From the 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
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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.
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
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 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 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).
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)).
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).
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, 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 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).
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
tubulin sequence and was used in
quantitative tandem competitive PCR assays as reported previously for
PfHT1, a malarial hexose transporter (12).
70 °C. Sarcoplasmic
reticulum (containing ~80% mammalian SERCA Ia) was prepared from
skeletal muscle of New Zealand White rabbits as described (15).
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (62K):
[in a new window]
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.
View larger version (16K):
[in a new window]
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.
View larger version (18K):
[in a new window]
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.
-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
-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
-tubulin. The ratio of PfATP4/
-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
-tubulin (~5-fold initial values) are seen in mature asexual stages (40 h after invasion).
View larger version (15K):
[in a new window]
Fig. 4.
Quantitative mRNA studies. The ratio
of PfATP4 (filled circles) or PfHT1 (the malarial hexose
transporter (12); open squares) to 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
-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/
-tubulin mRNA is ~20% that of PfHT1/
-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.
![]() |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | White, N. J. (1999) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 354, 739-749[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Krishna, S.
(1997)
Br. Med. J.
315,
730-732 |
3. | Krishna, S., Cowan, G., Meade, J. C., Wells, R. A., Stringer, J. R., and Robson, K. J. (1993) J. Cell Biol. 120, 385-398[Abstract] |
4. |
Kimura, M.,
Yamaguchi, Y.,
Takada, S.,
and Tanabe, K.
(1993)
J. Cell Sci.
104,
1129-1136 |
5. | Krishna, S., Cowan, G. M., Robson, K. J., and Meade, J. C. (1994) Exp. Parasitol. 78, 113-117[CrossRef][Medline] [Order article via Infotrieve] |
6. | Trottein, F., and Cowman, A. F. (1995) Eur. J. Biochem. 227, 214-225[Abstract] |
7. | Trottein, F., Thompson, J., and Cowman, A. F. (1995) Gene 158, 133-137[CrossRef][Medline] [Order article via Infotrieve] |
8. | Dyer, M., Jackson, M., McWhinney, C., Zhao, G., Day, K., and Mikkelsen, R. (1996) Mol. Biochem. Parasitol. 78, 1-12[CrossRef][Medline] [Order article via Infotrieve] |
9. | Bowman, S., Lawson, D., Basham, D., Brown, D., Chillingworth, T., Churcher, C. M., Craig, A., Davies, R. M., Devlin, K., Feltwell, T., Gentles, S., Gwilliam, R., Hamlin, N., Harris, D., Holroyd, S., Hornsby, T., Horrocks, P., Jagels, K., Jassal, B., Kyes, S., McLean, J., Moule, S., Mungall, K., Murphy, L., Barrell, B. G., et al.. (1999) Nature 400, 532-538[CrossRef][Medline] [Order article via Infotrieve] |
10. | Krishna, S., and Woodrow, C. J. (1999) in Transport and Trafficking in the Malaria-infected Erythrocyte (Cardew, G., ed), Vol. 226 , pp. 126-144, John Wiley & Sons, Ltd., London |
11. | Pittman, J. K., Mills, R. F., O'Connor, C. D., and Williams, L. E. (1999) Gene 236, 137-147[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Woodrow, C. J.,
Penny, J. I.,
and Krishna, S.
(1999)
J. Biol. Chem.
274,
7272-7277 |
13. | Ishii, T., and Takeyasu, K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8881-8885[Abstract] |
14. |
Geering, K.,
Theulaz, F.,
Verrey, F.,
Hauptle, M. T.,
and Rossier, B. C.
(1989)
Am. J. Physiol.
257,
C851-858 |
15. | East, J. M. (1994) Methods Mol. Biol. 27, 87-94[Medline] [Order article via Infotrieve] |
16. | Zhu, G., and Keithly, J. S. (1997) Mol. Biochem. Parasitol. 90, 307-316[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Webb, R. J.,
Khan, Y. M.,
East, J. M.,
and Lee, A. G.
(2000)
J. Biol. Chem.
275,
977-982 |
18. | Axelsen, K. B., and Palmgren, M. G. (1998) J. Mol. Evol. 46, 84-101[Medline] [Order article via Infotrieve] |
19. |
Sorin, A.,
Rosas, G.,
and Rao, R.
(1997)
J. Biol. Chem.
272,
9895-9901 |
20. | Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478[CrossRef][Medline] [Order article via Infotrieve] |
21. | Guerini, D., Foletti, D., Vellani, F., and Carafoli, E. (1996) Biochemistry 35, 3290-3296[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Norregaard, A.,
Vilsen, B.,
and Andersen, J. P.
(1994)
J. Biol. Chem.
269,
26598-26601 |
23. |
Sagara, Y.,
and Inesi, G.
(1991)
J. Biol. Chem.
266,
13503-13506 |
24. |
Curran, A. C.,
Hwang, I.,
Corbin, J.,
Martinez, S.,
Rayle, D.,
Sze, H.,
and Harper, J. F.
(2000)
J. Biol. Chem.
275,
30301-30308 |
25. |
Lytton, J.,
Westlin, M.,
Burk, S. E.,
Shull, G. E.,
and MacLennan, D. H.
(1992)
J. Biol. Chem.
267,
14483-14489 |
26. |
Lytton, J.,
Westlin, M.,
and Hanley, M. R.
(1991)
J. Biol. Chem.
266,
17067-17071 |
27. | Ishii, T., Lemas, M. V., and Takeyasu, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6103-6107[Abstract] |
28. |
Seidler, N. W.,
Jona, I.,
Vegh, M.,
and Martonosi, A.
(1989)
J. Biol. Chem.
264,
17816-17823 |
29. | Lafaire, A. V., and Schwarz, W. (1986) J. Membr. Biol. 91, 43-51[Medline] [Order article via Infotrieve] |
30. |
Ansah, T. A.,
Molla, A.,
and Katz, S.
(1984)
J. Biol. Chem.
259,
13442-13450 |
31. | Ma, H., Zhong, L., Inesi, G., Fortea, I., Soler, F., and Fernandez-Belda, F. (1999) Biochemistry 38, 15522-15527[CrossRef][Medline] [Order article via Infotrieve] |
32. | Staines, H. M., Chang, W., Ellory, J. C., Tiffert, T., Kirk, K., and Lew, V. L. (1999) J. Membr. Biol. 172, 13-24[CrossRef][Medline] [Order article via Infotrieve] |
33. | Adovelande, J., Bastide, B., Deleze, J., and Schrevel, J. (1993) Exp. Parasitol. 76, 247-258[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Tiffert, T.,
Staines, H. M.,
Ellory, J. C.,
and Lew, V. L.
(2000)
J. Physiol. (Lond.)
525,
125-134 |
35. | Garcia, C. R., Dluzewski, A. R., Catalani, L. H., Burting, R., Hoyland, J., and Mason, W. T. (1996) Eur. J. Cell Biol. 71, 409-413[Medline] [Order article via Infotrieve] |
36. | Alleva, L. M., and Kirk, K. (2000) in Molecular Approaches to Malaria (Macreadie, M., ed), Abstracts, p. 54, Lorne, Australia |
37. | Krishna, S., and Squire-Pollard, L. (1990) Parasitol. Today 6, 196-198[Medline] [Order article via Infotrieve] |
38. | Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647-655[CrossRef][Medline] [Order article via Infotrieve] |