the plasmodium falciparum clag9 gene encodes a rhoptry protein that is transferred to the host...

Molecular Microbiology (2004)

52

(1), 107–118 doi:10.1111/j.1365-2958.2003.03969.x

© 2004 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004

? 2004

52

1107118

Original Article

Clag9 is a rhoptry proteinI. T. Ling

et al

.

Accepted 8 December, 2003. *For correspondence. [email protected]; Tel. (+44) 20 8816 2335; Fax (+44) 20 88162730.

The

Plasmodium falciparum clag9

gene encodes a rhoptry protein that is transferred to the host erythrocyte upon invasion

Irene T. Ling,

1

* Laurence Florens,

2

Anton R. Dluzewski,

3,4

Osamu Kaneko,

5

Munira Grainger,

1

Brian Y. S. Yim Lim,

1

Takafumi Tsuboi,

5

John M. Hopkins,

3,4

Jeffrey R. Johnson,

2

Motomi Torii,

5

Lawrence H. Bannister,

3

John R. Yates, III

2

, Anthony A. Holder

1

and Denise Mattei

6

1

Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.

2

The Scripps Research Institute, Department of Cell Biology, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

3

Department of Anatomy, Cell and Human Biology, Guy’s, King’s and St Thomas’ School of Biomedical Science, Guy’s Hospital, London SE1 1UL, UK.

4

Department of Immunobiology, Guy’s, King’s and St Thomas’ School of Medicine, Guy’s Hospital, London SE1 9RT, UK.

5

Department of Molecular Parasitology, Ehime University School of Medicine, Shigenobu-cho, Ehime 791-0295, Japan.

6

Institut Pasteur, Biology of Host Parasite Interactions, URA 2581, F-75724, Paris Cedex 15, France.

Summary

The first gene characterizing the

clag

(cytoadherencelinked asexual gene) family of

Plasmodium falciparum

was identified on chromosome 9. The protein product(Clag9) was implicated in cytoadhesion, the bindingof infected erythrocytes to host endothelial cells, butlittle information on the biochemical characteristics ofthis protein is available. Other genes related to

clag9

have been identified on different chromosomes.These genes encode similar amino acid sequences,but

clag9

shows least conservation. Clag9 wasdetected in schizonts, merozoites and ring-stage par-asites after protease digestion and peptide analysisby mass spectrometry. Using antisera raised againstunique regions of Clag9 and against RhopH2, a com-ponent of the RhopH high-molecular-mass proteincomplex of merozoites, immunofluorescence co-

localized the two proteins to the apical region ofmerozoites. Immunoelectron microscopy co-localizedClag9 and RhopH2 exclusively to the basal bulbregion of rhoptries rather than to their apical ducts.The same Clag9-specific antibodies bound the RhopHcomplex, and the protein was detected in the complexpurified by antibodies to RhopH2. Clag9 protein wasalso shown to be present in ring-stage parasites, car-ried through from the previous cycle with the RhopHcomplex, in a location identical to that of RhopH2.Transcription of the

clag9

gene was shown to occurat the same time as the genes for other members ofthe RhopH complex,

rhoph2

and

3

. The results indi-cate that Clag9 is part of the RhopH complex andsuggest that, within this complex, the protein previ-ously designated RhopH1 is composed of more thanone protein product of the

clag

gene family. Theresults cast doubt on a direct role for Clag9 in cytoad-hesion; we suggest that the primary role of the RhopHcomplex is in remodelling the infected red blood cellafter invasion by the merozoite. The complex mayhave multiple functions dependent on its exact com-position, which may include, with respect to Clag9, acontribution to the mechanism of cytoadhesion.

Introduction

Plasmodium falciparum

is the parasite causing the mostsevere form of malaria in humans. The pathology ofmalaria infection is mediated by a number of differentmechanisms, which include the phenomenon of cytoad-hesion. Of particular interest are the molecules involvedin this process, both the receptors on host cells and par-asite ligands. A number of molecules that bind

P. falci-parum

-infected erythrocytes are expressed on the surfaceof endothelial cells. These include thrombospondin (TSP),CD36, intercellular adhesion molecule-1 (ICAM-1) andchondroitin sulphate A (CSA). CD36 is the most wide-spread receptor whereas ICAM-1 binding is important incerebral malaria, and CSA is involved in parasite bindingin the placenta (reviewed by Newbold

et al

., 1999). In thesearch for parasite factors involved in cytoadhesion, agene on chromosome 9,

clag9

(

c

ytoadherence

l

inked

a

sexual

g

ene on chromosome 9), was identified as havinga role in this process (Trenholme

et al

., 2000).

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I. T. Ling

et al.

© 2004 Blackwell Publishing Ltd,

Molecular Microbiology

,

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, 107–118

The

clag9

gene was initially identified through thestudy of parasite lines that no longer bound to C32 mel-anoma cells (which express CD36 on their surface). Theloss of this phenotype was associated with a subtelom-eric deletion in chromosome 9, a deletion that occursfrequently during parasite adaptation to

in vitro

cultureand appears to give a selective growth advantage

in vitro

(Biggs

et al

., 1989). These parasite lines expressed nei-ther the variant surface agglutination phenotype norPfEMP-1 (

P. falciparum

erythrocyte membrane protein 1,a product of the

var

gene family) on the surface of theinfected erythrocyte (Day

et al

., 1993). Fine structuremapping of the deleted region and sequence analysis(Barnes

et al

., 1994; Holt

et al

., 1998) revealed a candi-date gene that was transcribed in asexual blood stagesand consisted of nine exons. A genetic knock-out of thislocus (Trenholme

et al

., 2000) or antisense RNA inhibi-tion (Gardiner

et al

., 2000) resulted in the loss of theCD36 binding phenotype. Several related genes wereidentified as a result of the

P. falciparum

genomesequencing project (Gardner

et al

., 1998; 2002; Bowman

et al

., 1999), and there now appears to be five distinctgenes in the

clag

multigene family that encode similarproteins.

The high-molecular-mass rhoptry protein complex(RhopH) contains three non-covalently associated, butunrelated, proteins called RhopH1, 2 and 3 (Campbell

et al

., 1984; Holder

et al

., 1985; Cooper

et al

., 1988;Lustigman

et al

., 1988; Hienne

et al

., 1998). The genesencoding RhopH2 and 3 are single-copy genes on chro-mosome 9 (Brown and Coppel, 1991; Ling

et al

., 2003).Interestingly, RhopH1 was shown to be encoded by oneor both of the two

clag

genes on chromosome 3, C0110wand C0120w (Kaneko

et al

., 2001). The RhopH complexis located in the merozoite rhoptry organelles and isexternalized after erythrocyte invasion to the parasito-phorous vacuole or the surrounding parasitophorous vac-uolar membrane (PVM) (Hiller

et al

., 2003; Ling

et al

.,2003). The exact location of the RhopH complex withinthe rhoptries is not clear. Early immunoelectron micros-copy indicated that both RhopH2 and 3, and therefore byinference the whole RhopH complex, are located in rhop-tries (Holder

et al

., 1985; Coppel

et al

., 1987; Cooper

et al

., 1988; Jaikaria

et al

., 1993). A more recent studylocalized the complex to the rhoptry apical duct ratherthan the basal bulb of this organelle (Sam-Yellowe

et al

.,1995).

In view of the identification of RhopH1 as the productsof

clag

gene(s) on chromosome 3, we reinvestigatedClag9. The size and location of the Clag9 protein in theinfected erythrocyte has been examined (Gardiner

et al

.,2000; Trenholme

et al

., 2000), and it has been suggestedthat Clag9 may be involved in cytoadhesion by bindingdirectly to CD36, or indirectly, for example through a role

in PfEMP1 transport (Craig, 2000; Trenholme

et al

.,2000). Here, we investigate expression of the

clag9

geneand show that its protein product is part of the RhopHcomplex in the rhoptry organelles of merozoites and istransferred with the RhopH complex into the ring-stageparasite.

Results

Mass spectrometry-based proteomic analysis detected Clag9 in merozoites

In an initial analysis, Clag9 (PFI1730w) was identified introphozoites, merozoites, gametocytes and sporozoites(Florens

et al

., 2002). Here, we examined the expressionof Clag9 protein across the entire erythrocytic cell cycleusing the 3D7 cloned parasite line (Walliker

et al

., 1987);the Clag9 peptides detected in the merozoite preparationsare given in Table 1. These were identified as beingunique parasite-derived sequences that had no homologyto host proteins. The percentage of Clag9, RhopH2(PFI1445w) and RhopH3 (PFI0265c) sequences coveredby detected peptides was measured for each of the stagesanalysed (Fig. 1). When following the same protein (orproteins with similar molecular masses), this parametercan be considered a gross empirical measure of proteinabundance. Clag9 was identified in rings, trophozoites,schizonts and merozoites, and analysis of the percentageof specific peptides detected gave an expression profilesimilar to the proteins of the RhopH complex, RhopH2 and3 (Fig. 1). These profiles also reflect the known expressioncharacteristics of RhopH2 and 3, which are synthesizedfrom the early schizont stage onwards but are alsopresent in ring-stage parasites (Lustigman

et al

., 1988;Ling

et al

., 2003), and suggest similar expression charac-

Table 1.

Peptides derived from Clag9 identified in merozoite extracts.

Locus ID: MAL9P1.352 (Clag9)Peptide identified

Position in Clag9 amino acid sequence

K.

SILDNDELYNSLSNLENLLLQTLEQDELK

.I 35–63K.

SILDNDELYNSLSNLENLLLQTLEQDELKIPIMK

.G 35–68K.

ILNELNADGAEKPYIIPTSNCSANDIVKYEHTLK

.T 82–115K.

TQITLEYK

.P 116–123K.

KLNEHTINALR

.L 176–186K.

SDIIDYDDL.

L 216–224V.

GLKELYQNLVKCVEKCYIRNRK

.N 488–509Y.

TLNKAMLKEVVNDFFVIYKMNK

.D 860–881R.

LSVHDEPFLR

.F 983–992R.

NILYFPNHLPEELR

.K 1128–1141N.

HLPEELRKQ

.T 1135–1143Q.

EVQEDKGTDITPLPTFDIM

.D 1264–1282K.

DLLYYDDGIDR

.T 1313–1323K.

RYELIPLQR

.Y 1326–1334R.

YELIPLQR

.Y 1327–1334

The identified peptide sequence is in bold.

Clag9 is a rhoptry protein

109



,

52

, 107–118

teristics for Clag9. Although Clag9 and RhopH2 have sim-ilar molecular masses, the percentage of specific peptidesdetected for Clag9 was significantly lower than those ofRhopH2 (and RhopH3), suggesting that Clag9 waspresent in lower abundance.

Clag9 is located in the basal ends of rhoptries in

P. falciparum

schizonts

To determine the location of Clag9 within the mature sch-izont, three specific rabbit or mouse antisera were raisedagainst different sequences unique to Clag9 (using thesequence of 3D7 in the malaria genome project; Gardner

et al

., 2002) (Fig. 2). The coloured bars indicate the pep-tide regions: rabbit anti-Clag9 peptides in blue; mouseanti-Clag9 serum 1, green; and mouse anti-Clag9 serum2, orange. These were used in immunofluorescenceassays (IFA) together with monoclonal antibodies (mAbs)previously characterized as binding to either the RhopHcomplex (mAb 4E10) (Ling

et al

., 2003) or RhopH2 (mAb61.3) and rabbit anti-RhopH2 (Holder

et al

., 1985). Similarimages were obtained using rabbit anti-Clag9 serumtogether with either mAbs 4E10 or 61.3 in dual-labellingIFA; however, only the results for 4E10 are shown (Fig. 3).Clag9 within the mature parasite was visualized usingtetramethylrhodamine isothiocyanate (TRITC)-labelledanti-rabbit antibody (Fig. 3A, red), RhopH2 using OregonGreen anti-mouse antibody (Fig. 3B, green) and, wherethe two antibodies co-localize, the staining is yellow(Fig. 3C). DAPI staining of the nucleus is also shown(Fig. 3D). The staining of Clag9 appeared to be mainlypunctate and largely co-localized with that of RhopH2, aprotein previously shown to be present in the rhoptries(Holder

et al

., 1985). A similar punctate pattern typical ofrhoptries was produced by the two mouse anti-Clag9 anti-sera, which was identical and distinct from that obtainedwith antibodies against the microneme protein EBA175(data not shown). Both mouse anti-Clag9 sera also co-localized with rabbit anti-Clag9 in dual labelling experi-ments (data not shown). Specificity of the rabbit anti-Clag9 serum was determined by its lack of reactivity withT996 schizonts on thin smears and in Western blots ofparasite extracts (data not shown). T996 does not tran-scribe the

clag9

gene (data not shown). Normal mouseand normal rabbit sera gave no distinctive pattern underthe same conditions (data not shown).

The apical location of both RhopH2 and Clag9 wasconfirmed by immunoelectron microscopy of bothreleased merozoites (clone 3D7) and those present within

Fig. 1.

Sequence coverage of rhoptry proteins across the erythrocytic cycle in 3D7 parasites. The percentage of Clag9, RhopH2 and RhopH3 sequences covered by detected peptides was measured for each of the four stages of the

P. falciparum

erythrocyte life cycle analysed. When following the same protein (or proteins with similar molecular mass), this parameter can be considered as a gross empir-ical measure for protein abundance. The results loosely fit the tran-scription and expression profile: the genes are transcribed from early schizont stages onwards until parasite rupture. RhopH2 and3 have been detected previously in ring-stage parasites (Lustigman

et al

., 1988; Ling

et al

., 2003).

Rng Tpz Sch Mrz0

5

10

15

20

25

30

35

40

45

50

55S

eq

ue

nce

Co

vera

ge

(%

)

Stage

Fig. 2.

The deduced amino acid sequence of

clag9

(PFI1730w) was obtained from PlasmoDB (http://www.plasmodb.org). This representation shows the putative signal peptide sequence (in pink) (Trenholme

et al

., 2000) and the position of the cysteines (vertical lines) within the amino acid sequence. The coloured bars underneath indicate the peptide regions used to raise the various sera: rabbit anti-Clag9 peptides in blue; mouse anti-Clag9 serum1, green; and mouse anti-Clag9 serum 2, orange. The scale (in amino acids) is indicated.

http://www.plasmodb.org

110

I. T. Ling

et al.



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, 107–118

schizonts (cloned line C10; Hempelmann

et al., 1981).Antisera against Clag9 and RhopH2 showed clear label-ling of rhoptries, including immature forms within sch-izonts (Fig. 3E, F, H and I) and more mature elongatedforms of the organelle in free merozoites (Fig. 3G and J).Labelling for both molecules was confined to the basalbulb with no indication of apical duct labelling in theseorganelles (Fig. 3F and I), even in released merozoites(Fig. 3G and J). Control sera showed no distinctive pattern(data not shown).

Identification of Clag9 as part of the P. falciparum RhopH protein complex

In immunoprecipitation experiments, using 3D7 parasiteextracts prepared with buffer containing NP40, the rabbitanti-Clag9 peptide serum consistently precipitated a com-

plex containing three protein bands of similar size, thelargest being ª150 kDa whereas the smallest was thesame size as RhopH2 (Fig. 4, lane 3). A somewhat differ-ent pattern of bands was precipitated by the rabbit poly-clonal anti-RhopH2 (Fig. 4, lane 2) under similarconditions. This latter pattern is normally seen with anti-bodies against RhopH2 (Ling et al., 2003). Preparation ofa parasite extract with a buffer containing SDS essentiallydisrupted the RhopH complex, as seen in Fig. 4 (lane 1),where the anti-RhopH2 antibody precipitated only oneband, the second largest main band of the complex. Inthe extract prepared with SDS, the anti-Clag9 serum pre-cipitated more than one band, predominantly the largestone seen in the presence of mild detergent; and in additiontwo other smaller ones, the smallest being of similar sizeto RhopH2 (Fig. 4, lane 4). Preimmune rabbit serum failedto immunoprecipitate specific bands (data not shown).

A B C D

HE

F

G

I

J

Fig. 3. Localization of Clag9 to the rhoptry organelles.A–D. An identical field from a 1% formaldehyde-fixed thin smear of 3D7 schizonts: (A) rabbit anti-Clag9, TRITC; and (B) mAb 4E10, Oregon Green. Each antibody bound to P. falciparum schizonts gave a distinctive punctate pattern typical of apical proteins. The two antibodies were co-localized within schizonts (C) visual-ized as yellow; (D), DAPI, a nuclear stain. Con-trol normal mouse and rabbit sera produced no fluorescence.E–J. Electron micrographs illustrating immu-nogold labelling for Clag9 (E–G) and RhopH2 (H–J). Labelling in (E), (F), (H) and (I) shows antigen localization to the basal region of devel-oping rhoptries within schizonts (C10), and (G) and (J) depict similar basal labelling in released merozoites (3D7). No staining was detected in the neck of the rhoptries. Negative controls gave no distinctive pattern. A scale bar is indi-cated in (E–J).

Clag9 is a rhoptry protein 111

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 52, 107–118

In order to determine whether Clag9 forms a complexwith RhopH2, Western blot analysis of the immunoprecip-itates from antibodies against Clag9, as well as those fromantibodies of different RhopH2 specificities, was carriedout after SDS–PAGE. The blots were probed initially withthe secondary antibody only to determine the level ofbackground contributed by the presence of antibody

within the samples (data not shown). Then, the blots werewashed with PBS and probed with homologous and het-erologous sera. Figure 5 illustrates the results obtained byprobing one blot with rabbit anti-Clag9 serum (Fig. 5A)and mAb 49 (specific for RhopH2; Doury et al., 1994)(Fig. 5B). Clag9 is associated with RhopH2, because thelatter is present in the complex precipitated by rabbit anti-Clag9 serum (Fig. 5B, lane 2), and Clag9 is in the com-plexes precipitated by rabbit anti-RhopH2 and mAb 4E10(Fig. 5A, lanes 1 and 3). Rabbit anti-Clag9 serum consis-tently immunoprecipitates three bands detected by auto-radiography (Fig. 5C, lane 2), and the same polypeptideswere also detected in this complex by Western blottingwith the same serum (Fig. 5A, lane 2). A minor fourthband is detected on this blot by this antibody, which maybe the result of degradation or incomplete reduction.There are differences in the intensities of the Clag9 bandsrecognized by the rabbit anti-Clag9 antibody in the West-ern blot (Fig. 5A, lanes 1–3). In Fig. 5A (lane 2), onlycomplexes containing Clag9 are immunoprecipitated,whereas rabbit anti-RhopH2 and mAb 4E10 (Fig. 5A,lanes 1 and 3) potentially immunoprecipitate complexescontaining a mixture of Clags (Kaneko et al., 2001). Threebands were detected by rabbit anti-Clag9 in a similarexperiment (data not shown). The RhopH2 bands inFig. 5B are identical to the bands seen in the radiolabelledimmunoprecipitates (Fig. 5C) and detected by rabbit anti-RhopH2 in a similar experiment (Fig. 5D). The chemilumi-nescent bands were detected after 5–10 s exposure tofilm and were not detected under similar conditions whenno primary antibody was used (data not shown). Theapparent size of Clag9 appeared to be distinctly largerthan that of RhopH2 at ª150 kDa. In parallel experiments,the mouse anti-Clag9 serum 2 recognized Clag9 in anti-

Fig. 4. Identification of Clag9 as part of the RhopH complex. Immu-noprecipitation of proteins from NP40 and SDS extracts of 35S-radio-labelled parasites using a panel of antibodies. The immunoprecipitates were analysed by SDS–PAGE using a 5–12.5% gradient gel and visualized by fluorography using X-ray film. From NP40 schizont extracts (lanes 2 and 3) and SDS extracts (lanes 1 and 4), polyclonal rabbit anti-RhopH2 serum (lanes 1 and 2) and rabbit anti-Clag9 peptide serum (lanes 3 and 4) were used to precip-itate proteins. Both antibodies recognize a complex of several bands in NP40 extracts: RhopH2, indicated by the arrow on the left-hand side; and Clag 9, a set of three bands ª140–155 kDa, indicated by arrows on the right-hand side. No specific polypeptides were precip-itated by the preimmune rabbit antisera (data not shown). After extrac-tion in SDS, which disrupts the RhopH complex, RhopH2 alone is precipitated by anti-RhopH2 (lane 1), whereas three bands are still precipitated by rabbit anti-Clag9 (lane 4). This result was consistent over two separate experiments. The mobility of protein markers is indicated on the left according to their size (kDa).

Fig. 5. Western blot analysis of the protein complexes recognized by Clag9- and RhopH2-specific antibodies. Immunoprecipitates containing both non-radiolabelled and 35S-labelled material were reduced, subjected to electrophoresis and blotted on to nitrocellulose paper. Three antibodies were used for the immunoprecipitation: rabbit anti-RhopH2 (lanes 1), rabbit anti-Clag9 (lanes 2) and mAb 4E10 (lanes 3). The blots were probed with rabbit anti-Clag9 (A), and mAb 49 (B). RhopH2 was present in all samples (indicated by a triangle). The anti-Clag9 serum specifically recognized four bands in the homologous complex (A, lane 2), the major upper band indicated by an asterisk, and recognized bands in the 4E10 and RhopH2 complexes, predominantly the upper 150 kDa band. Specific proteins were identified within the respective complexes by autorad-iography (C).D. The result of a similar experiment in which the complexes were probed with rabbit anti-RhopH2 confirming the presence of RhopH2 in the rabbit anti-Clag9 complex. Counterstaining this blot with rabbit anti-Clag9 also showed the presence of Clag9 in all immunoprecipitates (data not shown). The mobility of protein markers is indicated at the side according to their size (kDa).

A B C D

112 I. T. Ling et al.


RhopH2 immunoprecipitates, and RhopH2 was detectedin mouse anti-Clag9 immunoprecipitates (data notshown).

Clag9 is present in ring-stage P. falciparum parasites

It has already been established that RhopH2 and 3 arepresent in ring-stage parasites in the intact RhopH com-plex (Lustigman et al., 1988; Ling et al., 2003). We inves-tigated whether Clag9, identified as part of the RhopHcomplex, is also present in this parasite stage andwhether Clag9 and RhopH2 are still associated. The loca-tion of the proteins was examined in dual-labelling IFAexperiments. The location of Clag9 was identified byTRITC-labelled secondary antibody (red, Fig. 6, 1A), andRhopH complex (mAb 4E10) by Oregon Green-labelledsecondary antibody (green, Fig. 6, 1B). The yellow colourin Fig. 6, 1C, indicates where the proteins co-localize, andthe nuclei are identified by the blue DAPI stain (Fig. 6, 1D).The precise subcellular location in the ring-stage parasitecannot be determined by these experiments, but it hasbeen suggested that the proteins could be located in thePVM (Hiller et al., 2003; Ling et al., 2003). Similar resultswere obtained using mAb 61.3 (specific for RhopH2) inplace of 4E10 (data not shown). Normal controls pro-duced no distinctive pattern under the same conditions(data not shown).

To confirm further the maintenance of the associationof Clag9 with the RhopH complex and the transfer of theseproteins from rhoptries, synchronous ring-stage parasitesthat had been radiolabelled in the previous cycle wereextracted with NP40 buffer and immunoprecipitated underthe same conditions as before (Ling et al., 2003). Twoextracts were prepared; mature radiolabelled parasiteswere allowed to invade for 4 h, and the remaining sch-izonts were removed. Half the culture was harvestedimmediately (R1); the remaining parasites were culturedfor 7 h before harvesting (R2). As reported before (Linget al., 2003), the RhopH complex was precipitated by mAb4E10 (Fig. 6, 2A) and rabbit anti-RhopH2 (Fig. 6, 2C) sim-ilar to that precipitated from schizont-stage parasiteextracts (Fig. 4C, lanes 1 and 3 respectively). We werealso able to immunoprecipitate a complex using rabbitanti-Clag9 and show that this complex persisted duringculture for 7 h (Fig. 6, 2B). The pattern of this complex isslightly different from that seen in the schizont stage(Fig. 4B) and will require further examination. In an effortto control for schizont contamination, the ring-stage prep-arations were immunoprecipitated with anti-MSP-1 anti-body (Ling et al., 2003). There were a small number ofschizonts present in R1, but no contamination in R2.There was a slight diminution in intensities of the bandsbetween R1 and R2 for all the antibodies, and againfurther work needs to be done to determine the fate of the

A B C D

Fig. 6.1. Rabbit anti-Clag9 reacts with P. falciparum ring-infected erythrocytes. The four panels show an identical field from a 1% formalde-hyde-fixed thin smear: (A) rabbit anti-Clag9, TRITC; (B) mAb 4E10, Oregon Green. Each antibody bound to ring-stage parasites clearly outlining the parasite within the erythrocyte. The two antibodies co-localized, with red and green fluorescence overlap being visualized as yellow (C).D. DAPI, a nuclear stain.Control normal mouse and rabbit sera pro-duced no fluorescence.2. Confirming the presence of Clag9 in ring-stage parasites. P. falciparum schizonts were labelled with 35S, merozoite invasion and ring formation was allowed to occur before parasites were harvested (R1) or cultured for 7 h (R2). NP40 extracts of R1 and R2 (lanes 1 and 2 respectively) were immunoprecipitated using mAb 4E10 (A), rabbit anti-Clag9 (B) or rabbit anti-RhopH2 (C). The immunoprecipitates were analysed by SDS–PAGE using 5–12.5% gradi-ent gels and visualized by fluorography using X-ray film. Each antibody recognizes a complex of several bands: RhopH2, indicated by the arrow on the right-hand side; and Clag 9, a set of three bands ª140–155 kDa, the largest indicated by an asterisk in (B). No specific polypeptides were precipitated by normal anti-body controls (data not shown). The mobility of protein size markers is indicated on the right according to their size (kDa).



complex in ring-stage parasites. Western blot analysis ofSDS-solubilized schizont and ring-stage parasites alsoshowed the presence of Clag9 in both parasite stages(data not shown).

Transcription of the clag9 gene

Of the RhopH proteins, both RhopH2 and 3 have beenshown previously to be present in ring-stage parasites.However, the genes for these proteins are transcribed inschizonts and not in earlier parasite stages (Lustigmanet al., 1988; Jaikaria et al., 1993; Ling et al., 2003). Toexamine at what point in parasite development Clag9 istranscribed, Northern blots with samples of RNA obtainedat different time points after invasion were probed withparts of the rhoph2 and 3 genes (Ling et al., 2003)(Fig. 7A and C) and a unique clag9-specific sequence(Fig. 7B). The clag9 probe hybridized to a single band ona chromosome blot (chromosome 9), indicating that it didnot cross-hybridize with other clag genes (data notshown). All probes detected transcripts over the sameperiod (34–46 h), with maximum transcription at about42 h after invasion. The transcripts were different in size(Fig. 7A–C), the transcript for clag9 being slightly smallerthan that of rhoph2. The signals detected using the rhoph2and 3 probes were of greater intensity than that using theclag9-specific probe. This technique cannot be useddirectly to estimate the amount of gene-specific message.However, it is clear from the differences in exposure times

(4 h for rhoph2 and 3, 4 days for clag9) that the clag9message is much less abundant.

Discussion

Clag9 was initially described as having a role in cytoad-hesion (Clag is an Australian branded glue). Cytoadhe-sion is a property of trophozoite- and schizont-stageparasites manifest from 16 to 20 h after invasion. How-ever, in this report, we demonstrate that the clag9 geneis transcribed late in the parasite’s asexual blood stagecycle, when the protein is also synthesized. Significantly,Clag9 is present in merozoites as part of the RhopHcomplex in the rhoptries. After erythrocyte invasion, theprotein is transferred to the ring stage still associated withthe RhopH complex. These results do not suggest a directrole for Clag9 in sequestration of the infected cell unlessit is mediated in the cycle after erythrocyte invasion, andthere is no evidence to suggest how this protein isinvolved mechanistically in cytoadhesion.

The clag9 gene was originally identified in a study ofparasite lines containing truncated chromosome 9 as aresult of subtelomeric deletions at both ends of the chro-mosome. The deleted sequence at the left end of thechromosome largely encompassed the rep20 structure,whereas at least 20% of the entire chromosome wasremoved as a result of deletions at the right end. Insequential mapping studies of these deletions, theregion(s) responsible for adhesion to CD36 and/or a crit-ical role in gametogenesis were identified (Shirley et al.,1990; Kemp, 1992; Chaiyaroj et al., 1994). The expressionof both phenotypes was mapped to a 0.3 Mb subtelomericregion (Day et al., 1993), and the gene associated withcytoadhesion was located in a 55 kb stretch within thisregion (Barnes et al., 1994). The breakpoints of deletionsin several independent parasite isolates were found clus-tered around an open reading frame (breakpoint openreading frame, bporf). The function of bporf is unknown,but the region of chromosome 9 distal to bporf appears tocontain the gene necessary for cytoadhesion to mela-noma cells, and this gene is not a var gene (Bourke et al.,1996). The clag9 gene was identified distal to bporf, andtargeted disruption of clag9 suggested that the gene prod-uct is essential for binding of infected erythrocytes toCD36 (Holt et al., 1999; Trenholme et al., 2000). In acomplementary approach, transfection with a plasmid toexpress antisense clag9 reduced binding to C32 mela-noma cells (expressing CD36), an effect that was reversedafter removal of the plasmid (Gardiner et al., 2000).Although these observations suggest that Clag9 is impor-tant in cytoadhesion, evidence of a direct mechanistic rolein this process is lacking.

In our study, the transcription of clag9 is shown to occurlate in the asexual blood-stage cycle, at the same time as

Fig. 7. The transcription of rhoph2 and 3 and clag9 is similar and stage specific. Northern blot analysis of RNA from 3D7 parasites throughout asexual blood-stage development is shown. RNA was harvested at 4 h intervals from synchronized parasites and analysed by hybridization with probes for rhoph3 (A), clag9 (B) and rhoph2 (C). All genes are transcribed between 34 and 46 h after invasion with maximum transcription at 42 h. The mobility of RNA markers is indi-cated on the right according to their size (kb).



other members of the RhopH complex, rhoph2 and 3. Thisoccurs during schizogony, rather than earlier as mighthave been expected if the clag9 gene product had a directrole in cytoadhesion. This timing would suggest a role forthe protein either in the merozoite or in the young devel-oping parasite after invasion, rather than in the trophozoiteand schizont stages of the parasite.

The data we present identify Clag9 as a merozoiteprotein. This assertion is based on the clear identificationof unique peptides derived from Clag9 in tryptic and pro-teinase K digests of merozoite protein lysates, and issupported by the localization of Clag9 in merozoites usingantibodies specific for protein sequences that are uniqueto Clag9. Using both fluorescence microscopy and immu-noelectron microscopy (IEM), Clag9 was located at theapical end of merozoites, more specifically in the rhoptryorganelles and co-localized with the known rhoptry proteinRhopH2. Interestingly, the IEM finding confirms thatRhopH2 (and, by inference, the RhopH complex) is clearlysituated in the basal bulb of the rhoptry. This is at variancewith the reports of an apical location within the rhoptryduct using antibodies against RhopH3 (Sam-Yelloweet al., 1995), but supports earlier studies showing a basalrhoptry location (Holder et al., 1985; Coppel et al., 1987;Cooper et al., 1988; Jaikaria et al., 1993). It is also inter-esting to note the location of both Clag9 and RhopH2 inthe bulb of rhoptries in released merozoites and not ontheir apical surfaces. This would appear to indicate thatthe function of the proteins is not associated with host cellrecognition. It would be useful to determine the stage atwhich these proteins are released from the rhoptries.

The results indicate that Clag9 is present in the RhopHcomplex. In a previous analysis of the RhopH complexaffinity purified using mAb 61.3, mass spectrometry anal-ysis identified RhopH1 as a product of one or both of theclag genes on chromosome 3 (Kaneko et al., 2001).Based on the results presented in this paper, we suggestnow that RhopH is a multimeric complex composed ofRhopH2 and 3, which are the products of single-copygenes, together with one or more Clag proteins that com-prise RhopH1. The organization of the RhopH complex,including the stoichiometry of the components and thepresence of other clag gene products, is currently underfurther investigation. The apparent absence of Clag9 inthe RhopH complex recognized by mAb 61.3 may beexplained by the fine specificity of this antibody, recogniz-ing only a subset of the RhopH complex and freeRhopH2. Alternatively, Clag9 may be less abundant inRhopH than the proteins encoded by the clag genes onchromosome 3 and is present in much smaller amountsthan RhopH2.

In earlier studies, Clag9 was identified as a 220 kDaTriton X-100-insoluble, SDS-soluble protein present inboth trophozoite- and schizont-stage parasites (Gardiner

et al., 2000; Trenholme et al., 2000). In contrast, our find-ings suggest that Clag9 has an apparent molecular massof about 150 kDa by SDS–PAGE. Antibodies to differentsequences unique to Clag9 bound to the ª150 kDa pro-tein, confirming its identity as Clag9. The two slightlysmaller bands consistently seen in the immunoprecipi-tates of the anti-Clag9 sera, still present after SDS treat-ment and recognized by the rabbit anti-Clag9 antibody inWestern blots, may be fragments of the protein.

After merozoite invasion of erythrocytes, the RhopHcomplex is transferred intact to the ring-stage infected redblood cell, and Clag9 is clearly seen within this complex.The proteins are secreted from the rhoptries and appearto be either in the parasitophorous vacuole or associatedwith the parasitophorous vacuolar membrane. We showedco-localization of RhopH2 and Clag9 immediately sur-rounding the parasite, and recent work has identifiedRhopH2 on the PVM (Hiller et al., 2003). In contrast, arecently identified 42 kDa rhoptry protein, involved inadhesion to endothelial cells and the placenta, reactedwith the ring-infected erythrocyte cell surface and wasshown to persist until 20 h after invasion (Douki et al.,2003). We suggest that one role of the RhopH complex isto begin the process of remodelling the infected erythro-cyte to accommodate the parasite. For example, the com-plex may be involved in the architecture necessary totransport other molecules into or out of the parasite. If theRhopH complex has such a function, this may provide alink to previous observations implicating a role in cytoad-hesion. For example, Clag9 has been suggested to beinvolved in PfEMP1 trafficking in the trophozoite (Holtet al., 1999; Craig, 2000). If the RhopH complex is a basiccomponent of the mechanisms transporting proteins tothe red blood cell cytoplasm, cytoskeleton or plasmamembrane, it would be necessary for this machinery tobe in place before the export of newly synthesized pro-teins from the parasite. A defect in the transport machin-ery may have a profound effect on the expression of otherproteins at the surface of the infected red blood cell andtherefore have an indirect effect on, for example,cytoadhesion.

We have shown that the RhopH protein complex con-tains the products of rhoph2 and 3 together with at leasttwo clag genes, but there are five members of the clagfamily in P. falciparum. clag9 has been most closely stud-ied and has been shown to be highly conserved acrossisolates (Manski-Nankervis et al., 2000). rhoph2 and 3also show a high degree of homology across isolates(Ling et al., 2003). Neither RhopH2, 3 or RhopH1/Claghave homology to any known protein nor do they containany recognizable domain. Between the members of theClag family, there is high degree of protein sequenceconservation with most of the variation in the N-terminalend of the molecule and in a small region towards the C-



terminus. The Clags have putative signal peptides, andClag9 has been suggested to have four transmembranedomains (Trenholme et al., 2000). The conserved regionsmay be involved in binding to other members of theRhopH complex, whereas the more variable regions couldgive the proteins their specificities of interaction with othermolecules. Currently, it is not known whether an individualRhopH complex contains one or more of the Clags. If theother members of the Clag family are present in an indi-vidual RhopH complex, this structure may be extremelydiverse, depending on the composition and stoichiometryof the components. Whether the different members of theclag multigene family have similar or related functions willrequire further analysis.

Experimental procedures

Parasites

Plasmodium falciparum lines 3D7 (Walliker et al., 1987) andC10 (Hempelmann et al., 1981) were maintained in vitro asdescribed, using either 10% human serum or 0.5% (w/v)AlbuMAX® I in RPMI-1640 medium (Guevara Patino et al.,1997). The cloned line 3D7 undergoes gametocytogenesis invitro, has been used extensively in cytoadhesion studies andwas used in the malaria genome sequencing project (Gard-ner et al., 2002). The clone C10 was isolated from an artificialmixture of parasites of different isotype and S-antigen char-acteristics (Hempelmann et al., 1981). 3D7 parasites wereused in all studies; C10 were only used in electron micros-copy studies. For synchronization of developmental stages,parasite collection with a magnet was used with an adapta-tion of the method of Staalsoe et al. (1999). Highly synchro-nous parasite preparations were produced using a MACStype D depletion column in conjunction with a SuperMACS IImagnetic separator (Miltenyi Biotec). Large numbers ofhighly purified schizonts and merozoites (purified in the pres-ence of EGTA) were obtained as described previously (Flo-rens et al., 2002; Taylor et al., 2002). To produce young ringstages, schizonts purified by retention on the magnetizedcolumn were mixed with uninfected erythrocytes and allowedto release merozoites to invade erythrocytes for 1 h beforeremoval of the remaining schizonts using the magnet.

Mass spectrometry-based proteomic analysis of parasite extracts

Whole-cell proteome analyses of P. falciparum trophozoites(five independent preparations) and merozoites (four inde-pendent preparations) were described previously (Florenset al., 2002). In addition, two more merozoite (free of otherstages), three schizont (95–100% pure) and seven schizont-free ring-stage (pretreated with either saponin or streptolysinO to release erythrocyte proteins) preparations were lysedby osmotic shock in 10 mM Tris-HCl, pH 8.5, for 1 h on ice.The pellet fraction was separated from the supernatant bycentrifugation for 30 min at 18 000 g at 4∞C. The membranepellet was solubilized in 0.1 M sodium carbonate, pH 11.5.After 1 h at 4∞C, centrifugation was performed as above to

separate the supernatant and sodium carbonate-extractedmembrane pellet. After denaturation in 8 M urea, reduction[5 mM Tris(2-carboxyethyl)phosphine hydrochloride, TCEP;Roche] and alkylation (20 mM iodoacetamide, IAM), proteinfractions were digested with proteinase K (Roche) for 4 h at37∞C in 0.1 M sodium carbonate, pH 11.5 (Wu et al., 2003).

As described previously (Washburn et al., 2001), peptidemixtures were concentrated and buffer exchanged to 5%acetonitrile (ACN), 0.5% acetic acid on Spec-Plus PTC18cartridges (Ansys). They were then loaded onto a 100 mminner diameter ¥ 365 mm outer diameter fused-silica micro-capillary column (Polymicro Technologies) with a 5 mm tip(Sutter Instruments P-2000 laser puller), packed first with5 mm of C18 reverse-phase (Aqua; Phenomenex), followed by5 mm of strong cation exchange material (Partisphere SCX;Whatman). Loaded microcapillary columns were installed in-line with a quaternary Agilent 1100 series high-performanceliquid chromatography (HPLC) pump, which allows directspraying into an LCQ-Deca ion trap mass spectrometerequipped with a nano-LC electrospray ionization source(ThermoFinnigan). The flow rate was 200–300 nl min-1.Three different elution buffers were used: buffer A (5% ACN,0.1% formic acid), buffer B (80% ACN, 0.1% formic acid) andbuffer C (500 mM ammonium acetate, 5% ACN, 0.1% formicacid). Fully automated 6- or 12-step chromatography runswere carried out. Full MS spectra were recorded on theeluting peptides over the 400–1600 m/z range. Tandem mass(MS/MS) spectra were acquired in a data-dependent manner,sequentially on the first, second and third most intense ionsselected from the full MS scan.

Trophozoite, merozoite, schizont and ring MS/MS data setswere searched against a database combining host proteins(human, mouse and rat sequences from NCBI RefSeq; http://www.ncbi.nlm.nih.gov/RefSeq/) with the latest release of theP. falciparum genome (Gardner et al., 2002) (complementedwith missing sequences of known Plasmodium proteins). ThePEP_PROBE algorithm (Sadygov and Yates, 2003), a modifiedversion of SEQUEST (Eng et al., 1994), was used to matchMS/MS spectra to peptides. The SEQUEST outputs wereparsed and filtered using DTASELECT (Tabb et al., 2002).Spectra/peptide matches were retained only if they had aminimum cross-correlation score (XCorr) of 1.8 for singlycharged, 2.5 for doubly charged and 3.5 for triply chargedspectra, and a normalized difference in correlation score(DCn) of at least 0.08. Any peptide hits had to have a mini-mum length of seven amino acids, were unique and were notdetected in searches against non-infected red blood cells, i.e.were not of host origin. In addition, the confidence for thematches to be non-random had to be at least 85% as definedby PEP_PROBE.

Peptides, recombinant proteins, DNA immunization and antibodies

A rabbit was immunized with a mixture of two peptidesfrom the 3D7 Clag9 amino acid sequence,ESDRFKQEQEKGIEFHD (residues 390–406) and LPT-FDIMDSKQNT (residues 1282–1294) from the deducedamino acid sequence of Clag9 (D. Mattei, unpublished), andthe serum was harvested. A gene-specific fragment of clag9(encoding amino acids 1080–1138, 3D7) was synthesized by



polymerase chain reaction (PCR) from P. falciparum genomicDNA (gDNA) using primers containing restriction sites, whichenabled cloning into pGEX-3X. Expression of the glutathioneS-transferase fusion protein was induced in 1 ml cultures,and the protein was found to be insoluble after treatment withBugBuster protein extraction reagent and benzonasenuclease (Merck Biosciences). The insoluble fraction waselectrophoresed under reducing conditions on NuPAGE 4–12% Bis-Tris polyacrylamide gels (Invitrogen Life Technolo-gies), and the fusion protein was excised. This was purifiedby electroelution (2.5 mM Tris, 19.2 mM glycine, 0.0001%SDS) at 5 W overnight (Green et al., 1982), followed by dial-ysis in PBS. Female BALB/c mice were immunized intraperi-toneally at 3 week intervals with the purified protein (50 mgprime, 100 mg first boost, 100 mg second boost, 75 mg thirdboost) as described previously (Taylor et al., 2002), and theserum was harvested (mouse anti-Clag9 serum1). A DNAfragment encoding amino acids 24–222 of Clag9 was ampli-fied by PCR from gDNA purified from the 3D7 parasite linewith the following primer pair: 5¢-GCTAGCACCTACAAAGGAGATAATATAAAT-3¢ and 5¢-CTCGAGATCATAATCAATTATATCAGATTTC-3¢ (NheI and XhoI restriction sites are in bold) andcloned into pJWE2 (Lu et al., 1996; Kaneko et al., 2002). Theplasmid was used to immunize mice, and serum was har-vested (mouse anti-Clag9 serum2) as described previously(Kaneko et al., 2002). Specificity was checked using therecombinant proteins corresponding to the equivalent regionof all Clag members expressed in COS7 cells (data notshown). The position of peptides and fusion proteins used inimmunization studies within the Clag9 sequence is shown bycoloured bars in Fig. 2: Clag9 peptides, blue; Clag9–GSTfusion protein, green; coding region of DNA expression vec-tor, orange.

The rabbit polyclonal antibodies against RhopH2 andMSP-1 and the mAbs 61.3, 4E10 and 49 have beendescribed previously (Holder et al., 1985; Doury et al., 1994;Ling et al., 2003).

Immunofluorescence assay (IFA) and immunoelectron microscopy (IEM)

For IFA, thin smears of P. falciparum-infected erythrocytes(containing both schizont and ring stages) were prepared andprobed with antibodies as described previously (Ling et al.,2003). These were probed with a panel of antibodies: mAbs61.3 and 4E10, both the mouse anti-Clag9 sera, rabbit anti-Clag9 and rabbit anti-RhopH2 sera. In dual-labelling experi-ments, slides were incubated with both rabbit anti-Clag9serum and mAb 4E10 (or mAb 61.3), and bound antibodywas detected using TRITC- and Oregon Green-labelled sec-ondary antibodies, respectively, as described previously (Linget al., 2003).

IEM was performed using rabbit serum raised againstClag9 sequences and the anti-RhopH2 serum. The methodwas as described previously (Bannister and Kent, 1993).Briefly, schizonts and free merozoites of P. falciparum (3D7and C10) were fixed in 0.1% (v/v) double-distilled glutaralde-hyde in RPMI 1640 (pH 7.3) with or without the addition of2% paraformaldehyde for 20 min at 4∞C. These were washedand processed by the progressive low-temperature dehydra-tion technique and embedded in Medium LR white resin

(Emscope), using ultraviolet light polymerization at room tem-perature. Thin sections were mounted on nickel grids andlabelled with either rabbit anti-Clag9 peptides serum or rabbitanti-RhopH2 serum. Labelling was detected with protein A-10 nm gold (a kind gift from Dr Pauline Bennett, King’s Col-lege London). Control samples were incubated with normalrabbit serum, or without primary antibody, before protein A–gold treatment. Sections stained with 2% (v/v) aqueous ura-nyl acetate were viewed in a Hitachi H7600 transmissionelectron microscope fitted with a CCD camera.

Metabolic labelling, immunoprecipitation and Western blot analysis of P. falciparum parasites

For analysis by immunoprecipitation, mature P. falciparumparasites (36–37 h after invasion) were biosyntheticallylabelled by incorporation of [35S]-methionine and cysteine for2 h. A portion of the parasites was harvested immediately,the remainder were returned to culture, allowed to matureand reinvade fresh erythrocytes. Remaining schizonts wereremoved by passing the culture through a magnet twice (Linget al., 2003). Half this culture was harvested immediately(R1), the remainder being cultured for 7 h before being har-vested (R2). For immunoprecipitation analysis, parasiteextracts were made using buffers containing Nonidet P40(NP40) (Ling et al., 1995), deoxycholate (DOC) or SDS (Tay-lor et al., 2002) and incubation for 30 min at 4∞C. The SDSextracts were diluted by the addition of DOC or NP40 buffer.After removal of any insoluble material and preadsorptionwith Protein G Sepharose (Pharmacia), antibodies wereadded to the extracts, and antigen–antibody complexes wereprecipitated using Protein G Sepharose at 4∞C. After wash-ing, the complexes were solubilized in reducing sample load-ing buffer and analysed by electrophoresis on 5–12.5%polyacrylamide gradient gels. Equal amounts of lysate wereused for each extraction, and proportional amounts of pre-cipitated sample were analysed. The gels were stained withCoomassie blue, treated with Amplify (Amersham) for 30 min,dried at 70∞C and exposed to X-ray film (Kodak) at -70∞C fora suitable period.

A combination of the immunoprecipitation and Westernblotting techniques was used to identify the polypeptidebands in the complexes recognized by the various antibodies.Immune complexes from NP40-solubilized radiolabelledmaterial were precipitated with Protein G Sepharose andresolved by SDS–PAGE on 5–12.5% gradient polyacrylamidegels under reducing conditions, as above. The proteins weretransferred to nitrocellulose, probed with antisera, and anti-body binding was visualized using ECL. After the decay ofchemiluminescence, the blot was exposed to BioMax film todetect the 35S signal. In this way, it was possible to comparethe bands specifically detected by antibodies on the Westernblot with the biosynthetically labelled components of theimmunoprecipitated RhopH complex.

Isolation of DNA and RNA and Northern blots

gDNA was isolated from P. falciparum parasites using theSNAP whole blood DNA isolation kit (Invitrogen Life Technol-ogies) or IsoQuickTM (Orca Research) according to the man-



ufacturers’ instructions. Total RNA was isolated from P.falciparum 3D7 parasites using the TRIzol method (Kyeset al., 2000). A Northern blot was prepared from RNA iso-lated at 4 h intervals from a synchronized parasite culture.Starting with schizonts, invasion was allowed to occur for 2 h,the remaining schizonts were removed, and aliquots werecollected every 4 h until merozoite release and the next asex-ual cycle. After RNA preparation from material collected ateach time point, each sample was quantified to allow approx-imately equal amounts of RNA to be loaded (Taylor et al.,2002). The blots were probed with parts of the rhoph2 and 3genes (Ling et al., 2003) and with a unique region of clag9(nucleotides 5049–5225 in the clag9 genomic sequence) asdescribed previously (Taylor et al., 2002).

Acknowledgements

We thank Helen Taylor for the Northern blot, Michael Black-man for the gift of the anti-MSP1 antibody, Jean-ClaudeDoury for mAb 49, Don Williamson and Peter Moore for thechromosome blot, Dr Pauline Bennett, King’s College Lon-don, for the kind gift of protein A-10 nm gold, and the Elec-tron Microscope Unit at Guy’s Hospital for technicalassistance. D.M. acknowledges support by grants from theIndo-French Centre for Promotion of Advanced Research(IFCPAR 2503-1) and the Institut Pasteur. J.R.Y. acknowl-edges the support of the Office of Naval Research (Co-operative Agreement N00014-01-2-0003), the US Army Med-ical Research and Material Command and the National Insti-tutes of Health. A.R.D., J.M.H. and L.H.B. acknowledgesupport from the Wellcome Trust (grant no. 069515) and theEuropean Commission (contract QLK2-CT-1999-01293).O.K. was supported in part by a Grant-in Aid for Encourage-ment of Young Scientists 15790215, and M.T. by Grants-inAid for Scientific Research 14370084 and 15406015, fromthe Ministry of Education, Culture, Sports, Science and Tech-nology, Japan. B.Y.S.Y.L. acknowledges the support of aMedical Research Council (UK) PhD studentship.

References

Bannister, L.H., and Kent, A.P. (1993) Immunoelectronmicroscopic localization of antigens in malaria parasites.Methods Mol Biol 21: 415–429.

Barnes, D.A., Thompson, J., Triglia, T., Day, K., and Kemp,D.J. (1994) Mapping the genetic locus implicated incytoadherence of Plasmodium falciparum to melanomacells. Mol Biochem Parasitol 66: 21–29.

Biggs, B.A., Kemp, D.J., and Brown, G.V. (1989) Subtelom-eric chromosome deletions in field isolates of Plasmodiumfalciparum and their relationship to loss of cytoadherencein vitro. Proc Natl Acad Sci USA 86: 2428–2432.

Bourke, P.F., Holt, D.C., Sutherland, C.J., Currie, B., andKemp, D.J. (1996) Positional cloning of a sequence fromthe breakpoint of chromosome 9 commonly associatedwith the loss of cytoadherence. Ann Trop Med Parasitol 90:353–357.

Bowman, S., Lawson, D., Basham, D., Brown, D., Chilling-worth, T., Churcher, C.M., et al. (1999) The complete

nucleotide sequence of chromosome 3 of Plasmodium fal-ciparum. Nature 400: 532–538.

Brown, H.J., and Coppel, R.L. (1991) Primary structure of aPlasmodium falciparum rhoptry antigen. Mol Biochem Par-asitol 49: 99–110.

Campbell, G.H., Miller, L.H., Hudson, D., Franco, E.L., andAndrysiak, P.M. (1984) Monoclonal antibody characteriza-tion of Plasmodium falciparum antigens. Am J Trop MedHyg 33: 1051–1054.

Chaiyaroj, S.C., Thompson, J.K., Coppel, R.L., and Brown,G.V. (1994) Gametocytogenesis occurs in Plasmodium fal-ciparum isolates carrying a chromosome 9 deletion. MolBiochem Parasitol 63: 163–165.

Cooper, J.A., Ingram, L.T., Bushell, G.R., Fardoulys, C.A.,Stenzel, D., Schofield, L., and Saul, A.J. (1988) The 140/130/105 kilodalton protein complex in the rhoptries of Plas-modium falciparum consists of discrete polypeptides. MolBiochem Parasitol 29: 251–260.

Coppel, R.L., Bianco, A.E., Culvenor, J.G., Crewther, P.E.,Brown, G.V., Anders, R.F., and Kemp, D.J. (1987) A cDNAclone expressing a rhoptry protein of Plasmodium falci-parum. Mol Biochem Parasitol 25: 73–81.

Craig, A. (2000) Malaria: a new gene family (clag) involvedin adhesion. Parasitol Today 16: 366–367; discussion 405.

Day, K.P., Karamalis, F., Thompson, J., Barnes, D.A., Peter-son, C., Brown, H., et al. (1993) Genes necessary forexpression of a virulence determinant and for transmissionof Plasmodium falciparum are located on a 0.3-megabaseregion of chromosome 9. Proc Natl Acad Sci USA 90:8292–8296.

Douki, J.B., Sterkers, Y., Lepolard, C., Traore, B., Costa, F.T.,Scherf, A., and Gysin, J. (2003) Adhesion of normal andPlasmodium falciparum ring-infected erythrocytes toendothelial cells and the placenta involves the rhoptry-derived ring surface protein-2. Blood 101: 5025–5032.

Doury, J.C., Bonnefoy, S., Roger, N., Dubremetz, J.F., andMercereau-Puijalon, O. (1994) Analysis of the high molec-ular weight rhoptry complex of Plasmodium falciparumusing monoclonal antibodies. Parasitology 108: 269–280.

Eng, J.K., McCormack, A.L., and Yates, J.R., III (1994) Anapproach to correlate tandem mass spectral data of pep-tides with amino acid sequences in a protein database. JAm Soc Mass Spectrom 5: 976–989.

Florens, L. Washburn, M.P. Raine, J.D. Anthony, R.M.Grainger, M., Haynes, J.D., et al. (2002) A proteomic viewof the Plasmodium falciparum life cycle. Nature 419: 520–526.

Gardiner, D.L., Holt, D.C., Thomas, E.A., Kemp, D.J., andTrenholme, K.R. (2000) Inhibition of Plasmodium falci-parum clag9 gene function by antisense RNA. Mol Bio-chem Parasitol 110: 33–41.

Gardner, M.J., Tettelin, H., Carucci, D.J., Cummings, L.M.,Aravind, L., Koonin, E.V., et al. (1998) Chromosome 2sequence of the human malaria parasite Plasmodium fal-ciparum. Science 282: 1126–1132.

Gardner, M.J., Hall, N., Fung, E., White, O., Berriman, M.,Hyman, R.W., et al. (2002) Genome sequence of thehuman malaria parasite Plasmodium falciparum. Nature419: 498–511.

Green, G.R., Poccia, D., and Herlands, L. (1982) A multisam-ple device for electroelution, concentration, and dialysis of



proteins from fixed and stained gel slices. Anal Biochem123: 66–73.

Guevara Patino, J.A., Holder, A.A., McBride, J.S., and Black-man, M.J. (1997) Antibodies that inhibit malaria merozoitesurface protein-1 processing and erythrocyte invasion areblocked by naturally acquired human antibodies. J ExpMed 186: 1689–1699.

Hempelmann, E., Ling, I., and Wilson, R.J. (1981) S-antigensand isozymes in strains of Plasmodium falciparum. TransRoy Soc Trop Med Hyg 75: 855–858.

Hienne, R., Ricard, G., Fusai, T., Fujioka, H., Pradines, B.,Aikawa, M., and Doury, J.C. (1998) Plasmodium yoelii:identification of rhoptry proteins using monoclonal antibod-ies. Exp Parasitol 90: 230–235.

Hiller, N.L., Akompong, T., Morrow, J.S., Holder, A.A., andHaldar, K. (2003) Identification of a stomatin orthologue invacuoles induced in human erythrocytes by malaria para-sites: a role for microbial raft-proteins in apicomplexanvacuole biogenesis. J Biol Chem 278: 48413–48421.

Holder, A.A., Freeman, R.R., Uni, S., and Aikawa, M. (1985)Isolation of a Plasmodium falciparum rhoptry protein. MolBiochem Parasitol 14: 293–303.

Holt, D.C., Bourke, P.F., Mayo, M., and Kemp, D.J. (1998) Ahigh resolution map of chromosome 9 of Plasmodium fal-ciparum. Mol Biochem Parasitol 97: 229–233.

Holt, D.C., Gardiner, D.L., Thomas, E.A., Mayo, M., Bourke,P.F., Sutherland, C.J., et al. (1999) The cytoadherencelinked asexual gene family of Plasmodium falciparum: arethere roles other than cytoadherence? Int J Parasitol 29:939–944.

Jaikaria, N.S., Rozario, C., Ridley, R.G., and Perkins, M.E.(1993) Biogenesis of rhoptry organelles in Plasmodiumfalciparum. Mol Biochem Parasitol 57: 269–279.

Kaneko, O., Tsuboi, T., Ling, I.T., Howell, S., Shirano, M.,Tachibana, M., et al. (2001) The high molecular mass rhop-try protein, RhopH1, is encoded by members of the clagmultigene family in Plasmodium falciparum and Plasmo-dium yoelii. Mol Biochem Parasitol 118: 223–231.

Kaneko, O., Mu, J., Tsuboi, T., Su, X., and Torii, M. (2002)Gene structure and expression of a Plasmodium falci-parum 220-kDa protein homologous to the Plasmodiumvivax reticulocyte binding proteins. Mol Biochem Parasitol121: 275–278.

Kemp, D.J. (1992) Antigenic diversity and variation in bloodstages of Plasmodium falciparum. Immunol Cell Biol 70:201–207.

Kyes, S., Pinches, R., and Newbold, C. (2000) A simple RNAanalysis method shows var and rif multigene family expres-sion patterns in Plasmodium falciparum. Mol Biochem Par-asitol 105: 311–315.

Ling, I.T., Ogun, S.A., and Holder, A.A. (1995) The combinedepidermal growth factor-like modules of Plasmodium yoeliimerozoite surface protein-1 are required for a protectiveimmune response to the parasite. Parasite Immunol 17:425–433.

Ling, I.T., Kaneko, O., Narum, D.L., Tsuboi, T., Howell, S.,Taylor, H.M., et al. (2003) Characterisation of the rhoph2gene of Plasmodium falciparum and Plasmodium yoelii.Mol Biochem Parasitol 127: 47–57.

Lu, S., Arthos, J., Montefiori, D.C., Yasutomi, Y., Manson, K.,Mustafa, F., et al. (1996) Simian immunodeficiency virusDNA vaccine trial in macaques. J Virol 70: 3978–3991.

Lustigman, S., Anders, R.F., Brown, G.V., and Coppel, R.L.(1988) A component of an antigenic rhoptry complex ofPlasmodium falciparum is modified after merozoite inva-sion. Mol Biochem Parasitol 30: 217–224.

Manski-Nankervis, J.A., Gardiner, D.L., Hawthorne, P., Holt,D.C., Edwards, M., Kemp, D.J., and Trenholme, K.R.(2000) The sequence of clag 9, a subtelomeric gene ofPlasmodium falciparum is highly conserved. Mol BiochemParasitol 111: 437–440.

Newbold, C., Craig, A., Kyes, S., Rowe, A., Fernandez-Reyes, D., and Fagan, T. (1999) Cytoadherence, patho-genesis and the infected red cell surface in Plasmodiumfalciparum. Int J Parasitol 29: 927–937.

Sadygov, R.G., and Yates, J.R., III (2003) A hypergeometricprobability model for protein identification and validationusing tandem mass spectral data and protein sequencedatabases. Anal Chem 75: 3792–3798.

Sam-Yellowe, T.Y., Fujioka, H., Aikawa, M., and Messineo,D.G. (1995) Plasmodium falciparum rhoptry proteins of140/130/110 kd (Rhop-H) are located in an electron lucentcompartment in the neck of the rhoptries. J Eukaryot Micro-biol 42: 224–231.

Shirley, M.W., Biggs, B.A., Forsyth, K.P., Brown, H.J.,Thompson, J.K., Brown, G.V., and Kemp, D.J. (1990)Chromosome 9 from independent clones and isolates ofPlasmodium falciparum undergoes subtelomeric deletionswith similar breakpoints in vitro. Mol Biochem Parasitol 40:137–145.

Staalsoe, T., Giha, H.A., Dodoo, D., Theander, T.G., andHviid, L. (1999) Detection of antibodies to variant antigenson Plasmodium falciparum-infected erythrocytes by flowcytometry. Cytometry 35: 329–336.

Tabb, D.L., McDonald, W.H., and Yates, J.R., III (2002) DTA-Select and contrast: tools for assembling and comparingprotein identifications from shotgun proteomics. J Pro-teome Res 1: 21–26.

Taylor, H.M., Grainger, M., and Holder, A.A. (2002) Variationin the expression of a Plasmodium falciparum protein fam-ily implicated in erythrocyte invasion. Infect Immun 70:5779–5789.

Trenholme, K.R., Gardiner, D.L., Holt, D.C., Thomas, E.A.,Cowman, A.F., and Kemp, D.J. (2000) clag9: a cytoadher-ence gene in Plasmodium falciparum essential for bindingof parasitized erythrocytes to CD36. Proc Natl Acad SciUSA 97: 4029–4033.

Walliker, D., Quakyi, I.A., Wellems, T.E., McCutchan, T.F.,Szarfman, A., London, W.T., et al. (1987) Genetic analysisof the human malaria parasite Plasmodium falciparum.Science 236: 1661–1666.

Washburn, M.P., Wolters, D., and Yates, J.R., III (2001)Large-scale analysis of the yeast proteome by multidimen-sional protein identification technology. Nature Biotechnol19: 242–247.

Wu, C.C., MacCoss, M.J., Howell, K.E., and Yates, J.R.(2003) A method for the comprehensive proteomic analysisof membrane proteins. Nature Biotechnol 21: 532–538.

the plasmodium falciparum clag9 gene encodes a rhoptry protein that is transferred to the host...

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