From the Malaria Research Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India
Received for publication, February 19, 2001
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ABSTRACT |
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Invasion of erythrocytes by malaria parasites is
mediated by specific molecular interactions. Plasmodium
vivax is completely dependent on interaction with the Duffy blood
group antigen to invade human erythrocytes. The P. vivax
Duffy-binding protein, which binds the Duffy antigen during invasion,
belongs to a family of erythrocyte-binding proteins that also includes
Plasmodium falciparum sialic acid binding protein and
Plasmodium knowlesi Duffy binding protein. The receptor
binding domains of these proteins lie in a conserved, N-terminal,
cysteine-rich region, region II, found in each of these proteins. Here,
we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli.
Recombinant PvRII is incorrectly folded and accumulates in inclusion
bodies. We have developed methods to refold and purify recombinant
PvRII in its functional conformation. Biochemical, biophysical, and
functional characterization confirms that recombinant PvRII is pure,
homogeneous, and functionally active in that it binds Duffy-positive
human erythrocytes with specificity. Refolded PvRII is highly
immunogenic and elicits high titer antibodies that can inhibit binding
of P. vivax Duffy-binding protein to erythrocytes,
providing support for its development as a vaccine candidate for
P. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.
The invasion of erythrocytes by malaria parasites is mediated by
specific molecular interactions between host receptors and parasite
ligands (1). Plasmodium vivax and the related simian malaria
parasite Plasmodium knowlesi require interaction with the
Duffy blood group antigen to invade human erythrocytes (2, 3). P. knowlesi can also invade rhesus erythrocytes using alternative Duffy-independent receptors (4). P. falciparum commonly uses sialic acid residues of glycophorin A as invasion receptors (5-9). Like P. knowlesi, P. falciparum also invades
erythrocytes by multiple pathways and is not completely dependent on
sialic acid residues of glycophorin A (8, 10, 12, 13).
Parasite ligands that bind host receptors to mediate erythrocyte
invasion include P. vivax and P. knowlesi
Duffy-binding proteins, P. knowlesi DBL domains are also found in the extracellular region of the P. falciparum erythrocyte membrane protein-1 (PfEMP-1) family of
proteins that are expressed on the surface of P. falciparum-infected trophozoites and schizonts (23-25). Some
members of the PfEMP-1 family bind endothelial receptors such as CD36,
ICAM-1, vascular cell adhesion molecule, E-selectin, CD31,
chondroitan sulfate A, and hyaluronic acid to mediate cytoadherence,
which is responsible for sequestration of P. falciparum
trophozoites and schizonts in the deep vasculature of various host
organs (26-31). Binding to ICAM-1 is thought to be important for
sequestration in brain capillaries and is implicated in the pathology
of cerebral malaria (32). Binding to hyaluronic acid and chondroitan
sulfate A is important for sequestration in the placenta, which often
leads to complications in pregnancy (30, 33). DBL domains derived from
PfEMP-1 have been shown to bind ICAM-1, chondroitan sulfate A, CD31,
and uninfected erythrocytes (34-39).
DBL domains are thus used by different Plasmodium species to
bind diverse host receptors to mediate erythrocyte invasion and cytoadherence, two mechanisms that are central to malaria pathogenesis. It is important to understand the structural basis of the interaction of DBL domains with host receptors. This may allow the development of
receptor-blocking agents that inhibit invasion or reverse
cytoadherence. Receptor binding DBL domains are also attractive vaccine
candidates because antibodies directed against such functional domains
may block their interaction with erythrocytes or endothelial receptors.
Here, we describe methods to produce milligram quantities of correctly
folded recombinant PvRII, the binding domain of P. vivax
Duffy-binding protein, which contains around 350 amino acids, including
12 cysteines. Recombinant PvRII has been expressed in Escherichia
coli purified from inclusion bodies under denaturing conditions
and refolded in vitro into its native conformation. We
demonstrate that refolded PvRII is highly immunogenic and elicits high
titer antibodies that can inhibit the binding of P. vivax Duffy-binding protein to erythrocytes, providing support for the development of recombinant PvRII as a vaccine candidate for P. vivax malaria. Large scale production of functional DBL domains is
now possible, opening avenues for more extensive biochemical, structural, and immunological studies.
Plasmid Constructs and E. coli Strains Used for Recombinant
Expression of PvRII--
DNA encoding PvRII (amino acids 194-521 of
P. vivax Duffy-binding protein) fused to hexa-histidine
(6-His) at the C-terminal end was amplified by polymerase chain
reaction using primers 5'-GCA TGC CAT GGA TCA TAA GAA AAC GAT CT-3' and
5'-CGA GTG TCG ACT CAG TGA TGG TGA TGG TGA TGT GTC ACA ACT TCC TGA
GT-3' and a plasmid containing the gene encoding P. vivax
Duffy-binding protein as template (18). The polymerase chain reaction
product was digested with NcoI and SalI and
cloned as a NcoI-SalI fragment downstream of the
T7 promoter in expression vector pET28a+ (Novagen) to yield plasmid
pVPET1. E. coli BL21 (DE3) strain (Novagen) was transformed with plasmid pVPET1 and used for expression of recombinant PvRII.
Expression of PvRII in E. coli--
Luria broth containing
kanamycin (50 µg/ml) was inoculated with E. coli
BL21(DE3)pVPET1 and cultured overnight at 37 °C. Fresh Luria
broth containing kanamycin (25 µg/ml) was inoculated with the
overnight culture at a dilution of 1:50 and cultured at 37 °C to an
A600 nm of 0.6-0.8. Expression of PvRII
was induced by adding isopropyl-1-thio- Isolation of Inclusion Bodies and Purification of Recombinant
PvRII by Metal Affinity Chromatography under Denaturing
Conditions--
AFter induction, E. coli cells were
harvested by centrifugation, washed in chilled wash buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl), resuspended in chilled lysis buffer (10 mM Tris, pH 8.0, 5 mM benzamidine-HCl, 2 mM phenylmethylsulfonyl fluoride, 10 mM EDTA,
100 mM NaCl, 200 µg/ml lysozyme), and lysed by
sonication. Inclusion bodies were collected by centrifugation of lysed
cells at 4 °C, solubilized in 10 mM Tris buffer, pH 8.0, containing 6 M guanidine hydrochloride (GdnHCl), and
treated with 10 mM dithiothreitol (DTT) for 2 h at
37 °C. After removal of DTT by ultrafiltration using filters with
10-kDa cutoff, recombinant PvRII was purified from solubilized
inclusion bodies by metal affinity chromatography under denaturing
conditions using a nickel nitrilotriacetic acid column as described by
the manufacturer (Qiagen). Solubilized inclusion bodies were loaded on
a nickel nitrilotriacetic acid column previously equilibrated with
equilibration buffer (10 mM Tris, pH 8.0, 100 mM NaH2PO4, 6 M
GdnHCl). The column was washed with equilibration buffer at pH 6.3, and
bound protein was eluted using a pH gradient starting at pH 6.3 and
ending at pH 4.3. The final concentration of the purified protein was
adjusted to ~4.5 mg/ml with equilibration buffer.
Refolding PvRII by Rapid Dilution--
Purified PvRII was
refolded by 100-fold dilution in refolding buffer containing 50 mM phosphate buffer, pH 7.2, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 1 M
urea, and 0.5 M arginine so that the final protein
concentration was ~45 µg/ml. Refolding was allowed to proceed at
10 °C for 36 h with stirring. At the end of 36 h, the
refolding solution was dialyzed for 48 h against dialysis buffer
(50 mM phosphate buffer, pH 6.5, 1 M urea) to
remove arginine before proceeding with purification by ion-exchange chromatography.
Purification of Refolded PvRII by Ion Exchange and Gel Filtration
Chromatography--
After removal of arginine by dialysis, the
refolded protein was loaded on an SP-Sepharose column equilibrated with
50 mM phosphate buffer, pH 6.5. The bound protein was
eluted with a linear gradient of NaCl (100 mM NaCl to 1.5 M NaCl). Fractions containing refolded PvRII were pooled,
and PvRII was further purified by gel filtration chromatography using a
Superdex 75 column (Amersham Pharmacia Biotech). For gel filtration
chromatography, 50 mM phosphate buffer, pH 7.2, containing
200 mM NaCl was used.
Analysis of Refolded, Purified PvRII by Reverse Phase
Chromatography--
Refolded PvRII was loaded on a reverse phase C8
column. The gradient used for elution was developed using Buffer A
(0.05% trifluoroacetic acid in water) and Buffer B (0.05%
trifluoroacetic acid in 90% acetonitrile, 10% water). The column was
initially equilibrated with 90% Buffer A and 10% Buffer B and reached
a composition of 10% Buffer A and 90% Buffer B in 40 min.
N-terminal Sequencing and Mass Spectral Analysis of Refolded,
Recombinant PvRII--
Automated Edman degradation was carried out
using an Applied Biosystems 491 protein sequencer using standard
methods. Approximately 100 pmol of purified refolded PvRII was used for
the N-terminal sequencing reaction. Mass spectral analysis of refolded,
purified PvRII was performed by electron spray ionization mass
spectroscopy using a Hewlett Packard HP100 Series LC/MSD mass
spectrometer by standard procedures.
Detection of Free Thiols in Refolded, Recombinant
PvRII--
Analysis of free protein thiol was carried out using
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) according to the method of Ellman (40). The sensitivity of the assay was determined using known
amounts of cysteine. Free thiol could be clearly detected at a
concentration of 30 µM. The presence of free thiols in
refolded, purified PvRII was determined in the presence of 6 M GdnHCl to promote side chain availability.
Circular Dichroism (CD) Spectra--
CD spectra were recorded on
a Jasco-J720 spectropolarimeter. Spectra of purified, refolded PvRII in
10 mM phosphate buffer, pH 7.2, were recorded as the
average of 10 individual spectral scans in the far-UV region from 184 to 260 nm using a cuvette with a path length of 0.1 cm and the
following instrument parameters: instrument sensitivity, 1 millidegrees; response time, 2 s; scan speed, 50 nm/min.
Concentration of PvRII used for CD spectroscopy was measured by the BCA
protein assay method (Pierce) using bovine serum albumin as a standard.
Deconvolution of the CD spectra was performed using the method of Bohm
et al. (41).
Erythrocyte Binding Assay--
Blood collected in 10% citrate
phosphate dextrose was stored at 4 °C for up to 4 weeks and washed
three times in RPMI 1640 (Life Technologies, Inc.) before use. Duffy
phenotypes of erythrocytes were determined by standard blood-typing
methods using two antisera (anti-Fya and anti-Fyb) (Ortho-Clinical
Diagnostics). Duffy negative erythrocytes, Fy(a Detection of Inactive PvRII by Immuno-precipitation and Western
Blotting after Preabsorption of Refolded PvRII with Duffy-positive
Erythrocytes--
Different quantities of refolded PvRII (100, 200, 400, 800, 2000 ng) were immuno-precipitated as described below using
polyclonal rabbit serum raised against refolded PvRII, separated by
SDS-PAGE, and detected by Western blotting using a commercially
available mouse monoclonal antibody raised against penta-histidine
(5-His) (Qiagen) to determine the sensitivity of detection.
Immunoprecipitation was performed by incubating refolded PvRII with a
rabbit polyclonal serum raised against PvRII at a dilution of 1:200 for
1 h on ice and with protein A-Sepharose beads (Amersham Pharmacia
Biotech) at room temperature for 1 h. The beads were collected by
centrifugation and washed once with 0.5% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 50 mM Tris, pH
7.4 (NETT) containing 0.5% bovine serum albumin and twice with NETT
buffer without bovine serum albumin. Bound proteins were eluted by
boiling, separated by SDS-PAGE, and detected by Western blot using a
mouse monoclonal antibody directed against 5-His (Qiagen). 100 ng of
refolded PvRII can be clearly detected by this method. Refolded PvRII
(8 µg) was preabsorbed four times with 800 µl of packed
Duffy-positive human erythrocytes to remove active PvRII before
immunoprecipitation. Preabsorption with an equal number of
Duffy-negative human erythrocytes was used as a control. Residual PvRII
left after preabsorption was detected by immunoprecipitation and
Western blotting as described.
Animals and Immunization--
Rabbits used in this study were
procured from the Animal Facility of the International Center for
Genetic Engineering and Biotechnology, New Delhi, India. Animals were
housed, fed, and used in experiments according to the guidelines set
forth in the National Institutes of Health manual titled Guide for the
Care and Use of Laboratory Animals (National Institutes of Health
Publication 86-23, United States Department of Health and Human
Services, Washington D. C.). Two 8-week-old New Zealand White rabbits
were immunized with 200 µg of recombinant PvRII emulsified in
complete Freund's adjuvant and delivered by the subcutaneous route.
The rabbits were boosted on day 28 with 100 µg of PvRII formulated in
incomplete Freund's adjuvant delivered by the subcutaneous route. One
rabbit was immunized with adjuvant alone according to the schedule
described above to provide control antiserum. Rabbits were bled on days
0 (preimmune), 27, and 42, and the sera were used for immunoassays.
ELISA--
Rabbit sera were tested for recognition of
recombinant PvRII using an ELISA. Briefly, wells of flat-bottom
Immulon-2 plates (Dynatech Laboratories) were coated with 0.1 µg of
PvRII and blocked with 5% milk powder solution in phosphate-buffered
saline (blocking buffer). Antigen-coated wells were incubated for 90 min at 37 °C with 100 µl of rabbit serum diluted in blocking
buffer. Serial dilutions (1:2-fold) of sera starting with a 1:1000-fold
dilution were tested. After washing with phosphate-buffered saline and 0.05% Tween 20, 100 µl of horseradish peroxidase-labeled anti-rabbit IgG antibody (Sigma) diluted 1:2000-fold was added to each well and
incubated for 60 min at 37 °C. The enzyme reaction was developed with o-phenylenediamine dihydrochloride as the chromogen and
hydrogen peroxide as the substrate. The reaction was terminated by the addition of sulfuric acid, and the absorbance at 490 nm
(A490) was recorded in each well using an ELISA
microplate reader (Molecular Devices). Preimmune sera as well as
adjuvant-alone control sera were used at similar dilutions. To
determine end point titers, the last dilution of test sera yielding an
A490 value 1.5 times that obtained with
preimmune serum was determined.
Inhibition of Erythrocyte Binding to PvRII Expressed on the
Surface of COS Cells with Antisera Raised against Refolded
PvRII--
COS7 cells were cultured as described previously (20). COS7
cells growing in 35-mm-diameter wells at 40-60% confluency were transfected using Lipofectin with 2.5 µg of plasmid pHVDR22 according to the manufacturer's instructions (Life Technologies, Inc.). Plasmid
pHVDR22 was designed to express PvRII on the surface of mammalian cells
(20); it contains DNA encoding PvRII fused to the signal sequence of
Herpes simplex virus glycoprotein D (HSV gD) at the N
terminus and the transmembrane region and cytoplasmic domain of HSV gD
at the C terminus in a mammalian expression vector (20, 42). The signal
sequence and transmembrane segment of HSV gD target the fusion protein
to the surface of COS cells. Expression of PvRII on the COS cell
surface was confirmed by immunofluorescence assays, as described
previously using a monoclonal antibody, DL6, directed against HSV gD
sequences in the fusion protein (20, 42). Binding of erythrocytes to
transfected COS cells expressing PvRII on the surface was tested using
an erythrocyte binding assay, as described earlier (20). Briefly, 200 µl of a 10% suspension of Duffy-positive human erythrocytes was
added to 2 ml of media in wells containing transfected COS cells.
Erythrocytes were allowed to settle for 2 h at 37 °C.
Non-adherent erythrocytes were removed by washing COS cells three times
with RPMI. Erythrocyte binding assays were performed in the presence of
different dilutions of rabbit sera raised against refolded PvRII.
Preimmune sera and rabbit sera raised against adjuvant alone were used
as controls. The number of COS cells covered with rosettes of adherent
erythrocytes was scored in 50 fields at a magnification of 40.
Expression, Purification, and Refolding of Recombinant
PvRII--
Recombinant PvRII accumulates in inclusion bodies as a
misfolded, insoluble aggregate when expressed in E. coli.
Misfolded PvRII was solubilized in 6 M GdnHCl, purified by
metal affinity chromatography under denaturing conditions, and refolded
by rapid dilution. Oxidized and reduced glutathione were used during
refolding to enable disulfide bond formation. Conditions such as final
concentration of the recombinant protein after dilution, pH,
temperature, duration of refolding, and concentrations of arginine,
urea, glutathione, and oxidized glutathione were optimized to maximize
yield of soluble PvRII after refolding. Refolded PvRII was purified by
ion-exchange chromatography and gel filtration chromatography as described.
Refolded, purified PvRII was separated by SDS-PAGE and detected by
silver staining (Fig. 1A).
Densitometry scanning of silver-stained gels indicates that the purity
of recombinant PvRII is greater than 98%. Yields of purified, refolded
PvRII are ~2 mg/liter E. coli culture. Recombinant PvRII
migrates with the expected mobility of ~39 kDa.
Biochemical, Biophysical, and Functional Characterization of
Refolded and Purified PvRII--
Refolded and purified PvRII was
characterized using a variety of biophysical, biochemical, and
functional assays. N-terminal sequencing of recombinant PvRII yields
the sequence MDHKKTISSAINHA, which is identical to the expected
sequence. No other sequence is detected. The molecular mass of
recombinant PvRII measured by electron spray ionization mass
spectroscopy is 39,803 Da. The predicted mass of PvRII with a
6-His fusion if all 12 cysteines are disulfide-linked is 39,802 Da.
N-terminal sequencing and mass spectroscopic data confirm the identity
of recombinant PvRII. The mobility of refolded PvRII by gel filtration
chromatography on a Superdex 75 column is consistent with an apparent
molecular mass of ~39 kDa, indicating that purified PvRII does not
form aggregates or multimers (data not shown).
Refolded PvRII migrates slower on SDS-PAGE gels after reduction with
DTT, indicating that disulfide linkages are present in the refolded
protein (Fig. 1B). The homogeneity of refolded PvRII was
analyzed by reverse phase chromatography, a method that can be expected
to separate different conformers of the same protein based on
differences in surface hydrophobicity. Refolded PvRII elutes as a
single symmetric peak by reverse phase chromatography on a C-8 column,
suggesting that the final purified product is homogeneous (Fig.
2). Reduction of refolded PvRII with DTT
results in an increase in elution time by 1 min, confirming the
presence of disulfide linkages in the refolded protein (Fig. 2). Free
thiol content in refolded PvRII was assayed using the method of Ellman (40) to further assess the oxidation state of the refolded protein. Free thiol could be clearly detected at a concentration of 30 µM in this assay. No free thiols were detected in
recombinant PvRII at a concentration of 50 µM.
Considering that PvRII contains 12 cysteines, this indicates that
greater than 95% of cysteines are disulfide linked.
CD spectroscopy was used to probe the secondary structure of refolded
PvRII. The CD spectrum of PvRII shows characteristic
An erythrocyte binding assay was used to test whether refolded PvRII is
functional. Recombinant PvRII was incubated with Duffy-positive and
Duffy-negative human erythrocytes to allow binding. Erythrocytes with
bound protein were collected by centrifugation. Bound protein was
eluted with 300 mM NaCl, separated by SDS-PAGE, and
detected by Western blot. Rabbit serum raised against a 42-amino acid
peptide derived from PvRII was used to detect bound PvRII. Refolded
PvRII binds Duffy-positive human erythrocytes but not Duffy-negative human erythrocytes (Fig. 4A).
Refolded PvRII thus binds erythrocytes with the same specificity as
P. vivax Duffy-binding protein, indicating that it is
correctly folded in its functional conformation. Rabbit serum reacts
with a high molecular weight erythrocyte-derived protein that is
released due to red cell lysis upon the addition of NaCl for elution of
bound protein. Preimmune rabbit serum collected before immunization
with PvRII also reacts with this erythrocyte-derived protein (data not
shown).
What fraction of refolded PvRII is functionally active and correctly
folded? Preabsorption of refolded PvRII with erythrocytes followed by
detection of residual PvRII by immunoprecipitation and Western blotting
was used to detect incorrectly folded PvRII. Approximately 100 ng of
PvRII is clearly detected by this method. Duffy-positive and
Duffy-negative human erythrocytes were used to preabsorb 8 µg of
refolded PvRII. No residual PvRII was detected when Duffy positive
erythrocytes were used for preabsorption (Fig. 4B). On the
other hand Duffy-negative erythrocytes failed to absorb PvRII.
Considering that sensitivity of detection of PvRII is 100, and 8 µg
of refolded PvRII was used, the inability to detect residual protein
after preabsorption indicates that at least 98.75% of refolded PvRII
is removed by preabsorption and is functional.
Immunogenicity of Refolded PvRII and Inhibition of Erythrocyte
Binding to PvRII with Antisera Raised against Refolded
PvRII--
Refolded PvRII formulated in Freund's adjuvant was used to
immunize rabbits. Titers for reactivity of rabbit sera with PvRII were
determined by ELISA. Sera from two immunized rabbits had titers of
1:64,000 and 1:32,000, indicating that PvRII is highly immunogenic.
The ability of rabbit sera to block binding of PvRII to erythrocytes
was tested in an erythrocyte binding assay. COS cells were transfected
with constructs designed to express PvRII fused with the signal
sequence and transmembrane domain of HSV gD at the amino and carboxyl
ends, respectively, to target the P. vivax domain to the
cell surface (20). Monoclonal antibody DL6 directed against HSV gD
sequences in the fusion protein confirmed expression of PvRII on the
COS cell surface (20, 42). Transfection efficiencies were in the range
of 15-20%. Erythrocyte binding assays were performed 48-60 h after
transfection. COS cells were incubated with Duffy-positive human
erythrocytes in the presence of either preimmune serum, serum from a
rabbit immunized with adjuvant alone, or different dilutions of serum
from a rabbit immunized with PvRII. The number of COS cells covered
with rosettes of erythrocytes was scored in 50 fields at a
magnification of 40. Results of one of three similar experiments are
shown in Table I. Rabbit serum raised against refolded PvRII completely blocks binding of erythrocytes to COS
cells expressing PvRII up to a dilution of 1:2500. These data indicate
that refolded PvRII can be used to elicit high titer antibodies that
are capable of inhibiting binding of P. vivax Duffy-binding
protein to erythrocytes.
The functional binding domains of erythrocyte-binding proteins of
Plasmodium sp. map to region II, the conserved, N-terminal, cysteine-rich regions that are also referred to as DBL domains (20,
21). Functional domains of PfEMP-1 that mediate rosetting as well as
binding to endothelial receptors such as ICAM-1, chondroitan sulfate A,
and CD31 have also been mapped to DBL domains (34-39). DBL domains
thus play central roles in two important pathogenic mechanisms, namely
erythrocyte invasion and cytoadherence. To dissect the structural basis
for the interaction of DBL domains with host receptors, it is necessary
to obtain the three-dimensional structures of DBL domains and map
receptor-binding sites within these functional domains. We have
previously shown that binding residues map to a central 170-amino acid
stretch between the fourth and seventh cysteines of PvRII (22). Here,
we describe methods to produce milligram amounts of recombinant PvRII
in its functional conformation and present biochemical, biophysical,
and functional characterization of the recombinant domain.
Recombinant PvRII was expressed in E. coli, purified from
inclusion bodies under denaturing conditions, refolded in
vitro by the method of rapid dilution, and further purified by ion
exchange chromatography and gel filtration chromatography. The final
product was shown to be pure, homogenous, and functional. Purified
PvRII is greater than 98% pure as determined by densitometry scanning of silver-stained SDS-PAGE gels. The mobility of purified PvRII by gel
filtration chromatography is consistent with an apparent molecular
weight of ~39 kDa, indicating that purified PvRII does not form
aggregates or multimers. Moreover, refolded PvRII elutes as a single,
symmetrical peak by reverse phase chromatography, suggesting that it
contains a single, homogeneous population of conformers. Importantly,
refolded PvRII binds Duffy-positive human erythrocytes but not
Duffy-negative human erythrocytes, indicating that it is folded in its
functional conformation. No residual PvRII is detected after
preabsorption of refolded PvRII with Duffy-positive human erythrocytes.
Considering that the detection limit for PvRII is 100 ng, and 8 µg of
refolded PvRII was used, the inability to detect residual PvRII after
preabsorption with Duffy-positive human erythrocytes indicates that
greater than 98.75% of refolded PvRII is functional. The CD spectrum
of refolded PvRII indicates the presence of significant The binding domain of P. vivax Duffy-binding protein, PvRII,
is a promising vaccine candidate since antibodies directed against this
functional domain may block erythrocyte binding and invasion by
P. vivax. Rabbit sera raised against refolded PvRII
recognize PvRII up to dilution of 1:64,000 by ELISA, indicating that
refolded PvRII is highly immunogenic. Sera from immunized rabbits
completely inhibit erythrocyte binding to COS cells expressing PvRII on
the surface up to a dilution of 1:2500. These data indicate that
recombinant PvRII can elicit high titer binding inhibitory antibodies,
providing support for the development of recombinant PvRII as a vaccine for P. vivax malaria. Due to the difficulty in culturing
P. vivax, it is not possible to test antibodies raised
against PvRII for inhibition of erythrocyte invasion by P. vivax. Antibodies raised against the binding domain of P. falciparum EBA-175 have been shown to inhibit erythrocyte invasion
by P. falciparum in vitro (44).2 By analogy, antibodies
directed against PvRII can be expected to effectively block erythrocyte
invasion by P. vivax.
Individuals living in endemic areas develop binding inhibitory
antibodies directed against PvRII after repeated exposure to P. vivax infection (45). However, naturally acquired binding inhibitory antibodies are of low titer. Moreover, human sera from individuals residing in endemic areas completely block binding of
erythrocytes to COS cells expressing PvRII only up to a dilution of
1:10 (45). Here, we have shown that rabbit sera raised against refolded
PvRII inhibit erythrocyte binding to COS cells expressing PvRII up to a
dilution of 1:2500. It remains to be determined if the presence of high
titer antibodies that inhibit binding of P. vivax
Duffy-binding protein to erythrocytes will confer protection against
P. vivax malaria.
Recombinant PvRII has been previously expressed in its functional form
as a secreted protein in insect cells using baculoviral vectors (46).
Recombinant proteins expressed as secreted proteins in eukaryotic cells
are commonly glycosylated, which is often a disadvantage for
crystallographic studies. Previous attempts to express PvRII in its
functional form using bacterial expression systems have been
unsuccessful (11, 46). This report is the first description of methods
developed for refolding and purification of functional PvRII produced
in a bacterial expression system. These methods should be applicable to
DBL domains derived from other erythrocyte-binding proteins and from
extracellular regions of PfEMP-1. Indeed, we have used similar methods
to express, purify, and refold P. falciparum region F2, the
binding domain of P. falciparum EBA-175. Refolded P. falciparum region F2 specifically binds sialic acid residues of
human glycophorin A.2 The availability of methods for large
scale production of correctly folded DBL domains is an important step
for structural studies as well as for malaria vaccine development
efforts based on these functional receptor binding domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
proteins,
which bind Duffy-independent receptors on rhesus erythrocytes, and
P. falciparum sialic acid-binding protein (also known as
EBA-175), which binds sialic acid residues on glycophorin A (4,
14-18). These parasite ligands share similar features and belong to a
family of erythrocyte-binding proteins (19). The extracellular domain
of each erythrocyte-binding protein contains two conserved
cysteine-rich regions, regions II and VI, at the amino and carboxyl
ends, respectively. P. falciparum EBA-175 contains a tandem
duplication (F1 and F2) of the N-terminal, conserved, cysteine-rich
region. The functional receptor binding domain of each
erythrocyte-binding protein lies in region II (20, 21). In the case of
EBA-175, region F2 was found to have receptor binding activity (21).
P. vivax region II
(PvRII)1 specifically binds
the human Duffy antigen, and P. falciparum region F2
specifically binds sialic acid residues of glycophorin A. Region II of
the P. knowlesi Duffy-binding protein binds both human and
rhesus Duffy antigens. P. knowlesi
region II binds sialic acid residues on rhesus erythrocytes, and P. knowlesi
region II binds as yet unidentified receptors on rhesus
erythrocytes (20, 22). These conserved, receptor binding domains are
referred to as Duffy binding-like (DBL) domains after the binding
domain of P. vivax Duffy-binding protein.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactopyranoside to the
culture at a final concentration of 1 mM. Induced cultures
were allowed to grow for 4 h at 37 °C.
b
), did not react
with either anti-Fya or anti-Fyb. Duffy-positive erythrocytes used in
the erythrocyte binding assays had the phenotype Fy(a+b+). Refolded
PvRII was incubated with Duffy-positive and Duffy-negative human
erythrocytes at room temperature for 1 h to allow binding. The
reaction mixture was layered over dibutylpthalate (Sigma) and
centrifuged to collect erythrocytes. Bound protein was eluted from the
erythrocytes with 300 mM NaCl, separated by SDS-PAGE, and
detected by Western blot using rabbit serum raised against a 42-amino
acid peptide derived from PvRII (amino acids 378-420 of P. vivax Duffy-binding protein).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
[in a new window]
Fig. 1.
Refolded and purified recombinant PvRII.
A, silver-stained SDS-PAGE gel of refolded and purified
PvRII. Different quantities (0.6 µg, 1.0 µg, and 2.0 µg) of
refolded and purified PvRII were reduced, denatured, separated by
SDS-PAGE, and detected by silver-staining. B, mobility of
refolded and purified PvRII by SDS-PAGE before and after reduction.
Refolded PvRII has slower mobility by SDS-PAGE after reduction with
dithiothreitol (+DTT), indicating the presence of disulfide
linkages. Molecular mass markers in kDa are shown.
View larger version (31K):
[in a new window]
Fig. 2.
Reverse phase high performance liquid
chromatography profile of refolded and purified PvRII before and after
reduction. Refolded, purified PvRII was analyzed by reverse phase
chromatography on a C8 column. The gradient used for elution was
developed using Buffer A (0.05% trifluoroacetic acid in water) and
Buffer B (0.05% trifluoroacetic acid in 90% acetonitrile, 10%
water). The column was initially equilibrated with 90% Buffer A and
10% Buffer B and reached a composition of 10% Buffer A and 90%
Buffer B in 40 min. Refolded PvRII elutes as a single, symmetric peak,
indicating that it contains a single, homogeneous population of
conformers. Reduction of refolded PvRII results in a shift in elution
time by 1 min. AU, absorbance units.
-helical
signature minima at 208 and 222 nm (Fig.
3). Deconvolution of the CD spectrum by
the method of Bohm et al. (41) indicates the
following distribution of secondary structure components for PvRII:
52.6%
-helices, 9.8%
-sheets, 14.0%
-turns, and 21.8% random-coils. The sum of secondary structural elements calculated from
the CD spectrum totals 98.2%, showing confidence in the measurement and deconvolution. Spectra recorded on three different batches of
refolded PvRII are identical. Denaturation of refolded PvRII with 6 M GdnHCl results in loss of minima at 208 and 222 nm,
indicating disruption of
-helical structures.
View larger version (12K):
[in a new window]
Fig. 3.
CD spectra of refolded and purified
PvRII. CD spectra of recombinant PvRII are shown before
(solid line) and after (broken line) denaturation
with 6 M GdnHCl. Minima near 208 and 222 nm and a maximum
near 190 nm indicate the presence of significant -helical content in
the refolded protein. Denaturation of refolded PvRII with 6 M GuHCl results in loss of minima at 208 and 222 nm.
View larger version (18K):
[in a new window]
Fig. 4.
Erythrocyte binding assay with refolded and
purified PvRII. A, erythrocyte binding assay. Refolded
PvRII was incubated with Duffy-positive (Fy(a+b+)) and Duffy-negative
(Fy(a b
)) erythrocytes to allow binding. Erythrocytes and bound
protein were collected by centrifugation. Bound PvRII was eluted with
300 mM NaCl, separated by SDS-PAGE, and detected by Western
blotting using a rabbit antiserum raised against a 42-amino acid
peptide derived from PvRII. Refolded PvRII specifically binds
Duffy-positive (Fy(a+b+)) but not Duffy-negative ((Fy(a
b
)) human
erythrocytes. B, detection of inactive PvRII by
immunoprecipitation and Western blotting after preabsorption with
Duffy-positive erythrocytes. Residual PvRII was immunoprecipitated,
separated by SDS-PAGE, and detected by Western blot either after
preabsorption with Duffy-positive (Fy(a+b+)) or Duffy-negative
(Fy(a
b
)) human erythrocytes or without preabsorption (
). MW,
Mr.
Inhibition of erythrocyte binding to PvRII expressed on the surface of
COS cells with rabbit serum raised against refolded, purified PvRII
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical
content, as has been predicted for DBL domains (43). It remains to be
experimentally determined if other DBL domains also contain high
-helical content. Importantly, CD spectra recorded for different
batches of refolded PvRII are identical, implying conformational
consistency in the refolded product produced by the method described.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Roselyn Eisenberg and Gary Cohen, University of Pennsylvania, PA for providing plasmid pRE4 and monoclonal antibody DL6, Sachchidanand for peptide synthesis, Dr. Alessandro Vindigni, International Center for Genetic Engineering and Biotechnology (ICGEB), Trieste for N-terminal sequencing, Prof. P. Balaram, Indian Institute of Science, Bangalore for mass spectroscopy, and Drs. Virander Chauhan and Amit Sharma, ICGEB, New Delhi for critical review of this manuscript.
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FOOTNOTES |
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* This investigation received support from the United Nations Developmental Program/World Bank/ World Health Organization Special Program for Research and Training in Tropical Diseases (TDR) and International Research Scholar's Program of Howard Hughes Medical Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M101531200
To whom correspondence should be addressed: Malaria Research
Group, International Center for Genetic Engineering and Biotechnology (ICGEB), P. O. Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India. Tel.: 91 11 618 7695; Fax: 91 11 616 2316; E-mail:
cchitnis@icgeb.res.in
2 K. C. Pandey, S. Singh, and C. E. Chitnis, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: PvRII, P. vivax region II; DBL, Duffy binding-like; PfEMP-1, P. falciparum erythrocyte membrane protein-1; ICAM-1, intercellular adhesion molecule 1; GdnHCl, guanidine hydrochloride; DTT, dithiothreitol; CD, circular dichroism; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; Herpes simplex virus glycoprotein D, VCAM, vascular cell adhesion molecule.
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REFERENCES |
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