pfspatr - a plasmodium falciparum protein containing an altered
TRANSCRIPT
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
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
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
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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.
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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.
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
8
sporozoites in negative controls)-(Mean number of invaded sporozoites in test)/(Mean
number of invaded sporozoites in negative controls)] x 100.
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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).
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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.
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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.
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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).
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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).
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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.
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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.
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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.
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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.
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from
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
by guest on April 14, 2018
http://ww
w.jbc.org/
Dow
nloaded from