molecular analysis of plasmodium falciparum hexokinase

Molecular and Biochemical Parasitology, 56 (1992) 89-102 89 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00

MOLBIO 01830

Molecular analysis of Plasmodium falciparum hexokinase

P6tur Olafsson, Hugues Matile and Ulrich Certa Department PRTB, F. Hoffmann-La Roche, Ltd., Basel, Switzerland

(Received 12 May 1992; accepted 23 July 1992)

Hexokinase, a key glycolytic enzyme, is involved in the initial phosphorylation reaction of imported glucose and specific blocking of this activity may therefore arrest the development of malaria parasites. We describe here the cloning of a single copy hexokinase gene of Plasmodium falciparum (PfHK) from cDNA or genomic DNA libraries. The deduced amino acid sequence of PfHK has 26% identity with human hexokinase I and its predicted molecular mass assigns it as an invertebrate type isoenzyme of hexokinase. A single 1.5-kb exon is translated from a 3-kb mRNA in asexual stages of the parasite. In contrast to aldolase and GPI, the gene for this glycolytic enzyme is located on chromosome 8. Poly- and monoclonal antibodies against recombinant PfHK support our cloning results at the protein level as they detect a protein of the predicted size and isoelectric point by Western blotting in parasite protein samples. Moreover, polyclonal rabbit lgG against recombinant PfH K partially inhibits the hexokinase activity of a P. falciparum lysate which provides direct proof that the gene cloned encodes hexokinase of the parasite.

Key words: Glycolysis; Hexokinase; Chromosome mapping; Chemotherapy

Introduction

Glycolysis is defined as an evolutionarily conserved pathway in which one molecule of glucose is cleaved in multiple steps into 2 molecules of pyruvate coupled with the generation of metabolic energy. The lack of a functional tricarboxylic acid (TCA) cycle [1] gives this pathway elevated importance in malaria parasites. The many-fold increased glucose consumption of Plasmodium falciparum infected erythrocytes [2] may ex- plain why some of the known glycolytic

Correspondence address: P6tur Olafsson, Dept. PRTB, F. Hoffmann-La Roche, Ltd., Grenzacherstr. 124, CH-4002 Basel, Switzerland.

Note." Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M92054.

Abbreviations: PfHK, Plasmodium falciparum hexokinase; GP1, glucosephosphate isomerase; PGK, phosphoglycerate kinase; PCR, polymerase chain reaction; IEF, isoelectric focusing.

enzymes of the parasite have different biochemical properties from the host equivalents [3,4].

The first enzyme of the glycolytic pathway is hexokinase (ATP:D-hexose 6-phosphotransfer- ase, E.C. 2.7.1.1.) which phosphorylates hex- oses in the initial glycolysis reaction. Its catalytic properties may have been optimized in plasmodia for increased glucose turnover. This assumption is consistent with the obser- vation that the hexokinase activity increases proportional with the parasitemia in Plasmo- dium falciparum cultures [4] and a higher Michaelis-Menten constant (Km) of PfHK [4]. These biochemical requirements make it likely that the primary structure differs from the human enzyme.

In higher vertebrates tissue specific hexokinases (isoenzymes) with different substrate specificities and kinetic properties have been described. They are probably required for optimal substrate turnover in the biochemical environment of a given tissue (for review see ref. 5). Although the life cycle of malaria

90

parasites is rather complex, it is surprising that all cloned glycolytic enzymes of P. falciparum are encoded by single copy genes, suggesting the absence of isoenzymes [6-8]. One exception may be GPI, which is believed to undergo post- translational modifications resulting in the appearance of 3 isoenzymes encoded by a single gene [9]. A considerable amount of biochemical and structural information about the reaction mechanism of hexokinase is provided by the work on yeast and mammalian enzymes. With the exception of isoenzyme IV (glucokinase), isoenzymes I, II and III in humans have arisen from a gene duplication and fusion event followed by independent evolution [10]. As a result mammalian hexokinases have twice the molecular weight of their invertebrate counterparts.

We describe here the cloning of a single hexokinase gene (PfHK) from cDNA and genomic DNA libraries of P. falciparum. We detect substantial primary sequence differences in PfHK compared to the human isoenzyme. The gene lacks any detectable intron/exon structures and is located on chromosome 8. Experiments with monoclonal or polyclonal antibodies against recombinant PfHK prove that the gene cloned encodes hexokinase of P. falciparum.

Materials and Methods

Parasites. P. falciparum isolates used in this study were K l, MAD-20, RO-33, Palo Alto, RO-71, NF-54, and FCR-3. The rodent parasites Plasmodium berghei (ANKA strain), Plasmodium yoelii (strain 17X), and Plasmo- dium chabaudi (strain AS) were used for blotting experiments. Parasite-infected blood was cultured by the method of Trager and Jensen [11] with some modifications [12].

DNA isolation J?om parasites. Saponin lysates containing about l01° parasites were used for DNA isolation as described elsewhere [13].

RNA isolation from parasites. About l01° parasites from a saponin lysate were suspend-

ed in 1 ml RNA-Zol T M (Cinna/Biotecx, Houston, TX). After 2-3 h on ice, 100 #l CHC13 was added per ml of solution and incubated on ice for 15 rain. After centrifugation at 12000 rev. min -~ for 5 min at 4°C, 2 vols. of isopropanol were added to the supernatant. After 1 h at - 2 0 ° C the RNA was pelleted (10 rain, 4°C, 12000 rev. min-~). The washed and dried pellet was suspended in 1 mM EDTA/0.5% SDS. The poly(A) + fraction was obtained using a commercial oligo(dT) ~ system following the instructions of the supplier (Pharmacia).

Polymerase chain reaction analysis. Degenerated oligonucleotide primers deduced from hexokinase amino acid consensus sequences were used to amplify the appropriate fragment by the polymerase chain reactior (PCR) utilizing commercial reagents and ar automated thermocycler (Perkin-Elmer). PrioJ to cycling, 0.5 #g of the genomic DN,~ template was denatured for 5 rain at 96°£ followed by 40 cycles of amplification (dena. turation 1 min at 96°C, annealing 2 min a 50°C, extension 3 rain at 72°C).

Primers used in this study had the followin~ sequences: Degenerate HK-1, 5'-AAT GTV~ GAY ACW GTW GGW ACW-3' (Fig. 2, nl 925-945). Degenerate HK-2, 5'-WGC WG(~ WCC YTT WCC WGA WCC ATC YTC-3 (Fig. 2, nt 1641-1615). Adapted HK-1, 5'-ATG TTG ATA CTG TTG GTA CTC TTA TGT- 3' (Fig. 2, nt 926-952). Adapted HK-2, 5'-CTG CTC CTT TWC CWG AWC CAT-3' (Fig. 2, nt 1639-1619). HK-3, 5'-ATG AGT GAG TAC GAT ATT GCA AAA AAT GAT GTA ACA-Y (Fig. 2, nt 211 246). HK-4, 5'-TTA TGG TAA TTG TGG AAT GTC CGC ATT TAA-Y (Fig. 2, nt 1692-1663). HK-lv, 5'- (AT)4 AGA TCT AGT GAG TAC GAT ATT GCA AAA AAT GAT GTA ACA-3' (Fig. 2, nt 21~246). HK-IH, 5'-(AT)4 AAG CTT AAT GAG GAA TCC ATA AAT TTG G-3' (Fig. 2, nt 433~415). HK-IIH, 5'-(AT)4 AAG CTT AAA ATC CAA CAC TCT TCA CTT CTT TA-3' (Fig. 2, nt 750-726). HK-IIIH, 5'-(AT)4 AAG CTT GTT ATG GTA ATT GTG GAA TGT CCG CAT TTA A-3' (Fig. 2, nt 1692-

1663). PGKv, 5'-ATG TTA GGC AAC AAA TTA AGC ATT AGT GAC-3' (ref. 7, nt 15- 44). PGKH, 5'-TTT GTT TGA AAG TGC TAA TAC ACC TGG TAA-3' (ref. 7, nt 1233- 1262). p41v, 5'-TTT TCC ATG GCT CAT TGC ACT GAA TAT ATG AAT-3' (ref. 8, nt 319-341). p41H, 5'-GTG CCA TGG TTA ATA GAC ATA TTT CTT TTC-3' (ref. 8, nt 1414-1443). Prior to PCR, the 5' ends of the primers were phosphorylated using T4-poly- nucleotide kinase.

Hybridization conditions. Nucleic acid blot- or plaque lift filters were prehybridized in 4 x SSC containing dolfed Denhardt 's reagents and 50 ~g m l - " denatured calf thymus carrier DNA [14] for 2 h at 60°C. Hybridization with 106 cpm m l - ~ was carried out overnight under the same conditions. The filters were washed 3 x 30 min in 2 x SSC/0.1% SDS at 60°C

followed by overnight exposure to Kodak X-o- mat AR film with intensifying screen.

Northern blot. About 5 pg/lane of total K1 RNA or 0.5 #g/lane poly(A) + K1 RNA were used for Northern blots. Probes used for hybridization were nick translated P. Jalciparum PCR fragments of either hexokinase (primers HK-1/2), aldolase (primers p41 v/ H), or phosphoglycerate kinase (primers PGKv/H).

Screening cDNA libraries. Two P. fah'iparum cDNA libraries were screened: library PG of isolate M25 [8] and library JH of isolate K1 in 2 NMl149 (obtained from John Hyde, UMIST, Manchester, UK). 40000 recombinant phage of the M25 library were screened with a nick-translated PCR probe (primer pair HK-1/2). Four positive clones (cP2-4/5/6/7) were amplified and the inserts were subcloned into M13mpl8 [15]. The radiolabeled insert of clone cP2-5 was used for the screening of 40 000 plaques of the K1 cDNA library. Three positive clones were named cH-4/6/8.

Construction and screening of genomic DNA libraries o f isolate K1. 5 /~g of EcoRI methylated genomic DNA (K1) were partially

91

digested with DraI, ligated to EcoRI linkers, redigested with EcoRI and separated on a 1% agarose gel. Fragments ranging from 2-6 kb were isolated by an anion exchange membrane technique (NA 45, Schleicher and Schuell). The fragments were ligated to dephosphorylated 2gtl l EcoRI arms (Promega Biotech) and packaged using a commercial in vitro packag- ing system (Protoclone T M 2gtll system; Pro- mega). 60000 recombinant plaques were screened with the radiolabeled PCR fragment HK-3/4 yielding 5 positive clones. Two clones (PfHK-2 and PfHK-3) were selected for further processing and turned out to be identical. To clone additional 5' sequences, we made an additional EcoRV/HincII 'bookshelf' library ranging from 3-5 kb (expected 'Southern- blot' fragment 4.0 kb). Screening of 60000 recombinant phage yielded 2 positive clones (PfHK-4 and PfHK-5); one (PfHK-4) was selected for sequencing and therefore an appropriate restriction fragment was subcloned into M 13mp 18.

Subcloning and DNA sequence analysis. DNA sequence analysis was performed by subcloning appropriate restriction fragments in M13mpl8 and generating nested exonuclease III deletions [16] for dideoxy sequencing [17]. All other DNA manipulations were performed according to standard protocols [14]. Restric- tion enzymes were all purchased from New England Biolabs, Inc.

Southern blot. 5 #g of genomic DNA of P. falciparum isolates K1, MAD-20, RO-33, Palo Alto, RO-71 and NF-54 or o fP . berghei and P. chabaudi were digested to completion with several restriction enzymes. Southern blotting was done as described [13] using the nick- translated 5' DraI fragment of PCR product HK-3/4. For low stringency probing, the blots were boiled in water for 5 min followed by rehybridization with the same probe at 50°C.

Chromosome blot. Samples (about 108 parasites/lane) were prepared as previously described [18]. Running conditions were: (1) 72 h/5 min pulse/100 V (90 130 mA)/14°C; (2) 48

92

h/10 min pulse/100 V (900130 mA)/14°C; (3) 48 h/15' min pulse/100 V (90-130 mA)/14°C. The gel was then stained with ethidium bromide, destained and photographed. Prior to blotting the gel was soaked 30 min in 0.3 N HC1, 45 min in denaturing solution (0.25 N NaOH/0.75 M NaC1) and 2 x 30 min in 1 M NH4Ac/0.02 N NaOH. The blot was probed with the same probes used for Northern blotting and additionally with a nick-translated hsp70 [19] probe as a specific marker for chromosome 8 [20].

Expression o f hexokinase in E. coli. We used PCR primer pairs HK-Iv/In (for hexokinase aa 2-75), HK-Iv/IIn (for aa 2-180) and HK- Iv/IIIn (for aa 2-493) with 0.5 pg genomic K1 DNA as template to amplify the appropriate sequences for expression. Utilising the restriction sites included in the PCR primers (BamHI, HindIII) the fragments were cloned into the vector pDS 781 containing the regulatable promoter/operator element PN25*/ 0 [21] and the synthetic ribosomal binding site RBSII [22]. The constructs pDS781-HKI, pDS781-HKn and pDS781-HKToT (coding for the proteins 6xHis-DHFR-HKaa2_75, 6xHis-DHFR-HKaa2_180 and 6xHis-DHFR- HKaa2_493, respectively) were used to trans- form E. coli/pDMI.1. Expression of the DHFR fusion proteins was monitored as described [21].

Nickel chelate chromatography. E. coli cells expressing recombinant proteins were harves- ted by centrifugation and lysed for 1 h at RT in 6 M guanidinium hydrochloride/0.1 M NaH2PO4. After centrifugation the supernatant was loaded onto a NTA column [23] (nickel-nitrilotriacetic acid) followed by washes with 8 M urea/0.1 M NaH2PO4/0.01 M Tris- HC1 at different pHs as described [24]: pH 8.0, pH 6.3, pH 5.9 and pH 4.5. The recombinant fusion protein is recovered in the pH 5.9 fraction. After analysis of the fractions by SDS-gel electrophoresis appropriate fractions were pooled, dialysed against H20 and lyophi- lized, yielding 15 mg of purified recombinant DHFR fusion protein per liter of culture.

Polyclonal antibodies. A rabbit was immu- nized 3 times with a 50-#g dose of the DHFR- HKToT fusion protein in complete Freund's adjuvant. IgG was purified by conventional protein A chromatography using Biorad's Econosystem cartridge and the instructions supplied.

Monoclonal antibodies. Monoclonal antibodies were produced against purified DHFR- HKI and DHFR-HKn fusion proteins. BALB/ c mice were given 3 s.c. injections (5 #g/ injection, in CFA) at one-week intervals. Three days after the last injection, the local lymph nodes were excised and the fusions were made with the PAI cell line (a nonsecretor variant of the P3-X63Ag8 myeloma). The hybridoma supernatant was screened by Western blotting (whole K1 parasite lysates). Bound antibodies were detected using horseradish peroxidase- conjugated goat anti-mouse IgG (H+ L) (Nordic Immunologicals). From each fusion specific hybridomas were cloned, leading to 4 lines producing anti-PfHK monoclonal antibodies (mAb).

Hexokinase extraction and activity assay. Parasite saponin iysates were extracted with 1 vol. of 10 mM monothioglycerol (MTG) followed by 3 cycles of freeze thawing. The extract was centrifuged for 15 min at 4°C and 14000 rev. min -1 and the supernatant was withdrawn. Hexokinase activity was deter- mined colorimetrically essentially as described [25]. The same procedure was used to prepare uninfected erythrocyte control lysates. Yeast hexokinase was purchased from Boehringer, FRG.

Isoelectric focusing. IEF was carried out as described [9] using commercial precast gel plates (LKB-Pharmacia; pH range 3.5-9.5). IEF markers came from the same supplier.

Western blot. Western blotting of recombinant and parasite proteins was carried out as described [26]. Culture supernatants (Hex-l.7/ 2.6/3.6/3.9) were used at a 1:10 dilution for monoclonal antibodies and the rabbit serum

was used at a l:1000 dilution. Proteins of IEF gels were blotted by capillary transfer (2 h).

Indirect immunoJluoreseence. IFA was performed on acetone-fixed parasite samples as described using serial dilutions of polyclonal rabbit serum [13].

Results

Cloning strategy. Based on conserved amino acid sequence blocks from known hexokinases we designed a degenerate oligonucleotide primer pair for the initial PCR amplification of the PfHK gene. In a control experiment using yeast genomic DNA as template the primer pair amplified a unique band of 771-bp DNA, which is consistent with the predicted size (data not shown; ref. 27). Using P. falciparum genomic DNA as template under the same conditions the specificity of the PCR was reduced as four bands ranging from about 0.2-1.4 kb in size appeared as the major reaction products (data not shown). The

93

uncertainty concerning the P. falciparum hexokinase gene and its product made it necessary to analyze these PCR products separately. We first focused on the 0.7-kb consensus fragment by subcloning and sequencing assuming that the PfHK gene lacks introns in that region. The DNA of 5 independent clones encoded an open reading frame of 239 amino acids. Although amino acid alignment of this candidate clone (PfHK- 1) with the equivalent part of yeast HK-A [27] gave 28% identity we considered this information insufficient to conclude that the amplified DNA fragment is part of a PfHK gene.

PfHK detects a 3-kb mRNA transcript. In order to detect gene transcription of clone PfHK-1 we probed Northern blots with the PfHK candidate clone using either total RNA or poly(A) ~ RNA isolated from asexual blood stage parasites. In total RNA, the probe detects a major 3-kb transcript and traces of an additional 2.0 kb RNA band (Fig. IA, lane 1). In the poly(A) + fraction only the major 3- kb transcript is detected suggesting that the

A 1 2 B 1 2 3

9 , 5 _ _

7 . 5 _ 7 . 5 - -

4 . 4 _ 4 4 - -

T e 2+, 1 . 4 _

1 . 4 _

0 . 2 _ _

Fig. I. Northern blot analysis of clone PfHK-I. (A) 5/ tg total (lane 1) or 0.5 ~g polyadenylated RNA (lane 2) were probed with a nick translated PfHK-1 probe. The molecular weight and position of single stranded RNA markers is indicated at the left. Note that the minor 2-kb transcript is not detected in the mRNA fraction. (B) Total K1 RNA was probed with either

PGK (lane 1), aldolase (lane 2), or PfHK-1 (lane 3) probes. Markers are indicated as in (A).

94

A~ TAA

DNAI Io Dtl AI DBJI ~ !I~ JI I I I B~p D

cDNA KI

M25

TATTATATATATATATTTATATATATATATATATATATATATATATATTTTTTTTTT~A 60

T~TTG~TTTGATCCTTATATCATATATAAATATTAGGTT~GCACATAAATTTTTTTA 120

TTGCCTTAGATAAATATAGGAAAAT~G~TATCTACATATATACATAGAGT~TTATTT 180 ..............................

ACCATATACTTTTCTGTATTT~TTTAAA~TGAGTGAGTACGATATTGCAAAA~TGAT 240 M S E Y D I A K N D i0

GT~CATATACC~GTTAGATAC~TAG~TGTGATATACC~TAAATG~GAGTTATCA 3OO V T Y T K L D T I E ~ D I P I N E E L S 30

TGGAG~TT~TAAATTTGTG~TCAGTT~G~TATCTTATTCAACATTAG~G~TTT 360 W R I N K F V N Q L R I S Y S T L E E F 50

GTAGAC~TTTTGTATATG~TTA~GAAAGGTTTAG~GCTCATCGTA~CATCCAAAT 420 V D N F V Y E L K K G L E A H R K H P N 70

TTATGGATTCCTCATGAGTGTAGTTTT~GATGTTGGATTCATGTATTGCTAATATTCCT 480 L W I P H E ~ S F K M L D S ~ I A N I P 90

ACTGGTC~GAG~GGGTACATATTATGCTATAGATTTCGGTGGTACT~TTTTCGAGCT 540 T G Q E K G T Y Y A I D F G G T N F R A I I 0

V R A S L D G K G K I K R D Q E T Y S L I 3 0

AAATTTACAGGATCCTATTCTCATGAGA~GGTTTATTAGATAAGCATGC~CAGCATCA 660 K F T G S Y S H E K G L L D K H A T A S I 5 0

C~TTA~TGATCATTTTGCTGAAAG~TTAAATATATTATGGGGG~TTT~TGATTTA 720 Q L F D H F A E R I K Y I M G E F N D L I 7 0

GAT~T/LAAG~GTG~GAGTGTTGGATTTACCTTTTCTTTCCCTTGTACATCACCTTCA 780 D N K E V K S V G F T F S F P ~ T S P S I g 0

m m m i m i m ATT~TTGTTC~TTCTTATTGATTGGACAAAGGGATTTGAAACAGGTAGAGCTACG~T 840 I N ~ S I L I D W T K G F E T G R A T N 2 1 0

GATCCAGTAG~GGTCGTGACGTA~GTA~TT~TG~TGATGCTTTTGT~GAGCTGCT 900 D P V E G R D V ~ K L M N D A F V R A A 2 3 0

ATTCCAGCGAAAGTATGTTGTGTATTG~TGATGCTGTTGGTACTCTTATGTCATGTGCA 960 I P A K V ~ V L N D A V G T L M S ~ A 2 5 0

TaTC~WG~TAGAaGTACCCCaCCa~T~ATAGGTATTa~TAC~T 1 0 2 0 Y Q K G R G T P P ~ Y I G I I L G T G S 270

~TGGTTGTTATTATG~CCTG~TGG~GAAATAT~GTATGCTGG~ATTAT~AT 1080 N G ~ Y Y E P E W K K Y K Y A G K I I N 290

ATCG~TTTGGT~TTTTGATAAAGATTTACCTACATCACCCATCGATTTAGTTATGGAT 1140 I E F G N F D K D L P T S P I D L V M D 310

TGG~CAGCT~TCGTAGTAGAC~TTGTTTG~TGATATCTGGTGCTTACTTA 1200 W Y S A N R S R Q L F E K M I S G A Y L 330

GGTG~TCGT~G~GAT'I~ATGGTA/~TGTTT~AC~kAGTGCATGTTCTA~GATG 1260 G E I V R R F M V N V L Q S A ~ S K K M 350

TGGATTAGTGATAGTTTC~TTCAG~TCTGGTAGTGTTGTATTAAATGATACTTCAAAA 1320 W I S D S F N S E S G S V V L N D T S K 370

~T~TG~GATAGTAGGAAAGTTGCT~GGCTGCTTGGG TATGGATTTTACTGATG~ N F E D S R K V A K A A W ~ M D F T D E 138°390

CA~T~ATGTCTTACGTAA~TTTGTG~GCTGTATAT~TAGATCTGCAGCTCTTGCT 1440 Q I Y V L R K I ~ E A V Y N R S A A L A 410

CGTGGTACTATAGCTGCTATAGCTAAAAG~TCAAAATTATTG~CATTCTAAATTTACT 1500 R G T I A A I A K R I K I I E H S K F T 430

TGTGGTGTTGATGGCTCC~ATTTGTTAA~TGCTTGGTATTGTAAAAGATTAC~G~ 1560 ~ G V D G S L F V K N A W Y ~ K R L Q E 458

CATTTA~GTTATCTTAGCTGACAAAGCTGA~ATTT~TTATTATTCCAGCAGATGAT 1620 H L K V I L A D K A E N L I I I P A D D 470

GGTTCAGG~.~.GGGAGCAGCCATCACAGCAGCAGTCATCGCATT~TGCGGACATTC~A 1680 G S G K G A A I T A A V I A L N A D I P 490

Q L p *** 493

minor RNA has a reduced affinity for the poly(dT) affinity resin. (Fig. 1A, lane 2). This result indicates that PfHK-1 is part of an actively transcribed gene. In addition, the amount of PfHK transcripts in total RNA is in the same range as the transcripts of either PGK [7] or aldolase [8] (Fig. I B).

Isolation and analysis of PfHK cDNA clones. The results above made it likely that the m R N A detected can be isolated from c D N A libraries. We therefore probed c D N A of 2 P. /'alciparum isolates with a PfHK-1 probe assuming that the gene sequence of hexokinase is conserved. In both libraries positive signals appeared and the inserts of the clones were subcloned and partially sequenced. Three clones of the K1 library (cH-4/6/8) had identical insert sequences and, in addition, typical poly(A) tails. Comparison of the deduced amino acid sequence with known hexokinases defines the encoded protein with high probability as a hexokinase (Fig. 2). Lack of a putative start codon in the K1 c D N A clones made it likely that the 5' end with the initiation site was not cloned yet and was possibly located further upstream. The 5' sequence lacking in the K1 c D N A clones was found in the positive clones isolated from the M25 c D N A library (cP2-4/5/6/7). In contrast to the K1 clones, the M25 clones included a conventional ATG initiation codon and a typical AT-rich upstream sequence. The following 33 nucleotides downstream of the candidate start codon are different in the

Fig. 2. Complete nucleotidc and deduced amino acid sequences of the P. Jalciparum hexokinase gene. A partial restriction map of the hexokinase gene with sites relevant for this study appears on top followed by a schematic presentation of the clones used to obtain the sequence shown below (A, Accl; B, BamHI; Bg, B~,lll; C, Clal; D, Dral; E, EcoRV; K, Kpnl; P, PstI). Four consensus adenosine residues [40] proceeding the ATG initiation codon are underlined and an asterisk triplet marks the stop codon. The 33 nucleotide sequence different in the M25 and K1 c D N A clone is overlined by dashes and cysteine residues are double underlined. Conserved amino acid residues located in functional hexokinase consensus

domains are marked with a black square.

M25 and K1 cDNAs (Fig. 2). An elevated GC content in the K1 sequence suggested artefacts during the reverse transcription of K1 RNA especially since the downstream coding sequence is entirely identical in M25 and K1 (data not shown). This potential artefact made it necessary to isolate genomic DNA clones from K1. An additional benefit of this approach is the detection of potential intron/ exon structures and possibly the isolation of clones encoding P. falciparum hexokinase isoenzymes.

Analysis of PfHK clones from genomic K1 libraries. The occurrence of only 2 DraI restriction sites in the coding sequence of the K1 cDNA clones made it likely that the fragment containing the initiation site can be isolated from a partial Dral or a gene specific "bookshelf' library of genomic K1 DNA (see Materials and Methods). After screening of the libraries with a 5' end specific probe we isolated and sequenced clones containing that region. As expected, the genomic K1 sequence was identical to the M25 cDNA around the putative start codon, confirming a reverse transcription error. In addition, the downstream sequence of the genomic clones (PfHK- 2/3/4/5) was identical to the cDNA clones for either K1 or M25. The single exon of PfHK is embedded in AT-rich non-coding sequences. As in other P. falciparum genes, the ATG initiation codon is preceded by a stretch of 4 consensus adenosine residues [28] and a eukaryotic TATA-box is located about 30 nucleotides upstream of the initiation codon (Fig. 2). A putative parasite polyadenylation consensus signal (AATAAA) is lacking at the expected position [7]. The single exon encodes a 54.3-kDa protein which classifies the hexokinase of P. falciparum as an invertebrate type isoenzyme.

Genomic organization of the hexokinase gene. Isoenzymes can be detected at the genetic level by restriction site polymorphism and low stringency hybridizations [29]. We prepared Southern blots with genomic K1 DNA digested with various restriction en-

A

2 3 0

9.42

6 .6 - 4 4 "

2.3

2 0

95

1 2 3 4 5 6 7 8 9 10 11 12 13 !4 15 16 17 18 19 20

B 1 2

2 3 0 9 4 6.6 4 4 0 ~

2.3 2 0

0 5 -

e

= ' e

3 4 5 6

D

7 8

Fig. 3. Southern blot analysis of the hexokinase gene. (A) Restriction enzyme digested genomic K1 DNA was probed with the 5' Dral fragment of the PfHK gene (PCR product HK-3/4). The enzymes used were Dral (lane 1), EcoRV (lane 2), Eco47111 (lane 3), HaellI (lane 4), Hindll (lane 5), Hpal (lane 6), NaeI (lane 7), NruI (lane 8), Pvull (lane 9), Scal (lane 10), SnaBl (lane 11), SspI (lane 12), Stul (lane 13), EcoRV/SnaBI (lane 14), EcoRV/Haelll (lane 15), EcoRV/Hindll (lane 16), EcoRV/Hpal (lane 17), EcoRV/ ScaI (lane 18), SnaBl/Hpal (lane 19)and SnaBl/Scal (lane 20). The DNA in tracks 7 and 8 was degraded probably due to nuclease contamination of NaeI and Nrul. (B) Sspl digested genomic DNA of 6 P. [alciparum isolates (lanes 1 6), P. berghei (lane 7) and P. chabaudi (lane 8) was probed with the same probe as in (A). The P. jalciparum isolates used were K1 (Thailand; lane 1), MAD-20 (Papua New Guinea; lane 2), RO-33 (Ghana; lane 3), Palo Alto (Uganda; lane 4), RO-71 (Ghana; lane 5) and NF 54 (Amsterdam Airport, origin unknown; lane 6). In lanes 3 and 6 the 0.5-kb Sspl fragment appears only after overexposure due to insufficient amounts of DNA loaded.

96

zymes. A banding pattern consistent with a single gene appears after hybridization at high stringency with a hexokinase probe (Fig. 3A). No additional bands appeared when the blot was washed and rehybridized with the same probe at 50°C or 40°C (data not shown). This is consistent with the cloning results and we conclude that hexokinase is a single copy gene in P. falciparum. The result independently confirms the lack of hexokinase isoenzymes in this organism [4].

We next examined possible PfHK polymorphism by probing restricted genomic DNA from 6 geographically independent P. falciparum isolates with the PfHK Kl-probe. The banding pattern detected was identical, suggesting that no major polymorphism occurs in this essential gene (Fig. 3B). In addition, the probe hybridizes to the same extent with DNA ofP . berghei and P. chabaudi (Fig. 3B, lanes 7 8). The different banding pattern suggests an altered gene structure in the rodent malarias although the primary sequence may not

deviate drastically from P. falciparum.

Chromosome mapping oJ" the hexokinase gene. The previously described GPI [6] and aldolase [8] genes of P..lalciparum are both localized on the stable chromosome 14. This genetic link opens the possibility that the genes for the first glycolytic enzymes catalyzing 6 carbon sugar reactions are organized in an operon like cluster. We directly tested that model by probing chromosome blots with hexokinase or aldolase and PGK [7] or hsp70 [19] as chromosomal markers. However, both the hexokinase and the hsp70 probe label chromosome 8 in two strains (Fig. 4). Thus the glycolytic enzymes of P. Jalciparum are not organized in a transcription unit.

Reactivity o1" poly- and monoclonal antibody against recombinant P.. /alciparum hexokinase. For the current and future studies we wished to raise monoclonal antibodies for the detection of hexokinase in parasite samples.

1 2 3 4

r~ 2 r, r, 2 r, .~

h ~p t() p41 I ' ( , K H k

Fig. 4. Chromosome mapping of the hexokinase gene. Chromosome preparations of P. Jalciparurn isolates K I and FCR-3 were separated by pulse field electrophoresis (stained gel, left). After transfer, filter strips with 2 samples were probed with

different genes (right): hsp70 (blot 1), aldolase (p41; blot 2), PGK (blot 3) and hexokinase (blot 4).

1 2 3 4

106.0 -- ~

80.0 _

4 9 . 5 _

32 .5_

2 7 . 5 _

18.5_ ~

Fig. 5. Western blotting of whole parasite lysates. Parasite proteins were separated by SDS-page, electroblotted and probed with mouse monoclonal antibody Hex-3.9. Isolates used for this blot were KI (lane I), MAD-20 (lane 2), P. herghei (lane 3) and P. yoelii (lane 4). Numbers indicate the position and molecular weight of marker proteins in kDa.

We expressed parts of the coding region in Escherichia coli as a D H F R fusion protein using the inducible pDS vector system [22]. Expression yield after affinity purification by nickel chelate chromatography was about 15 mg recombinant protein per liter of culture with an estimated homogeneity of roughly 80% (data not shown). The purified recombinant protein was insoluble and had no detectable hexokinase activity (data not shown). The protein preparation was used to immunize a rabbit or mice to produce polyclonal rabbit or monoclonal mouse antibodies (mAb). Four mAbs were obtained which detect a single band of the expected size by Western

97

blotting of blood stage parasite lysates of K l and MAD-20 (54.3 kDa; Fig. 5, lanes 1 and 2). In contrast to the polyclonal rabbit serum, the mAbs detect the antigen only in the denatured state in Western blots (data not shown). The polyclonal rabbit serum appears monospecific for P. falciparum hexokinase as reacts with neither leukocytes nor uninfected erythrocytes by indirect immunofluorescence (data not shown). Interestingly, only one of the 4 monoclonal antibodies (Hex-3.9) reacted with a considerably larger polypeptide in the 2 rodent malaria parasites P. berghei and P. yoelii (Fig. 5, lanes 3 and 4). The cross- reactivity of P. falciparum DNA or antibody probes with the rodent malaria parasites indirectly suggests that the primary sequence and epitope structure of hexokinase is probably conserved in these organisms.

Detection and inhibition of active parasite derived hexokinase by polyclonal rabbit IgG against recombinant P. falciparum hexokinase. For future studies, we wished to estab- lish if the polyclonal rabbit serum against insoluble recombinant PfHK interacts with the parasite derived enzyme in the non-denatured active state. We first focused a lysate containing active hexokinase of P. falciparum by IEF. Yeast hexokinase and noninfected erythrocytes were included as controls. Hexokinase was then detected either by activity staining or by Western blotting. As calculated from the deduced amino acid sequence, PfHK activity is focused at pH 7, a region of the gel which is also detected by IgG against recombinant PfHK (Fig. 6, panel A and B). Only marginal hexokinase activity focusing at pH 6 was detected in a sample of non-infected human erythrocytes (data not shown).

Since the antibodies against recombinant PfHK interact with the parasite derived enzyme in its native state, we measured hexokinase activity in the presence and absence of purified IgG against PfHK or P. falciparum aldolase as control [8]. Inhibition of hexokinase activity increases proportional to the amount of PfHK IgG added reaching a maximum at 50% inhibition (Fig. 7). No

9 8

A B 1 2 3

® ® 9 . 6 - - 9 . 6 - -

8.2 _-- " . 8.2 - -

8.0 8.0 - -

7 . 8 - - ~ ~ " 7 . 8 - -

7 . 5 - - ~ 7 .5 - -

7.1 - - 7.1 - 7 . 0 - 7 . 0 - -

6 . 5 - - 6 . 5 -

6 . 0 - - 6 . 0 - -

4 . 7 c : ~ , ~ 4 . 7

1 2 3

Fig. 6. Detection of native P. falciparum hexokinase by activity staining and Western blotting. Samples with active P. falciparum (lane 1) or yeast (lane 3) hexokinase were electrofocused followed by activity staining (A) or antibody probing with polyclonal IgG against recombinant PfHK (B). The strong signal in lane 1 (and 3) at pH 7.5 is an artefact of loading and the band between pH 7.5 and 7.1 is not a positive band but visible (red) hemoglobin. Lane 2 contains IEF marker proteins, some

of which are visible after focusing and during Western blotting.

inhibition occurred with the same amounts of aldolase IgG included in the assay (data not shown). Finally, these data provide definite proof that the gene cloned and analyzed encodes hexokinase of P. falciparum.

Discussion

Using a PCR technique, we have cloned a single gene for hexokinase, a key glycolytic enzyme of P. falciparum. The complete gene is composed of one exon located on chromosome 8 which encodes a protein of 54.3 kDa. Monoclonal antibodies against recombinant PfHK detect a single protein of the expected size in asexual blood stage parasite lysates by Western blotting. Polyclonal rabbit serum against recombinant PfHK partially inhibits

the hexokinase activity of a parasite lysate. In Fig. 1 we show that the PfHK gene is

actively transcribed at levels comparable with aldolase or PGK, 2 other glycolytic enzymes of P. falciparum. For these genes, about half of the transcript length would be sufficient to encode the exons and the additional, presum- ably untranslated regions, may thus contain regulatory or signal sequences. Such oversized mRNAs have also been found in rat brain hexokinase and mammalian glucokinase [30,31]. Aldolase expression, for example, is developmentally regulated reaching a peak in the schizont stage of P. falciparum [32]. The messenger RNA levels, however, remain un- changed during blood stage development which suggests indirectly a post-transcription- al control mechanism of aldolase gene expression (U. Certa, unpublished results).

e-

a 0

x

" I "

1.0

0.5

I i i i i i i I i

/-

0.1

I ! I I I I I I t

0 5 10

Incubation time (minutes)

Fig. 7. Inhibition of native PfHK activity by polyclonal lgG against recombinant PfHK. Constant amounts of an active parasite lysate were assayed colorimetrically for hexokinase activity. Open circles indicate the activity in the absence of inhibitory lgG. The activity is inhibited by increasing amounts of rabbit IgG against recombinant PfHK included in the assay (black dots, 2 pg; open triangles, 10 pg; black triangles, 100 pg). Black squares show the hexokinase activity present in sample of uninfected erythrocyte using the same amount of soluble

protein (80/~g). Values are plotted in A units.

The minor 2.0-kb RNA band detected by the PfHK probe is most likely a breakdown product of the mature transcript (Fig. 1A, lane 2). It is not present in the polyadenylated RNA fraction, and both the M25 and K1 derived cDNA clones represent truncated transcripts (see Fig. 2, top). Knowledge and analysis of the complete non-coding sequences of the glycolytic enzyme transcripts may provide insight into the regulation of these genes, as their expression appears to underly similar control mechanisms.

The hexokinase structural gene has certain properties typical for glycolytic enzyme genes of P. falciparum. It is a single copy gene and the structure appears highly conserved within P. falciparum and similar genes are detected in

99

P. berghei and P. chabaudi (Fig. 3). The non- coding flanking sequences have an elevated AT content of more than 90% which decreases to 68.4% in the exon due to GC-rich codons. Unlike other mapped genes for glycolytic enzymes, PfHK is located on chromosome 8 [6-8]. A bacterial operon-like organization of these genes is, thus, ruled out.

Until recently, glycolytic enzymes of P. falciparum were considered poor targets for rational anti-malarial agents [33]. In particular, it was supposed that the sequence and structure of these enzymes is quite similar to those of the host because they catalyze the same enzymatic reactions. Cloning of the glucose phosphate isomerase, phosphoglycerate kinase, and aldolase genes of P. falciparum revealed that the homology of these proteins to the human equivalents is surprisingly low [6- 8]. The differences in the primary structures of human and malarial aldolases, for example, are directly reflected in different catalytic properties [32]. As shown here, hexokinase is another example of a glycolytic enzyme which is different from that of the host. The overall amino acid identity is only 26.2%. As expected, the highest homology scores appear in functionally essential domains such as the putative ATP [34] and glucose binding sites [35,36] (Fig. 2). In the aligned human [37] and parasite hexokinase amino acid sequences only 5 out of 15 cysteine residues appear at the same relative position (data not shown). The deduced amino acid sequence lacks a signal peptide but contains a cluster of hydrophobic residues at the carboxy terminus. The opposite situation is found for the human hexokinase isozyme I where a potential mitochondrial membrane anchor sequence is present at the amino terminus [38,39]. The polyclonal antibodies described here will be useful tools to determine the cellular localization of P. falciparum hexokinase in cell fractions or in situ by using immune gold electron micro- scopy. These monoclonal antibodies against P. falciparum hexokinase fail to cross-react with erythrocyte hexokinase by immunofluorescence or Western blotting. The epitopes recognized are therefore parasite-specific,

100

which apparently reflects the detected primary sequence and structural differences at the immunological level. In addition, polyclonal rabbit IgG against recombinant PfHK inhibits the hexokinase activity of parasites lysates. We thus expect that PfHK specific drugs can be developed which would block the first step in the glycolysis of P. falciparum.

Acknowledgements

We thank Dr. J. Hyde for the gift of the K1 cDNA library and a PGK clone and Dr. P. Ghersa for the M25 cDNA library. We are especially grateful to Y. Bfirki, P. Petitjean, A. Soederberg, C. Feil and G. Kloepfer for expert technical assistance and Dr. A2. Skar- phed-insd6ttir (University of Reykjavik) and Prof. R.M. Franklin (Biocentre, University of Basel) for critical reading of the manuscript and suggestions.

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molecular analysis of plasmodium falciparum hexokinase

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