analysis of a cation-transporting atpase of plasmodium falciparum

MOLECULAR

iik%HEMIcAL PMASITOLOGY

ELSEVIER Molecular and Biochemical Parasitology 78 (1996) 1~ 12

Analysis of a cation-transporting ATPase of Plasmodium falciparum ’

Michael Dyer a-b,*, Michael Jacksonb, Chris McWhinneyb, Gang Zhaob, Ross Mikkelsenb

“Department of’ Zoology, Umioersity of’ 0.\ford3 South Parks Road. Oxjbrd. UK

hDepartmcnt qf Radiation Omcolog~. Medical College of Virginia, P.O. Box 980058, Richmond, VA -73298-0058. USA

Received 2 November 1995: revised I2 February 1996: accepted 19 February 1996

Abstract

We have cloned and characterised one gene, PfATPase4 which encodes a P-type ATPase containing all the primary

sequence motifs characteristic of this class of transmembrane ion transporters, and also a fragment of a second P. ,fakiparunz P-type ATPase pseudogene (PfATPaseS). Analysis of conserved domains and motifs of specific ATPases reveals that PfATPase4 is most analogous to Ca’ + ATPases of the endoplasmic reticulum. The PfATPase4 gene gives

rise to a transcript of 8 kb shortly after erythrocyte invasion. Although this mRNA is not detected in later stages, the protein detected immunologically at 190 kDa persists throughout and is detected in free merozoites. Immunofluores- cence microscopy reveals that the PfATPase4 protein is concentrated in discrete compartments at the periphery of the parasite. Detailed sequence and structural analyses of these and the other P-type ATPases of P. ,fic/ciparurn described

previously, reveals that they comprise an unusual family in several respects. Firstly, the large number of non-homologous genes so far characterised reflects the complexities of ionic regulation in the diverse environments

encountered by the parasite. Secondly, the plasmodial P-type ATPase family may be classified both at primary

sequence and structural levels into two distinct groups - those typical of P-type ATPases (including PfATPase4) and those which are much more divergent. A third complexity is illustrated by the fact that one of the other members [l] here termed PfATPase6, has an even greater similarity to the sarcoplasmic reticulum Ca’+. ATPases than does PfATPase4. which raises questions about the possible functional relationship between these two members.

Kevnwrds: ATPase; Cation: Ion; Plasmodium falciparum; Transport

1. Introduction

Ahhreaiations: aa. amino acid; GST, glutathione S-transferase: PBS, phosphate-buffered saline; PCR, polymerase

chain reaction; PM, plasma membrane: SERCa’+ ATPase,

sarcoplasmic reticulum Ca’ + ATPase; TM, transmembrane.

* Corresponding author. Tel.: + 44 865 281234; fax: + 44

865 281245; e-mail: [email protected]

’ Note: Nucleotide sequence data reported in this paper are

available in the Genbank TM databases under the accession

numbers U.39298 and U39299.

The intraerythrocytic stages of development of the virulent human malaria parasite Plasmo&um

fulcipurum confer upon the parasite the advan- tages of facilitated host immune system evasion, while the parasite ingests haemoglobin as a source of amino acids and utilizes the red blood cell reducing system to avoid the oxidant stress pro-

Ol66-6851/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved

PII SOl66-6851(96)02593-S

2 M. Dyer et al. / Molecular and Biochemical Parasitology 78 (1996) I 12

duced by the parasite [1,2]. However, several as- pects of intraerythrocytic existence are thus com- plicated for the parasite; notably metabolism and transport. During its 48 h asexual cycle within the erythrocyte, P. falciparum increases its volume an estimated 25 times [3] and divides asexually. This rapid growth demands large amounts of nutrients.

Transmembrane electrochemical gradients (of Na + and H + in most cells) are maintained at the expense of considerable energy input and provide the driving force for transport of ions, nutrients such as sugars and amino acids into the cell and also secretion of toxic waste products. While erythrocytes maintain a low concentration of cytoso- lit Ca’+ (< 100 nM) [4], by actively pumping out Ca2+ by means of a Ca2+ ATPase, there is evidence that uptake of extracellular Ca’+ by P. falciparum -infected erythrocytes is necessary for parasite development [5,6]. In all eukaryotes, maintenance of an appropriate free cytosolic Ca’ + is essential for cell growth and Ca2 + tran- sients represent an important second messenger for cellular responses to extracellular stimuli and regulation of cell cycle transitions. Blocking Ca2 + influx in early erythrocytic Plasmodium inhibits maturation beyond the ring stage similar to the Ca’+-sensitive late Gl arrest in other eukaryotic cells [6,7].

The erythrocyte cytoplasm (although morpho- logically separated from the parasite by the vacuolar membrane) also presents the parasite with an environment of low [Na+] as well as low [Ca’+]. While Ca* + and also Na+ are readily available extracelluarly, current evidence favours the view that Plasmodia do not have direct access to the serum (for a review of the evidence for and against the existence of a direct access channel, see [8,9]) and so the parasite must regulate its ionic composition in general through control of ion transport processes across three membranes: the erythrocyte plasma membrane, the parasitophorous vacuolar membrane and the parasite plasma membrane.

Central to regulation of internal cation concen- trations, are the multipass membrane cation mo- tive P-type ATPases, which contribute to maintenance of electrochemical gradients by ac-

tively pumping ions using energy derived from ATP hydrolysis.

This family of P-type ATPase cation transporters include; the Nat/K +-; the plasma membrane Ca* + (PMCa’ + )-; the sarcoplasmic-endoplasmic reticulum Ca2 + (SERCa* +)-. the gastric H+/K + -. K+- and H+-ATPases. The enzymatic activities are confined to the large (100-140 kDa) subunits, which all share a number of structural and functional features, including a similar arrangement of predicted transmembrane domains and 10 conserved sequence domains including the phosphorylation and nucleotide - binding sites which are contained in a large cytoplasmic domain [lO,ll].

Several genes encoding these proteins have now been isolated from P. falciparum; PfATPasel (encoding MALl) [12] which has characteristics of a Na + /K + ATPase; a gene (here assigned PjAT- Pase6) which is highly homologous to endoplasmic and sarcoplasmic Ca2 + ATPases [13] and PfATPase2 which has features in common with a yeast Ca 2 + ATPase [14]. In addition, partial sequences have been determined for two additional genes which have characteristics of P-type AT- Pases; PfATPase3 [12,15] and most recently PfATPase4 [14,16] which previous to this present study was considered to be full length.

In this study, we describe the molecular cloning of two P. falciparum P-type ATPases: PfATPuse4 and a pseudogene, PfATPaseS, and investigate the expression of PfATPase4. We also carried out analysis of all the P. fulciparum ATPase genes isolated to date and present our findings.

2. Materials and methods

2.1. Parasite cultures

The T9/96 strain of P. falciparum was maintained in culture by standard methods [17]. Para- site cultures were synchronized by sorbitol lysis [ 181. For certain experiments, infected erythrocytes were purified on a Percoll density step gradi- ent prepared in RPMI-1640 [19]. Free parasites were obtained by nitrogen cavitation [20].

M. Dyer et al. I Molecular and Biochemical Parasitology 78 (1996) 1~ 12

2.2. DNA and RNA blot anal_vses

Infected human erythrocytes with parasitemia of 5 - 10% were sedimented at 2000 x g for 5 min and washed twice in phosphate-buffered saline (PBS: 135 mM NaCl, 2.5 mM KCl, 10 mM Na,HPO,, 2 mM KH,PO,, pH 7.4). Cells were resuspended in PBS to 25% original culture volume and mixed with an equal volume of 0.04% saponin in PBS to lyse erythrocyte membranes. After 30 s at 37°C parasites were pelleted by centrifugation at 4000 x g for 10 min and washed twice in PBS. Free parasites were treated with proteinase K (0.2 mg ml - ‘) in the presence of 2% SDS at 37°C for 16 h. Genomic DNA was extracted with phenol-chloroform, ethanol precipi- tated and then treated with RNAase A (20 ,ug ml ~ ‘). RNAase was removed by phenol-chloroform extraction and genomic DNA re-precipi- tated. For DNA blotting experiments, 5 bug purified genomic, or 0.1 /tg plasmid DNA were incubated with restriction enzymes (2-5 units /Lg. ‘), fragments resolved by agarose gel elec- trophoresis and denatured in 0.5 M NaOH, 1.5 M NaCl and neutralized with 0.5 M Tris-HCl, pH 7, 1.5 M NaCl before transfer to Hybond-nylon (Amersham) membrane by capillary blotting in 20 x SSC (1 x SSC: 150 mM NaCl, 15 mM Na,- citrate). DNA was fixed to the filters by UV crosslinking.

RNA was extracted from saponin-released parasites using acid guanidium thiocyanate-phenol- chloroform extraction [21]. Total RNA (5 pg) was resolved on formaldehyde agarose denaturing gels and ethidium bromide stained to check loading before transfer to nylon membrane (Hybond-N, Amersham International) by capillary blotting in 20 x SSC. Size markers (Gibco-BRL) were included.

Prehybridization and hybridization for both DNA and RNA blots were carried out in 7% SDS, 0.5 M sodium phosphate, pH 7.5. Probes were labelled by the random hexanucleotide prim- ing method using a kit (Stratagene) in the presence of [a-‘2P]dATP and purified over G50 sephadex columns. All probes were incubated with filters overnight at 65°C and unbound probe removed by washing filters at a final stringency of 0.1 x SSC, 0.1% SDS at 65°C.

2.3. AmpliJication of P. j~lciparum P-type ATPase fragments by polymerase chain reaction

Two degenerate oligonucleotides were designed from two highly conserved domains of all P-type ATPases found in both prokaryotic and eukaryotic organisms [lo, 111, incorporating P. j&i- parum codon bias [22]. The upstream sense primer was derived from the conserved phosphorylation site I/LCSDKTGTLT (Fig. 1) where D is the phosphorylated aspartyl residue. It was designated PAT1 and comprised the sequence 5’-TTA TGT ACA(TGT) GAT AAA ACA(T) GGA(T) ACA(T) TTA AC-3’. The sequence of the anti- sense oligonucleotide primer PAT2 was 5’-ATC TGC TTT TTT IAA IGA IGG IGC ATC ATT IAC ICC ATC ICC IGC CAT-3’ and corre- sponded to MTGDGVNDAPALK within the ATP-binding domain (Fig. 1). The polymerase chain reaction (PCR) was performed using Taq DNA polymerase (Gibco-BRL) on 100 ng Kl genomic DNA in the presence of 0.5 JIM of each oligonucleotide under the manufacturer’s recom- mended conditions. Optional thermal cycling comprised an initial denaturation at 95°C for 5 min, 3 cycles of: 95°C for 1.5 min. 45°C for 1.5 min, 72°C for 1.5 min and then 27 cycles of 95°C for 1 min, 50°C for 1.5 min and 72°C for 1.5 min. The two PCR products generated were gel purified and subcloned into the SfnuI site of pBluescript (Stratagene). DNA sequencing was carried out on multiple independent clones of each PCR product using the Sequenase Kit (US Biochemical) and computational analysis of the data obtained was carried out using the Univer- sity of Wisconsin Genetics Computer Group (UWGCG) programs [23] and those of the Seqsee ~1.2 package [24]. Both fragments comprised a reading frame with homology to P-type ATPases.

2.4. Screening of’ P. _falciparum genomk and cDNA libraries

An oligo-dT-primed cDNA library constructed in igtl 1 from poly(A) + RNA from P. jirlciparum isolate Kl, was screened with the 1.2-kb cloned PCR product generated as described above. Three bacteriophage clones were isolated. The longest

M. Dyer et al. / Molecular and Biochemical Parasitology 78 (1996) l-12

1 MSSQNNNKQG GQDIBJNKKDS DDIKPSNSKE DLINSLKNDE LNKNTTbiDQN DNKKNEXMIK

61 KNEXLNNSNN VEDGDNENSK FMNKSKEGLN NINGEKNDDN NSIVKhESP KSIGYNYYAS

121 ESIENLCKEF GLESINTGLN SEQVKINRDK YGENFIEKDE WPVWLIFLS QycspwLLL

181 J,VwVASJ,AJ, NEVVEGVAII SIVTJWCLA TYMEKSSGDA IGKLAEMASP QCTVLRNGQK

241 TM1 TM2

WIPSREVW GDVVLINTGD SISADLRLFD VIELKTNESL LTGESEDIKK TIVADNLSTP 301 FATNLCFATT SVTSGSGKGI VISTGLDTQV GKIASQLKKS SKGSKLTPLQ VALNKLGGU

361 IVJ,W IISJAVIIKY RDPAHAQKDP TFVT;LSIGVG FAVSSIPEGL PMVVTITLSA

421 TM3 TM4 GAKDMVKKNA NVRKLPAVET LGCCSVICSD KTGTZZ'EGKM

481 PAT1 LTKTFDFYPT KGFEPCGGLF DSNELTSEKK KEIVIAKNQN 541 DKTRSLMFAA YLNSYDTTLS RDPKTLKYGI HGNMSEGPIV

601 DNFQRLDDLE VTFNSSRKMK ITFYKLKTVN VFENVYLDKP

661 STHLLEETSM KKVQVSYNST ITQEERNVLI KKNLELSQKA

721 KLEDADADER LKYVNYDENG GFIPMGYVAS FDPPRPGVKE

781 PTAVAIGKLI GLIEEKSEQV EDINSLAIEC SELHINKNPN

841 RAQQEDKITI VQSLKRKGYL VAMTGDGVND APALKAADIG

901

TAINAVTICK NSSLSDENNK

TSYDKVLYNY GNPSNKSVIV

VAAAKVGYSF INNPNHKSYL

RKEYTHIALI KGAPDRLLDR

LRVLSICIKP LTDQNIEELK

AIQTCREAQV KVIMITGDQK

PAT2 EPILPNDQLD EFTDKILIYS

VAMGINGTEV AKGASEMILI

DDNFCTVVSA IDVGRTIFSN IQKLPFP LEALQILFLN

961 lw5 LMTDGCPAVA LSREPPNDDN MKTPPRPKKQ PIMTKRWWFY GILmEA LCVJtTrSu

1021 TM6 JBYICTGFYNL NGIHNLCKTV NLVDVNDANV YHEYKYFCSS YEYRISTDW GWVTNVSFWD 1081 PQNNEAVNFW GAAKGKVENI NPLSDIVHPE LRLRMQDGCS GDLTLDENRW CRPKDNKTSD 1141 GYNDELEGIL KKGFEDVTAK GSKRGRTMAF ISAVWCEMLR AYTVRRREPF YKVFNRNMWM 1201 HLACSISATL TFLSTCIPGI TSILNTT.

Fig. 1. The translated amino acid sequence of P. firlciporum ATPax is a composite of genomic, cDNA and PCR clones as

described in the text. The regions from which the initial PCR primers were designed are highlighted, italicised and labelled PATI

and PATZ. Transmembrane domains defined by the computer programme Alexis [24] are underlined and labelled TM I to TM6. The

position at which this sequence diverges from that published [16] is denoted ‘*‘.

M. Dyer el al. I Molecular and Biochemical Paru,ritolog~ 78 (1996) I I2 5

insert (3 kb) was excised and subcloned into the

EcoRI site of pBluescript and sequenced in its entirety on both strands. However, it was found to lack both the 3’ and 5’ ends of the coding sequence.

The 3’ end of the gene was then isolated by screening a i,NM1149 genomic library of HindIII- digested genomic DNA. (Hybridisation of the

PCR product to a blot of P. jdciparum genomic DNA indicated that the 3’ sequence was located

within a 3.5-kb HindIII fragment (data not shown)). The 5’ end of the gene eluded isolation by

a variety of methods including; construction and

screening of sized-enriched genomic libraries, in- verse PCR and screening of a random-primed

cDNA library. However it was finally confirmed by

PCR cloning made possible by the publishing of the sequence of the 5’ end of the gene [16]. The

sequence of the cDNA, genomic and PCR clones

together gave an uninterrupted open reading

frame, designated PfATPase4, whose sequence is available under GenEMBL accession number

U39298. The translated sequence is presented in

Fig. 1.

2.5. Comtruc’tion of LI PfATPuse4 ,fusion protein ~ md antiserum unal~~sis

In order to raise polyclonal antibodies against the P. f~dcipurum P-type ATPase (PfATPase4). a

750-bp fragment of PfATPase4 (aa 447-700) was

excised from the pBluescript cloned PCR product by first digesting with HitzdIII (and filling in the ends with the Klenow fragment of DNA polymerase l), and then BnnzHI. This fragment was gel

purified and ligated into pGEX 3X, cut first with EcoRI (and the ends made flush with Klenow) and

then Bun?HI to produce a fusion protein with

glutathione-S-transferase (GST) expressed in E. coli [25]. The GST.ATPase4 fusion protein induced by isopropyl-thiogalactosidase was of the predicted molecular mass (56 kDa), but was insoluble

and was therefore purified by first releasing the protein aggregates in TES (50 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl) containing 1 mg ml ~ ’ lysozyme, pelleting at 5000 x g for 10 min and carrying out a series of washing steps in TES buffer plus 0.1% sodium deoxycholate, then 1% NP40, and finally 8 M urea. After washing in TES, the

resuspended pellet was DNAse 1 treated and the

fusion protein purified by preparative SDS-PAGE,

electroeluted and concentrated by centrifugation in an Amicon filter. Mice were immunized initially with 50 pg of fusion protein in Freund’s complete

adjuvant, followed by equivalent doses adminis- tered in Freund’s incomplete adjuvant at 3-week

intervals. Responses were tested by ELISA and immunoblot analysis on the fusion protein and animals bled after the fourth boost.

-7.6. Ajinit>, pur$cation of mtibodies

Antisera from immunized mice were affinity

purified over GST.ATPase4 which had been solu- bilized by stirring for 12 h in 8 M guanidine

hydrochloride and bound by overnight incubation

to an activated (2 h at 56°C in 0.025% glutaralde-

hyde) polystyrene multi-well dish. Antibodies spe-

cific for the GST portion of the fusion protein were then repeatedly absorbed out over a polystyrene

surface coated with GST. In both purification procedures, antibody was bound in the presence of 5% milk powder, 0.5% bovine serum albumin

(BSA) in Tris saline, pH 7.4 containing 0.05%

Tween 20 (TBST), washed extensively in TBST and

eluted in 0.1 M glycine, pH 2.51150 mM NaCl.

Antibodies were immediately neutralized with 1 M Tris, pH 7.4, and diluted IO-fold in TBST.

For immunofluorescence assay (IFA) on free

parasites released by nitrogen cavitation, slides were first immersed in a solution of 100 /lg ml ’ poly-L-lysine and allowed to air dry. Free parasites at various dilutions were applied in 25 j11 aliquots

to each slide well in PBS containing 1 mM Mg’$

(PBSM) and allowed to settle and attach for 10 min. Unattached cells were washed off by immer- sion in PBSM and the slides acetone-fixed. Prepa-

ration of slides with infected erythrocytes was carried out by washing the cells (1% haematocrit) in PBS, making smears and acetone fixing. Texas Red-conjugated (sheep) anti-mouse antibodies (Sigma) were visualized with a Nikon Dapkot fluorescence microscope. All slides were counter- stained with 2 Ilg ml ’ Hoes&t dye, which was

6 hl. Dyer et al. / Molecular and Biochemical Parasitology 78 (1996) I - 12

co-incubated with the secondary antibody to per- mit visualization of parasite nuclei.

P. falciparum proteins were separated by SDS- PAGE [26] and electroblotted [27] for detec- tion with appropriate dilutions of affinity puri-

fied mouse antibody. Both peroxidase-conjugated anti-mouse IgG with enhanced chemilumin-

escence reagents (Amersham) and alkaline phos-

phatase-conjugated anti-mouse IgG with the bro- mochlorindolyl phosphate/nitro-blue tetrazolium

substrate system (Promega) were used for detec-

tion of bound primary antibody.

3. Results

With few exceptions (e.g., Ref. [12]), the CY

subunits of all P-type ATPases, regardless of their ion transport capacity, are approximately lOO- 140 kDa in size and contain a predicted 8- 10 membrane spanning domains [lO,l I]. While over-

all between-species homology is lower than for

most housekeeping genes, there are 9-10 regions of relatively high homology (Fig. 5), most of

which are cytoplasmic, although region (E) has been designated as a transmembrane conserved

domain and as such is considered likely to be directly involved in the trdnsmembrane transfer of ions. We utilized a PCR-based strategy using

degenerate oligonucleotides (as outlined in Mate- rials and methods) to amplify fragments of P.

fulcipuru~n P-type ATPase genes. The two frag-

ments amplified were 850 and 1000 bp in size,

which was within the expected size range for the region flanked by the two primers. Both fragments were further analyzed by sequencing multi-

ple cloned representatives of each. The 850-bp fragment contained a reading frame which, although demonstrating similarity to the family of P-type ATPases (53% amino acid (aa) identity to Nat K +ATPase [28]), was interrupted by multiple stop codons. This fragment, PfATPaseS, whose nucleotide sequence may be retrieved under Genbank accession number U39299 was concluded to represent a P-type ATPase pseudogene.

The sequence of the lOOO-bp fragment comprised a single open reading frame of 343 aa which had highest identity at the amino acid level

to the corresponding regions from Na+ /I(’ AT-

Pases (max. 43% identity [28]) and Ca* + ATPases

(max. 40% identity [29]). It also had total identity

with the partial sequence of PjATPase4 recently

described by Trottein and Cowman [14,16]. This

product was used to probe P. fulciparum genomic

and cDNA libraries, resulting in the determina-

tion of the 3684-bp sequence which can be re-

trieved under GenEMBL accession number

U39298. The sequence contains a single open

reading frame throughout, from nucleotide 1 to a

stop codon at position 368224. It translates into a

polypeptide chain of 1227 residues with a pre-

dicted molecular mass of 135 kDa.

During the preparation of this manuscript, the

sequence of an apparently full length cDNA en-

coding PfATPase4 was published [16]. This se-

quence aligns to the sequence published here from

residue 1 (methionine) to residue 1097 (‘*’ in Fig.

1). From this position the two sequences diverge,

the open reading frame of Cowman et al. [16]

ending after 14 bp, whereas our own reading

frame extends a further 389 bp. Since our 3’

sequence has been verified over this junction by

sequencing both cDNA and genomic clones, and

genomic DNA blotting has shown efATPase4 to

be a single copy gene, then this sequence diver-

gence must be due to either: (1) alternative splic-

ing whereby the 3’ sequence of Cowman et al. [16]

is a downstream exon and is fused to the rest of

the transcript during processing through splicing

out of our published 3’ sequence; or (2) a recom-

binational rearrangement occurred during the

construction of the library used by Cowman et al.

[16]. There is no precedent in P. jdciparum for

alternative splicing and we have demonstrated

using PCR to distinguish between the two models

that splicing is not taking place (data not shown).

Thus the 3’ of Cowman’s sequence is most likely

to represent a recombinational event occurring during library construction, resulting in the fusion

of two unlinked loci.

Searches of protein databases with the sequence of the PfATPase4 polypeptide, revealed significant

similarities with the a-subunits of P-type ATPases

across the spectrum of evolutionary diversity from E. coli and c~~~~obacterin, to mammals.

M. Dyer et al. ! Molecular and Biochemical Parasitologic 78 (1996) I - I.? 7

8K :b >

- 9.5 kb

- 7.5

- 4.4

- 2.4

- 1.4

- 0.24

Fig. 2. RNA blot of 5 ,ug total RNA per track, using a

fragment of f/2 TPase4 as a hybridisation probe under condi-

tions of high stringency. After synchronisation by sorbitol

lysis, parasites were microscopically assessed and harvested 50,

21, 35 and 44 h later to yield RNA from rings (lane I ),

trophozoites (lane 2), schizonts (lane 3) and segmenters (lane

4). The size of the hybridising band was calculated using RNA

size standards (Gibco-BRL).

3.1. E.upressionlsuhcellular localisaton

We analysed the expression of PfATPase4 mRNA throughout the asexual phase of the life

cycle by blotting RNA purified from synchronised cultures at various timepoints and using a fragment of PfATPase4 as a probe (Fig. 2). A pre- dominant transcript of approximately 8.0 kb, was present primarily in early stages, following mero-

zoite invasion - rings and trophozoites, indicat-

ing temporal regulation of expression throughout the asexual cycle. This would imply that the non-

coding 5’ and 3’ regions of the mRNA would together consist of 4300bp - extremely long even

for a P. fulcipurunz transcript. This approximate size was confirmed by a second blot of RNA from asynchronous parasites.

Electroblotting a protein preparation from an asynchronous asexual culture of P. j&iparurn Kl with affinity-purified antibodies specific for the PfATPase4 250 aa portion of the fusion protein, showed a band at 190 kDa not present in the erythrocyte control (Fig. 3C) ~ (and also not recognised by pre-immune serum processed through the same affinity purification procedure

(data not shown)). This molecular mass is larger

than predicted from the primary sequence (13.5 kDa). The discrepancy in molecular mass may be due to modification such as O-linked glycosyla-

tion and phosphorylation and/or hindered mobil- ity through the gel by presence of charged residues. It is also possible that the 8-kb transcript

is either polycistronic or contains additional AT- Pase exon(s) which may be removed by alternative

splicing in certain circumstances.

Immunofluorescence studies (Fig. 4) demon-

strate expression of the protein throughout the asexual cycle --- all parasites fluoresce - includ-

ing as shown, trophozoites and early schizonts (Fig. 4A,B) as well as free merozoites (Fig. 4C). Antibody staining is markedly concentrated in

specific locations around the periphery of the parasites (Fig. 4B), and could be associated with

the plasma membrane itself and/or vesicles located in the cytoplasm, concentrated beneath the

plasma membrane.

4. Discussion

4.1. Sequence malJvis of PjA TPuse4

Using the FASTA programme [23], PfATPase4 has the highest overall similarity with a P-type

ATPase of the cyanobacterium Synechocystis sp. (33.4%) which is postulated to be involved in

Ca’+ influx [30], although in general PfATPase4 has highest amino acid similarity to the Na +/K +

class of transporting ATPases.

Alignment of PfATPase4 with representative

members of P-type ATPase transporter family of various ion specificities, as well as those of P. falciparwn which have been sequenced, was carried out with the assistance of the CLUSTALV programme [23]. Ten regions (A)---(J) which are ubiquitous amongst P-type ATPases were iden- tified within PfATPase4, thus confirming its identity as a member of the P-type family of cation pumps (Fig. 5). Most conserved are the domains known to be functionally critical [lo, 1 l] ~ notably the energy transduction domain (within region C).

the phosphorylation site (within region F, around the phosphorylated aspartic acid residue, ASPIC’),

M. Dver et al. / Molecular and BiochemL,:! Pa uito1og.v 78 (1996) l-13

56kDa > 56kDa >

C

190kDa >

Fig. 3. (A) Expression of glutathione S-transferaseePfA TPase4 fusion construct. Coomassie blue-stained SDS-PAGE of total E. co/i lysate in the absence (lane 1) and presence (lane 2) of the inducing agent 1 mM isopropylthio-B-D-galactoside. (B) Immunoblot of

0.5 /Lg purified GST (lane 1) and GST-PfATPase4 fusion protein (lane 2) using mouse antiserum affinity purified first over the fusion

protein and then by repeated absorption over GST. (C) Immunoblot of protein prepared from an asynchronous asexual culture of

P. fblciparum Kl (lane 2) and on control red blood cell preparation (lane I). separated by SDS-PAGE. Sizes were calculated from

molecular mass standards (Sigma).

and the fluorescine isothiocyanate (FITC)-binding

site (region G) which forms part of the ATP-

binding site along with region (I).

Within the conserved domains, PfATPase4 has

greatest homology with the SERCa’ + ATPase

(51% aa identity/60% similarity [31]) and secondly

with the P. falciparum ATPase here referred to as

PfATPase6 (50% identity/58% similarity) and

concluded to be an organellar type Ca2 + ATPase

of P. falcipavum [13]. However, PfATPase6 has a

higher homology to mammalian SERCa’ + AT-

Pase than does PfATPase4 (63% identity c/f

51%). PfATPases4 and -6 as well as being closely

related to each other, are more typical of mem-

bers of the family of P-type ion transporters than

either PfATPasesl or -2. (PfATPasel has highest

homology = 30% identity to Na + K + ATPase and

PfATPase 2 has highest homology = 31% to

SERCa’+ ATPase.) This raises two intriguing

questions. Firstly, what is the functional relation-

ship between PfATPase6 and PfATPase4 and,

secondly, what may the functions of such diver-

gent transporters as PfATPasesl and -2 be? Supporting the view that both PfATPases4 and

-6 may be Ca2+ transporters is their possession at appropriate positions of eight amino acids which

have been characterised as essential for Ca’+ transport by in vitro mutagenesis [32] (PfAT- Pase6: p314, E315, N’OO8, T’O”, D’O”, plOl5, E1131;

PfATPase4: p407 E408 E935 NY60 ~963 ~964 ~967 . ,

E’06’. Neither PfATPase6 ‘nor PfATbase4 have

motifs required for the interaction of calmodulin or phospholamban (features of plasma membrane Ca2 + ATPase (PMCa2 + ATPase) and SERCa’ + ATPase, respectively), which suggests that their activity may be under control of different regula- tory mechanisms. Zf both proteins transport Ca’ + across a membrane, then they must differ either in their cellular locations or in regulation of activity. The expression of PfATPase6 has not been stud- ied.

Fig. 4. Immunofluorescence assay on acetone-fixed free parasites P. fblciparunz, released from the host erythrocytes by nitrogen cavitation. The primary antibody was afinity-purified anti-PfATPase4 (1:200) and the secondary was Texas Red-conjugated (sheep) anti-mouse antibody (panels A,B.C). Panel D was incubated with control (prebleed) serum (1:200). Panels E, F, G and H show the same field as A. C. E and G but reveal the nuclear stain Hoescht dye. Panels A/E, B:F and D/H show trophozoites and early schizonts; panels C/G show free merozoites. Size bar = lO/tm.

A translation of the pseudogene fragment

P_iI TPusr5 is included in the alignment in Fig. 5.

It encodes a typical P-type ATPase and is closely

related to PfATPase4 (51% identity over 77 aa)

and PfATPase6 (47% identity) and unlike the

much more divergent PfATPasel (29% identity)

and PfATPase2 (32% identity).

4.1. Structurul unal~~ses: transmernbrane dormins

This divergence of PfATPasesl and -2 from the

typical P-type ATPases extends also to structural

considerations. Fig. 6 shows the arrangement of

conserved domains (A)-(J) in relation to the pre-

dicted transmembrane domains (as defined by the

Alexis programme [24]). All mammalian P-type

ATPases display an almost identical arrangement,

the primary variation being the length of the

carboxy terminus and the number of transmem-

brane domains contained therein.

Of the Plasmodial P-type ATPases, PfATPase4 is most similar in arrangement to the typical

P-type ATPases. There are only two regions within the carboxy terminus which are predicted

to form membrane-spanning structures. In accordance with typical P-type ATPases,

both PfATPases4 and -6 are predicted to have

conserved domain (E) largely buried in transmembrane pass 4 (TM4). This domain is considered likely to be involved directly in ion passage, and in the case of both PMCa’+ATPases and SERCa’+ ATPases, contains residues crucial for high affinity Ca’+ binding [32]. In contrast, domain (E) of PfATPasesl and -2 is both poorly

M. Dyer et al. 1 Molecular and Biochemical Parasitology 78 (1996) I-12

Fig. 5. Alignment using the CLUSTALV programme [23] of the IO conserved regions (A)-(J) of all the plasmodial P-type ATPases sequenced to date and representative members of the main classes of ion transporters. Pf4, P. falciparum ATPase4; Pfl, P. fulciparum ATPaseI (or MALI, [ 121); Pf2, P. fhlciparum ATPase2 [ 141; Pf3, P. falciparum ATPdse3 [ 151; Pf5, P. fblciparum ATPase5: SrCa, sarcoplasmic reticulum Ca’+ATPase [31]; PMCa, plasma membrane Ca’+ATPase [29]; NaK, Na+/K + ATPase [28]; H. H + ATPase [IO,1 I]; HK, H +/K + ATPase [JO.1 I]; K. K+ATPase [35]. The amino acid sequence number for each ATPase is shown at the beginning of each conserved domain. Gaps were introduced for maximal alignment and are indicated by a dash (-). Identical residues to PfATPase4 are indicated with a dot (.). A termination codon in pseudogene PfATPase5 is represented by (*).

conserved and is predicted not to be membrane- spanning, which is contrary to the proposed structure of a P-type ATPase. Also, domain (D) which is normally cytoplasmic, is predicted to be contained within TM4 in PfATPase2. While there may be error in prediction of transmembrane domains of these multi-pass channels, it is also possible that their ion binding/transport domain(s) differ in sequence and location from other P-type ATPases.

This study along with the data from others shows that the plasmodial P-type ATPase gene family is large, suggesting that the parasite’s homeostatic mechanisms are complex. The proteins may be clearly subdivided into those that

are typical P-type ATPases (Group 1, PfATPase4, PfATPase6 and the pseudogene PfATPase5) and those that have diverged (Group 2, PfATPasesl and -2).

These divergent P-type ATPases may be suitable for exploitation as novel drug targets, since their functioning is most likely vital to cell survival and selectivity is very possible since there are no appar- ent host homologues known. Selective inhibitors of P-type ATPases are already in clinical use [33,34]. However only functional studies will be able to determine positively the ion transport specificity and mechanism of these plasmodial transporters.

M. Dyer et al. 1 Molecular and Biochemical Parasitology 78 (1996) I --I2 II

FY4 +iJl~ 1227 aa

pfl +ll lllllcl,ln_-.=.-+, 1956aa

Pf2 1553aa

Pf6 a-0 1228aa

StCa *I 994aa

PCs ,_#MIM~ iOOOaa

NaK +JIlll+e 1013aa -

H +lk#l/I 916aa

HK +#lMJ+w 1026aa

K m 681aa

conserved domain a-j

transmembrane domain l-10

repetitive or Asn/Lys rich domain

0 100aa I&u

Fig. 6. Scale drawing of all P. Jhlciparum P-type ATPases and representative members of each ion class of ATPase (as listed under

Fig. 5). Conserved domains (A)-(J) are shown above the line (empty boxes), transmembrane domains below the line (solid boxes)

and repetitive or asparagine/lysine-rich domains as a white dash within a black line.

Acknowledgements

This study was supported by the National Insti- tutes of Health (A124307).

References

[‘I

PI

[31

[41

[Sl

Eckman, J.R. and Eaton, J.W. (1979) Dependence of

plasmodial glutathione metabolism on the host cell. Na-

ture 278. 7544756.

Friedman. M.J. (1979) Oxidant damage mediates variant

red cell resistance to malaria. Nature 280, 2455247.

Desai. S.A., Krogstad, D.J. and McCleskey, E.W. (1993)

A nutrient-permeable channel of the intraerythrocytic

malaria parasite. Nature 362, 6433646.

David-Dufilho, M.. Montenay-Garestier. T. and De-

vynyck, M.A. (1988) Fluorescence measurements of free

Ca’+ concentration in human erythrocytes using the

Ca’+indicdtor fura-2. Cell Calcium 9, 1677179.

Schiebel, L.W., Colombani. P.M., Hess, A.D., Aikawa,

M., Atkinson, C.T.. Igarashi, I., Matsumoto, Y. and

Milhous, W.K. (1989) Calcium/calmodulin functions in

P. ,f&iparum in vitro ~ implications for antiprotozoal

drug design. Prog. Clin. Parasitol. I, 21-56.

[6] Krogstad. D.J., Sutera, S.P.. Marvel, J.S., Gluzman, I.Y..

Boylan, C.W.. Colca, J.R., Williamson, J.R. and

Schlesinger, P.H. (1991) Calcium and the malaria para-

site; parasite maturation and the loss of red blood cell

[71

PI

[91

[lOI

[I ‘I

[I21

u31

deformability. Blood Cells 17, 2299241.

Lu, K.P. and Means, A.R. (1993) Regulation of the cell

cycle by calcium and calmodulin. Endocrine Rev. 14.

40-58.

Pouvelle. B., Spiegel. R., Hsiao. L.. Howard, R.J., Mor-

ris, R.L., Thomas. A.P. and Taraschi, T.F. (1991) Direct

access to serum macromolecules by intraerythrocytic

malaria parasites. Nature 353. 73- 75.

Elford, B.C., Cowan, G.M. and Ferguson, D.J.P. (1995)

Parasite-regulated membrane transport processes and

metabolic control in malaria-infected erythrocytes.

Biochem. J. 308, 361-374.

Serrano, R. (1988) Structure and function of proton

translocating ATPase in plasma membranes of plants and

fungi. Biochim. Biophys. Acta 947. I-28.

Green, N.M. (1989) ATP-driven cation pumps: alignment

of sequences. Sot. Trans. 17, 9722989.

Krishna, S.. Cowan, G., Meade. J.C.. Wells, R-A.,

Stringer, J.R. and Robson, K.J. (1993) A family of

Cation ATPase-like molecules from Pkasmodiwn j&i- parum. J. Cell Biol. 120, 2. 385-395.

Kimura, M., Yagamaguchi, Y.. Takada, S. and Tanabe,

K. (1993) Cloning of a Ca’ + ATPase of Plamodium fizlcipuntm and comparison with Vertebrate Ca’ + AT-

Pases. J. Cell Sci. 104, 112991136.

[14] Trottein, F. and Cowman. A.F. (1995) Molecular cloning

and sequence of two novel P-type ATPases from P/as- modiumj&iparum. Eur. J. Biochem. 227. 214 225.

[15] Krishna, S.. Cowan, G.C., Robson. K. and Meade, J.C.

(I 994) Plasmodium ,/&ipurum: Further characterisation

of putative cation ATPases. Exp. Parasitol. 78. 113- 117.

12 M. Dyer et al. 1 Molecular and Biochemical Parasitology 78 (1996) l-12

[16] Trottein, F.. Thompson, J. and Cowman, A.F. (1995) Cloning of a new Cation ATPase from Plasmodium falciparum: conservation of critical amino acids involved in Ca’ + -binding in mammalian organellar Ca* + ATPases. Gene 158, 133-137.

[17] Choi, I. and Mikkelsen, R.B. (1990) Plasmodium f&i- parum: ATP/ADP transport across the parasitophorous vacuole and plasma membranes. Exp. Parasitol. 71, 452- 462.

[18] Lambros, C. and Vandenberg, J.P. (1979) Synchronisa- tion of Plasmodium falciparum erythrocytic cultures in culture. J. Parasitol. 65, 4188420.

[19] Aley, S.B., Sherwood, J.A., Marsh, K., Eidelman, 0. and Howard, R.J. (1986) Identification of isolate-specific proteins on sorbitol-enriched Plasmodium falciparum infected erythrocytes from Gambian patients. Parasitology 8, 511-525.

[20] Read. L.K. and Mikkelsen. R.B. (1990) Cyclic AMP and Ca2 + -dependent protein kinases in Plasmodium jhlci-

parum. Exp. Parasitol. 71, 39948. [21] Chomcyzynski, P. and Sacchi, N. (1987) Single step

method of RNA isolation by acid guanidium thiocyandte- phenol-chloroform extraction. Anal. Biochem. 162, 1566 159.

[22] Hyde, J.E., Kelly, S.L., Holloway, S.P., Snewin, V.A. and Sims, P.F.G. (1989) A general approach to isolating Plasmodium falciparum genes using non-redundant oligonucleotides inferred from protein sequences of other organisms. Mol. Biochem. Parasitol. 32, 247-262.

[23] Devereux, J., Haeberli, P. and Smithies, 0. (1984) A comprehensive set of sequence analysis programmes for the VAX. Nucleic Acids Res. 12, 3877395.

[24] Bokyo, R., Willard, L., Wishart, D., Richards, F. and Sykes, B. SEQSEE suite of programmes. University of Alberta, Protein Engineering Network of Centres of Ex- cellence.

[25] Smith, D.B. and Johnson, KS. (1988) Single-step purification of polypeptides expressed in E. coli as fusions with

glutathione S-transferase. Gene 67, 31-40. [26] Laemmli, U.K. (1970) Cleavage of structural proteins

during the assembly of bacteriophage T4. Nature 227, 680-685.

[27] Towbin, H., Staehelin, T. and Gordon, J. (1979) Elec- trophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354.

[28] Shull, G.E., Schwartz, A. and Lingrel, J.B. (1986) Amino acid sequence of the catalytic subunit of the Na + /K + ATPase deduced from a complementary DNA. Nature 316, 691-695.

[29] Shull. G.E. and Greeb. J. (1988) Molecular cloning of two isoforms of the plasma membrane Ca’+ transporting ATPase from rat brain, J. Biol. Chem. 262, 8846-8857.

[30] Gersler, M., Richter, J. and Schumann, J. (1993) Molecu- lar cloning of a P-type ATPase from the Cyanobacterium, Synechocystis sp. Homology to eukaryotic Ca2 + AT- Pases. J. Mol. Biol. 234, 1284-1289.

[31] MacLennan, D.H., Brandl, C.J., Korczak, B. and Green, N.M. (1986) Amino acid sequence of a Ca’+ and Mg’+- dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature 316, 6966700.

[32] Clarke, D.M., Loo, T.W., Inesi, G. and MacLennan, D.H. (1989) Location of high affinity Ca* + binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca’ + ATPase. Nature 339, 476-478.

[33] Allen, D.G., Eisher, D.A. and Wray, S.C. (1985) Birthday for Digitalis. Nature 316. 674-675.

[34] Sachs. G., Carlsson, E., Lindberg. P. and Wallmark, B. (1988) Gastric H + ,K + -ATPase as therapeutic target. Annu. Rev. Pharmacol. Toxicol. 28, 269-284.

[35] Hesse. J.E., Wieczork, L., Altendorf, K., Reicin, AS., Dorus, E. and Epstein, W. (1984) Sequence homology between two membrane transport ATPases. Proc. Natl. Acad. Sci. USA 81. 4146-4750.

analysis of a cation-transporting atpase of plasmodium falciparum

Documents