pfspatr - a plasmodium falciparum protein containing an altered

1

PfSPATR - A Plasmodium falciparum protein containing an altered thrombospondin type I repeat domain is expressed at several stages of the parasite life cycle and is the target of inhibitory antibodies

Rana Chattopadhyay, Dharmendar Rathore1, Hishasi Fujioka2, Sanjai Kumar, Patricia de La Vega, David Haynes, Kathleen Moch, David Fryauff, Ruobing Wang, Daniel J. Carucci and Stephen L. Hoffman3,4

Malaria Program, Naval Medical Research Center, Silver Spring, MD 20910-7500; 1Laboratory of Malaria & Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425; 2 Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106

3Corresponding Author 4 Present Address: Sanaria 308 Argosy Drive Gaithersburg, MD 20878. Email: [email protected] Phone: 240-299-3178 Fax: 301-990-6370

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 25, 2003 as Manuscript M300865200 by guest on A

pril 14, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

2

ABSTRACT

The annotated sequence of chromosome 2 of Plasmodium falciparum was examined for

genes encoding proteins that may be of interest for vaccine development. We describe

here the characterization of a protein with an altered thrombospondin type-I repeat

domain (PfSPATR) that is expressed in the sporozoite, asexual and sexual erythrocytic

stages of the parasite life cycle. Immuno-electron microscopy indicated that this protein

was expressed on the surface of the sporozoites and around the rhoptries in the asexual

erythrocytic stage. An E. coli-produced recombinant form of the protein bound to

HepG2 cells in a dose dependent manner and antibodies raised against this protein

blocked the invasion of sporozoites into a transformed hepatoma cell line. Sera from

Ghanaian adults and from a volunteer who had been immunized with radiation-attenuated

P. falciparum sporozoites specifically recognized the expression of this protein on

transfected COS-7 cells. These data support evaluation of this protein as a vaccine

candidate.

Abbreviations: TSR: thrombospondin type I repeat; RT-PCR: Reverse transcription-

Polymerase chain reaction; CSP: Circumsporozoite protein; TRAP: Thrombospondin

related anonymous protein; PfSPATR: Plasmodium falciparum Secreted Protein with

Altered Thrombospondin Repeat

by guest on April 14, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3

INTRODUCTION

Plasmodium parasites cause malaria and are transmitted by the bite of infected

mosquitoes. Numerous candidate genes and strategies have been evaluated in an attempt

to develop malaria vaccines (1-5), yet there is currently no licensed malaria vaccine and it

is unlikely that there will be one for at least a decade (6). The complex life cycle of the

parasite with its distinct morphological and antigenic stages has been a major hurdle in

developing such vaccines. It is anticipated that use of data from the recently completed

genomic sequence of Plasmodium falciparum (7) will lead to an increased understanding

of parasite biology that will eventually be translated into new drugs and vaccines for

malaria (8).

Chromosome 2 was the first chromosome of P. falciparum to be sequenced and initial

analysis indicated that there were 209 genes on this chromosome (9). In an effort to

discover additional protein candidates for vaccine development we sought to characterize

one of the genes from chromosome 2 of P. falciparum, which had been annotated as a

putative secreted protein containing a thrombospondin type I repeat (TSR) domain (9).

The TSR is an ancient eukaryotic domain (10) now known to be present in more than 300

different proteins (11), including surface antigens of pathogenic microorganisms (12).

Numerous Plasmodium surface antigens have been shown to possess the TSR domain

(13-15) and these proteins have been shown to be involved in ookinete and sporozoite

motility, host cell attachment and invasion (16-19), thus making them potentially good

vaccine targets. In addition to a TSR domain we found that the predicted protein also


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

4

possessed a cysteine rich signature that could represent a Type II EGF-like domain. The

orthologue of this protein is present in the murine malaria parasite P. yoelii and named

‘secreted protein with altered thrombospondin repeat’ or SPATR (20).

We have characterized its expression, localization and function at different stages of the

Plasmodium life cycle and report that this protein is expressed at several stages of the life

cycle, binds to hepatoma cells, and antibodies to the protein inhibit P. falciparum

sporozoite invasion of these liver cells.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

5

EXPERIMENTAL PROCEDURE

RT-PCR and cloning: Total RNA was isolated from P. falciparum sporozoites,

synchronized erythrocytic stages (ring, trophozoite and schizont) and gametocytes using

High Pure RNA Isolation Kit (Roche). 2 ug RNA from each of these stages was reverse

transcribed using random hexamers and Superscript II RNase H-- reverse transcriptase

(Invitrogen). 5 ul of the reverse transcribed product of each of the above stages was

subjected to PCR using PfSPATR specific primers. Primer design was based on the

published sequence with GenBank accession number AE001404 (9). The amplified

products were cloned into TA Cloning vector (Invitrogen) and sequenced.

Recombinant protein expression and purification: For protein expression, a 690 bp

cDNA encoding the mature form of PfSPATR (without signal sequence) was cloned, as a

BamHI and EcoRI fragment, into pGEX-3X (Amersham Pharmacia Biotech), a GST-

based E. coli expression vector. The recombinant protein was expressed in BL21 E. coli

cells and the expression was induced with 1mM IPTG. The protein was purified on a

glutathione-agarose column as per the manufacturer’s instructions (Amersham Pharmacia

Biotech).

Generation of anti-PfSPATR serum in mice: Outbred CD-1 mice were immunized

intra-peritoneally with 10 µg of the purified protein in Freund’s complete adjuvant and

boosted 3 and 6 weeks after the first immunization, with 10 µg of protein in Freund’s

incomplete adjuvant. Sera were collected 12 days after the third dose. Anti-GST


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

6

antibodies were depleted by passing the sera through an immobilized GST column

(Pierce), which was confirmed by Western Blot.

Immunofluorescence assay: Spots of P. falciparum sporozoites and smears of

erythrocytic stages and gametocytes were made on glass slides. Anti-PfSPATR serum at

1:20 to 1:6400 dilutions were added and incubated in a moist chamber at 370C. After 1

hour unbound material was removed by washing and anti-mouse IgG-FITC conjugate

was added. Unbound conjugate was removed and the slides were observed under UV

light in a fluorescence microscope. Pre-immune mice sera were used as controls.

Immuno-electron Microscopy: Immuno-electron microscopy was carried out on

sporozoites isolated from infected mosquito salivary glands and in vitro cultured blood

stages of P. falciparum (Clone 3D7) using 1:40 anti-PfSPATR antiserum as described

(21). Pre-immune sera were used as control.

Expression of PfSPATR on COS-7 cells: DNA encoding the full length ORF of

PfSPATR was cloned in plasmid pRE4 (22), a mammalian expression vector, and the

endotoxin free plasmid was transfected into COS-7 cells using Lipofectin. Expression of

PfSPATR was evaluated by immunofluorescence using murine anti-PfSPATR antibodies,

and human serum samples obtained from 1) naturally-immune adult, lifelong residents of

P. falciparum hyper-endemic area in Ghana, 2) malaria-naïve volunteers immunized with

irradiated P. falciparum sporozoites and 3) their controls exposed to the bite of

uninfected mosquitoes (23). All sera were used at a dilutions ranging from 1:50 to 1:400.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

7

The use of human serum samples for this experiment was approved by the institutional

review board at the Naval Medical Research Center.

Cell binding assay: The hepatoma cell line HepG2 was used to evaluate the binding

activity of PfSPATR. Cells were seeded in 96 well plate a day before the start of the

experiment. The next day, 0 to 1.00 µM of the recombinant proteins were added on to the

paraformaldehyde-fixed cells and incubated at 37oC for one hour. Unbound material was

removed followed by the addition of murine anti-protein antibodies. After 1 hr.

incubation at 37oC, alkaline phosphatase-conjugated goat anti-mouse antibody was added

and the bound protein was measured by a fluorescence based assay using 4-

methyllumbelliferyl phosphate as substrate (24).

In vitro Inhibition of Sporozoite Invasion (ISI) in HepG2 cells: The ISI assay was

performed as described (19). Briefly, 50,000 HepG2 cells were placed in each of the

eight wells of tissue culture slides two days before the experiment. P. falciparum (NF54)

sporozoites were isolated from mosquito salivary glands using a discontinuous gradient as

described (25). 20,000 sporozoites were added to the cells along with anti-PfSPATR

serum at a final dilution of 1:50 in the presence (20µg/ml or 10 µg/ml) or absence of

PfSPATR protein. Anti-P. falciparum circumsporozoite protein (PfCSP) monoclonal

antibody, NFS1, at a concentration of 10 µg/ml was added as a positive control (NFS1

mAb was diluted 1:600 to achieve this concentration). After 3 hours incubation at 37°C

the numbers of sporozoites that had invaded the hepatoma cells were counted. Percent

inhibition was calculated by the following formula: [(Mean number of invaded


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

8

sporozoites in negative controls)-(Mean number of invaded sporozoites in test)/(Mean

number of invaded sporozoites in negative controls)] x 100.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

9

RESULTS

Selection, cloning and expression of PfSPATR: Analysis of the published DNA

sequence of chromosome 2 of P. falciparum identified a 1069 nucleotide sequence

containing a two-exon gene encoding a 250 amino acid long putative secreted protein

with an altered TSR domain (Figure 1). Application of RT-PCR to assess its expression

in sporozoites, infected erythrocytes (rings, trophozoites, schizonts and gametocytes)

revealed that the gene is transcribed in all the evaluated stages of the parasite life cycle

(Figure 2). The amplified fragment was 753 bp in length and sequencing of the cDNA

confirmed that the correct mRNA had been amplified (data not shown). To obtain

recombinant PfSPATR protein, a 690 bp cDNA fragment encoding the mature protein

was cloned in plasmid pGEX-3X, a GST-based E.coli expression vector. The construct

was expressed in BL21 cells and induced with 1mM IPTG for 3 hours. Though, the

coding sequence had a high AT content, a characteristic feature of P. falciparum genes,

we could detect the expression of the fusion protein on a Coomassie blue stained

polyacrylamide gel (data not shown). The fusion protein was purified to homogeneity on

a glutathione agarose column (data not shown). Purified protein was used to immunize

outbred CD-1 mice and anti-PfSPATR serum was obtained.

Localization of PfSPATR protein in different parasite stages: To determine if the

transcribed mRNA was associated with protein expression, we evaluated the cellular

expression and localization of PfSPATR at different stages of the parasite life cycle.

Immunofluorescence assays using anti-PfSPATR antibodies produced in mice

demonstrated binding in all the evaluated stages viz., sporozoites, asexual erythrocytic


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

10

stages and gametocytes suggesting that the protein is expressed in several stages of

parasite life cycle (Figure 3). The strongest reactivity was observed against sporozoites

where the protein was detectable even at dilutions of 1:6400 of the anti-serum.

Sporozoites and asexual erythrocytic stages were further evaluated by immuno-electron

microscopy to determine the specific location of PfSPATR. In longitudinal and cross

sections, PfSPATR was localized on the surface of sporozoites, and was not detected in

the intracellular organelles like micronemes (Figure 4A, B). In asexual erythrocytic

stages, PfSPATR was detectable around the rhoptries and to a lesser extent on the

infected erythrocyte membrane (Figure 4C). Western Blot using anti-PfSPATR antiserum

on sporozoite and blood-stage parasite lysates detected PfSPATR protein at its expected

size of ~30 kDa (data not shown).

Reactivity of sera from malaria endemic regions with PfSPATR on COS-7 cells:

Having established expression, we investigated whether sera from individuals exposed to

P. falciparum parasites recognized PfSPATR. Employing a plasmid expressing PfSPATR

to transiently transfect COS-7 cells, it was found that the protein was expressed on the

cells surface and was readily recognized by the anti-PfSPATR serum (Figure 5, Panel A)

but not by the pre-immune serum (Panel B). Sera from a malaria-naïve volunteer who

had been immunized with radiation-attenuated P. falciparum sporozoites (Panel C) and

five clinically immune adults (Panel E-I) from a region of Ghana with intense P.

falciparum malaria transmission recognized the PfSPATR expressed on COS-7 cells.

However, serum from a volunteer who was also immunized with irradiated sporozoites


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

11

but with low anti-sporozoite antibodies (Panel D) as characterized by IFA and sera from

two non- immune adults (Panel J and K) did not recognize PfSPATR expression.

Biological function of PfSPATR: As PfSPATR showed strong surface localization on

sporozoites, we investigated its possible involvement in cell-cell interaction by

evaluating its potential for binding to the human hepatocyte cell line, HepG2. The

protein demonstrated potent binding to HepG2 cells that was dose dependent (Figure 6)

and comparable to that of PfCSP. In contrast, serum albumin, used as negative control

showed no binding (data not shown). This result suggested that PfSPATR could function

as another parasite ligand involved in the interaction of sporozoites with liver cells, and

that a receptor for PfSPATR was present on HepG2 cells.

In vitro inhibition of sporozoite invasion: As PfSPATR efficiently bound HepG2 cells,

we investigated the ability of antibodies against PfSPATR to prevent sporozoite invasion

of human liver cells. Anti-PfSPATR serum at a final dilution of 1:50 inhibited

sporozoite invasion by more than 80%. Non-immune control serum showed no

inhibition, suggesting that the inhibitory property of anti-PfSPATR serum was specific.

The inhibitory activity was comparable to that of an anti-PfCSP monoclonal antibody

which prevented invasion by more than 90% at 10 µg/ml. This invasion inhibition was

antigen-specific as shown by the addition of free recombinant protein in the assay which

completely reversed the inhibition (Figure 7).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

12

DISCUSSION

We investigated a protein of P. falciparum that is encoded by a gene located on

chromosome 2 of the parasite (9). Based on protein sequence homology, the protein had

been suggested to possess an altered TSR domain towards the carboxyl terminus (20).

The central motif present in most TSR family proteins is WSXW (where ‘X’ can be any

amino acid) followed by CSXTCG (26). As the CSXTCG motif is absent in the SPATR

sequence, it has been referred to as an ‘altered thrombospondin domain’(20). In

Plasmodium, TSR-containing genes show synteny linkage conservation between different

species (27). Along with the altered TSR domain, the protein has a 15 amino acid region

(72 NSRNCWCPRGYILCS 86) which has characteristics suggestive of a Type II EGF-like

signature sequence. The BLOCKS protein domain database and search tool

(http://blocks.fhcrc.org) that predicted this characteristic indicated that the PfSPATR

sequence was not typical of the type II EGF signature sequence that is found in a diverse

range of proteins(28). Interestingly, no other protein domain database and search tool

predicted this type II EGF-like signature. Although the precise role of the TSR and

potential EGF domains are as yet unclear, they are located in the extra-cellular region of

membrane-bound proteins or in proteins known to be secreted (29). While TSR (13-15)

or EGF domains (30-32) are individually present in a number of Plasmodium proteins, no

Plasmodium antigen has been reported in which these two domains are predicted to be

present together.

We found that the PfSPATR gene is transcribed during the sporozoite and the major

erythrocytic stages of the parasite life cycle (Figure 2). Sporozoites for our studies were


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

13

produced in mosquitoes, and the erythrocytic stage parasites were produced in cultures of

erythrocytes, so there could not have been any cross-contamination. The expressed

sequence matched perfectly with the predicted gene structure of the protein, which also

verified the predicted exon-intron boundaries of the gene (Figure 1). Only a few

Plasmodium proteins have been reported to be expressed during multiple stages of the

parasite life cycle (33).

We evaluated the cellular expression of PfSPATR by IFA and by immuno-electron

microscopy and found that the protein is present in sporozoite, asexual erythrocytic and

gametocyte stages of the life cycle (Figure 3 and 4). In sporozoites it was exclusively

located on the surface, while in asexual blood stages, it was present around the rhoptries

of merozoites and on the membrane of infected erythrocytes (Figure 4). The presence of

this protein on the surface of the parasite in different stages, led us to investigate whether

this protein was recognized by the host immune system in P. falciparum-infected

individuals. The PfSPATR protein that was expressed on the cell surface of transfected

COS-7 cells was recognized by sera from naturally-infected, clinically-immune adult

Africans, indicating that this protein was recognized by the host immune system (Figure

5). The fact that the serum from an individual immunized with irradiated sporozoites,

recognized the protein, corroborates the expression and immunogenicity of the protein

during the early pre-erythrocytic stage of the parasite infection in humans. Serum from a

volunteer with low levels of anti-sporozoite antibodies and control sera from two non-

immune donors did not recognize PfSPATR expression on COS-7 cells, indicating that

this reaction was specific.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

14

The expression of PfSPATR protein on the sporozoite surface and its recognition by the

host immune system in infected individuals led us to investigate its biological role in the

parasite life cycle. Other known Plasmodium antigens with similar properties have been

shown to be involved in cell-cell interactions (16-18). We hypothesized a role for this

protein in the binding of sporozoites to liver cells, a property known to be associated with

other sporozoite proteins possessing a TSR domain. PfSPATR bound to human liver cells

and its binding was comparable to that of PfCSP, the predominant sporozoite surface

protein (Figure 6). The binding of PfSPATR appeared to be specific and is presumed to

be involved in the sporozoite invasion of liver cells as evidenced by inhibition of invasion

by anti-PfSPATR antibodies, and reversal of inhibition by the addition of recombinant

PfSPATR protein (Figure 7). It will be interesting to determine whether there are anti-

PfSPATR antibodies that block the invasion inhibiting activity of anti-PfSPATR

antibodies as has been demonstrated for MSP-19 (34).

The presence of antibodies that partially inhibit the sporozoite invasion of

hepatocytes does not indicate that an individual will be protected against P. falciparum

infection. If a mosquito injects 20 sporozoites and 19 of them are inhibited from invading

hepatocytes by antibodies to PfSPATR, or against any other sporozoite protein, that

individual will not be protected against developing P. falciparum infection, as within one

week a single successfully invaded sporozoite can give rise to 10,000 merozoites, each of

which can invade erythrocytes. Immunization of volunteers with a number of PfCSP

recombinant protein vaccines has elicited antibodies to sporozoites that successfully

inhibit sporozoite invasion of hepatoma cells in vitro, but fail to protect the volunteers


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

15

against challenge (35). Nonetheless, we know from passive transfer studies in mice and

monkeys (36) that antibodies against sporozoites can completely protect against

sporozoite challenge. In those cases the invasion inhibitory activity was generally > 95%.

We are currently investigating the potential of anti-SPATR antibodies to protect against

infection in the P. yoelii rodent malaria model.

The SPATR protein is present in multiple Plasmodium species. It is present in the

transcriptome of P. yoelii sporozoites (20) and we have also identified its orthologue in

P. knowlesi and P. vivax species (unpublished results). The presence of this protein in

human, simian and rodent malaria parasite species suggests that the protein plays an

important role in the biology of the parasite. Numerous efforts are currently underway to

develop an effective vaccine against malaria (34). The complex life cycle of the parasite,

with distinct sets of antigens expressed during various stages of development, has made

vaccine design and development a major challenge to malaria researchers. We have

herein described a molecule which holds potential for investigation as a malaria vaccine

candidate. Its multi-stage expression by sporozoites, asexual erythrocytic forms, and

gametocytes, along with its possible role in liver cell invasion, suggest that PfSPATR

could be a valuable new vaccine component.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

16

Acknowledgements

We thank Dr. Gary H. Cohen and Dr . Roselyn J. Eisenberg, University of Pennsylvania,

Philadelphia for providing the pRE4 plasmid, DL6 and ID3 monoclonal antibodies used

in expression of PfSPATR in COS-7 cells work and Dr. Yupin Charoenvit of Malaria

Program, Naval Medical Research Center, Maryland for providing sera from irradiated

sporozoite trial volunteers. We also acknowledge Dr. Stefan H.I. Kappe, Department of

Pathology, New York University School of Medicine, New York and Dr. Mani

Subramanian, Human Genome Sciences, Rockville, Maryland for their critical comments

and suggestions.

This work was supported by the Naval Medical Research & Development Command

work unit 6000.RAD1.F.A0309. The views of the authors are their own and do not

purport to reflect those of the US Navy or the US Department of Defense.

The experiments reported herein were conducted according to the principles set forth in

the "Guide for the Care and Use of Laboratory Animals, "Institute of Laboratory Animal

Research, National Research Council, National Academy Press (1996).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

17

Figure Legends

Figure 1: Schematic representation of PfSPATR gene and protein. The mature mRNA,

formed after removal of the intron region, is 753 nucleotide in length and translates into a

250 amino acid polypeptide. The boxed sequence represents the probable Type II EGF-

like domain while the altered thrombospondin domain is underlined. The first twenty one

amino acids represent the putative signal sequence and are shown in lower case letters.

Figure 2: Transcription of PfSPATR gene at different stages of the P. falciparum life

cycle. The reaction was performed in the absence (-RT) and presence (+RT) of reverse

transcriptase.

Figure 3: Immunofluorescence assay using anti- PfSPATR sera from mice immunized

with the recombinant protein to detect PfSPATR protein expression in different stages of

P. falciparum. (A) Sporozoite (1:6400), (B) Trophozoite (1:640), (C) Asexual

erythrocytic stage schizont (1:640), (D) Merozoites escaping from a late stage schizont

(1:640), (E) Gametocyte (1:640). Dilutions of antiserum used are in parentheses.

Figure 4: Localization of PfSPATR in P. falciparum by immuno-electron microscopy.

(A) Longitudinal section of sporozoite, (B) Cross section of sporozoite, (C) Cross section

of an infected erythrocyte containing a schizont (R: Rhoptry; Mz: Merozoites; Fv: Food

vacuole; Hz: Hemozoin pigments; E: Erythrocyte membrane; Mn: Micronemes).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

18

Figure 5 : Recognition of PfSPATR expression on COS-7 cells. (A) Serum from mice

immunized with PfSPATR recombinant protein, (B) Serum taken prior to immunization

with PfSPATR recombinant protein, (C) Serum from a volunteer immunized with

radiation attenuated P. falciparum sporozoites who had high levels of anti-sporozoite

antibodies, (D) Serum from a volunteer who had been immunized with irradiated P.

falciparum sporozoites whose serum was negative for anti-sporozoite antibodies, (E-I)

Sera from semi-immune adults from Ghana, Africa, and (J-K) sera from non-immune

adults from the United States.

Figure 6: Binding of PfSPATR protein to hepatoma cells. Binding activity of PfSPATR

(filled circles) and recombinant PfCSP (open circles) was evaluated on HepG2 cells in a

fluorescence-based assay.

Figure 7 : In vitro Inhibition of sporozoite invasion into HepG2 cells by anti- PfSPATR

serum. The final dilution of anti-PfSPATR serum and control antisera used were 1:50 and

the NFS1 monoclonal antibody against PfCSP was diluted 1:600 to give a final

concentration of 10µg/ml. Numbers of P. falciparum sporozoites invading liver cells

were counted in the presence of anti-PfSPATR serum alone or with the recombinant

PfSPATR protein (20 µg/ml or 10 µg/ml) used as a competitor to neutralize the inhibitory

affect of anti-PfSPATR antibodies.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

19

References:

1. Amador, R., Moreno, A., Valero, V., Murillo, L., Mora, A. L., Rojas, M., Rocha,

C., Salcedo, M., Guzman, F., Espejo, F., and et al. (1992) Vaccine 10, 179-184

2. Herrington, D. A., Clyde, D. F., Losonsky, G., Cortesia, M., Murphy, J. R., Davis,

J., Baqar, S., Felix, A. M., Heimer, E. P., Gillessen, D., Nardin, E., Nussenzweig,

R. S., Nussenzweig, V., Holligdale, M. R., and Levine, M. M. (1987) Nature 328,

257-259

3. Stoute, J. A., Slaoui, M., Heppner, D. G., Momin, P., Kester, K. E., Desmons, P.,

Wellde, B. T., Garcon, N., Krzych, U., and Marchand, M. (1997) N Engl J Med

336, 86-91

4. Wang, R., Doolan, D. L., Le, T. P., Hedstrom, R. C., Coonan, K. M., Charoenvit,

Y., Jones, T. R., Hobart, P., Margalith, M., Ng, J., Weiss, W. R., Sedegah, M., de

Taisne, C., Norman, J. A., and Hoffman, S. L. (1998) Science 282, 476-480

5. Nardin, E. H., Oliveira, G. A., Calvo-Calle, J. M., Castro, Z. R., Nussenzweig, R.

S., Schmeckpeper, B., Hall, B. F., Diggs, C., Bodison, S., and Edelman, R. (2000)

J Infect Dis 182, 1486-1496

6. Long, C. A., and Hoffman, S. L. (2002) Science 297, 345-347.

7. Gardner, M. J., Hall, N., Fung, E., White, O., Berriman, M., Hyman, R. W.,

Carlton, J. M., Pain, A., Nelson, K. E., Bowman, S., Paulsen, I. T., James, K.,

Eisen, J. A., Rutherford, K., Salzberg, S. L., Craig, A., Kyes, S., Chan, M. S.,

Nene, V., Shallom, S. J., Suh, B., Peterson, J., Angiuoli, S., Pertea, M., Allen, J.,

Selengut, J., Haft, D., Mather, M. W., Vaidya, A. B., Martin, D. M., Fairlamb, A.

H., Fraunholz, M. J., Roos, D. S., Ralph, S. A., McFadden, G. I., Cummings, L.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

20

M., Subramanian, G. M., Mungall, C., Venter, J. C., Carucci, D. J., Hoffman, S.

L., Newbold, C., Davis, R. W., Fraser, C. M., and Barrell, B. (2002) Nature 419,

498-511.

8. Hoffman, S. L., Subramanian, G. M., Collins, F. H., and Venter, J. C. (2002)

Nature 415, 702-709.

9. Gardner, M. J., Tettelin, H., Carucci, D. J., Cummings, L. M., Aravind, L.,

Koonin, E. V., Shallom, S., Mason, T., Yu, K., Fujii, C., Pederson, J., Shen, K.,

Jing, J., Aston, C., Lai, Z., Schwartz, D. C., Pertea, M., Salzberg, S., Zhou, L.,

Sutton, G. G., Clayton, R., White, O., Smith, H. O., Fraser, C. M., Adams, M. D.,

Venter, J. C., and Hoffman, S. L. (1998) Science 282, 1126-1132

10. Hutter, H., Vogel, B. E., Plenefisch, J. D., Norris, C. R., Proenca, R. B., Spieth, J.,

Guo, C., Mastwal, S., Zhu, X., Scheel, J., and Hedgecock, E. M. (2000) Science

287, 989-994.

11. Apweiler, R., Attwood, T. K., Bairoch, A., Bateman, A., Birney, E., Biswas, M.,

Bucher, P., Cerutti, L., Corpet, F., Croning, M. D., Durbin, R., Falquet, L.,

Fleischmann, W., Gouzy, J., Hermjakob, H., Hulo, N., Jonassen, I., Kahn, D.,

Kanapin, A., Karavidopoulou, Y., Lopez, R., Marx, B., Mulder, N. J., Oinn, T.

M., Pagni, M., and Servant, F. (2001) Nucleic Acids Res 29, 37-40.

12. Naitza, S., Spano, F., Robson, K. J. H., and Crisanti, A. (1998) Parasitol. Today

14, 479-484

13. Ozaki, L. S., Svec, P., Nussenzweig, R. S., Nussenzweig, V., and Godson, G. N.

(1983) Cell 34, 815-822


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

21

14. Robson, K. J., Hall, J. R., Jennings, M. W., Harris, T. J., Marsh, K., Newbold, C.

I., Tate, V. E., and Weatherall, D. J. (1988) Nature 335, 79-82

15. Trottein, F., Triglia, T., and Cowman, A. F. (1995) Mol Biochem Parasitol 74,

129-141

16. Cerami, C., Frevert, U., Sinnis, P., Takacs, B., Clavijo, P., Santos, M. J., and

Nussenzweig, V. (1992) Cell 70, 1021-1033

17. Menard, R., Sultan, A. A., Cortes, C., Altszuler, R., van Dijk, M. R., Janse, C. J.,

Waters, A. P., Nussenzweig, R. S., and Nussenzweig, V. (1997) Nature 385, 336-

340

18. Templeton, T. J., Kaslow, D. C., and Fidock, D. A. (2000) Mol Microbiol 36, 1-9.

19. Rathore, D., Sacci, J. B., de la Vega, P., and McCutchan, T. F. (2002) J Biol

Chem 277, 7092-7098.

20. Kappe, S. H., Gardner, M. J., Brown, S. M., Ross, J., Matuschewski, K., Ribeiro,

J. M., Adams, J. H., Quackenbush, J., Cho, J., Carucci, D. J., Hoffman, S. L., and

Nussenzweig, V. (2001) Proc Natl Acad Sci U S A 98, 9895-9900.

21. Nguyen, T. V., Fujioka, H., Kang, A. S., Rogers, W. O., Fidock, D. A., and

James, A. A. (2001) J Biol Chem 276, 26724-26731.

22. Cohen, G. H., Wilcox, W. C., Sodora, D. L., Long, D., Levin, J. Z., and

Eisenberg, R. J. (1988) J Virol 62, 1932-1940.

23. Hoffman, S. L., Goh, L. M., Luke, T. C., Schneider, I., Le, T. P., Doolan, D. L.,

Sacci, J., de la Vega, P., Dowler, M., Paul, C., Gordon, D. M., Stoute, J. A.,

Church, L. W., Sedegah, M., Heppner, D. G., Ballou, W. R., and Richie, T. L.

(2002) J Infect Dis 185, 1155-1164.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

22

24. Rathore, D., and McCutchan, T. F. (2000) Infect Immun 68, 740-743

25. Pacheco, N. D., Strome, C. P., Mitchell, F., Bawden, M. P., and Beaudoin, R. L.

(1979) J Parasitol 65, 414-417.

26. Lawler, J., and Hynes, R. O. (1986) J Cell Biol 103, 1635-1648

27. Carlton, J. M., Vinkenoog, R., Waters, A. P., and Walliker, D. (1998) Mol

Biochem Parasitol 93, 285-294

28. Henikoff, S., and Henikoff, J. G. (1994) Genomics 19, 97-107.

29. Davis, C. G. (1990) New Biol 2, 410-419.

30. Wang, L., Black, C. G., Marshall, V. M., and Coppel, R. L. (1999) Infect Immun

67, 2193-2200.

31. Black, C. G., Wu, T., Wang, L., Hibbs, A. R., and Coppel, R. L. (2001) Mol

Biochem Parasitol 114, 217-226.

32. Wu, T., Black, C. G., Wang, L., Hibbs, A. R., and Coppel, R. L. (1999) Mol

Biochem Parasitol 103, 243-250.

33. Florens, L., Washburn, M. P., Raine, J. D., Anthony, R. M., Grainger, M.,

Haynes, J. D., Moch, J. K., Muster, N., Sacci, J. B., Tabb, D. L., Witney, A. A.,

Wolters, D., Wu, Y., Gardner, M. J., Holder, A. A., Sinden, R. E., Yates, J. R.,

and Carucci, D. J. (2002) Nature 419, 520-526.

34. Richie, T. L., and Saul, A. (2002) Nature 415, 694-701.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

23


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

24


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

25

Figure 3


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

26

Figure 4


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

27

Figure 5


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

28

Figure 6


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

29

Figure 7

% Inhibition

0 20 40 60 80 100

Anti-CS Antibody

Anti-PfSPATR serum + 10 ug/ml PfSPATR

Anti-PfSPATR serum +20 ug/ml PfSPATR

Anti-PfSPATR serum

Control Serum


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Stephen L. HoffmanandVega, David Haynes, Kathleen Moch, David Fryauff, Ruobing Wang, Daniel J. Carucci

Rana Chattopadhyay, Dharmendar Rathore, Hishasi Fujioka, Sanjai Kumar, Patricia de Latarget of inhibitory antibodies

type I repeat domain is expressed at several stages ofthe parasite life cycle and is the PfSPATR - A Plasmodium falciparum protein containing an alteredthrombospondin

published online April 25, 2003J. Biol. Chem.

10.1074/jbc.M300865200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M300865200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M300865200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/04/25/jbc.M300865200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2003/04/25/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/04/25/jbc.M300865200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

pfspatr - a plasmodium falciparum protein containing an altered

Documents