Expression and Functional Characterization of a Plasmodium falciparum Ca2+-ATPase (PfATP4) Belonging to a Subclass Unique to Apicomplexan Organisms*

Sanjeev KrishnaDagger §, Charles WoodrowDagger , Richard WebbDagger , Jeff PennyDagger , Kunio Takeyasu||, Masatsugu Kimura**, and J. Malcolm EastDagger Dagger

From the Dagger  Department of Infectious Diseases, St. George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, United Kingdom, || Faculty of Integrated Human Studies, Kyoto University, Sakyo-ku, Kyoto 606, Japan, ** Laboratory of Biophysics, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno-ku, Osaka 545-8585, Japan, and Dagger Dagger  Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, United Kingdom

Received for publication, November 22, 2000, and in revised form, December 21, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have obtained a full-length P type ATPase sequence (PfATP4) encoded by Plasmodium falciparum and expressed PfATP4 in Xenopus laevis oocytes to study its function. Comparison of the hitherto incomplete open reading frame with other Ca2+-ATPase sequences reveals that PfATP4 differs significantly from previously defined categories. The Ca2+-dependent ATPase activity of PfATP4 is stimulated by a much broader range of [Ca2+]free (3.2-320 µM) than are an avian SERCA1 pump or rabbit SERCA 1a (maximal activity < 10 µM). The activity of PfATP4 is resistant to inhibition by ouabain (200 µM) or thapsigargin (0.8 µM) but is inhibited by vanadate (1 mM) or cyclopiazonic acid (1 µM). We used a quantitative polymerase chain reaction to assay expression of mRNA encoding PfATP4 relative to that for beta -tubulin in synchronized asexual stages and found variable expression throughout the life cycle with a maximal 5-fold increase in meronts compared with ring stages. This analysis suggests that PfATP4 defines a novel subclass of Ca2+-ATPases unique to apicomplexan organisms and therefore offers potential as a drug target.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Apicomplexan organisms in the genera Plasmodium, Toxoplasma, Cryptosporidium, Eimeria, and Babesia are intracellular pathogens of great medical and veterinary significance. Plasmodium falciparum is the most important representative of these genera as it causes 400 million episodes of malaria and kills more than 1 million children in sub-Saharan Africa each year. Conventional therapies for malaria are failing because of the spread of multidrug-resistant parasites (1), whereas prevention of malaria is impeded by a lack of effective vaccines, which will take many years to develop. Molecular and cellular studies of P. falciparum are therefore essential to identify potential drug targets and to increase our understanding of how intracellular parasites maintain homeostasis, obtain nutrients, and produce up to 36 progeny during a 48-h asexual stage life cycle (2).

Parasite-encoded membrane transport proteins are crucial for these processes as they supply essential nutrients and remove toxic waste products, regulate the intracellular ionic microenvironment, and generate plasma membrane electrochemical gradients. These membrane transporters present opportunities for interventions designed to disrupt critical cellular functions. The study of transporters encoded by different apicomplexan organisms also provides opportunities for developing drugs aimed at biochemical targets shared by these pathogens but confined to this taxon.

P type ATPase membrane proteins of P. falciparum were first proposed for study as drug targets when sequences encoding four representatives (PfATP 1-3 and PfATP 6) were identified (3-5). Subsequently, a further member of this family (PfATP4) was described (6-8), and most recently a sixth example (PfATP 7, also designated MAL3P6 (9)) has been recognized by the malaria genome sequencing initiative.

These malarial sequences contain signature motifs that are common to all P type ATPases, including highly conserved phosphorylation site- and nucleotide-binding motifs and a transduction domain, as well as similarities in predicted hydropathy profiles. However, functional assay of malarial P type ATPases has not been reported, because malarial sequences (particularly those of integral membrane proteins) are difficult to express in heterologous systems (10). Some malarial P type ATPase-like sequences also contain segments that are not found in orthologues (3, 6). These inserts are hydrophilic and may contain tandemly repeated amino acid motifs that are of undefined function. Their presence increases uncertainty regarding both the function of malarial P type ATPase-like sequences and the ability to express these sequences in heterologous systems. Heterologous expression of P type ATPases has hitherto been limited to a few mainly mammalian examples.

We have studied a Ca2+-ATPase of P. falciparum (PfATP4), because Ca2+-ATPases are vital components of the machinery employed by eukaryotic cells to regulate intracellular calcium concentrations. Sequence analysis of Ca2+-ATPases suggests the following three categories of transporter: SERCA (sarco/endoplasmic reticulum)-type, PMR1 (yeast Golgi)-type, and PMCA (plasma membrane)-type ATPases (11). PfATP4 has not yet been assigned to any of these categories on the basis of primary sequence analysis, although its localization to the plasma membrane of asexual stage parasites has suggested that it may be more like a PMCA than a SERCA pump (8). Comprehensive analysis of primary sequence (and function) has also been hindered, because the full sequence for PfATP4 was not elucidated in two previous reports (7, 8).

Here we complete the ORF1 for PfATP4, analyze its relationship to current classifications of Ca2+-ATPases, and quantitate expression of its mRNA during the asexual stage of the life cycle of the parasite. We also functionally characterize PfATP4 using Xenopus laevis oocytes. These studies suggest that PfATP4 does not belong to any conventional category of Ca2+-ATPase (SERCA-, PMCA-, or PMR1-type) but rather defines a novel subclass of Ca2+-ATPases that is also represented in other apicomplexan organisms.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All materials were obtained from Sigma except where stated otherwise.

DNA Constructs-- All PCR experiments used thermostable polymerase (Pfu; Stratagene) and genomic DNA (clone T9/96) as template, unless stated otherwise. PCR products were verified by sequence analysis.

mRNA Quantitation Studies-- Parasites (clone 3D7) synchronized to an 8-h time window as described previously (12) were collected every 8 h into RNA isolatorTM (GenoSys Biotechnologies, Cambridge, UK) and RNA extracted according to the manufacturer's recommendations.

A competitor plasmid containing a novel asymmetrical restriction endonuclease site was generated (by converting a PstI to a HindIII site at nucleotide 2760) in PfATP4 sequence by PCR. This mutated fragment (contained between nucleotide positions 2475 and 3040) was ligated into a construct containing a previously mutated beta -tubulin sequence and was used in quantitative tandem competitive PCR assays as reported previously for PfHT1, a malarial hexose transporter (12).

PfATP4 Sequence Analysis-- Dyer et al. (8) (accession number U39298) extended the ORF initially reported for PfATP4 (7) (accession number U16995). However, hydropathy analysis of this extended PfATP4 sequence predicted only six transmembrane helices, an unusually low number for this class of ATPase. Comparison of PfATP4 sequences with expanding malarial sequence data bases indicated that a premature stop codon had been incorrectly identified (8). The first correct in frame stop codon in the ORF deduced from the malaria genome sequencing initiative therefore extended the previously published PfATP4 sequence (8) by 111 nucleotides allowing prediction of four additional membrane-spanning helices in this fully completed sequence. This suggestion was verified by independent sequence analysis of a 3795-nucleotide PCR product made from genomic DNA that included both published and new sequences (accession number AF203980). This encodes a derived amino acid sequence of 1264 amino acids (predicted molecular mass = 140 kDa).

Sequence and hydropathy analyses and alignments were performed using DNASTARTM (version 1.6; DNASTAR Inc., Madison, WI) and MacVectorTM (version 4.5.1; Eastman Kodak Co., Rochester, NY) software.

Constructs Used for Expression Studies-- For functional studies in X. laevis oocytes, full-length sequence encoding PfATP4, together with a strong Kozak consensus sequence (CACCATG, where the initiation codon is underlined) and BglII restriction sites, was cloned into the SmaI site of pUC18 and sequenced. PfATP4 was then cloned into BglII sites of pSP64, and capped cRNA was transcribed (MEGAscriptTM SP6 mMESSAGE mMACHINETM kit; Ambion, Austin, TX) from XbaI-linearized template (12). Control experiments included cRNA made from a clone encoding a previously characterized avian SERCA I pump (cSERCA1) (13) and tPfATP4, which encodes a prematurely truncated version of PfATP4 (nucleotides 1-3684) described in Ref. 8.

Expression of RNA in Xenopus oocytes-- Xenopus oocytes were harvested, and connective tissue was removed with collagenase (2 mg/ml Type IA, shaken for 2 h) (12). Stages V and VI oocytes were selected and microinjected with cRNA (5-30 ng) encoding cSERCA1, PfATP4, or tPfATP4 in RNase-free water (25-50 nl) or a corresponding volume of RNase-free water. Oocytes were subsequently incubated (at 19 °C) in Barth's solution (12) for 3-7 days.

Membrane Preparation-- Total membrane preparations from oocytes were obtained as in Ref. 14 with minor modifications as follows: oocyte homogenates were centrifuged at 1000 × g for 5 min; the resulting yolk granule/melanosome pellet was recentrifuged. Supernatants from the low speed spins were pooled and centrifuged (100,000 × g, 90 min) to obtain a membrane fraction, which was stored at -70 °C. Sarcoplasmic reticulum (containing ~80% mammalian SERCA Ia) was prepared from skeletal muscle of New Zealand White rabbits as described (15).

Ca2+-ATPase Assay-- Ca2+-ATPase activity was monitored by a coupled enzyme assay as described previously (15). Briefly, membranes (2-50 µg of protein) were mixed at 25 °C in a volume of 2.5 ml containing ATP (2 mM), phospho(enol)pyruvate (2.5 mM), NADH (0.25 mM), pyruvate kinase (7.5 units), and lactate dehydrogenase (8.0 units). The resynthesis of ATP consumed by Ca2+-ATPase activity is coupled by the pyruvate kinase and lactate dehydrogenase to NADH oxidation. The oxidation rate of NADH was recorded at A340 (Shimadzu UV---160; Shimadzu Corporation, Kyoto, Japan), and ATPase activity (expressed as IU or µmol of ATP hydrolyzed/mg of total protein/min) was calculated using an extinction coefficient for NADH of 6200 liter mol-1 cm-1. Ca2+-ATPase activity was defined as the activation seen on addition of calcium; [Ca2+]free was calculated as described previously (15). ATPase activity was measured after a 30-min preincubation of membranes (25 °C) with ouabain (final concentration = 200 µM), vanadate (1 mM), thapsigargin (0.8 µM), or cyclopiazonic acid (1 µM) before addition to the reaction mixture.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sequence Analyses-- The hydropathy profile of the full-length PfATP4 sequence is compared with representatives of the major subclasses of Ca2+-ATPases and with an orthologue encoded by Cryptosporidium parvum; see Fig. 1A. Ten transmembrane helices are predicted for PfATP4 as for other Ca2+-ATPases including the C. parvum orthologue (in contrast to the reported prediction of eight transmembrane helices for the latter sequence (16)). The hydropathy profile of another malarial Ca2+-ATPase, PfATP6 (4), resembles most closely the profile of SERCA I (data not shown). This observation is consistent with a high degree of conservation of the primary sequence between SERCA 1a and PfATP6 (61.7% sequence identity in core analysis; see below). In contrast, the predicted hydropathy profile of PfATP4 does not resemble either that of SERCA pumps (e.g. PfATP4 contains an extended hydrophilic N-terminal region, indicated by a filled oval in Fig. 1A, and an extended M7/M8 extracellular loop, shown under a hatched box) or that of plasma membrane ATPases (e.g. PfATP4 lacks a long hydrophilic C-terminal region, indicated by an open bar in PMCA in Fig. 1A). The extended M7/M8 loop of PfATP4 may be of functional significance, as this region is predicted to protrude from the noncytoplasmic face of the membrane, and may contribute to Ca2+ translocation in mammalian orthologues (17). This region is rich in acidic residues (21/137 (15.3%) glutamate/aspartate residues in PfATP4) as is the shorter M7/M8 loop in SERCA (e.g. 7/35 (20%) acidic residues for cSERCA1). P type ATPase core domains are ubiquitous on cytosolic loops between transmembrane regions M2 and M3 and M4 and M5 and are therefore often used in partial alignment analyses (18). As shown in Fig. 1, B and C, comparison of core regions of Ca2+-ATPases shows that PfATP4 has only moderate (<50%) sequence identity with representatives from all three functionally characterized classes of P type Ca2+-ATPases (viz. SERCA, PMCA, or PMR1 sequences (19)), and <25% identity if full-length sequences are compared (data not shown). PfATP4 is therefore phylogenetically distinct from existing subclasses of Ca2+-ATPases and occupies a position separate from them on a phylogenetic tree (Fig. 1C).



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Fig. 1.   Hydropathy profiles of representative Ca2+-ATPases. A, hydropathy profiles (14 amino acid windows for all analyses; generated using MacVectorTM (version 4.5.1; Kodak) software) of the following are displayed: SERCA, chicken SERCA2a (accession number A40812); PMCA, rat plasma membrane Ca2+-ATPase isoform 2 (accession number J03754); PMR1, yeast Golgi Ca2+-ATPase (accession number P13586); PfA4, PfATP4 (accession number AF203980); and Cp1, C. parvum Ca2+-ATPase (accession number U65981). Predicted membrane-spanning segments are indicated by a number. Conserved Ca2-binding residues (Glu or Asp) are indicated by asterisks. Hydrophilic segments between M7 and M8 and malarial inserts are highlighted by hatched boxes and an oval, respectively. A hydrophilic C-terminal region in PMCA is shown under an open bar. The number of amino acids is shown at the base of the figure. B, core regions (as described in Ref. 18) of the above sequences and that of Pv1 (P. vivax putative Ca2+-ATPase; accession number X98484) are aligned as outlined under "Experimental Procedures." Each sequence is numbered individually, and an asterisk in Core E marks the phosphorylation site. C, dendrogram showing relationships between Ca2+-ATPase sequences shown above. Branch lengths are proportional to sequence distances in arbitrary units. Bootstrap values (for n = 100 iterations) are also shown to provide a measure of the robustness of the inferred topology. The tree was rooted at the midpoint of the most widely separated taxonomic units. All alignments were carried out using the ClustalW (version 1.7) program.

Orthologues of PfATP4 were identified in other apicomplexan genomes using NCBI tBLASTn software. Sequences (often incomplete) that are similar to PfATP4 are also encoded by Toxoplasma gondii (36% sequence identity compared with PfATP4 amino acid residues 682-731 and 1159-1176), Plasmodium vivax (89% sequence identity compared with PfATP4 amino acid residues 282-455), and C. parvum (43% sequence identity compared with PfATP4) (16) (accession numbers W66177, X98484, and U65981, respectively; the latter 2 sequences were included in the analyses illustrated in Fig. 1B). No orthologues of PfATP4 were found in nonapicomplexan organisms.

Ca2+-, Thapsigargin-, and Calmodulin-binding Motifs-- Six amino acid residues (Glu309, Glu771, Asn796, Thr799, Asp800, and Glu908) in transmembrane regions M4, M5, M6, and M8 comprise the high affinity sites responsible for the binding of a pair of Ca2+ ions to SERCA 1a (20). In contrast, PMCA- and PMR1-type Ca2+-ATPases contain only four amino acid residues in similar positions (Glu309, Asn796, Thr799, and Asp800 in positions corresponding to SERCA 1a notation), indicating that they bind one, rather than two, Ca2+ ions per enzyme molecule (19, 21). Five of the six Ca2+-binding amino acid residues of SERCA pumps were identified in M4, M5, and M6 of PfATP4, whereas a glutamate residue that had been previously aligned to Glu908 (of SERCA 1a) (8) is located in a predicted hydrophilic segment and is therefore unlikely to participate in a high affinity Ca2+-binding site. However, a glutamate residue (Glu1176) that aligns to Glu908 is also present in the full-length PfATP4 sequence (M8; see Fig. 1A, indicated by a boxed star). Both this residue and the hydrophobic region in which it is located are also conserved in PfATP4 orthologues of T. gondii and C. parvum (Fig. 1A). We therefore suggest that this novel M8, as identified in our sequence analysis, contributes to a further Ca2+-binding site (boxed in Fig. 1A) in apicomplexan enzymes. Thapsigargin selectively inhibits SERCA enzymes by binding at M3 (22, 23). PfATP6 shows >60% sequence identity in M3 to SERCA1a suggesting that it may be inhibited by thapsigargin. PfATP4 and its orthologues resemble non-SERCA enzymes such as PMCA1 and PMR1 in having <25% sequence identity to SERCA1a in this region, suggesting that PfATP4 will not be inhibited by thapsigargin. Conversely, there are no PMCA-like C-terminal calmodulin-binding motifs in the full-length PfATP4 sequence, which also lacks the recently proposed N-terminal calmodulin-binding motif (24).

Malarial sequences often contain hydrophilic inserts, and one is present at the N terminus of PfATP4 (indicated by an oval in Fig. 1A). M1 in SERCA and PMCA enzymes begins 60-100 amino acids from their respective N termini, whereas in PfATP4 M1 is predicted to begin at Gln173. Furthermore, the proportion of asparagine and lysine residues in the region before M1 in PfATP4 is elevated (21% Asn/13% Lys) compared with 6% Asn/6% Lys and 4% Asn/9% Lys for SERCA or PMCA, respectively. No insert could be identified in the orthologue of C. parvum suggesting that these features are peculiar to malarial ATPases rather than to apicomplexan pumps in general.

Taken together, these observations suggest that PfATP4 and its orthologues constitute a new subfamily of P type Ca2+-ATPases that we propose as Type 4 (apicomplexan) according to Pittman's classification (11). PfATP4 contains 4 potential Ca2+-binding sites and lacks a calmodulin-binding domain like SERCA pumps, while localizing to the parasite plasma membrane (perhaps as well as internal structures), and lacking a thapsigargin-binding sequence as do PMCA- or PMR1-like transporters. Moreover, PfATP4 also contains an extended M7/M8 loop and a long hydrophilic N-terminal region (features not found in SERCA, PMCA, or PMR1 ATPases).

Ca2+-ATPase Assays

SERCA-- To confirm that cSERCA1 (an avian SERCA 1 pump) was expressed, we compared Ca2+-dependent ATPase activity in membrane preparations from oocytes injected with cRNA encoding cSERCA1 with that in sarcoplasmic reticulum preparations from rabbit muscle. Results are shown in Fig. 2. As expected, maximal activities were higher in SR preparations (mean ± S.E. = 3.030 ± 0.300 IU for SR (n = 4) versus 0.048 ± 0.007 IU for cSERCA1 (n = 14)), but calcium dependences for the two preparations were almost identical, with EC50 values (i.e. [Ca2+] required for half-maximal activation) of 0.23 and 0.26 µM for SR and cSERCA1, respectively. Membrane preparations from water-injected oocytes gave mean ± S.E. Ca2+-ATPase activities of 0.014 ± 0.005 IU (n = 19) and calcium dependences similar to those seen for SR and cSERCA1 (data not shown), indicating that ~30% of calcium-dependent ATPase activity measured in cSERCA1-injected oocytes is due to endogenous Xenopus Ca2+-ATPases. This background activity has not been subtracted in data shown in Fig. 2. Ca2+-ATPase activities for membrane preparations from cSERCA1-injected oocytes correspond closely with those of microsomes prepared from SERCA-transfected COS-1 cells (range between 0.01 and 0.10 IU (25, 26)).



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Fig. 2.   Calcium dependence of ATPase activities. Percentages of maximum Ca2+-ATPase activity (normalized to the maximum value) are displayed for rabbit skeletal muscle SR (star), PfATP4-injected (diamond), and cSERCA1-injected (filled circle) oocyte membrane preparations at the indicated [Ca2+]free values (where pCa is -log10[Ca2+]free). Results represent at least three experiments for each value.

PfATP4-- A Ca2+ dependence plot for PfATP4 is shown in Fig. 2. Maximal Ca2-dependent ATPase activities (0.067 ± 0.019 IU; n = 20) were similar in magnitude to those seen for cSERCA1, suggesting comparable expression of the two cRNA species in oocytes (assuming the same stoichiometry of ATP hydrolysis for PfATP4 as for SERCA). However, a broader activity peak with an EC50 of 0.40 µM was observed for PfATP4 compared with SR/cSERCA1 preparations (p < 0.05 comparing maximal ATPase activities for PfATP4 with SR/cSERCA1 at pCa values of 3.5 and 4.5; see Fig. 2). Thus, near-maximal activation of the enzyme was preserved at higher free calcium concentrations (>30 µM) than those seen with SERCA pump preparations, where free [Ca2+] becomes inhibitory at >10 µM (27).

The addition of bovine brain calmodulin (1 µM) did not increase the sensitivity of PfATP4 to [Ca2+] in two independent experiments (data not shown). tPfATP4 did not increase ATPase activity in oocyte membrane preparations (mean ± S.E. activity = 0.005 ± 0.007 IU or 10.6 ± 15.9% of full-length PfATP4 sequence (n = 3; p < 0.05) compared with PfATP4 activity). This residual Ca2+-ATPase activity was fully inhibitable by thapsigargin, confirming that it was because of endogenous Xenopus Ca2+-ATPases.

Inhibitor Susceptibilities of Membrane Preparations-- Fig. 3 summarizes inhibition profiles of Ca2+-ATPase activity in different membrane preparations. As expected, all preparations are resistant to ouabain (a Na+/K+-ATPase inhibitor), and conversely all are inhibited by the pentacoordinate phosphate analogue, vanadate. PfATP4 is insensitive to thapsigargin in contrast to SR, cSERCA1, or control samples. Preincubation with thapsigargin (0.8 µM) reduces Ca2+-ATPase activity to 2.4 ± 1.4% (SR; n = 6), 5.2 ± 3.2% (cSERCA1; n = 5), and 10.0 ± 5.6% (water-injected controls; n = 6) of maximal levels. For PfATP4 significantly greater ATPase activity is maintained (74.7 ± 8%; n = 7; p = 0.003 comparing thapsigargin inhibition of cSERCA1 and PfATP4). The small (25%) inhibition seen in PfATP4 preparations probably represents an abolition of endogenous (oocyte) Ca2+-ATPase activity, rather than an effect on PfATP4. As thapsigargin inhibits SERCA specifically, this lack of susceptibility of PfATP4 to thapsigargin supports our earlier suggestion that PfATP4 is not a SERCA-type enzyme but rather represents a novel class of Ca2+-ATPases. Preincubation with cyclopiazonic acid (a metabolite from Aspergillus and Penicilium that inhibits SERCA and PMR1 pumps but not PMCA (19, 28)) inhibited SR, cSERCA1, and PfATP4 samples. Mean ± S.E. ATPase activities of SR, cSERCA1, and PfATP4 preparations in the presence of cyclopiazonic acid (1 µM) compared with uninhibited controls were, respectively, as follows: 5.8 ± 0.6% (n = 3), 14.1 ± 8.55 (n = 3), and 22.6 ± 4.3% (n = 4).



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Fig. 3.   Inhibitor susceptibilities of rabbit skeletal muscle SR and oocyte membrane preparations. Percentages of Ca2+-ATPase activity (normalized to control values) are displayed for rabbit skeletal muscle (SR), PfATP4-injected (PfATP4), and cSERCA1-injected (cSERCA1) oocyte membrane preparations after preincubation (30 min; 25 °C) with the following: no inhibitor (open bars), thapsigargin (0.8 µM; cross-hatched bars), sodium orthovanadate (1 mM; shaded bars), ouabain (0.1 mM; filled bars), and cyclopiazonic acid (1 µM; hatched bars). Results represent at least three experiments in each case.

mRNA Quantitation Studies-- We characterized the pattern of PfATP4 mRNA expression using synchronized cultures of asexual stage P. falciparum parasites and compared this expression with that of PfHT1, the malarial hexose transporter. The technique of tandem competitive PCR was used to quantitate precisely the amount of PfATP4 mRNA relative to that of a housekeeping gene, beta -tubulin (12), and results are shown in Fig. 4. These data are shown in comparison with published data on PfHT1. The precision and reproducibility of this ratiometric method were confirmed by assessing the gene copy number of PfHT1 relative to that for beta -tubulin using genomic DNA as template in five independent tandem competitive PCR experiments (mean ratio ± S.E. = 1.07 ± 0.12; see Fig. 4, open circle). Soon after invasion of red cells, there is relatively low expression of PfATP4 relative to beta -tubulin. The ratio of PfATP4/beta -tubulin mRNA rises ~4-fold to a peak at 8-16 h, after which it falls slightly to approximately twice the initial level by 32 h. Maximal levels of PfATP4 relative to beta -tubulin (~5-fold initial values) are seen in mature asexual stages (40 h after invasion).



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Fig. 4.   Quantitative mRNA studies. The ratio of PfATP4 (filled circles) or PfHT1 (the malarial hexose transporter (12); open squares) to beta -tubulin cDNA in samples obtained from synchronized parasite cultures following invasion (0 h) is shown. Samples were collected at 8-h intervals, and the ratios of mRNAs were calculated from a minimum of six determinations for each value displayed, as described under "Experimental Procedures." Data for PfHT1 and the estimates of variability and precision for this method (open circle ± S.E.) have been published previously (12).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The recent functional characterization of a malarial high affinity facilitative hexose transporter (PfHT1) has confirmed that X. laevis oocytes are a valuable tool for heterologous assay of malarial transport proteins (10, 12). We have used this system to express PfATP4 and measure Ca2+-ATPase activity in membrane preparations. PfATP4 activity is distinguished from the endogenous Ca2+-ATPases of oocytes by virtue of its magnitude (up to 20-fold higher than water-injected controls), lack of susceptibility to thapsigargin, and activation by a broader range of [Ca2+] (from pCa 3.5-5.5, corresponding to [Ca2+]free = 3.2-320 µM). The absence of induced Ca2+-dependent ATPase activity in oocytes injected with cRNA encoding tPfATP4 (a truncated version of PfATP4 that lacks terminal transmembrane segments) also confirms that full-length PfATP4 sequence is required for function and further that this induced activity is not due to nonspecific effects of microinjecting cRNA. Susceptibility to inhibition by vanadate of all the ATPases assayed in our experiments confirms that these activities are mediated by P type ATPases (Fig. 3). The lack of inhibition of ATPase activity by ouabain (a specific Na+/K+-ATPase inhibitor) excludes this class of ATPase from contributing to increases in ATPase activity in experimental oocyte membrane preparations, because oocyte Na+/K+-ATPases are inhibited by ouabain (29)). We estimate that PfATP4 constitutes ~1% of oocyte membrane protein, based on the assumption that maximal rates of ATP hydrolysis by PfATP4 and SERCA 1 (i.e. cSERCA1 activity) are similar. The measured cSERCA1 activity is equivalent to ~0.7% of oocyte membrane protein, as calibrated against SERCA 1a activity in rabbit muscle preparations where it constitutes 80% of total protein. This estimate for the level of expression of PfATP4 accords well with estimates for heterologous expression of SERCA in COS-1 cells (0.2-2% of total membrane protein, giving activities of 0.01-0.1 IU (25, 26)).

Detailed sequence analysis of PfATP4, and comparison with orthologues, suggests that PfATP4 defines a new subclass of Ca2+-ATPase found in apicomplexan organisms. Comparison of core sequences to discern phylogenetic relationships between P type ATPases from different organisms provides a useful approach for defining major subclasses (18) but does not distinguish between SERCA and PMR1 subclasses as identified by Pittman et al. (11). Therefore, analysis of the full ORF for PfATP4 was necessary to distinguish many defining characteristics of the apicomplexan subclass of Ca2+-ATPases. These were found to include six putative Ca2+-binding amino acid residues and an extended M7/M8 loop of >130 amino acids (the equivalent region in SERCA 1a being a low affinity (Km ~2.5 mM) Ca2+-binding site involved in releasing Ca2+ ions into the endoplasmic reticulum/SR lumen (17)). In addition, functional characterization revealed a relatively broad Ca2+ dependence that is comparable with the profile of PMCA (30) and also both thapsigargin insensitivity and inhibition with cyclopiazonic acid. As thapsigargin and cyclopiazonic acid both bind to the M3 region of SERCA (31), these findings implicate differences in that region of PfATP4 as being of potential importance in determining this pattern of susceptibility to inhibition.

PfATP6 is a SERCA-type ATPase (4) that may also be important in Ca2+ homeostasis in P. falciparum. No other examples of the Ca2+-ATPase family of sequences have been identified in the malarial genome, of which >90% is now sequenced in whole or part. Taken together with published immunolocalization data (including staining of PfATP4 on free merozoites (8)), our functional characterization of PfATP4 suggests that PfATP4 is likely to perform a key function as a plasma membrane Ca2+-ATPase in asexual stages of the life cycle of P. falciparum. These observations do not exclude an intracellular role for PfATP4, as in plants PMCA-type and SERCA-type ATPases may be found in both the plasma membrane and endoplasmic reticulum (11).

PfATP4 is expressed throughout the asexual stages of the life cycle of the malaria parasite (Fig. 4) (8). mRNA expression levels vary by ~5-fold relative to the housekeeping gene beta -tubulin and generally increase as parasites mature. This pattern of mRNA expression differs from that observed with PfHT1, a facilitative malarial hexose transporter, with which it is directly comparable, because the same cDNA samples were used for both experiments. Overall, the ratio of PfATP4/beta -tubulin mRNA is ~20% that of PfHT1/beta -tubulin (peak ratio > 1 at 8 h post-invasion). After PfHT1 mRNA peaks at 8 h, levels fall sharply 16 h after invasion, whereas they continue to rise for PfATP4 (12). The divergent expression of these two transporters possibly reflects differing substrate requirements as the parasites develop and exemplifies stage-specific control of gene expression.

Intraerythrocytic [Ca2+]free is ~50 nM and is primarily regulated by PMCA (32). When rings mature into meronts, fluorimetrically assayed intraparasitic [Ca2+]free undergoes a small increase in concentration from 45 nM (24 h post-invasion) to 125 nM (in meronts 44 h after invasion) (33), even though erythrocyte PMCA activity is retained (34). This independent regulation of intraparasitic [Ca2+]free depends upon the ability to export Ca2+ or to sequester it into membrane-bound stores, probably by the actions of both PfATP6 and PfATP4. PfATP6 has not been localized within parasites, functionally assayed, or found to be sensitive to thapsigargin, but thapsigargin has been reported to increase [Ca2+]I in parasites (35), consistent with a role for SERCA-type activity in intraparasitic Ca2+ homeostasis. In contrast, [Ca2+] in freed parasites monitored with Fura-2 does not increase following treatment with thapsigargin (1 µM) but is increased by cyclopiazonic acid (1 µM; see Ref. 36), findings consistent with the inhibitor susceptibility profile of PfATP4 in Xenopus oocytes (see Fig. 3 and "Results"). These latter observations suggest that PfATP4 may therefore also be important in maintaining relatively low intraparasitic [Ca2+]free in the face of up to ~1 mM [Ca2+]free, encountered as merozoites enter plasma (37). The importance of other secondary mechanisms for Ca2+ homeostasis, such as the action of Ca2+/H+ antiporters, remains to be determined.

The transition from an intracellular to an extracellular environment is common to many apicomplexan organisms that encode orthologues of PfATP4 and that may therefore share similar mechanisms for [Ca2+] homeostasis. As P type ATPases are susceptible to highly selective inhibition, this subclass of [Ca2+] pump presents an enticing target for antiparasitic drug development. Identification of potential inhibitors will undoubtedly be accelerated by the recent solution of the crystal structure for SERCA 1a (38).


    ACKNOWLEDGEMENTS

We thank Dr. Lisa Alleva, Prof. Kiaran Kirk, Dr. Ursula Eckstein-Ludwig, and Prof. Tony Lee for helpful discussions. This work was partly funded by MRC co-operative grant G9800300


    FOOTNOTES

* This work was partly funded by MRC co-operative Grant G9800300.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Wellcome Trust Senior Research Fellow in Clinical Science. To whom correspondence should be addressed. Tel.: 44-208-725-5836; Fax: 44-208-725-3487; E-mail: s.krishna@sghms.ac.uk.

Wellcome Trust Clinical Training Fellow.

Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M010554200


    ABBREVIATIONS

The abbreviations used are: ORF, open reading frame; PCR, polymerase chain reaction; SR, sarcoplasmic reticulum; tPfATP4, truncated PfATP4.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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