni-nitrilotriacetic acid
(pre-equilibrated with 50 mM sodium phosphate, pH 8.0,
300 mM NaCl) slurry (8 mL per litre of culture) for 1 h
with gentle agitation. The slurry was passed through a
column and washed with 50 bed vols 50 mM sodium
phosphate, 40 mM imidazole, 300 mM NaCl, 1 mM
phenylmethanesulfonyl fluoride, 5 mM 2-mercaptoethanol,
pH 6.0, to remove nonspecifically bound proteins.
r-Pfen was eluted with 250 mM imidazole in the same
buffer.
FEBS 2004
Characterization of P. falciparum enolase (Eur. J. Biochem. 271) 4847
The oligomeric state of r-Pfen was analyzed by gel-filtration
chromatography on a Superdex-75 Hiload-16/60 column
on an Amersham-Pharmacia Biotech (Kwai Chung,
Hong Kong), AKTA FPLC system. The column was preequilibrated with 2 column vols buffer (50 mM sodium
phosphate, 150 mM NaCl, pH 7.4). Then 0.5 mg protein in
500 lL was applied to the column, and 2 mL fractions were
collected at a flow rate of 1 mLÆmin)1. The column was
calibrated using appropriate molecular mass gel-filtration
markers.
Manchester, UK), fitted with a 337-nm laser. Protein
[5 pmol in 0.5 lL 40% acetonitrile/0.1% trifluoroacetic
acid (v/v)] was mixed with an equal volume of matrix
[saturated solution of sinapinic acid in 40% acetonitrile/
0.1% trifluoroacetic acid (v/v) in water] and applied to the
MALDI target plate. This was allowed to dry at room
temperature to form cocrystals of protein and matrix. BSA
was used as an external mass standard. Single and double
charged peaks arising from BSA were used for calibration.
The operating parameters were: operating voltage, 20 kV;
sampling rate, 500 MHz; sensitivity, 50 mV. Typically
20–25 scans were averaged to obtain the spectrum.
Electrophoresis and Western blotting
Primary sequences and 3D structure modeling
Proteins were resolved on an SDS/12% polyacrylamide gel
[21] and visualized by staining with Coomassie Brilliant Blue
R-250. For Western blotting, crude cellular extracts and
purified r-Pfen separated by SDS/PAGE (12% gel) were
transferred to nitrocellulose membrane using semidry
Western transfer apparatus (Bio-Rad Laboratories, Inc.,
Hercules, CA, USA) at constant voltage (20 V) for 35 min.
The membranes were blocked with 5% skimmed milk in
phosphate buffered saline (NaCl/Pi; 137 mM NaCl, 2.7 mM
KCl, 10.0 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) containing 0.05% Tween 20 for 1 h. The blots were treated
with the mouse anti-His serum and horseradish peroxidaseconjugated anti-mouse secondary IgG, respectively
(1 : 1000 dilution for both). The immunoblots were developed using dianilinobenzene substrate.
The enolase sequences were aligned using CLUSTAL W for
homology comparisons [24]. The 3D structures of r-Pfen
and rabbit muscle enolases were modeled according to the
known 3D structure of T. brucei enolase (PDB:1OEP)
published previously, using the SWISS-MODEL server [25] and
structures were viewed with VIEWERPRO 5.0 (Accelerys, San
Diego, CA, USA).
Gel-filtration chromatography
Protein measurements and enzyme assay
Protein concentrations were determined by the Bradford
method using Bio-Rad protein assay dye reagent with
BSA as standard [22]. All kinetic measurements were made
at 20 ± 1 C. Enolase activity was measured in the forward
(formation of PEP from 2-PGA) and reverse (formation of
2-PGA from PEP) direction by monitoring the increase or
decrease respectively in PEP absorbance at 240 nm in a
continuous spectrophotometric assay on a Perkin-Elmer
lambda 40 spectrophotometer. The change in PEP concentration was determined using an absorption coefficient
(e240nm) ¼ 1400 M)1Æcm)1. As the absorption coefficient of
PEP varies with pH and concentration of Mg2+, in
experiments where pH or Mg2+ were varied, appropriate
values of molar absorptivity for PEP were used [23].
Typically, 540 lL of assay mixture containing 1.5 mM
2-PGA (for the forward reaction) or 1.1 mM PEP (for the
reverse reaction) and 1.5 mM MgCl2 in 50 mM Tris/HCl,
pH 7.4, was used. One unit of enzyme was defined as the
amount of enzyme that converts 1 lmol substrate (2-PGA
or PEP) into product (PEP or 2-PGA) in 1 min at 20 C.
Kinetic parameters were determined from [substrate] vs.
velocity curves by fitting the data to the Michaelis–Menten
equation using the SIGMAPLOT software.
MALDI-TOF analysis
For determination of the exact molecular mass of the
expressed recombinant protein, MALDI-TOF mass spectra
were recorded in linear mode on Tof-Spec 2E (Micromass,
Reaction with thiol-modifying reagents
r-Pfen or rabbit muscle enolase (0.1 lM) was placed in
buffer (1 mM KH2PO4, 5 mM MgCl2, 0.1 mM dithiothreitol
and 50 mM triethanolamine/HCl, pH 8.0) and incubated
for 30 min at 37 C. Different amounts of iodoacetamide or
N-ethylmaleimide were added to the enzyme samples and
allowed to react at 37 C. Enzyme activity was assayed at
different time intervals.
Generation of antiserum and ELISA
Standard protocols were followed to raise rabbit polyclonal
antiserum [26]. Briefly, 500 lg r-Pfen was emulsified with
Freund’s complete adjuvant and injected into a 2-monthold New Zealand White rabbit. Two boosts of 100 lg each
of the r-Pfen emulsified with incomplete Freund’s adjuvant
were given at an interval of 21 days. Ten days after the
second booster, the rabbit serum was collected. All animal
experiments were carried out as per the Guidelines of the
Committee for the purpose of control and supervision of
experiments on animals (CPCSEA), Animal Welfare
Division, Government of India. The specific immunization
experimental protocol was examined and cleared by the
Institutional Animal Ethics Committee.
For ELISA, the r-Pfen, rabbit muscle and yeast enolases
were coated (100 lL of 0.6 lM per well) on a Maxisorp plate
overnight at 4 C. Unbound antigen was removed by
washing with NaCl/Pi. The wells were blocked with 5%
skimmed milk in NaCl/Pi containing 0.05% Tween 20
(NaCl/Pi/Tween) for 1 h at 37 C. This was washed twice
with NaCl/Pi/Tween. Antibodies raised in rabbit were
diluted (2000–128 000-fold), and 100 lL of this was added
to each well. Each dilution was coated in duplicates. This
was allowed to bind to the antigens for 2 h at 37 C and then
washed 6–7 times with NaCl/Pi/Tween. To this, goat antirabbit secondary IgGs conjugated with horseradish peroxidase (1 : 2000 dilution; 100 lL per well) in NaCl/Pi/Tween
containing 0.01% BSA was added and allowed to incubate
FEBS 2004
4848 I. Pal-Bhowmick et al. (Eur. J. Biochem. 271)
for 45 min at 37 C. This was thoroughly washed with
NaCl/Pi/Tween (7–8 times). Then 100 lL of 1 mgÆmL)1
2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid), prepared in 20 mM citrate/80 mM Na2HPO4, pH 4.3, containing 1 lLÆmL)1 30% H2O2, was added to each well and
incubated for 10 min in the dark. The absorbance was read
at 405 nm on an EL808 Ultra Microplate reader (Biotek
Instruments Inc., Winooski, VT, USA).
Indirect immunofluorescence assay
An immunofluorescence assay was performed on the blood
smears obtained from Plasmodium yoelii-infected mouse as
described previously [27]. Briefly, the smears were fixed for
30 s using chilled methanol and treated with preimmune
(control) or anti-(r-Pfen) serum at a dilution of 1 : 50 at
room temperature for 1 h. This was then stained for 45 min
with Alexa Fluor 488-conjugated anti-rabbit IgG. Parasite
nuclei were stained with DAPI at a final concentration of
1 lgÆmL)1. The necessary washes were given after each
antibody incubation step, and slides were mounted under
glass coverslips in 5 lL vectashield mounting medium.
Slides were examined using a Nikon fluorescence microscope.
Results and Discussion
Clone sequence and recombinant protein purification
Native enolase from P. falciparum strain K1 [17] and
strain 3D7 (NCBI: NP_700629) are predicted to contain
446 amino acids. The PCR amplification of the enolase
gene from the gametocyte cDNA library of the NF54
strain of E. coli resulted in a fragment of the expected size
of 1.4 kb. This fragment was cloned in pQE30 vector, and
E. coli cells were transformed with the recombinant
plasmid as described above (Materials and methods).
The cloned gene was subjected to DNA sequencing, and
the full amino-acid sequence of the recombinant protein
was deduced. The amino-acid sequence was found to be
identical with the 3D7 strain. However, these two strains
differ from the K1 strain at position 131 in having an
alanine residue in place of a proline. Figure 1 shows a
comparison of amino-acid sequences of enolases from
P. falciparum strains NF54 (this work), K1 [17] and
P. yoelii (NCBI: AA1892).
The pQE30 vector is specifically designed for the overexpression of heterologous proteins in E. coli. It allows the
expression of the recombinant protein and results in the
addition of a short noncleavable His tag sequence at its
N-terminus. Cloning resulted in incorporation of an additional 18 (MRGSHHHHHHGSACELGT-) and seven
(-LQPSLIS) residues to the N-terminus and C-terminus,
respectively, of Pfen. This would yield a r-Pfen protein of
mass 51 389.73 Da in contrast with 48 677 Da for the
native enzyme.
For purification of r-Pfen, typically 1 L culture was
grown at 37 C, yielding 2 g wet cell pellet. Cells were
lysed, and the extract was subjected to centrifugation to
obtain soluble supernatant and pellet fractions. Both
fractions contained r-Pfen (Fig. 2A, lanes 1 and 2). As the
soluble fraction contained a decent amount of r-Pfen,
recombinant protein was purified from this fraction by
affinity chromatography using an agarose/Ni-nitrilotriacetic
acid column as described in Materials and methods. As
expected, most of the enolase bound to the resin, and a wash
with 40 mM imidazole removed nonspecifically bound
proteins (lanes 3 and 4 of Fig. 2A). Finally pure enolase
P. falciparum NF54 ------MAHVITRINAR---EILDSRGNPTVEVDLETNLGIFRAAVPSGASTGIYEALEL 51
P. falciparum K1
------MAHVITRINAR---EILDSRGNPTVEVDLETNLGIFRAAVPSGASTGIYEALEL 51
P. yoelii
MLVKYWLASYFMIINPKNYEHIFYSRGNPTVEVDLETTLGIFRAAVPSGASTGIYEALEL 60
:* : **.:
.*: *************.**********************
P. falciparum NF54 RDNDKSRYLGKGVQKAIKNINEIIAPKLIGMNCTEQKKIDNLMVEELDGSKNEWGWSKSK 111
P. falciparum K1
RDNDKSRYLGKGVQKAIKNINEIIAPKLIGMNCTEQKKIDNLMVEELDGSKNEWGWSKSK 111
P. yoelii
RDNDKSRYLGKGVQQAIKNINEIIAPKLIGLDCREQKKIDNMMVQELDGSKTEWGWSKSK 120
**************:***************::* *******:**:******.********
P. falciparum NF54 LGANAILAISMAVCRAGAAANKVSLYKYLAQLAGKKSDQMVLPVPCLNVINGGSHAGNKL 171
P. falciparum K1
LGANAILAISMAVCRAGAAPNKVSLYKYLAQLAGKKSDQMVLPVPCLNVINGGSHAGNKL 171
P. yoelii
LGANAILAISMAICRAGAAANKTSLYKYVAQLAGKNTEKMILPVPCLNVINGGSHAGNKL 180
************:******.**.*****:******::::*:*******************
P. falciparum NF54 SFQEFMIVPVGAPSFKEALRYGAEVYHTLKSEIKKKYGIDATNVGDEGGFAPNILNANEA 231
P. falciparum K1
SFQEFMIVPVGAPSFKEALRYGAEVYHTLKSEIKKKYGIDATNVGDEGGFAPNILNANEA 231
P. yoelii
SFQEFMIVPVGAPSFKEAMRYGAEVYHTLKSEIKKKYGIDATNVGDEGGFAPNILNAHEA 240
******************:**************************************:**
P. falciparum NF54 LDLLVTAIKSAGYEGKVKIAMDVAASEFYNSENKTYDLDFKTPNNDKSLVKTGAQLVDLY 291
P. falciparum K1
LDLLVTAIKSAGYEGKVKIAMDVAASEFYNSENKTYDLDFKTPNNDKSLVKTGAQLVDLY 291
P. yoelii
LDLLVASIKKAGYENKVKIAMDVAASEFYNSETKTYDLDFKTPNNDKSLVKTGQELVDLY 300
*****::**.****.*****************.******************** :*****
P. falciparum NF54 IDLVKKYPIVSIEDPFDQDDWENYAKLTAAIGKDVQIVGDDLLVTNPTRITKALEKNACN 351
P. falciparum K1
IDLVKKYPIVSIEDPFDQDDWENYAKLTAAIGKDVQIVGDDLLVTNPTRITKALEKNACN 351
P. yoelii
IELVKKYPIISIEDPFDQDDWENYAKLTEAIGKDVQIVGDDLLVTNPTRIEKALEKKACN 360
*:*******:****************** ********************* *****:***
P. falciparum NF54 ALLLKVNQIGSITEAIEACLLSQKNNWGVMVSHRSGETEDVFIADLVVALRTGQIKTGAP 411
P. falciparum K1
ALLLKVNQIGSITEAIEACLLSQKNNWGVMVSHRSGETEDVFIADLVVALRTGQIKTGAP 411
P. yoelii
ALLLKVNQIGSITEAIEACLLSQKNNWGVMVSHRSGETEDVFIADLVVALRTGQIKTGAP 420
************************************************************
P. falciparum NF54 CRSERNAKYNQLLRIEESLGNNAVFAGEKFRLQLN 446
P. falciparum K1
CRSERNAKYNQLLRIEESLGNNAVFAGEKFRLQLN 446
P. yoelii
CRSERNAKYNQLFRIEESLGANGSFAGDKFRLQLN 455
************:******* *. ***:*******
Fig. 1. Amino-acid sequence alignment of
enolases from P. falciparum strain NF54 with
P. falciparum strain K1 [17] and P. yoelli
(NCBI:AA18892) using CLUSTAL W [24].
Enolase from strain NF54 differs from that of
strain K1 in having a P131A mutation (shown
in bold).
FEBS 2004
Characterization of P. falciparum enolase (Eur. J. Biochem. 271) 4849
M
1
2
3
4
5
1
2
3
4
5
kDa
205
116
97
66
50 kDa
45
29
Fig. 2. Analysis of proteins from transformed E. coli XL1 Blue cells over-expressing r-Pfen. Cells were induced with 0.5 mM isopropyl thio-b-Dgalactoside for 6 h and harvested. (A) Analysis on SDS/PAGE (12% gel). Lane M, Molecular mass markers; lanes 1 and 2, insoluble and soluble
fractions, respectively, of the E. coli extract; lane 3, flow through after binding of the r-Pfen supernatant fraction to Ni-nitrilotriacetic acid; lane 4,
40 mM imidazole wash of the protein bound to Ni-nitrilotriacetic acid resin; lane 5, elution of r-Pfen with 250 mM imidazole. (B) Immunoblot of
cells over-expressing r-Pfen probed with 1 : 1000 anti-His serum. The arrow shows the position of r-Pfen.
Oligomeric state of r-Pfen
The oligomeric state of r-Pfen was examined by gel-filtration
chromatography. Figure 3 shows an elution profile of
0.5 mg r-Pfen in 500 lL 50 mM sodium phosphate/150 mM
NaCl, pH 7.4, on a Superdex-75 column. The column was
calibrated using appropriate molecular mass markers. The
apparent molecular mass determined for native r-Pfen was
100 kDa. Purified r-Pfen when analyzed on SDS/PAGE
showed a single band at 50 kDa (Fig. 2A, lane 5),
indicating that it forms a homodimer in the native state. It is
also interesting to note that, in the MALDI-TOF spectrum,
a peak was observed at m/z 102 782 corresponding to a
singly charged dimeric form of r-Pfen. Enolases from most
organisms form dimers of 40–50-kDa subunits [10,12],
exception for octameric enolases from thermophilic [12] and
sulfate-reducing bacteria [28]. The oligomeric state of none
of the apicomplexan enolases has been reported so far.
Kinetic characterization
Purified r-Pfen was assayed for enolase activity by measuring either the conversion of 2-PGA into PEP (forward
reaction) or PEP into 2-PGA (reverse reaction). The enzyme
had a specific activity of 30 ± 3 UÆ(mg protein))1 in the
forward direction and 10 ± 2 UÆmg)1 in the reverse
direction. For the determination of Km, initial reaction
rates were measured at several different concentrations of
2-PGA (Fig. 4A) and PEP (Fig. 4B). Data were fitted to the
30
OD280 (mAU)
protein was eluted with 250 mM imidazole. The eluted
protein showed a single band at the expected molecular
mass ( 50 kDa) on SDS/PAGE (Fig. 2A, lane 5). The
identity of the protein was further established by Western
blotting using anti-His serum (Fig. 2B). About 50 mg active
r-Pfen was purified from 1 L E. coli culture.
The molecular mass of the recombinant protein was also
analyzed by MS. The MALDI-TOF spectrum of purified
r-Pfen contained three peaks at m/z 25707, 51 383.04 and
102 782. The peak at m/z 51 383.04 can be attributed to a
singly charged monomeric species of r-Pfen, which is in
good agreement with the calculated average mass of
51 389.73 Da. The peak at m/z 25 707 represents a doubly
charged monomeric species, and the one at m/z 102 782 is
attributed to the presence of a singly charged dimeric species
of r-Pfen.
The r-Pfen sequence gave a theoretical absorption
coefficient (e280) of 41400 M)1Æcm)1. The concentration of
purified r-Pfen determined by Bradford assay using BSA
as standard was in good agreement with that obtained
by measuring A280 and using the theoretical absorption
coefficient.
20
10
0
40
50
60
70
Elution Volume (ml)
80
90
Fig. 3. Gel-filtration chromatogram of r-Pfen. Protein (0.5 mg in
500 lL) was run on a Superdex-75 column precalibrated using
appropriate molecular mass markers (chymotrypsinogen A, 25 kDa;
ovalbumin, 43 kDa; BSA, 67 kDa; yeast enolase, 93 kDa; alcohol
dehydrogenase, 150 kDa). Blue Dextran 2000 was used to measure the
void volume. The molecular mass obtained for r-Pfen from this
experiment was 98 ± 5 kDa.
FEBS 2004
4850 I. Pal-Bhowmick et al. (Eur. J. Biochem. 271)
50
40
B
40
Activity (milliUnits)
Activity (milliUnits)
A
30
20
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30
20
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0
0
0.0
0.2
0.4
0.6
0.8
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1.2
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1.6
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[2-PGA](mM)
20
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
[PEP](mM)
25
C
D
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Activity (milliUnits)
Activity (milliUnits)
18
16
14
12
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8
6
15
10
5
0
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
8.2
pH
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
pH
Fig. 4. Kinetic characterization of r-Pfen. (A) Plot of [2-PGA] vs. activity; and (B) plot of [PEP] vs. activity for the determination of Km. A 5 lL
sample of enzyme containing 1.5 and 3.0 lg of r-Pfen, respectively, were used for the 2-PGA and PEP assay, respectively. Experimental data were
fitted according to the Michaelis–Menten equation using SIGMAPLOT. The best fit gave Km2PGA ¼ 0.041 ± 0.004 mM and KmPEP ¼
0.25 ± 0.03 mM. pH was plotted against activity using (C) 2-PGA and (D) PEP as substrates. A 5 lL sample of enzyme containing 0.5 lg r-Pfen
was used for the 2-PGA assay and 2.5 lg was used for the PEP assay.
Michaelis–Menten equation {v ¼ Vmax[S]/(Km + [S])}
using SIGMAPLOT software. The best nonlinear fit gave
Km2PGA ¼ 0.041 ± 0.004 mM and KmPEP ¼ 0.25 ±
0.03 mM. These values for Km2PGA and KmPEP are similar
to those reported for mammalian, yeast and other enolases
[18]. The variation of r-Pfen activity as a function of pH was
also analysed. Figure 4C,D shows plots of enzyme activity
vs. pH when 2-PGA or PEP was used as substrate. Maximal
r-Pfen activity is observed in the range pH 7.4–7.6 irrespective of the substrate used. Most mammalian enolases
have their activity maxima in the range pH 6.8–7.1, whereas
the plant ones are around pH 8.0 [10].
The effect of univalent cations on the activity of r-Pfen
was also investigated. Figure 5A shows the variation in
r-Pfen activity with increasing concentrations of NaCl and
KCl. NaCl inhibits the enzyme with 50% inhibition around
0.3–0.4 M. This inhibitory effect of Na+ is very similar to
that observed for mammalian enolases [14]. In contrast,
KCl showed a slight activating effect on r-Pfen. The activity
of all three rabbit isozymes (aa, bb and cc) are significantly
stimulated (40–100%) by KCl at lower concentrations
(< 400 mM), whereas in the higher concentration range the
activation effect is lost [14]. KCl has a mild activating effect
on yeast enolase at concentrations < 200 mM, but strongly
inhibits activity at higher concentrations [14]. This kinetic
response of r-Pfen to various concentrations of KCl is at
variance to those of mammalian and yeast enolases.
Figure 5B shows the effect of increasing concentrations of
Mg2+ on the activity of r-Pfen, rabbit and yeast enolases. In
the low concentration range, Mg2+ acts as an activating
cofactor for all the enolases. Data from the low concentration range (£ 1 mM) were fitted to the Michaelis–Menten
equation to derive the apparent activation coefficient. The
activation constant derived for r-Pfen from the data
presented here is 0.18 ± 0.02 mM. Higher concentrations
of Mg2+ have an inhibitory effect on r-Pfen activity. The
maximal inhibition observed for r-Pfen is much less
(< 40%) than that observed for the yeast and rabbit
muscle enzymes (60–70%) (Fig. 5B). Previous kinetic studies have suggested the presence of three bivalent cationbinding sites on enolase, with the first two high-affinity sites
involved in activation and a third low-affinity site
involved in inhibition [13]. In the crystal structure, two
Mg2+-binding sites have been detected. These are believed
to be involved in assembly of the active site and catalysis
[29,30]. Recently, a third bivalent cation-binding site has
been identified in the structure of T. brucei enolase. It has
been suggested that binding of Mg2+ at this site may be
FEBS 2004
Characterization of P. falciparum enolase (Eur. J. Biochem. 271) 4851
A
B
Fig. 5. Effect of univalent and bivalent cations on r-Pfen activity.
(A) Effect of NaCl (d) and KCl (s). Data are plotted as percentage
activity vs. [salt]. A 540 lL volume of assay mixture containing 1.1 mM
PEP and 1.5 mM MgCl2 in 50 mM Tris/HCl, pH 7.4, was used. A 5 lL
volume of enzyme solution containing 2.5 lg enolase protein was used
for each assay. (B) A comparison of the effect of MgCl2 on the activity
of r-Pfen (d), yeast enolase (s) and rabbit muscle enolase (.). The
assay mixture consisted of 1.1 mM PEP in 50 mM Tris/HCl, pH 7.4.
The residual activity in the absence of Mg2+ is due to contaminating
bivalent cations in the assay mixture. For comparison, data for each
enzyme were normalized taking highest observed activity as 100%.
responsible for the observed inhibition at high metal ion
concentrations [19].
Homology-based structure modeling
Enolase is highly conserved across species. The overall
structure of enolase comprises an eightfold a/b barrel
domain preceded by an N-terminal a + b domain [19]. A
highly conserved catalytic site is located between the two
domains. It will be interesting to model the parasite enzyme
on the basis of the known enolase structure and examine the
structural differences between Pfen and the mammalian
enzyme in the vicinity of the conserved active site. Such an
exercise may lead to identification of parasite-specific
residue(s), which may be amenable to specific chemical
modifications and hence selective inactivation. We modeled
the 3D structure of r-Pfen and rabbit muscle enzymes on the
basis of T. brucei enolase (PDB: 1OEP) which is 60%
homologous to Pfen. Figure 6 shows the active-site regions
of these enzymes along with some of the residues in the
vicinity. In a recent study on the T. brucei enzyme, it was
shown that modification of Cys241 and Cys147 with
iodoacetamide leads to partial inactivation of the Trypanosoma enzyme [18,19]. This inactivation was attributed to the
perturbation caused to active-site structure by the addition
of a carboxamidomethyl group to Cys147 and/or Cys241.
Analogous positions in Pfen are occupied by Ala251 and
Cys157. Ala148 replaces Cys157 in Pfen in rabbit muscle
enolase. It will be interesting to examine the effect of thiolmodifying regents on r-Pfen. It is expected that similar to
Cys147 in T. brucei, Cys157 in Pfen will be carboxamidomethylated, causing partial inactivation. As the rabbit
enzyme does not have a similar Cys, it may not be affected.
To determine whether Cys157 is accessible to chemical
modification, which may lead to inactivation (similar to
T. brucei [19]), we treated the enzyme with iodoacetamide
(Fig. 6D). There was no effect on the activity of r-Pfen even
after 2 h of treatment with 10 mM iodoacetamide. As
expected, the addition of iodoacetamide to rabbit muscle
enolase also did not have any effect on the activity.
Although Cys157 occupies a position similar to Cys147 in
T. brucei (Fig. 6A,B), the microenvironment in the two
cases may be quite different. It is likely that either the
Cys157 is not accessible to iodoacetamide or the carboxamidomethyl group fits into the cavity around the Cys without
any perturbation of the arrangement of the active-site
residues. The latter possibility would suggest that the use of
larger thiol-modifying reagents (e.g. N-ethylmaleimide)
might lead to inactivation. In the case of T. brucei enolase,
complete inactivation by N-ethylmaleimide has been
observed [19]. The addition of N-ethylmaleimide to r-Pfen
did lead to partial inactivation of the enzyme (Fig. 6D).
However, similar inactivation was also observed for rabbit
enolase, which does not have analogous Cys157 near the
active site (Fig. 6C), suggesting that N-ethylmaleimideinduced inactivation is probably due to modification of
other Cys residues in the protein. Although these preliminary attempts have not succeeded in achieving speciesspecific inactivation, efforts will be made to design
substrate-based active-site-directed affinity reagent(s) for
selective inactivation of the parasite enzyme.
Reactivity and specificity of anti-(r-Pfen) evaluated
by ELISA
Antibodies raised in rabbit after two boosts of r-Pfen
protein showed quite high titer and reactivity with r-Pfen.
Reactivity was observed even at a dilution of > 64 000
(Fig. 7A). In comparison, when equimolar quantities of
rabbit muscle and yeast enolases were used as antigens,
almost no significant reactivity was observed beyond an
antiserum dilution of 1 : 16 000. To rule out the possibility
that this antiserum may contain a significant fraction of
antibodies directed against the His6 tag of r-Pfen, we used
an unrelated His6-tagged protein (rOS-F, a recombinant
odorant-binding protein from Drosophila) as control. No
significant cross-reactivity was observed against this protein
(data not shown). Although there is 61–68% homology
FEBS 2004
4852 I. Pal-Bhowmick et al. (Eur. J. Biochem. 271)
A
C
B
D
120
% Activity
100
80
60
40
20
0
20
40
60
Time (min)
80
100
Fig. 6. Comparison of the active-site regions of (A) T. brucei (PDB code 1OEP), (B) P. falciparum and (C) rabbit muscle enolase. P. falciparum and
rabbit muscle (P25704; ENOB_rabbit) enolases were modeled using the T. brucei X-ray crystallographic structure. Residues involved in substrate
and metal binding are shown in green and magenta, respectively. (D) Effect of iodoacetamide (open symbols) and N-ethylmaleimide (filled symbols)
on r-Pfen (circles) and rabbit muscle enolase (squares). Enolase (20 lg) was incubated with 10 mM iodoacetamide or 8 mM N-ethylmaleimide.
Enzyme activity was assayed at various time points.
among yeast, rabbit and P. falciparum enolases, the polyclonal antibodies raised here exhibit considerably higher
specificity for r-Pfen.
We further assessed the specificity of the antiserum by
performing an indirect immunofluorescence assay on blood
smears obtained from P. yoelii-infected mice. The gene
sequences of enolase from murine malarial parasite, P. yoelii
and P. falciparum, exhibit 90% identity and 94% similarity
in their amino-acid sequences (Fig. 1). On the basis of such a
large sequence homology, it is expected that polyclonal
antibodies raised against r-Pfen would cross-react with the
P. yoelii enolase protein. As shown in Fig. 7B, the immune
serum reacted with the parasite-infected mouse red blood
cells and not with uninfected red blood cells. The parasiteinfected cells can be identified by using DAPI staining. As
uninfected red cells do not have a nucleus, they do not pick
up DAPI. DAPI-positive cells (parasite-infected) are the
only ones stained by anti-(r-Pfen). All the erythrocytic stages
of the parasite (rings, trophozoites and schizonts) reacted to
anti-(r-Pfen). A control immunofluorescence assay experiment was also performed using preimmune rabbit serum.
As expected, no staining of the parasite-infected cells was
observed (Fig. 7C). These experiments also demonstrate
that anti-(r-Pfen) sera did not have any cross-reactivity
towards the mammalian red blood cell enolase protein.
Conclusions
We have cloned and developed an over-expression system for
P. falciparum enolase. This has allowed us to obtain decent
amounts of pure protein (50–60 mg per litre of culture). The
measured physicochemical parameters (molecular mass and
absorption coefficient at 280 nm) for the expressed protein
are in good agreement with those predicted on the basis of the
cloned sequence. The presence of a 50-kDa band on SDS/
PAGE for purified r-Pfen and 100 kDa on gel-filtration
FEBS 2004
Characterization of P. falciparum enolase (Eur. J. Biochem. 271) 4853
and the fact that they fail to react with mammalian enolases
(Fig. 7B). This recombinant protein is highly immunogenic,
as only two booster doses were sufficient to give titers of
> 1 : 64 000 for specific reactivity with the antigen. This
polyclonal antibody is being used to investigate subcellular
localization of enolase at different stages in the life cycle of the
parasite. The availability of large quantities of r-Pfen will also
facilitate structural investigations on this apicomplexan
glycolytic enzyme.
2.0
A 405 nm
1.6
a
1.2
b
0.8
c
0.4
Acknowledgements
0.0
2,000
4,000
8,000
16,000
32,000
64,000
128,000
Antiserum (fold dilution)
We are grateful to Dr Nirbhay Kumar of Johns Hopkins University,
Baltimore, MD, USA for the gift of k Orient P. falciparum strain NF54
gametocyte asexual stage library. We thank Mr Prateek Gupta and
Mr Yogesh Gupta for help with some of the experiments.
References
a: anti-r-pfen
a: pre-immune
b: DAPI
b: DAPI
Fig. 7. Specificity of polyclonal antibodies raised against r-Pfen in
rabbit. (A) ELISA reactivity of anti-(r-Pfen) with (a) r-Pfen, (b) rabbit
muscle enolase and (c) yeast enolase, measured as A405 and plotted
against increasing dilutions of antibody. (B) Immunofluorescence
assay with P. yoelii-infected mouse red blood cells treated with (a)
anti-(r-Pfen) serum (1 : 50 dilution) and (b) DAPI (1 lgÆmL)1).
(C) P. yoelii-infected cells were treated with (a) preimmune sera
(1 : 50 dilution) and (b) DAPI (1 lgÆmL)1).
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forms an active homodimer similar to the enolases from
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presence of a peak at m/z 102 782 in the MALDI spectrum.
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enolases in its extent of inhibition caused by high Mg2+
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muscle and P. falciparum exhibit a high degree of sequence
homology (67–69%), antibodies raised against r-Pfen in
rabbit are quite specific, as evident from ELISA (Fig. 7A)
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