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Prepublished online as a Blood First Edition Paper on May 1, 2003; DOI 10.1182/blood-2002-11-3513.
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Blood, 1 September 2003, Vol. 102, No. 5, pp. 1911-1914
RED CELLS
Brief report
Mature parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum binds to the 30-kDa domain of protein 4.1 in malaria-infected red blood cells
Karena L. Waller,
Wataru Nunomura,
Xiuli An,
Brian M. Cooke,
Narla Mohandas, and
Ross L. Coppel
From the Department of Microbiology and the Victorian Bioinformatics
Consortium, Monash University, Melbourne, Victoria, Australia; Department of
Biochemistry, School of Medicine, Tokyo Women's Medical University, Tokyo,
Japan; and New York Blood Center, New York, NY.
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Abstract
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The Plasmodium falciparum mature parasite-infected erythrocyte
surface antigen (MESA) is exported from the parasite to the infected red blood
cell (IRBC) membrane skeleton, where it binds to protein 4.1 (4.1R) via a
19-residue MESA sequence. Using purified RBC 4.1R and recombinant 4.1R
fragments, we show MESA binds the 30-kDa region of RBC 4.1R, specifically to a
51-residue region encoded by exon 10 of the 4.1R gene. The 3D
structure of this region reveals that the MESA binding site overlaps the
region of 4.1R involved in the p55, glycophorin C, and 4.1R ternary complex.
Further binding studies using p55, 4.1R, and MESA showed competition between
p55 and MESA for 4.1R, implying that MESA bound at the IRBC membrane skeleton
may modulate normal 4.1R and p55 interactions in vivo. Definition of minimal
binding domains involved in critical protein interactions in IRBCs may aid the
development of novel therapies for falciparum malaria.
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Introduction
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During maturation of Plasmodium falciparum within human red blood
cells (RBCs), several parasite proteins are synthesized and transported into
the RBC cytoplasm where some associate with the RBC membrane skeleton
(reviewed in Cooke et
al1). One of these
proteins, the mature parasite-infected erythrocyte surface antigen (MESA), has
a 19-residue sequence
(DHLYSIRNYIECLRNAPYI2)
that binds the RBC membrane skeleton protein 4.1
(4.1R).3 Although
the precise function of MESA within infected RBCs (IRBCs) is unknown, studies
have shown that interruption of the MESA-4.1R interaction results in
accumulation of unbound MESA within the RBC cytoplasm and parasite death
through an unknown mechanism that may be related to the presence of unbound
MESA.4 Interruption
of this and other protein interactions at the IRBC membrane skeleton in vivo
offers as yet unexplored avenues for the development of novel antimalarial
therapies. Here, using recombinant 4.1R and MESA proteins, we mapped the
region of 4.1R that binds the 19-residue region of MESA and also demonstrated
competition between MESA and p55 for 4.1.
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Study design
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Fragments of 4.1R and MESA were amplified by polymerase
chain reaction (PCR) as previously
described5
(Figure 1), either from cDNAs
of 4.1R8 or
appropriate genomic DNA subfragments of the MESA
gene.3,7
The resulting fragments were cloned into the Escherichia coli protein
expression plasmids
pGEX-KG10 or
pET-31b(+) (Novagen, Darmstadt, Germany), and glutathione
S-transferase (GST)– and histidine-tagged fusion proteins were
expressed and purified using standard affinity chromatography under native
purification
conditions.3,8,11
Additionally, 4.1R was purified from healthy human
RBCs.9 Purified
proteins (Figure 1C) were
dialyzed extensively against phosphate-buffered saline (PBS; 0.15 M NaCl
containing 10 mM Na2HPO4/NaH2PO4,
pH 7.4) and then used in protein interaction assays using an IAsys resonant
mirror biosensor (Affinity Sensors, Cambridge, United Kingdom), as previously
described5,8
or in GST-pull down assays (Figure
2 and Table 1;
methods are described in figure legend and table footnote). Approval for the
study was obtained from the institutional review boards of Monash University,
Tokyo Women's Medical University, and the New York Blood Center. Informed
consent was provided according to the Declaration of Helsinki.
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Figure 1.. Schematic representation of MESA and 4.1R fragments and purified GST
fusion proteins. (A) Schematic of the amino terminal region of MESA and
the relative locations of the various MESA fragments used in this study. The
first exon of the MESA gene encodes a putative signal peptide
containing a hydrophobic core that is thought to be cleaved from the mature
MESA polypeptide.6
The second exon encodes a highly charged protein that is characterized by 7
peptide repeat
regions,6 of which
only the GESKET peptide repeat region is shown. Amino acid (aa) residue
numbers are shown adjacent to the MESA fragment names in the protein
schematic. Previously, MESA fragment 3 (MF3) and MF4 have been described as
binding and nonbinding regions for 4.1R, respectively, with MF3 containing the
sequence (DHLYSIRNYIECLRNAPYI) mapped as the 4.1R binding
region.3,7
Oligonucleotide primer sequences used in the construction of the MESA
DNA fragments in this study are as follows: MF3(S)+19 and MF3(S) 19 (F),
cgc gga tcc GAT ATC TAT ACG AAT TGT; MF3(S)+19 and MF3(S)19 (R), ccg
gaa ttc CAT TAC ATT CAC ATG TTT TCT A; MF4(S) (F), cgc gga tcc GCT AAT ACT GAA
AAA AAT GAT; and MF4(S) (R), ccg gaa ttc ACT TGT TTT TTA ATT TCT TC. A
MF319 DNA fragment was originally amplified by splice-overlap extension
PCR to delete the region encoding the 19-residue
sequence7 and was
later used as a template to amplify the shorter MF3(S)19 fragment.
Sequence shown in upper case is complementary to the sequence amplified; lower
case sequence is noncomplementary. Forward and reverse primers are designated
F and R, respectively. (B) Schematic of the full-length 4.1R molecule and the
relative locations of the various 4.1R fragments used in this study. Amino
acid (aa) numbers are shown adjacent to the 4.1R fragment names and the
protein schematic. Oligonucleotide primer sequences used in the construction
of the recombinant 4.1R DNA fragments in this study are as follows:
30-kDa F1 and F2 (F), GGG CTG GCA AGC CAC GTT TGG TG; 30-kDa F1 and F2 (R),
ccg gaa ttc TGT AAA ATT CCA AGG GAC; 30-kDa F3 (F), cgc gga tcc TTT AAT GTA
AAG TTT TAT CC; 30-kDa F3 (R), ccg gaa ttc CGG GGC CAG TTT AAA ATC; 30-kDa F4
(F), cgc gga tcc AAT CAG ACC AAG GAA CTT; 30-kDa F4 (R), ccg gaa ttc GCG GTT
AAT TCT CAG CTT; 30-kDa F5, F6, and F7 (F), cgg gat ccA TGA CTC CAG CTC AGG
CT; 30-kDa F5 and F6 (R), gag cgc tcg agt caA AAT TTG GAT CCT AGC GCA AGA AAT
TTG CTT TTG GG; 30-kDa F7 (R), gct agc TCG AGT CAC AAG TCC TTT GCT TTA TGA AG;
30-kDa F8 (F), cgg gat ccC AAG AGC AGT ATG AAA GTA CC; 30-kDa F8 (R), gag cgc
tcg agt caA AAA TTT GGA TCC TAG CGC AAG AAA TTT GCT TTT GGG; 30 + 16-kDa (F),
ccg gaa ttc TAA TGC ACT GCA AGG TTT CT; 30 + 16-kDa (R), ccg ctc gag TGG CTC
AGC TTG CTC AGG; 22/24-kDa (F), cgc gga tcc CCT CCC CTG GTG AAG ACA C; and
22/24-kDa (R), gcc caa gct tCT CAT CAG CAA TCT CGG T. Construction of the
pGEX-KG plasmids encoding the 30-kDa, 30-kDa F1, 16-kDa, and 10-kDa regions of
4.1R has previously been
described.8 (C) RBC
4.1R and recombinant GST fusion proteins were purified as previously
described.3,9
Each purified protein (approximately 1-2 µg total protein) was resolved by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in
15% (wt/vol) gels before staining with Coomassie brilliant blue. The protein
samples are 80-kDa RBC 4.1R (lane 1), GST-30 + 16 kDa (lane 2), GST-30 kDa
(lane 3), GST-16 kDa (lane 4), GST-10 kDa (lane 5), GST-22/24 kDa (lane 6),
GST–30-kDa F6 (lane 7), GST–30-kDa F5 (lane 8), GST–30-kDa
F7 (lane 9), GST–30-kDa F8 (lane 10), GST (lane 11), GST–30-kDa F2
(lane 12), GST–30-kDa F3 (lane 13), GST–30-kDa F4 (lane 14),
GST-MF3(S)+19 (lane 15), GST-MF3(S)19 (lane 16), and GST-MF4(S) (lane
17).
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Figure 2.. Three-dimensional structure of 30-kDa 4.1R and competition of MESA
binding to 4.1 by p55. (A) The putative 24-residue MESA binding site of
protein 4.1 is located within a 51-residue region encoded by exon 10 of
4.1R, located in the C-lobe of the protein molecule. The binding
affinity between 4.1R and MESA was enhanced by sequences within the 16-kDa
domain, although MESA did not bind to the 16-kDa domain alone. The binding
residues within this 30-kDa region for phosphatidylserine (PS), p55,
calmodulin (CaM), band 3, and GPC are also shown. The representation is drawn
on the basis of the 3D crystal structure described by Han et
al.12 (B)
Competition of binding was demonstrated using GST-pull down assays, in which
GST-4.1 80 kDa (coupled to glutathione Sepharose beads; Amersham Pharmacia
Biotech, Piscataway, NJ) pre-incubated (30 minutes at room temperature) with
increasing concentrations of p55 (ranging from 0.0 µM to 2.0 µM), before
the addition of 1.0 µM His-tagged MESA(S)+19 (30 minutes at room
temperature). Subsequently, beads were washed and collected, and the proteins
resolved by SDS-PAGE (10% [wt/vol]). The binding of MESA to 4.1R was detected
by Western blot using monoclonal anti-His antibody (Roche, Indianapolis, IN).
The first lane contains 0.45 µg His-tagged MESA that was included as an
immunoblot MESA-positive control. Competition of MESA for 4.1 by p55 was
indicated by the decreasing levels of MESA detected in samples with increasing
concentrations of p55 used in the preincubations.
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Results and discussion
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Quantitative binding data showed that GST-MF3(S)+19 bound purified 4.1R
with moderate affinity (KD(kin) = 2.7 x
10–7 M; Table
1). Binding studies using recombinant GST-30 kDa, -16 kDa, -10
kDa, and -22/24 kDa proteins of 4.1R showed that, whereas GST-MF3(S)+19 bound
the 30-kDa domain with moderate affinity (KD(kin)
= 1.9 x 10–6 M), it did not bind the 10-kDa,
16-kDa, or 22/24-kDa domains. To map the MESA binding sequences within the
30-kDa region more precisely, 8 smaller subfragments of the 30-kDa domain were
generated (Figure 1B).
GST-MF3(S)+19 bound with moderate affinity to GST-30 kDa F5
(KD(kin) = 1.17 x
10–6 M) but not to GST-30 kDa F1, F2, F3, F4, F6,
F7, or F8 recombinant proteins. These data enabled us to identify the 51
residues of the 30-kDa domain encoded by exon 10 of 4.1R to be
responsible for the interaction of 4.1R with MESA. Comparison of the sequences
of the nonbinding fragments F4, F6, and F8 with the binding fragment F5
suggests that the binding domain could be further mapped to the central 24
residues of the 4.1R 30-kDa region, although such an assignment would need to
be further confirmed by a direct binding assay. All interactions presented
here were confirmed, with similar affinities, using cuvettes coated with the
synthetic 19-residue MESA peptide (data not shown). No binding of GST-4.1R
fusion proteins or purified RBC 4.1R to GST-MF3(S)19, GST-MF4(S) (data
not shown), GST, or BSA was detected. Further, although the 16-kDa domain of
4.1R did not bind GST-MF3(S)+19, we found the 30 + 16-kDa fusion protein bound
with higher affinity (KD(kin) = 1.3 x
10–7 M) to MESA than GST-30 kDa alone
(KD(kin) = 1.9 x
10–6 M).
Protein 4.1R functions to stabilize horizontal protein interactions between
spectrin tetramers and actin filaments in the RBC membrane skeleton, as well
as participating in several interactions that link the underlying
spectrin-based membrane skeleton to the lipid membrane, including the
4.1R-glycophorin C (GPC)–p55 and 4.1R-band 3 interactions (reviewed in
Gascard and
Mohandas13 and
Pinder et al14). In
these and other membrane skeleton interactions, 4.1R has been identified as an
important modulator of protein binding affinities. 4.1R has been shown to
modulate the 4.1R-GPC-p55 and spectrin-actin–4.1R ternary complexes, by
increasing the affinity of the
p55-GPC8 and
spectrin-actin
interactions.15
Several studies have identified the specific residues in the 30-kDa domain of
4.1R that bind to GPC and
p55.8,16
The GPC binding site in 4.1R was mapped to 40 residues encoded by exon 8 of
the 4.1R gene, whereas the region of 4.1R that binds to p55 was
mapped to a 33-residue region encoded by exon
10.8 Our findings
show that there is an overlap between the binding domains of MESA and p55 on
the 4.1R protein. Recently, the 3D crystal structure of the 30-kDa domain
(also known as the FERM [4.1-ezrin-radaxin-moesin]
domain17) has been
resolved (Figure
2A12),
in which the exon 10 encoded sequences are located in the C-lobe of the 30-kDa
domain. The location of the MESA binding domain in this structure is also
shown. On the basis of our earlier findings, we hypothesized that binding of
MESA to 4.1R may act as a competitive inhibitor in the p55-4.1R interaction.
GST-pull down assays, using GST-4.1R 80 kDa coupled to glutathione Sepharose
beads were performed to assess the binding of MESA to 4.1R in the absence or
presence of p55. These data showed greatly reduced binding of MESA to 4.1R
with increasing concentrations of p55
(Figure 2B). The competitive
interaction of MESA and p55 for 4.1R may, therefore, result in modulation of
the ternary complex between 4.1R-GPC-p55 and alter the stability of the
membrane skeleton of P falciparum IRBCs.
Interestingly, fusion of the 30-kDa and 16-kDa regions of 4.1R enhanced the
affinity of the interaction between 4.1R and MESA by an order of magnitude.
Sequences that have little or no direct binding ability, but enhance the
affinity of other binding sequences, perhaps through altered protein
conformation, have previously been reported. For example, sequences from the
repeat regions of the P falciparum circumsporozoite protein (CSP)
have been shown to enhance the affinity of binding of CSP-derived peptides
with human hepatoma cell
lines.18 This,
however, is the first report in which the affects of the 16-kDa domain of 4.1R
on binding of the 30-kDa domain with a partner protein have been
described.
The death of P falciparum when cultured in 4.1R-deficient RBCs is
a clear indication of the extreme importance of the MESA-4.1R interaction for
parasite survival.4
Here, we show data that MESA binds to a 51-residue region of the 4.1R 30-kDa
domain and also that MESA and p55 compete for interaction with 4.1R. The death
of parasites in 4.1R-deficient RBCs may, therefore, result from the
accumulation of toxic levels of unbound MESA in the IRBC cytoplasm because of
MESA's inability to associate with 4.1R in 4.1R-deficient
RBCs4 and/or the
subsequent inability of the parasite to correctly modulate the protein
interactions that form the RBC membrane skeleton and facilitate parasite
survival in vivo. Although the precise function of MESA at the membrane
skeleton of IRBCs is unresolved (parasites that do not express MESA [resulting
from spontaneous deletion of chromosome 5] cultured in normal human RBCs show
that MESA is not critically required for
growth4 or
cytoadhesion19 in
vitro), the experiments of Magowan et
al4 have
conclusively demonstrated that interruption of the MESA-4.1R interaction
results in parasite death. Consequently, increased understanding of the
MESA-4.1R interaction and its potential interruption in vivo may offer an as
yet unexplored avenue for the future development of novel antimalarial
therapies.
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Acknowledgements
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We thank Dr Philippe Gascard and Ms Marilyn Parra (Life Sciences Division,
Lawrence Berkeley Laboratories, Berkeley, CA) and Ms Lisa M. Stubberfield
(Department of Microbiology, Monash University, Melbourne, Victoria,
Australia) for the gift of plasmids.
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Footnotes
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Submitted December 2, 2002;
accepted April 24, 2003.
Prepublished online as Blood First Edition Paper, May 1, 2003; DOI
10.1182/blood-2002-11-3513.
Supported by Grant-in Aid for Scientific Research from the Ministry of
Education of Japan (grant 12680702), the National Institutes of Health (grant
DK-32094), the Howard Hughes International Scholars Program, and the National
Health and Medical Research Council of Australia.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked "advertisement" in accordance with 18 U.S.C. section
1734.
Reprints: Karena L. Waller, Department of Medicine (Cardiology), Albert
Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461; e-mail:
kwaller{at}aecom.yu.edu.
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Abstract
Introduction