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 a nd purified enolase from Plasmodium falciparum strain NF54 in Escherichia coli in active form, as an N- terminal His 6 -tagged protein. The sequence o f the cloned enolase f rom the NF54 strain is identical to that of s train 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 agre ement w ith t h e mass calculated from the sequence. The molecular mass of r-Pfen determined in gel-filtration experiments was  100 k Da, indicating that P. falciparum enolase is a homodimer. Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of  30 UÆmg )1 and K m2PGA ¼ 0.041 ± 0.004 m M .The Michaelis constant for the reverse reaction (K mPEP )is 0.25 ± 0.03 m M . pH-dependent activity measurements gave a maximum at pH 7.4–7.6 irrespective o f the direction of catalysis. The activity of this enzyme is inhibited by Na + , whereas K + has a slight activating effect. The cofactor Mg 2+ has an apparent activation constant of 0.18 ± 0.02 m M . However, at higher co ncentrations, it has an inhibitory effect. P olyclonal antibody raised against pure recombinant P. falciparum enolase in rabbit showed high specificity towards recombinant protein and is a lso a ble to recognize enolase from the murine malarial parasite, Plasmodium yo elii, which shares 90% id entity with the P. falc iparum protein. Keywords: enolase; homodimer; localization; P lasmodium falciparum; purification. Malaria remains one of the most infectious diseases in the third world with abou t 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 i n uninfected cel ls, and t he activity of many of the glycolytic enzymes is higher in t he 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 wo rk 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 phospho enolp yruvate (PEP), h as not yet been characterized. Enolases are highly conserved across species [9]. In most species, i t exists a s a symmetric homodimer [10]. However, in several bacterial species, octameric enolases have been reported [11,12]. Conservation is particularly pronounced for the active-site r esidues, leading to similar kinetic properties among enolases from diverse sources. F or activity, enolase r equires the binding of 2 m ol bivalent cations (in vivo this is usually Mg 2+ )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 + ,whereasthe 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: e nolase (EC 4.2.1.11). *Present address: Section of Infe ctious Diseases/Internal Medicine, Yale Unive rsity, New Haven, CT 0 6511, USA. (Received 4 September 2004, ac cepted 22 Oc tober 2004) Eur. J. Biochem. 271, 4845–4854 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04450.x There have been repo rts 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, P fen p rotein has not yet been c haracterized. The d educed sequence o f Pfen exhibits high homology with mammalian enolases (68–69%), but differs in containing a plant-like pentapeptide ( EWGWS), a d ipeptide insertion, and s ome different residues [17]. These i nclude Cys157. T he analogous residue in Trypanosoma b rucei enolase (Cys147) has recently b een shown to be m odified with iodoacetamide [18,19]. Reaction with iodoacetamide also leads to partial inactivation o f t he enzyme. It w ill be interesting t o e xamine 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 k inetic properties of parasitic and m amma- 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 p resented in this paper. Materials and methods Materials Taq DNA polymerase, T4 DNA ligase, endonucleases (KpnIandPstI), 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 s econdary 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 p rotein 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 w as from Molecular Probes, I nc. (E ugene, 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 a nd 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 w ere: PfenoEcoRIKpn I (32-mer) 5¢-CCGGAATTCGGTACCATGGCTCATGT AATAAC-3¢ and PfenoPstIXhoI (30-mer) 5¢-CATTCT CGAGCTGCAGATTTAATTGTAATC-3¢. A gametocytic cDNA library constructed from the NF54 strain was u sed 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 s tandard Robocycler Gradient S tratagene machine (Stratagene, La Jolla, CA, USA) in a reaction consisting of 400 ng of each of the primers, 100 l M dNTP mix, pH 8.8 buffer, 2 m M MgCl 2 , 50 m M 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 KpnIandPstI 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 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 m M isopropyl thio-b- D -galactoside. Before induction, cultures were grown at 37 °CtoanA 600 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 5 000 g for 10 min and stored at )80 °C. The cells were lyse d by incubation in 50 m M sodium phosphate (10 mL per g wet weight), pH 8.0, containing 300 m M NaCl, 1 mgÆmL )1 lysozyme and 1 m M phenylmethanesulfonyl fluorid e for 30minoniceandsonicatedforsixcycles,15seachwith 15 s cooling between successive bursts at 5 output in a Branson sonifier 450. The lysate was centrifuged at 45 000 g for 3 0 min in a Beckman Ultracentrifuge (model LE-80K, 70 Ti rotor). Affinity chromatography His 6 -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 mixe d with Ni-nitrilotriacetic acid (pre-equilibrated with 50 m M sodium phosphate, pH 8 .0, 300 m M 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 m M sodium phosphate, 4 0 m M imidazole, 300 m M NaCl, 1 m M phenylmethanesulfonyl fluoride, 5 m M 2-mercaptoethanol, pH 6.0, to remove nonspecifically bound proteins. r-Pfen was eluted with 250 m M imidazole in the same buffer. 4846 I. Pal-Bhowmick et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Gel-filtration chromatography The oligomeric st ate 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 w ith 2 column vols buffer ( 50 m M sodium phosphate, 1 50 m M NaCl, p H 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 .Thecolumnwas calibrated using appropriate molecular mass gel-filtration markers. Electrophoresis and Western blotting Proteins were resolved on an SDS/12% polyacrylamide g el [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 n itrocellulose membrane using semidry Western transfer apparatus (Bio-Rad Laboratories, Inc., Hercules, C A, USA) at constant voltage (20 V) f or 35 min. The membranes were blocked with 5% skimmed milk in phosphate buffered saline (NaCl/P i ;137m M NaCl, 2 .7 m M KCl, 10.0 m M Na 2 HPO 4 ,1.8m M KH 2 PO 4 , pH 7.4) con- taining 0.05% Tween 20 for 1 h. The blots were treated with the mouse anti-His s erum and hors eradish peroxidase- conjugated anti-mouse secondary IgG, respectively (1 : 1000 dilution for both). The immunoblots w ere d evel- 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 s tandard [22]. All kinetic m easurements w ere made at 20 ± 1 °C. Enolase activity was measured i n the forward (formation of PEP from 2-PGA) a nd reverse (formation of 2-PGA from P EP) direction by monitoring the increase o r decrease respectively in PEP absorbance at 240 nm in a continuous spectrophotometric assay on a Perkin-Elmer lambda 40 spectrophotometer. T he change in PEP concen- tration was determined using an absorption coefficient (e 240nm ) ¼ 1400 M )1 Æcm )1 . As the absorption coefficient of PEP varie s with pH a nd concentration of Mg 2+ ,in experiments where pH or Mg 2+ were varied, appropriate values of molar absorptivity for PEP were used [23]. Typically, 540 lL of assay mixture containing 1.5 m M 2-PGA (for the forward reaction) or 1.1 m M PEP (for the reverse reaction) and 1.5 m M MgCl 2 in 50 m M 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 t he Michae lis–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 mo de 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) i n water] and applied to the MALDI t arget plate. This was allowed to dry at room temperature to form cocrystals of p rotein and matrix. BSA was used as an external mass standard. S ingle 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, u sing the SWISS - MODEL server [25] and structures were viewed wi th VIEWERPRO 5.0 (Accelerys, S an Diego, CA, USA). Reaction with thiol-modifying reagents r-Pfen or rabbit muscle enolase (0.1 l M ) was placed in buffer (1 m M KH 2 PO 4 ,5m M MgCl 2, 0.1 m M dithiothreitol and 50 m M 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 a t different time intervals. Generation of antiserum and ELISA Standard protocols were followed to r aise rabbit polyclo nal antiserum [ 26]. B riefly,  500 lg r -Pfen w as emulsified w ith Freund’s complete adjuvant and injected into a 2-month- old N ew Zealand White rabbit. Two boosts of 100 lgeach of the r -Pfen e mulsified with incomplete Freund’s adjuvant were given at an interval of 21 days. Ten days after the second booster, the r abbit 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, t he r -Pfen, r abbit muscle and yeast enolases were coated ( 100 lLof0.6 l M per well) on a Maxisorp plate overnight at 4 °C. Unbound antigen was removed by washing with NaCl/P i . The wells were blocked with 5% skimmed milk in NaCl/P i containing 0.05% Tween 20 (NaCl/P i /Tween) for 1 h at 37 °C. This was washed twice with NaCl/P i /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 wasallowedtobindtotheantigensfor2 hat37 °Candthen washed 6–7 times with NaCl/P i /Tween. T o this, goat anti- rabbit secondary I gGs c onjugated with horseradish peroxi- dase (1 : 2000 dilution; 100 lL p er well) in NaCl/P i /Tween containing 0.01% BSA was added a nd al lowed t o i ncubate Ó FEBS 2004 Characterization of P. falciparum enolase (Eur. J. Biochem. 271) 4847 for 45 m in at 37 °C. This was thoroughly washed with NaCl/P i /Tween (7 –8 times). Then 1 00 lLof1mgÆmL )1 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid), pre- paredin20m M citrate/80 m M Na 2 HPO 4 , pH 4.3, contain- ing 1 lLÆmL )1 30% H 2 O 2 ,wasaddedtoeachwelland incubated f or 10 min in t he dark. T he absorbance was read at 405 nm on an EL808 Ultra Microplate reader (Biotek Instruments Inc., Winooski, V T, USA). Indirect immunofluorescence assay An imm unofluorescence assay w as performed o n 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 an ti-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 glasscoverslipsin5lL 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 f ragment of t he expected size of 1.4 kb. This fragment was cloned in pQE30 vector, and E. coli cells were transformed w ith 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. falc iparum 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 a ddi- 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 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 m M 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 fr om P. falciparum strain NF54 with P. falciparum strain K1 [17] and P. yoelli (NCBI:AA18892) u sing CLUSTAL W [24]. Enolase from s train N F54 diff ers fro m that of strain K1 in having a P131A mutation (shown in bold). 4848 I. Pal-Bhowmick et al.(Eur. J. Biochem. 271) Ó FEBS 2004 protein was eluted with 250 m M 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 f urther established b y Western blotting using anti-His serum (Fig. 2B). About 50 mg active r-Pfen was purified from 1 L E. coli culture. The m olecular m ass 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 a ttributed 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 (e 280 ) of 41400 M )1 Æcm )1 . The concentration of purified r-Pfen determined by Bradford assay using BSA as standard was in good agreement with that obtained by measuring A 280 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 -P fen in 500 lL50m M sodium phosphate/150 m M NaCl, pH 7.4, o n a Superdex-75 column. The column was calibrated u sing appropriate molecular mass markers. The apparent molecular mass determined for native r-Pfen was  100 kDa. Purified r-Pfen when a nalyzed 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 f or enolase activity b y measur- ing either t he 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 K m , initial reaction rates were measured at several different concentrations of 2-PGA (Fig. 4A) and PEP (Fig. 4B). Data were fitted to the 40 50 60 70 80 90 0 10 20 30 Elution Volume (ml) OD 280 (mAU) Fig. 3. Gel-filtration chromatogram of r-Pfen. Protein (0.5 mg in 500 lL) was r un on a Superdex-75 column precalibrated using appropriate molecular mass markers ( chymotrypsin ogen 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 experimentwas98±5kDa. 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 B lue c ells 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, respect ively, of the E. coli extract; lane 3, flow through after binding of the r-Pfen supernatant fraction to Ni-nitrilotriacetic a cid; 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 prob ed with 1 : 1000 anti-His serum. Th e arrow shows the position of r-Pfe n. Ó FEBS 2004 Characterization of P. falciparum enolase (Eur. J. Biochem. 271) 4849 Michaelis–Menten equation {v ¼ V max [S]/(K m +[S])} using SIGMAPLOT software. The best nonlinear fit gave K m2PGA ¼ 0.041 ± 0.004 m M and K mPEP ¼ 0.2 5 ± 0.03 m M . These values for K m2PGA and K mPEP are similar to those r eported f or mammalian , yeast and other enolases [18]. The variation of r-Pfen activity as a function of pH was also analysed. F igure 4 C,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 inh ibits the enzyme w ith 50% inhibition around 0.3–0.4 M . This inhibitory e ffect of Na + is very similar to that observed for mammalian enolases [14]. In contrast, KCl showed a s light activating effect on r-Pfen. T he activity of all three rabbit isozymes (aa, bb and cc) a re significantly stimulated (40–100%) by KCl at lower concentrations (< 400 m M ), whereas in t he higher concentration range the activation e ffect i s lost [14]. KCl has a mild activating eff ect on yeast enolase at concentrations < 200 m 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 Mg 2+ on the activity of r-Pfen, rabbit and yeast enolases. In the low concentration range, Mg 2+ acts as an activating cofactor for all the enolases. D ata from the low c oncentra- tion range (£ 1m M ) 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 m M . Higher concentrations of Mg 2+ 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 o n enolase, with the first two h igh-affinity sites involved in activation and a third low-affinity site involved in inhibition [13]. In the crystal structure, two Mg 2+ -binding sites h ave 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 i n the structure of T. brucei enolase. It has been suggested that binding of Mg 2+ at this site may be 0.00.20.40.60.81.01.21.41.6 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 Activity (milliUnits) p H 5.56.06.57.07.58.08.5 9.0 0 5 10 15 20 25 pH Activity (milliUnits) [PEP](mM) Activity (milliUnits) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0 10 20 30 40 50 Activity (milliUnits) [2-PGA](mM) A D B C Fig. 4. Kinetic c haracterization of r -Pfen. (A) Plot o f [2-PGA] vs. a ct ivity; and (B) plot of [PE P] vs. activity for the determ ination of K m .A5lL sample of enzyme containing 1.5 and 3.0 lg of r-Pfen , respect ively, were u sed for the 2-PGA and PEP assay, respectively. Experimental data were fitted according to the Michaelis–Menten equation using SIGMAPLOT . The best fit gave K m2PGA ¼ 0.041 ± 0.004 m M and K mPEP 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 f o r the 2 -PGA assay and 2.5 lg was used for the PEP assay. 4850 I. Pal-Bhowmick et al.(Eur. J. Biochem. 271) Ó FEBS 2004 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 b y a n N-terminal a + b domain [19]. A highly conserved catalytic site is located between the two domains. It w ill be interesting to model t he parasite enzyme on the basis of the known enolase structure and examine the structural differences between Pfen and t he mammalian enzyme in the vicinity of the conserved a ctive site. Such an exercise may lead to identification of parasite-specific residue(s), which may be amenable to specific chemical modifications and hence selective i nactivation. 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 t he 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 C ys241 and Cys147 with iodoacetamide leads to partial inactivation o f the Trypano- soma enzyme [18,19]. This inactivation was attributed to the perturbation caused to active-site structure by the addition of a c arboxamidomethyl 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 t o 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 e ffect on the activity of r-Pfen even after 2 h of treatment with 10 m M 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 o r the carboxam- idomethyl group fits into the cavity ar ound the Cys without any perturbation of the arrangement of the active-site residues. T he latter possibility would s ugge st that the use of larger thiol-modifying reagents (e.g. N-ethylmaleimide) might lead to inactivation. In the c ase of T. brucei enolase, complete inactivation by N-ethylmaleimide has been observed [19]. The addition of N-ethylma leimide t o r -Pfen did lead to partial inactivation of the enzyme (Fig. 6D). However, similar inactivation w as also observed for rabbit enolase, which does not have analogous Cys157 near the active site (Fig. 6 C), 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 e ven at a dilution of > 64 000 (Fig. 7A). I n comparison, when equimolar quantities of rabbit muscle and yeast enolases were u sed 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 t he His 6 tag of r-Pfen, we used an unrelated His 6 -tagged protein (rOS-F, a recombinant odorant-binding protein from Drosophila) as control. No significant cross-reactivity was observed against this pr otein (data not shown). Although there is 61–68% homology A B Fig. 5. Effect of univalent and bivalent cations on r-Pfen activity. (A) E ffect of NaCl (d)andKCl(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 MgCl 2 in 50 m M Tris/HCl, pH 7.4, was used. A 5 lL volume of enz yme solution containing 2.5 lg enolase protein was used for each assay. (B) A comparison of the effect of MgCl 2 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 r esidual activity i n the absence of Mg 2+ is due to contaminating bivalent cation s i n the assay m ixture. For compariso n, d ata for each enzyme were norm alized taking highest observed activity a s 100%. Ó FEBS 2004 Characterization of P. falciparum enolase (Eur. J. Biochem. 271) 4851 among 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 b lood smears obtained from P. yoelii -infected mice. The gene sequences of enolase from murine malarial parasite, P. yoelii and P. falciparum, exhibit 90% i dentity a nd 94% s imilarity 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 c ross-react with the P. yoelii enolase protein. A s s hown in Fig. 7B, the i mmune serum reacted with the parasite-infected mouse red blood cells and not with unin fected red blood cells. T he parasite- infected cells can be identified by using DAP I staining. As uninfected r ed 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 a nd 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. falc iparum 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 A B 010020 40 60 80 20 40 60 80 100 120 Time (min) % Activity 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 ra bbit muscle enolase (squares). E nolase (20 lg) was i ncubate d with 10 m M iodoacetamide or 8 m M N-ethylmaleimide. Enzyme activity was assayed at vari o us time points. 4852 I. Pal-Bhowmick et al.(Eur. J. Biochem. 271) Ó FEBS 2004 chromatography 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 Mg 2+ concentration (Fig. 5B) and inability of K + to activate it significantly (Fig. 5A) [14]. A lthough e nolases 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. 7 B). This recombinant protein is highly immunogenic, as only two booster doses were sufficient to give titers of > 1 : 6 4 000 for specific reactivity with the antigen. 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( 1994) Chelation of serine 39 to Mg 2+ latches a gate at the active-site o f enolase: structu re of the bis (Mg 2+ ) complex of yeast enolase and the intermediate analog phosphoacetohydroxamate at 2.1 A ˚ resolution. Biochemistry 33 , 9333–9342. 30. Kuhnel, K . & Luisi, B.F. (2001) Crystal structure o f t he E s cher- ichia coli RNA degradosome component enolase. J. Mol. Biol. 313, 5 83–592. 4854 I. Pal-Bhowmick et al.(Eur. J. Biochem. 271) Ó FEBS 2004 . Cloning, over-expression, purification and characterization of Plasmodium falciparum enolase Ipsita Pal-Bhowmick, K. Sadagopan,. have been repo rts of antibodies to enolase detected in high titers in Japanese and Thai P. falciparum patient sera and use of yeast enolase for immunodiagnostic