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 9
Trang 1Cloning, over-expression, purification and characterization of
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 falciparumstrain 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
sequen-cing 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 Km2PGA¼ 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 falciparumprotein
Keywords: enolase; homodimer; localization; Plasmodium falciparum; purification
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
drug-resistant phenotypes, the requirement of alternative
pre-ventive 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 selec-tively 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 metallo-enzyme that catalyzes the inter conversion of 2-phospho-glyceric 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: gkj@tifr.res.in
Abbreviations: DAPI, 4¢,6¢-diamidinophenylindole; PEP,
phospho-enolpyruvate; 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)
Trang 2There 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
falciparum-specific 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
mamma-lian 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)
pow-der were purchased from Roche Diagnostics Corp
(Indianapolis, IN, USA) Mouse anti-His sera were from
Qiagen, Hilden, Germany Horseradish
peroxidase-conju-gated 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
Super-dex-75 HiLoad 16/60 (Prep grade) column were from
Amersham Pharmacia Oligonucleotide primers,
dianilino-benzene, 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
Labora-tories, 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 lMdNTP mix, pH 8.8 buffer, 2 mMMgCl2,
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 recomb-inants, which were screened by PCR and plasmid DNA preparation, and finally sequencing was performed (Mac-rogen Inc., Seoul, South Korea) using standard protocols [20]
Expression inE 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 37C to an A600of 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 mMsodium 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 mM2-mercaptoethanol,
pH 6.0, to remove nonspecifically bound proteins r-Pfen was eluted with 250 mM imidazole in the same buffer
Trang 3Gel-filtration chromatography
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
pre-equilibrated with 2 column vols buffer (50 mM sodium
phosphate, 150 mMNaCl, 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
Electrophoresis and Western blotting
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 mMNaCl, 2.7 mM
KCl, 10.0 mM Na2HPO4, 1.8 mMKH2PO4, pH 7.4)
con-taining 0.05% Tween 20 for 1 h The blots were treated
with the mouse anti-His serum and horseradish
peroxidase-conjugated anti-mouse secondary IgG, respectively
(1 : 1000 dilution for both) The immunoblots were
devel-oped using dianilinobenzene substrate
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 ± 1C 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
concen-tration was determined using an absorption coefficient
(e240nm)¼ 1400M )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 mMPEP (for the
reverse reaction) and 1.5 mM MgCl2in 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 20C
Kinetic parameters were determined from [substrate] vs
velocity curves by fitting the data to the Michaelis–Menten
equation using theSIGMAPLOTsoftware
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,
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 Primary sequences and 3D structure modeling 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 theSWISS-MODELserver [25] and structures were viewed withVIEWERPRO5.0 (Accelerys, San Diego, CA, USA)
Reaction with thiol-modifying reagents r-Pfen or rabbit muscle enolase (0.1 lM) was placed in buffer (1 mMKH2PO4, 5 mMMgCl2,0.1 mMdithiothreitol and 50 mM triethanolamine/HCl, pH 8.0) and incubated for 30 min at 37C Different amounts of iodoacetamide or N-ethylmaleimide were added to the enzyme samples and allowed to react at 37C 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-month-old 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 lMper well) on a Maxisorp plate overnight at 4C 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 37C 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 37C and then washed 6–7 times with NaCl/Pi/Tween To this, goat anti-rabbit secondary IgGs conjugated with horseradish peroxi-dase (1 : 2000 dilution; 100 lL per well) in NaCl/Pi/Tween containing 0.01% BSA was added and allowed to incubate
Trang 4for 45 min at 37C 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),
pre-pared in 20 mMcitrate/80 mMNa2HPO4, pH 4.3,
contain-ing 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
micro-scope
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 over-expression 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 addi-tional 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 37C, 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).
Trang 5protein 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 41400M )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
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 mMsodium 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 measur-ing 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))1in 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
0 10 20 30
Elution Volume (ml)
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.
205 116 97
M 1 2 3 4 5 1 2 3 4 5
66
45 29
kDa
50 kDa
Fig 2 Analysis of proteins from transformed E coli XL1 Blue cells over-expressing r-Pfen Cells were induced with 0.5 m M isopropyl thio-b- D -galactoside 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 m M imidazole wash of the protein bound to Ni-nitrilotriacetic acid resin; lane 5, elution of r-Pfen with 250 m M imidazole (B) Immunoblot of cells over-expressing r-Pfen probed with 1 : 1000 anti-His serum The arrow shows the position of r-Pfen.
Trang 6Michaelis–Menten equation {v¼ Vmax[S]/(Km+ [S])}
using SIGMAPLOT software The best nonlinear fit gave
Km2PGA¼ 0.041 ± 0.004 mM and KmPEP¼ 0.25 ±
0.03 mM These values for Km2PGAand KmPEPare 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
irres-pective 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
0.3–0.4M 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 m , 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 concentra-tion 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 stud-ies have suggested the presence of three bivalent cation-binding 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
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0
10 20 30 40
6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2
6
8
10
12
14
16
18
20
pH
5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 0
5 10 15 20 25
pH
[PEP](mM)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
0
10
20
30
40
50
[2-PGA](mM)
A
D
B
C
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 m M and KmPEP ¼ 0.25 ± 0.03 m M 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.
Trang 7responsible 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 Trypano-somaenzyme [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 thiol-modifying regents on r-Pfen It is expected that similar to Cys147 in T brucei, Cys157 in Pfen will be carboxamido-methylated, 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 carboxam-idomethyl 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-ethylmaleimide-induced inactivation is probably due to modification of other Cys residues in the protein Although these prelim-inary attempts have not succeeded in achieving species-specific 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 His6tag 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
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 m M
PEP and 1.5 m M MgCl2 in 50 m M 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 m M PEP in 50 m M Tris/HCl, pH 7.4.
The residual activity in the absence of Mg 2+ is due to contaminating
bivalent cations in the assay mixture For comparison, data for each
enzyme were normalized taking highest observed activity as 100%.
Trang 8among yeast, rabbit and P falciparum enolases, the
poly-clonal 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 yoeliienolase 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
parasite-infected 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 experi-ment 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 falciparumenolase 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
20 40 60 80 100 120
Time (min)
D
C
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 m M iodoacetamide or 8 m M N-ethylmaleimide Enzyme activity was assayed at various time points.
Trang 9chromatography suggests that, in its native state, r-Pfen
forms an active homodimer similar to the enolases from
several other sources [10,12] This is further supported by the
presence of a peak at m/z 102 782 in the MALDI spectrum
Kinetic measurements showed substrate affinity to be similar
to that of mammalian enolases r-Pfen differs from rabbit
enolases in its extent of inhibition caused by high Mg2+
concentration (Fig 5B) and inability of K+to activate it
significantly (Fig 5A) [14] Although enolases from rabbit
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)
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
Acknowledgements
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.
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