INFECTION AND IMMUNITY, Sept. 2005, p. 5402–5409
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 9
Identification, Cloning, Expression, and Characterization of the Gene
for Plasmodium knowlesi Surface Protein Containing an Altered
Thrombospondin Repeat Domain
Babita Mahajan,1Dewal Jani,2Rana Chattopadhyay,3Rana Nagarkatti,2Hong Zheng,1
Victoria Majam,1Walter Weiss,3Sanjai Kumar,1* and Dharmendar Rathore2
Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics
Evaluation and Research, Food and Drug Administration, Rockville, Maryland1; Virginia Bioinformatics Institute,
Virginia Polytechnic Institute and State University, Blacksburg, Virginia2; and Malaria Program,
Naval Medical Research Center, Silver Spring, Maryland3
Received 3 December 2004/Returned for modification 16 January 2005/Accepted 27 April 2005
Proteins present on the surface of malaria parasites that participate in the process of invasion and adhesion
to host cells are considered attractive vaccine targets. Aided by the availability of the partially completed
genome sequence of the simian malaria parasite Plasmodium knowlesi, we have identified a 786-bp DNA
sequence that encodes a 262-amino-acid-long protein, containing an altered version of the thrombospondin
type I repeat domain (SPATR). Thrombospondin type 1 repeat domains participate in biologically diverse
functions, such as cell attachment, mobility, proliferation, and extracellular protease activities. The SPATR
from P. knowlesi (PkSPATR) shares 61% and 58% sequence identity with its Plasmodium falciparum and
Plasmodium yoelii orthologs, respectively. By immunofluorescence analysis, we determined that PkSPATR is a
multistage antigen that is expressed on the surface of P. knowlesi sporozoite and erythrocytic stage parasites.
Recombinant PkSPATR produced in Escherichia coli binds to a human hepatoma cell line, HepG2, suggesting
that PkSPATR is a parasite ligand that could be involved in sporozoite invasion of liver cells. Furthermore,
recombinant PkSPATR reacted with pooled sera from P. knowlesi-infected rhesus monkeys, indicating that
native PkSPATR is immunogenic during infection. Further efficacy evaluation studies in the P. knowlesi-rhesus
monkey sporozoite challenge model will help to decide whether the SPATR molecule should be developed as a
vaccine against human malarias.
An effective vaccine that reduces malaria-related mortality
and morbidity would indeed alleviate the suffering of millions
of people around the world (17). The onset and progression of
malaria infection require a complex sequence of recognition,
adhesion, and invasion events between the parasite and host
cells. To accomplish this, the parasite expresses a multitude of
proteins on its surface that serve as ligands that interact with
the receptors present on the host cells. For example, circum-
sporozoite protein and thrombospondin-related anonymous
protein (two sporozoite-stage proteins) participate in the bind-
ing of malaria sporozoites to liver cells (6, 30). In the erythro-
cytic-stage cycle, erythrocyte-binding antigen 175 is recognized
as a merozoite ligand that binds to glycophorin A on erythro-
cytes to initiate the process of merozoite invasion (33). Like-
wise, the products of the var genes expressed on the surface of
infected erythrocytes facilitate the adherence of mature forms
of parasites to deep venules of endothelial cells and thus shield
them from clearance by the host immune system.
The standard steps in preclinical vaccine development prior
to phase I clinical trials in humans are antigen identification, its
biochemical/biological characterization, and efficacy evalua-
tion in animal models. For malaria, animal studies are gener-
ally performed using pertinent orthologs of Plasmodium falci-
parum (the most lethal form of human malaria) in the mouse
(Plasmodium yoelii/Plasmodium berghei) and monkey (Plasmo-
dium knowlesi) models. An analysis of preclinical efficacy stud-
ies performed over the last 20 years suggests that the high
degree of protective efficacy observed in mice is generally dif-
ficult to achieve in humans, and studies on simian models may
be more appropriate to predicate the vaccine potency in hu-
Infection with P. knowlesi in the Old World rhesus monkey
Macaca mulatta is uniformly fatal and considered a reliable
model to determine the efficacy of candidate vaccines against
challenge with sporozoite or erythrocytic-stage parasites (31).
While in nature the macaque monkey is its natural host, cross-
species natural transmission of P. knowlesi to humans has been
reported (21, 34), demonstrating the relevance of the rhesus-P.
knowlesi model in evaluating the efficacy of malaria vaccines.
Recently, the genome sequences of P. falciparum and P.
yoelii parasites have become available (5, 11), and efforts to
generate partial or complete genome information for several
other Plasmodium species are currently under way. The recent
advances in bioinformatics have made it possible to assign
putative biologic function to the majority of malarial antigens
and, as a result, a large number of new antigens have become
available for evaluation as vaccine candidates. However, in the
absence of in vitro assays that could be used to predict vaccine
efficacy, in vivo immunization-challenge studies remain the
* Corresponding author. Mailing address: Bacterial and Parasitic
Diseases Section, Division of Emerging and Transfusion Transmitted
Diseases, Center for Biologics Evaluation and Research, Food and
Drug Administration, 1401 Rockville Pike, Rockville, MD 20852.
Phone: (301) 827 7533. Fax: (301) 827 4622. E-mail: kumars@cber
only credible method to identify novel protective antigens. This
brings the question regarding how to predict antigens for fur-
ther preclinical studies.
The complex life cycle of malaria parasites and stage-specific
expression of the majority of malarial antigens present a
unique challenge for vaccine development. Many malaria re-
searchers believe that for a vaccine to be effective, it would be
necessary to attack the parasite during multiple stages of its
development. By this criterion, it is reasonable to assume that
multistage, surface-expressed parasite proteins that are in-
volved in the process of adhesion to and/or invasion of the host
cells deserve special consideration as vaccine candidates.
Recently, we characterized a multistage P. falciparum se-
creted protein with an altered thrombospondin repeat
(SPATR) (8) that is expressed at sporozoite, erythrocytic form,
and gametocyte stages of the parasite. During the sporozoite
stage, this protein is expressed on the cell surface and plays a
role in the invasion of sporozoites into liver cells. Native P.
falciparum SPATR is immunogenic, since immune sera from
Ghanaian adults and from a volunteer who had been immu-
nized with irradiated P. falciparum sporozoites recognized the
recombinant P. falciparum SPATR expressed in transfected
COS-7 cells (8).
The P. yoelii ortholog of this protein identified earlier (22)
contains an altered thrombospondin repeat (TSR) domain,
which is an ancient protein module that existed before the
evolutionary separation of nematodes and vertebrates (18). In
Plasmodium spp., the TSR domain is present in several surface
proteins, and proteins encoding this domain have been impli-
cated in diverse biologic functions, including parasite mobility,
attachment to host cells, and host cell invasion (6, 19, 30). Two
of the TSR domain-containing proteins, circumsporozoite pro-
tein and thrombospondin-related anonymous protein, are cur-
rently undergoing clinical trials as vaccine candidates (1, 24),
suggesting that other Plasmodium proteins containing a TSR
domain could also be potential vaccine targets.
In this report, we describe the identification, cloning, recom-
binant expression in E. coli, and biological characterization of
a gene encoding a novel P. knowlesi protein with an altered
TSR domain. Based on its homology to the P. falciparum
SPATR protein, we named this protein PkSPATR. We believe
that the availability of a well-characterized recombinant
PkSPATR will expedite the preclinical efficacy determination
of this biologically important molecule and help guide the
decision as to whether this molecule should be further devel-
oped for clinical testing in humans.
MATERIALS AND METHODS
Parasites. P. knowlesi (Malaysian H strain) parasites were obtained from
blood-stage infections of rhesus monkeys. Parasitized blood was passed through
leukocyte reduction filters (Sepacell, Baxter, IL) to remove leukocytes. Infected
red blood cells were used to isolate total RNA of P. knowlesi parasites using the
High Pure RNA isolation kit (Roche Applied Science, Indianapolis, IN).
Reverse transcription, amplification, and cloning of DNA encoding PkSPATR.
Total RNA from P. knowlesi was used for reverse transcription-PCR, using
random hexamers and Superscript II RNase H?reverse transcriptase (Invitro-
gen). The primer design was based on the BLAST search results in P. knowlesi
database using published sequence of P. falciparum SPATR (GenBank accession
number AE001404). The forward primer (5? ATGAAAAAAAGTCGCTTTTTT
3?) and reverse primer (5? ATTCTGATTGGTCGCTTCCAA 3?) were used to
amplify the full-length 786-bp cDNA and it was cloned in the TOPO TA cloning
vector (Invitrogen). The DNA sequence of PkSPATR was determined by auto-
Expression and purification of recombinant PkSPATR. For recombinant pro-
tein expression the forward primer (5? CG GGATCC CCTTGAGTAAGAAA
TTGTCCGGA 3?) and reverse primer (5? CG GAATTC TTAATTCTGATTG
GTCGCTTCCAA 3?) were used to amplify a 720-bp DNA fragment encoding
the mature (Leu23to Asn262) PkSPATR protein. To facilitate cloning, BamHI
and EcoRI restriction sites were introduced in the forward and reverse primers,
respectively. The PCR-amplified fragment was cloned in-frame as a glutathione
S-transferase (GST) fusion protein in pGEX-3X, a T7 promoter-based E. coli
expression vector (Amersham Pharmacia Biotech), using BamHI and EcoRI
For expression in E. coli, BL-21 cells were transformed with the PkSPATR
plasmid and the expression was induced at an optical density at 600 nm of 1.0,
with 1 mM isopropyl-1-thio-?-D-galactopyranoside, for 4 h. The total cell pellet
was harvested by centrifugation at 4,000 ? g for 20 min and resuspended in wash
buffer (50 mM Tris, pH 7.5, 20 mM EDTA) containing lysozyme at a concen-
tration of 0.5 mg/ml. Cell suspension was incubated at room temperature for 1 h
with intermittent shaking. NaCl and Triton X-100 were added to obtain a final
concentration of 0.5 M and 2.5%, respectively, and the suspension was further
incubated at room temperature for 30 min, with vigorous shaking. This suspen-
sion was then centrifuged at 13,000 ? g at 4°C, for 50 min, and the resultant
pellet was resuspended in wash buffer containing 1% Triton X-100, using a
Tissuemizer, and centrifuged at 13,000 ? g for 50 min.
The pellet was washed repeatedly in wash buffer, without Triton X-100. After
four washes, the pellet containing inclusion bodies was dissolved in 6 M guani-
dine hydrochloride and incubated for 2 h at room temperature followed by
centrifugation at 50,000 ? g at 4°C, for 30 min. Supernatant, containing dena-
tured protein, was collected and the protein concentration was adjusted to 10
mg/ml with 6 M guanidine hydrochloride. Denatured protein was reduced by
adding dithioerythritol, to a final concentration of 65 mM, and incubating at
room temperature for 2 h. Protein was renatured by diluting 100-fold, in refold-
ing buffer (100 mM Tris, pH 8.0, 0.5 M L-arginine-HCl, 2 mM EDTA, and 0.9
mM oxidized glutathione). After incubation at 10°C for 36 h, renatured material
was dialyzed against 20 mM Tris, pH 8.0, and containing 100 mM urea. The
renatured protein was loaded onto a Q-Sepharose column, and eluted by a salt
gradient using a fast-protein liquid chromatography system. Relevant fractions
were pooled and purified to homogeneity by gel filtration chromatography on the
TSK-GEL G2000SW column. The purity of recombinant PkSPATR was deter-
mined on 4 to 20% polyacrylamide gels.
Generation of anti-PkSPATR antibodies. Anti-PkSPATR antibodies for im-
munofluorescence analysis were raised in mice by immunization with recombi-
nant PkSPATR in Freund’s adjuvant. Female 4- to 6-week-old CD1 mice were
purchased from Jackson Laboratories (Bar Harbor, Maine) and were housed,
fed, and used in the experiments in accordance with the guidelines set forth in
the National Institutes of Health manual Guide for the Care and Use of Labora-
tory Animals; 5 ?g of recombinant PkSPATR in 100 ?l phosphate-buffered saline
was emulsified in 100 ?l of complete Freund’s adjuvant or incomplete Freund’s
adjuvant and was delivered by subcutaneous route at 3-week intervals. Nonhe-
parinized whole blood was collected from the lateral tail vein of mice prior to
immunization and 14 days after the last boost and serum samples were isolated.
Immunofluorescence assay. An indirect immunofluorescence assay was used
to detect the expression of PkSPATR protein in sporozoite and erythrocytic
stages of P. knowlesi parasites. Briefly, twofold diluted test sera were reacted with
air-dried P. knowlesi-infected monkey erythrocytes. Antibodies were detected
using fluorescein isothiocyanate-labeled anti-mouse immunoglobulin G (South-
ern Biotechnology, Birmingham, AL). Slides were mounted using VectaShield
mounting medium for fluorescence (Vector Laboratories Inc., Burlingame, CA)
and evaluated using a fluorescent microscope.
Hepatocyte binding assay. HepG2, a hepatoma human cell line, was grown in
minimal essential medium, supplemented with 2 mM glutamine, 10% heat-
inactivated fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100
?g/ml). The assay was performed as described (27). Briefly, cells at a density of
100,000 cells per well were plated in 96-well plate 36 h before the experiment.
The cells were fixed with 4% paraformaldehyde, followed by blocking with
Tris-buffered saline containing 1% bovine serum albumin. Recombinant GST-
PkSPATR or recombinant GST protein of Schistosoma japonicum expressed in
E. coli at various concentrations was incubated with cells for 1 h followed by
anti-PkSPATR antibody for 30 min and anti-mouse alkaline phosphatase-cou-
pled conjugate for 30 min; 1 mM 4-methylumbelliferyl phosphate was used as the
substrate, and fluorescence was measured in a fluorometer with excitation at 350
nm and emission at 460 nm.
VOL. 73, 2005CHARACTERIZATION OF P. KNOWLESI SPATR PROTEIN5403
Reactivity of P. knowlesi-infected Macaca mulatta serum to recombinant
PkSPATR. Recombinant PkSPATR at 1 ?g/ml concentration in bicarbonate
buffer (15 mM Na2CO3, 35 mM NaHCO3,pH 9.6) was used to coat each well of
a 96-well polyvinylchloride microtiter plate by overnight incubation at 4°C. The
wells were blocked with 1% bovine serum albumin in 50 mM phosphate-buffered
saline for 2 h at 37°C, washed three times with washing buffer (phosphate-
buffered saline containing 0.05% Tween 20) and incubated for 60 min at 37°C
with various dilutions of a pool of hyperimmune sera obtained from M. mulatta
monkeys that had received multiple infections with P. knowlesi followed by drug
cure. Unbound antibodies were washed out followed by an incubation with an
alkaline phosphatase conjugate for 1 h. The plate was developed using 4-nitro-
phenylphosphate tablets (Sigma Chemical Co., St. Louis, MO) and the optical
density at 412 nm was measured using an enzyme-linked immunosorbent assay
Gene isolation and sequence analysis of PkSPATR. We
searched the P. knowlesi genome sequence database (http://www
.sanger.ac.uk/cgi-bin/BLAST/submitblast/p_knowlesi), using the
P. falciparum SPATR protein sequence as a TBLASTN query,
and identified a contig (pkn907d01.q1c) that encoded two open
reading frames showing reasonable sequence identity with P.
falciparum SPATR. We have recently reported the character-
ization of SPATR, a multistage P. falciparum protein with an
altered thrombospondin domain (8).
pkn907d01.qic, a 941-bp PkSPATR gene was PCR amplified
using P. knowlesi genomic DNA prepared from asexual blood-
stage parasites. Expression of a 786-bp PkSPATR transcript in
P. knowlesi asexual-stage parasites was confirmed by reverse
transcription-PCR. The genomic PCR and reverse transcrip-
tion-PCR products were cloned in the TOPO-TA cloning vec-
tor (Invitrogen, San Diego, Calif.) and their nucleotide se-
quences were determined. The difference in the transcript size,
as revealed by agarose gel electrophoresis (data not shown)
and nucleotide sequencing, shows that PkSPATR is encoded
by a two-exon gene separated by a short intron of 156 (Fig. 1a).
Bioinformatic analysis of the nucleotide sequence predicted
that the PkSPATR mRNA of 786 bases (GenBank accession
number AY952327) translates into a 262-amino-acid-long
polypeptide (Fig. 1b), with a predicted molecular mass of
?30.4 kDa (DNASTAR).
Analysis of the PkSPATR protein sequence using SignalP
3.0 (http://www.cbs.dtu.dk/services/SignalP/) revealed that the
first 21 amino acids encode a putative signal peptide with
cleavage site between amino acids Lys21and Glu22. The car-
boxyl terminus of the protein has been predicted to have an
altered thrombospondin repeat (TSR) domain that starts at
Phe203and ends at Asn260(http://smart.embl-heidelberg.de/)
(Fig. 1c). We compared the TSR domain of PkSPATR with
that from several known and hypothetical P. falciparum pro-
teins (Fig. 2). The TSR domain of PkSPATR has the con-
served WSXW motif but differs from other TSR domains as it
lacks the CSXTCG motif (where X is any amino acid). The
predicted protein is rich in asparagine (6.87%) and lysine
The ScanProsite (12) algorithm reconfirmed the presence of
the thrombospondin domain from amino acids 200 to 253 with
the thrombospondin module WSPW (WSXW module, where
X is any amino acid). This prediction algorithm also detected
a putative N-glycosylation site [amino acids 31 to 34 (NSSL)]
identified on contig
and a putative N-myristoylation site [amino acids 29 to 34
(GANSSL)] (Fig. 1c). Similar to P. falciparum and P. yoelii,
PkSPATR is cysteine-rich and contained 12 cysteine residues.
If the 262-amino-acid sequence of PkSPATR is divided in
three equal parts of ?87 amino acids each, the N terminus
(amino acids 1 to 87) has four cysteine residues, the central
(amino acids 88 to 175) has two cysteine residues, and the C
terminus (amino acids 176 to 262) has six cysteine residues.
Cross-species sequence comparison of Plasmodium SPATR.
The amino acid sequence alignment of SPATR protein or-
thologs from P. knowlesi (GenBank accession number
AAX51302), P. falciparum, P. vivax (GenBank accession num-
ber AAX53168), and P. yoelii was done using ClustalW.
SPATR protein sequences analyzed from four Plasmodium
spp. share ?46% identity and ?57% sequence similarity based
on charge, hydrophobicity, polarity, etc. The number and po-
sition of all the cysteine residues are conserved among these
orthologs, indicating the conserved structure of this molecule
within Plasmodium spp. Furthermore, the WSXW motif of
TSR domain in SPATR is conserved in the Plasmodium genus
with amino acid X being proline in PkSPATR and P. vivax
SPATR, aspartic acid in P. falciparum SPATR, and asparagine
in P. yoelii SPATR (Fig. 3a).
A neighbor-joining systematic tree of the SPATR molecule
was constructed to study its phylogenetic relationship.
PkSPATR and P. vivax SPATR cluster together and share
?83% sequence identity. P. falciparum SPATR and P. yoelii
SPATR cluster independently. PkSPATR shares ?62% and
57% identity with P. falciparum SPATR and P. yoelii SPATR,
respectively (Fig. 3b).
Expression of recombinant PkSPATR in E. coli. To produce
PkSPATR as a recombinant protein in E. coli, we amplified the
sequence coding for the mature protein (without signal se-
quence) by reverse transcription-PCR. A 720-bp (Leu23to
Asn262) fragment obtained from the asexual stage parasite by
reverse transcription-PCR was cloned as a BamHI/EcoRI-di-
gested fragment in the pGEX-3X, a GST-based E. coli expres-
sion vector. The expression of recombinant PkSPATR in E.
coli BL21 cells was induced with a 1 mM concentration of
IPTG. The protein was overexpressed and its expression was
readily detectable on a Coomassie blue-stained sodium dode-
cyl sulfate-polyacrylamide gels. Further analysis revealed that
PkSPATR was predominantly expressed as insoluble aggre-
gates in the form of inclusion bodies.
In E. coli, the aggregation of PkSPATR could be due to the
misfolding caused by incorrect disulfide linkage among the 12
cysteine residues. To purify the recombinant PkSPATR, inclu-
sion bodies were isolated, denatured, and reduced, and the
protein was subsequently renatured in vitro under redox con-
ditions by diluting 100-fold, in refolding buffer (100 mM Tris,
pH 8.0, 0.5 M L-arginine-HCl, 2 mM EDTA, and 0.9 mM
oxidized glutathione). The renatured protein was subsequently
purified to a high degree of homogeneity by column chroma-
tography (Q-Sepharose column), and eluted by a salt gradient
using a fast-protein liquid chromatography system. Since
PkSPATR was expressed as a GST fusion, on purification, a
single band of the expected molecular mass (?58 kDa) was
visible on a Coomassie blue-stained gel (Data not shown).
Cellular localization of PkSPATR. To demonstrate the ex-
pression of PkSPATR on malaria parasites and to determine
5404MAHAJAN ET AL.INFECT. IMMUN.
its cellular localization, we performed immunofluorescence
analysis of P. knowlesi sporozoite and blood-stage parasites. To
develop PkSPATR-specific antibodies for use in immunofluo-
rescence assay, groups of five CD-1 outbred mice were immu-
nized with 5 ?g of purified recombinant PkSPATR in Freund’s
adjuvant (see Materials and Methods). In the immunofluores-
cence assay, a distinct reactivity of anti-PkSPATR antibodies
was detected on the surface of P. knowlesi sporozoites (Fig. 4a)
and intraerythrocytic stage parasites (Fig. 4b) showing the mul-
tistage expression of SPATR. The reactivity pattern observed
in P. knowlesi sporozoites and intraerythrocytic-stage parasites
was similar to that observed for P. falciparum parasites (8).
Biological function of PkSPATR in P. knowlesi sporozoites.
Proteins containing one or more copies of TSR domains are
crucial components of both the locomotive and invasion ma-
chineries (6, 25, 28, 36). The structural features of PkSPATR
and its localization on the sporozoite surface suggested that
this molecule might play a role in the sporozoite invasion of the
FIG. 1. Structural and compositional analysis of PkSPATR. (a) Structure of 941-bp PkSPATR gene showing two exons and the intron; (b)
786-base transcript of PkSPATR; (c) amino acid sequence of PkSPATR. The first 21 amino acids comprise the signal peptide (underlined). The
thrombospondin domain (TSR), between amino acids 203 and 260, shown in shadow at the C terminus and the TSR module, WSXW, within the
TSR domain is boxed. The putative N-glycosylation site at amino acids 31 to 34 (NSSL) is italicized and underlined, and a putative N-myristoylation
site at amino acids 29–34 (GANSSL) is boxed. The 12 cysteine residues are underlined.
VOL. 73, 2005 CHARACTERIZATION OF P. KNOWLESI SPATR PROTEIN5405
hepatocytes. To test this hypothesis, we investigated its ability
to bind to HepG2, a human hepatocyte cell line, in an in vitro
liver cell-binding assay. The binding of recombinant PkSPATR
to HepG2 cells was determined at different protein concentra-
tions. At a concentration of 2,000 nM, PkSPATR gave a fluo-
rescence signal of 23,099, showing a high degree of binding to
hepatocytes (Table 1). In comparison, at 2,000 nM, E. coli
produced GST (fusion partner in recombinant PkSPATR),
had a fluorescence signal of only 896 (Table 1), suggesting that
the binding of PkSPATR to HepG2 cells was highly specific.
Recognition of PkSPATR by P. knowlesi infection-induced
antibodies. Having established the biological role of PkSPATR
in the liver cell-parasite interaction, we next wanted to deter-
mine whether PkSPATR was immunogenic during the course
of infection with erythrocytic stage P. knowlesi parasites.
Pooled sera from rhesus monkeys infected repeatedly with P.
FIG. 2. Multiple amino acid sequence alignment of thrombospondin domains of various malarial proteins.
FIG. 3. (a) Multiple amino acid sequence alignment of SPATR proteins from four different Plasmodium species. (b) Phylogenetic relationship
among SPATR proteins from Plasmodium spp.
5406 MAHAJAN ET AL.INFECT. IMMUN.
knowlesi parasites reacted with recombinant PkSPATR in an
enzyme-linked immunosorbent assay (Fig. 5). This showed that
native PkSPATR in P. knowlesi parasites is immunogenic and
induced antibodies that are recognized by epitopes present on
The recent availability of the genome sequence of several
Plasmodium species has paved the way for the identification of
novel vaccine antigens. However, thus far, the genomics-based
research has not led to the discovery of new vaccine candi-
dates, and the majority of antigens under clinical development
were identified at least a decade ago. Our ability to rapidly
exploit the genome database for vaccine development is im-
peded by the lack of reliable criteria to select antigens for
further preclinical development. One logical approach is to
focus on molecules with assigned biologic functions such as
invasion, sequestration, and their association with the progres-
sion of pathogenesis in the host.
In the present study, we report the gene cloning, recombi-
nant expression, and biologic characterization of SPATR of P.
knowlesi and its structural relationship with other Plasmodium
species. The sequence analysis of PkSPATR revealed that it
has a TSR domain at its carboxyl terminus. The TSR domain
of PkSPATR has the WXSW motif but lacks the CSXTCG
motif like its P. falciparum and P. yoelii orthologs. The other
malarial proteins known to have thrombospondin domain are
circumsporozoite protein, SSP2, and circumsporozoite protein/
SSP2-related protein. These molecules predominantly play im-
portant roles in ookinete and sporozoite motility and host cell
attachment and invasion (6, 25, 28, 36).
In malaria parasites, the precise motif(s) of TSR involved in
cell adhesion is not conclusively identified. The CSXTCG mo-
tif was first identified in circumsporozoite protein of P. falci-
parum, which has been shown to play a role as a cell adhesion
motif for leukocytes (29). Other studies have identified the
WSXW motif (amino-terminal to CSVTCG) as a heparin
binding motif (13, 14) and the heparin binding affinity has been
shown to be increased when a canonical BBXB motif (where B
represents a basic residue, Arg, Lys, or His, and X represents
any residue) was present adjacent to the WSXW motif. Other
studies have found that the downstream basic amino acid res-
idues are required for cell adhesion and not the CSVTCG
motif. Another study showed that circumsporozoite protein
binding to hepatocytes requires downstream basic amino acids
but not the VTCG motif (10).
Given the complex life cycle of malaria parasites, many ex-
perts concur that a successful malaria vaccine may require the
inclusion of several molecules expressed at various stages of
the parasite development. The other alternative is to focus on
biologically relevant antigens that are expressed during multi-
ple stages of the parasites development. For example, SSP2/
thrombospondin-related anonymous protein, one of the major
surface proteins of the sporozoite stage, binds to both insect
cells and human liver cells through a common glycosaminogly-
can receptor (23). Recently, we have described P. falciparum
SPATR, a multistage malaria protein having an alternative
TSR domain that binds to human liver cells and plays a role in
the invasion of sporozoite in the liver cells (8). P. falciparum
FIG. 4. Expression of PkSPATR during sporozoite and erythro-
cytic stages of P. knowlesi by immunofluorescence analysis. Sporozoite
and blood-stage parasites were probed with anti-PkSPATR antibodies
raised in mice using the recombinant protein. (a) Sporozoite; (b)
FIG. 5. Immune recognition of PkSPATR by hyperimmune sera of
P. knowlesi-infected rhesus monkeys. Recombinant PkSPATR at a 1
?g/ml concentration was coated on to an enzyme-linked immunosor-
bent assay plate and various dilutions of hyperimmune serum were
added, followed by the addition of anti-human immunoglobulin G
conjugated to alkaline phosphatase. Œ, normal rhesus monkey serum;
F, pooled sera from immune rhesus monkeys generated by repeated P.
knowlesi infections and drug cure.
TABLE 1. Binding of PkSPATR to HepG2 cellsa
Protein Concn (nM)
(relative units) ? SE
PkSPATR500 10,085 ? 1,443
17,237 ? 1,003
23,099 ? 1,285
GST alone2,000 896 ? 38
aCells were incubated with different concentrations of PkSPATR for 1 h,
followed by the addition of anti-PkSPATR and anti-mouse IgG-alkaline phos-
phatase conjugate. The plate was developed by using 4-methylumbelliferyl phos-
phate as a substrate. The data represent the average of triplicate determinations.
VOL. 73, 2005 CHARACTERIZATION OF P. KNOWLESI SPATR PROTEIN 5407
SPATR also binds to human erythrocytes and an anopheline
mosquito larvae multipotent cell line (Chattopadhyay et al.,
unpublished data). Likewise, antibodies against apical mem-
brane protein 1 can inhibit the invasion of sporozoites into
liver cells and also block the merozoite invasion of human
erythrocytes (2, 16, 32, 37). This suggests that the same malar-
ial protein, expressed during different parasite stages, can rec-
ognize different receptors present on eukaryotic cells of both
insect and mammalian origin.
Another multistage antigen called MB2, a 120-kDa protein,
has been found to be present on the sporozoite surface and is
imported into the nucleus of erythrocytic-stage parasites as a
66-kDa processed fragment (26). Although the receptor for
MB2 protein is yet unknown, its differential processing and
localizations at different stages of parasite development indi-
cate that MB2 is capable of interacting with host receptors on
both liver cells and erythrocytes. In this regard, we believe that
SPATR, a multistage antigen present in several Plasmodium
species, serves as a parasite ligand during the process of inva-
sion into host cells.
Asparagine-rich motifs in proteins are targets of opsonizing
antibodies that promote phagocytosis of parasites by immune
cells (3, 4, 15). The abundance of the asparagine residue
(6.87%) and the recognition of the recombinant PkSPATR by
immune sera from multiple P. knowlesi infections and drug-
cured rhesus monkeys in enzyme-linked immunosorbent assay
indicate that PkSPATR could be a target of opsonizing anti-
In nature, cysteine is one of the least abundant amino acids
but it is frequently found in the catalytic sites of the proteins.
Malarial proteins rich in cysteine residues have often been
implicated in parasite attachment and/or the invasion of the
host cells. The primary examples of this observation are con-
served cysteine-rich motif containing Duffy binding protein of
P. vivax (9), Duffy-binding-like domain containing P. falcipa-
rum EMP1 (35), epidermal growth factor-like domain in
MSP119(20), and ecto-domain in AMA-1 (37). It has also been
observed that specific position of the cysteine residue in a
protein, like circumsporozoite protein, is important for main-
tenance of its biologic function (7, 27). PkSPATR has 12 cys-
teine residues, the majority of which are localized at the car-
boxyl and amino termini and less abundant in the central part
of the protein. The abundance of cysteine residues and the
ability to bind with liver cells strongly indicate that PkSPATR
is a novel parasite ligand that plays an important role in at-
tachment and/or invasion in host cells.
In summary, the data presented in this paper, and from our
previous work, suggest that SPATR is expressed during mul-
tiple stages of parasite development and belongs to a family of
malarial antigens that participate in the process of cell invasion
and adhesion. Furthermore, the observation that P. knowlesi
infection in rhesus monkeys induced anti-SPATR antibodies
supports the argument that SPATR is a target of protective
immune responses. We believe that the isolation, biological
characterization, and production as a recombinant protein of
SPATR from P. knowlesi will facilitate the preclinical efficacy
evaluation of this biologically important molecule in a primate
We thank the veterinary and animal care staff of the Walter Reed
Army Institute for Research for their help in this study.
The views expressed in this article are those of the authors and do
not necessarily reflect the official policy or position of the United
States Food and Drug Administration or the Department of the Navy,
Department of Defense, United States of America.
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VOL. 73, 2005CHARACTERIZATION OF P. KNOWLESI SPATR PROTEIN5409