From the
The circumsporozoite protein (CSP), a major antigen of
Plasmodium falciparum, was expressed in the slime mold
Dictyostelium discoideum. Fusion of the parasite protein to a
leader peptide derived from Dictyostelium contact site A was
essential for expression. The natural parasite surface antigen,
however, was not detected at the slime mold cell surface as expected
but retained intracellularly. Removal of the last 23 amino acids
resulted in secretion of CSP, suggesting that the C-terminal segment of
the CSP, rather than an ectoplasmic domain, was responsible for
retention. Cell surface expression was obtained when the CSP C-terminal
segment was replaced by the D. discoideum contact site A
glycosyl phosphatidylinositol anchor signal sequence. Mice were
immunized with Dictyostelium cells harboring CSP at their
surface. The raised antibodies recognized two different regions of the
CSP. Anti-sporozoite titers of these sera were equivalent to
anti-peptide titers detected by enzyme-linked immunosorbent assay.
Thus, cell surface targeting of antigens can be obtained in
Dictyostelium, generating sporozoite-like cells having
potentials for vaccination, diagnostic tests, or basic studies
involving parasite cell surface proteins.
The development of safe and effective vaccine is important to
control diseases such as malaria, which kills 1-2 million persons
(mostly children) per year. Vaccination with irradiated Plasmodium sporozoites, the infectious stage inoculated by mosquitoes,
confers protection against malaria infection in mammals (1-4).
This protection is mediated by specific T lymphocytes (helper and/or
cytotoxic) and antibodies directed against the immunodominant repeat
region of the circumsporozoite protein (CSP),(
The first 17 amino acids of the CSP, as
deduced from the cDNA sequence (6), constitute a putative signal
sequence essential for translocation into the parasite endoplasmic
reticulum (ER). Two regions (I and II) conserved in different plasmodia
species
(7) flank a large central repeat domain. Region II is
essential for the binding to hepatocytes (8) and is followed by an
hydrophobic C terminus possibly involved in membrane anchoring. The
Plasmodium falciparum CSP repeat domain, consisting of about
40 Asn-Ala-Asn-Pro (NANP) units, is an immunodominant B cell
epitope
(6, 9) . The repeat domain, together with carrier
molecules, was used as subunit vaccine but conferred only limited
protection
(10, 11) . Elements important for
CD4
Advances in molecular genetics have
allowed to reintroduce in vitro modified genes within D.
discoideum, thus enabling heterologous proteins
expression
(21) . We decided to express the P. falciparum CSP in Dictyostelium due to the resemblance in the high
AT content of the two genomes. We choose two Dictyostelium
discoideum promoters for expressing CSP which are induced at
different developmental stages. The gene encoding the lectin-binding
protein discoidin I is expressed either early after starvation, when
cells have been grown on bacteria
(22) , or during vegetative
growth when cells are grown on a semi-synthetic medium
(HL-5)
(23) . The Dd ras D gene from
Dictyostelium, which encodes a protein resembling mammalian
ras oncogenes
(24) , is not transcribed during growth,
and its expression requires starvation and extracellular cAMP, thus
allowing essentially conditional expression
(25) . In previous
work, we showed CSP accumulation in Dictyostelium using such
promoters
(26) . In this work, we obtained secretion of an almost
complete CSP, as well as cell surface expression of a GPI-modified
version. Finally, we show in this study that cells presenting CSP at
their surface are capable of eliciting antibody response in mice.
For protein
sequencing, 10 liters of cells grown in HL-5 up to 10
To confirm the nature of the
produced polypeptide and exclude cross-reactivity with other epitopes,
we inhibited the binding of the monoclonal antibody Sp3E9 with
synthetic (NANP)
These results demonstrate that leader peptides are
not interchangeable between P. falciparum and D.
discoideum and suggest that the presence of an inadequate leader
peptide dramatically affects the expression of heterologous proteins.
Considering that in other expression systems, CSP was sensitive to
proteolytic degradation, we partially sequenced CSP produced in D.
discoideum. Immunopurified CSP was gel eluted and subjected to
amino acid sequencing. The N terminus of the protein varied between two
independent preparations, but was located within the first 25 amino
acids downstream of the signal peptide cleavage site. This indicates
the presence of exopeptidase activity rather than of a specific
protease-sensitive site in the CSP polypeptide as reported in other
expression systems
(16, 17, 18) . Due to
difficulties encountered during the sequencing of other
Dictyostelium proteins isolated in parallel, we suspect the
presence of a protected N terminus on the completed size CSP.
With the challenging view of developing sporozoite-like cells
with potential as live vaccine or in diagnostic tests, we expressed the
CSP of P. falciparum at the surface of D. discoideum cells. D. discoideum shares an AT-biased preferential
codon usage with numerous mammalian parasites and has a potential in
biotechnology applications
(44) . These reasons prompted us to
define the various requirements for cell surface expression of a
parasite antigen in Dictyostelium and to analyze the immune
response toward this cell surface antigen.
As shown in this study,
expression of CSP was successful only after replacement of its original
signal peptide by a Dictyostelium derived sequence. The CSP
signal sequence seems also non-functional in mammalian cells, since it
had to be replaced by a mammalian leader peptide for expression in
mouse mastocytoma P815 cells
(45) . Although the sequences
required for a peptide to function as a translocation signal have been
well defined (46), it is not clear why some signals are better
recognized and why signal peptides are in many instances not
interchangeable between organisms
(47) .
The CSP produced in
D. discoideum showed an apparent molecular mass of 62 kDa
(CS-49) either by silver staining, Ponceau, or metabolic labeling. The
size of the CSP made in P. falciparum is about 50
kDa
(6) , whereas the expected size based on the gene sequence is
43,340 Da. This difference in migration on SDS gels remains unclear,
since no putative glycosylation sites or phosphorylation sites can be
found on the protein sequence itself. Removal of the potential GPI
anchoring site in CS-150, or GPI anchor addition due to the CsA
sequence in pERIV-CS did not change the size of the protein to a large
extent. These results can be compared to the situation in P.
falciparum, where the apparent molecular weight of CSP is also
larger than expected for still unknown reasons.
Quantitative
estimation by ELISA indicates that CSP may represent between 0.03% and
0.3% of total D. discoideum proteins (data not shown). This is
in the range obtained in yeast cells for the CSP of Plasmodium
knowlesii(50) but is lower than the amount obtained for
CSP produced with baculovirus in insect cells. The level of expression
was increased by the replacement of the stop codon UAG with an ochre
stop codon to facilitate translation termination. Most codons used by
P. falciparum to encode the CSP are also preferred by
Dictyostelium with the exception of the arginine codon (AGG)
and of the UAG and UGA stop codons. Thus, additional changes in other
codons could further improve the yield of the CSP in
Dictyostelium.
CSP with its original C terminus did not
accumulate at the surface of Dictyostelium cells, even though
the protein is present at the surface of sporozoites. Parallel
experiments
(45) showed that a complete CSP with its C-terminal
hydrophobic peptidic segment was not expressed at the surface of
mastocytoma cells either. Instead, it was degraded intracellularly and
specific peptides were presented in association with major
histocompatibility class I molecules. These results are reminiscent of
the intracellular retention of non-processed GPI anchored molecules as
a result of either non-functional signal sequences or the use of GPI
metabolism deficient cell
lines
(40, 41, 42, 43) . Even though no
GPI anchor has been detected on CSP in vivo, the C-terminal
hydrophobic region contains all amino acids required for such
modification
(51, 52, 53, 54, 55) and such post-translational modification has been reported,
although at low level, in COS cells
(19) . Systematic studies on
interspecies requirements are necessary to define the structural
requirements for the GPI anchor addition enzyme.
Dictyostelium cells are able to add GPI anchors, which are either cleavable by
bacterial phosphatidylinositol-specific phospholipase C (e.g. PsA glycoprotein)
(56) or not, due to the presence of a
ceramide within the anchor (e.g. contact site A)
(39) .
Since both GPI-PLC and PLD treatment did not release a soluble form of
CSP, we suspected that the CSP hydrophobic region was unable to
function as GPI addition sequence in D. discoideum.
Accordingly, labeling experiments using either inositol or ethanolamine
(data not shown) failed to reveal any CSP modification. We circumvented
this problem by removing the hydrophobic domain and fusing CSP to a
sequence responsible for the addition of a GPI anchor to the contact
site A protein in Disctyostelium (31). As a result, the
CSP/CsA fusion protein accumulated at the cell surface. Furthermore,
the GPI anchor was then cleavable by GPI-PLD, indicating proper
modification of the CSP/CsA fusion protein expressed in D.
discoideum. Recent evidence
(57) shows that GPI anchoring
tends to stabilize proteins at the surface of Dictyostelium cells. The patchy appearance of CSP by immunofluorescence is
reminiscent of capping processes
(58) . Even though we cannot
fully exclude a staining procedure artifact, the patchy appearance may
indicate a tendency of CSP to aggregate at the cell surface of D.
discoideum, as it does at the parasite surface in P.
falciparum.
Raising parasite-reactive antibodies using
recombinant proteins or peptides as immunogens has been difficult,
possibly due to differences between native and recombinant protein
conformations
(59) . The GPI-anchored CSP at the surface of
Dictyostelium cells adopts a peculiar conformation since the
protein is recognized by anti-NANP and by anti-C terminus but not by
anti-N terminus antibodies raised against synthetic peptides. In
addition, antibodies obtained by immunization with CSP-harboring
Dictyostelium cells reacted with the NANP repeat, but not with
the N-terminal synthetic peptide. Thus, the NANP repeats are most
likely localized at the surface of the CSP produced in D.
discoideum, whereas the N-terminal region could either be
localized internally and/or be non-immunogenic in a H2
In
conclusion, our ability to obtain membrane-bound CSP indicates that
Dictyostelium is a promising system to express a variety of
Plasmodium parasite proteins. The absence of a cell wall, as
well as the relatively simple culture conditions
(28) , renders
D. discoideum suitable for large scale protein preparation of
such immunogenic cell surface antigens. Producing a live vaccine using
Plasmodium protein harboring Dictyostelium cells can
now be envisaged, particularly since Dictyostelium is
non-pathogenic and non-toxic, at least in mice as seen during the
immunizations performed in this study. Furthermore, the low cost of
growing and maintaining this organism may offer economic advantages for
producing membrane-bound recombinant proteins.
Sera were obtained 7 days after a single boost of
BALB/c mice immunized with 2
We thank Dr. G. Gerisch for advice, Dr. U. Certa for
critical reading of the manuscript, Dr. P. Caspers for the NF54 CSP
gene, Dr. A. Noegel for the CsA cDNA clone, Dr. F. Sinigaglia for the
(NANP)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
an abundant surface protein. However, the preparation of
sporozoite-based vaccines has been hampered by the difficulty in
obtaining sufficient amounts of sporozoites. Thus, it is important to
develop heterologous expression systems allowing for large scale
production of properly processed recombinant antigens. In previous
studies, parts of the CSP were produced in various systems and shown to
elicit both antibodies and T cells which mediated partial
protection
(1, 5) . Production of a complete protein, in
either soluble or membrane-bound form, should enhance both immunization
and protection potential.
and CD8
T cell responses have
been mapped outside the repeat region, indicating that the entire CSP
or segments thereof could be used to improve vaccine
efficacy
(1, 5, 12) . Moreover, partial or
complete protection against parasite infection was obtained in mice
injected with an attenuated strain of Salmonella typhimurium expressing the Plasmodium berghei CSP
gene
(13, 14) , with a mixture of mastocytoma P815 cells
transfected with the Plasmodium yoelii CSP, or the sporozoite
surface protein 2
(15) . Full-length or partial P. falciparum recombinant soluble CSP have been produced using different
expression systems, but production of a complete CSP was often limited
by a low level of expression, or endogenous proteolytic
cleavages
(16, 17, 18) . The role of the
hydrophobic CSP C-terminal domain remains unclear, despite its
resemblance to a GPI anchor addition region. No GPI modification of CSP
has yet been described in vivo, even though CSP expressed in
COS cells seems attached to the membrane by a GPI anchor (19). We,
therefore, decided to obtain cell surface expression of CSP and analyze
its immunogenicity in mice.
Chemicals and Cell Cultures
Bacterial media and
current cloning procedures are described
(27) . (NANP) peptide was obtained from Dr. F. Sinigaglia (Hoffman La Roche
Ltd., Basel, Switzerland). Dictyostelium cells were cultured
in shaking suspensions in HL-5 media and starved in PDF
(28) .
Electroporation and selection of expressing cells were done as
described
(21, 29) . For discoidin I promoter-dependent
expression, D. discoideum cells were starved for 4 h in
shaking suspension (160 rpm) in PDF at a density of 5
10
/ml unless otherwise stated
(26) . For ras promoter constructs, the cells were starved for 6 h in PDF at
about 5
10
/ml and transcription was induced by
addition of 200 µM cAMP for 1 h unless otherwise
stated
(25) .
DNA Constructs
To obtain expression of different
forms of CSP in D. discoideum, we used the vectors pVEII
(discoidin I promoter)
(30) and pERI (ras promoter)
(26) . The construction of both pEDII-CS and
pERI-CS based on the CSP gene isolated of P. falciparum NF54
strain has been described earlier
(26) . New constructs are shown
in Fig. 1A. The leader peptide of CSP was replaced by a
leader peptide of D. discoideum, the CsA leader peptide
(31) (Fig. 1B). The natural stop codon of the CSP
was changed to UAA by replacing most of the CSP coding region by a DNA
fragment amplified using specific oligonucleotides carrying adequate
restriction sites. The 3` amplimer contained an in-frame UAA instead of
the UAG creating construct pEDII-CS 49. The secreted form of the CSP
(CSP-150) was obtained by deleting the 69 nucleotides encoding the last
23 amino acids of the C-terminal segment. The construct pEDII-CS 150
was obtained by replacing most of the CSP coding region by an amplified
fragment lacking the C-terminal segment encoding information but
carrying useful restriction fragment for insertion. The hepatocyte
binding sequence (part of conserved region)
(8) is present on
the polypeptide. The last amino acid is now a serine (position 310)
(32) following the fourth cysteine of CSP. To express a
GPI-anchored form, we modified the pERII vector, which contains the
ras promoter, by adding a multiple cloning site, the actin 6
termination sequence, and the actin 15 neo cassette.(
)
A DNA fragment encoding the
last 40 amino acids of the CsA sequence corresponding to the GPI
addition recognition sequence was obtained by polymerase chain reaction
amplification and cloned between the EcoRV and XhoI
in the multiple cloning site of pERII. A DNA fragment comprising the
CSP gene but excluding the N-terminal signal peptide and the C-terminal
hydrophobic encoding segments was amplified and inserted into the
modified pERII vector between the Asp718 and EcoRV
sites, leading to pERIV-CS. The region of fusion between the amino acid
sequence CSP and the CsA C-terminal segment is shown in
Fig. 1C.
Figure 1:
Circumsporozoite protein expression
vectors. A, pEDII is derived from pVEII (30) and contains the
Tn903 neoR gene between an actin 15 promoter and termination
sequences, allowing G418 selection in D. discoideum (dottedline). CSP lacking its first 18 amino
acids was fused in-frame to the CsA leader peptide (see panelB) leading to pEDII-CS 2. Replacement of the original
P. falciparum UAG to UAA resulted in construct pEDII-CS 49.
Removal of the last 23 amino acids comprising the hydrophobic
C-terminal peptidic region lead to pEDII-CS 150. pERIV-CS was derived
from pERII (see ``Materials and Methods''), which contains
the Tn5 neoR gene between actin 15 promoter and termination
sequences. The CSP and CsA signal peptides were placed behind the
D. discoideum ras promoter fragment 3.1 (25). The GPI
anchoring domain of the CsA protein was placed in frame at the C
terminus of CSP (see panelC). L, CsA leader
peptide; CSP, P. falciparum circumsporozoite surface
antigen; DiscoidinI and ras, sequences
promoting transcription in Dictyostelium. Arrows indicate transcription start sites. I, II, and
III indicate highly conserved domains of the CSP (6).
B, the discoidin I promoter in the pEDII series was fused to a
D. discoideum contact site A (CsA) synthetic leader
peptide keeping the original discoidin I AUG (M). This
sequence differs from the original sequence (31, 49) by the replacement
of the lysine in position 2 by serine and arginine. An asterisk indicates the probable cleavage site of the CsA leader peptide.
The amino acids of the P. falciparum CSP are
underlined. C, in pERIV-CS, the CSP hydrophobic
domain was replaced by the last 48 amino acids of the CsA protein. An
extra proline (P) was added during cloning between the CSP
(underlined) and CsA sequences.
Protein Analysis
The proteins from 2
10
cells (per 4-mm-wide slot), boiled in 1
Laemmli
buffer for 5 min, were separated by 10% SDS-PAGE
(27) . Proteins
were electrotransferred onto nitrocellulose (immunoblots). Fifty
µg/ml anti-NANP mAb (Sp3E9) (33) was added to the filter and
incubated overnight at room temperature.
I-Protein A or
alkaline phosphatase-conjugated protein A and chemiluminescence
reaction (Amersham Corp.) were used to reveal anti-NANP binding.
Partially purified CSP was obtained from Triton X-114-soluble proteins
(34) loaded on an anti-NANP affinity column
(26) . In
purity assays, CSP synthesizing D. discoideum cells were
pulse-labeled by [
S]methionine for 2 h after an
initial starvation period of 2 h
(35) .
/ml
were starved for 4 h, lysed in 200 ml of Triton X-114
(34) , and
phase separated. Four volumes of a solution containing 25 mM
Tris-HCl, pH 8.2, 50 mM NaCl, 0.5% Nonidet P-40, and 0.5%
deoxycholic acid were added to the detergent fraction, and the samples
were passed over an anti-NANP immunoaffinity column. The material was
eluted in glycine, pH 2.5, and lyophilized. The sample was loaded on a
10% SDS-PAGE and, after Coomassie staining, the 62-kDa band was eluted
from the gel using an elution device from Life Technologies Inc. The
eluted material was concentrated using a Centricon filter, lyophilized,
and resuspended for amino acid sequencing.
FACS Analysis
Fluorescence-activated cell sorter
(FACS) analysis was performed on approximately 1 10
pERIV-CS and control Ax2 cells. After starvation and cAMP
induction, the cells were centrifuged at about 1,000
g and resuspended in 200 µl of medium containing 5% fetal calf
serum. Cell suspensions were incubated for 45 min at 4 °C with a
1:500 dilution of Sp3E9. Cells were washed by centrifugation through a
cushion of fetal calf serum (FCS), resuspended in 110 µl of medium
containing 5% FCS and 10 µl of commercial biotin-conjugated donkey
anti-rabbit IgG (Amersham). After a 45-min incubation at 4 °C, the
cells were washed and resuspended in 100 µl of medium containing 5%
FCS. Finally, cells were incubated for 1 h at 4 °C with 10 µl
of fluorescein-conjugated streptavidin (Amersham), washed, and analyzed
on a fluorescence-activated cell sorter (FACS II System, Becton
Dickinson).
GPI-Phospholipase D Assay
The GPI-PLD sensitivity
of GPI-anchored CSP was tested by lysing pERIV-CS cells in 20
mM Tris/HCl, pH 7.5, 0.1 M CaCl, 0.008%
Triton X-100 by four cycles of freezing and thawing. One or 5 units of
GPI-PLD enzyme (Boehringer Mannheim) was added and the extracts
incubated for 1 h at 37 °C. Triton X-114 in 1
Tris-buffered
saline containing 1 mM EDTA was then added to a final
concentration of 1%, and the aqueous and detergent phases were
separated. The samples were resolved on a 10% SDS-polyacrylamide gel
and analyzed by immunoblotting using the Sp3E9 monoclonal antibody as
described previously.
Immunofluorescence Staining
5 10
cAMP-induced starved pERIV-CS cells were preincubated for 5 min
in 200 µl of 1% skim milk in 1
PBS and kept at 4 °C.
After centrifugation at 1,000
g for 1 min., the cells
were resuspended in 200 µl of 50 µg/ml Sp3E9 antibody in 1%
skim milk and incubated for 30 min. After centrifugation, the cells
were rinsed three times in 1
PBS. The cells were then
resuspended in 200 µl of fluorescein isothiocyanate-conjugated
anti-mouse antibody (diluted 1:40) (Nordic, DAKO Immunoglobulin) and
incubated for 30 min. After three washes in 1
PBS, the cells
were mounted for microscopy and observed under a Leitz microscope.
ELISA
Serum and monoclonal antibodies produced
against the N-terminal (amino acids 22-125), the NANP repeat
peptide, or the C-terminal segment peptides were assayed by ELISA.
Briefly, vinyl plates were coated with different peptides, washed, and
blocked with 1% bovine serum albumin in PBS. Monoclonal or serum
antibodies were serially diluted in 1% bovine serum albumin/PBS
containing 0.05% Tween 20. Diluted sera were added to antigen-coated
wells and incubated for 1 h at room temperature. Plates were washed
with PBS with 0.05% Tween 20, and an appropriate dilution of
peroxidase-conjugated, species-specific anti-IgG was added and
incubated for 1 h at room temperature. One hundred microliters of
peroxidase substrate solution were added to each well, and the
A was determined. The end point of ELISA titers
for the mice sera was designated to be the serum dilution producing an
absorbance value 2 S.D. greater than the average of the control mice.
Immunization of Animals and Analysis of the
Antisera
Two 25 µl or 1
50 µl of a 1:1
sonicated mixture of incomplete Freund's adjuvant and 2
10
cells were injected into BALB/c mice either
subcutanously or intraperitonally, respectively. After 4 weeks, a boost
was performed with an equivalent material, and sera were collected 10
days afterward and analyzed by ELISA. The same antisera were analyzed
by immunofluorescence on air-dried, unfixed sporozoites of isolates
NF54 using fluorescein-labeled rabbit anti-mouse IgG as second
antibody
(36) .
A Dictyostelium Leader Peptide Is Required for CSP
Expression
The genome of the P. falciparum malaria
parasite is particularly rich in adenosine and thymidine resulting in a
biased codon usage, reminiscent of the slime mold D.
discoideum(37) . Thus, we expressed the Plasmodium CSP gene in D. discoideum under the discoidin I promoter,
which was selected to take advantage of its high level of expression
both during vegetative growth and early starvation
(23) . When
merging the complete CSP coding region with the discoidin I promoter up
to the AUG (pEDI-CS, Fig. 1), no recombinant protein was
detectable, even though we detected CSP mRNA in the transfected cells
(data not shown). We questioned the ability of the first 16 amino acids
of the CSP to allow translocation through the ER in D.
discoideum, since alterations of leader peptide sequences have
been shown to affect dramatically the level of protein synthesis in
other systems
(38) . We thus replaced the CSP leader peptide
encoding region by a synthetic oligonucleotide sequence based on the
D. discoideum contact site A leader peptide
(31) (pEDII-CS, Fig. 1B). Fig. 2A shows the detection of CSP using a monoclonal antibody (Sp3E9)
against the NANP repeat motif. A 62-kDa protein was detected in
pEDII-CS cells (Fig. 2A, lanec) but
not in cells containing either the vector pVEII alone
(Fig. 2A, lanea) or pEDI-CS
(Fig. 2A, laneb).
Figure 2:
Immunoblot analysis of CSP produced in D.
discoideum cells. A, cells were transformed with the
expression vector pVEII alone (P1, lane a), the
expression vector pEDI-CS (C1B, laneb), and
the expression vector with the contact site A leader peptide pEDII-CS
(CSP, lanec). Stably transformed cells were
harvested after 4 h of starvation to ensure optimal transcription from
the discoidin I promoter and CSP was revealed by immunoblotting using
(NANP) monoclonal antibody Sp3E9. The apparent molecular
mass of the CSP (62 kDa) was estimated using molecular mass standards.
B, proteins from pVEII-CS cells were analyzed as in
A, except that the membrane strips were probed with an
anti-NANP monoclonal antibody preincubated for 10 min at room
temperature in presence of (NANP)
peptide. Numbers at bottom of the figure represent molar ratios of (NANP)
to mAb (e.g.1:1 in lanea).
Lanee, no (NANP)
peptide added.
Molecular mass standards were as follows: phosphorylase b (97
kDa), bovine serum albumin (66 kDa), egg white ovalbumin (42.7
kDa).
Although both
D. discoideum and P. falciparum share an AT-biased
preferential codon usage, the CSP stop codon is UAG, whereas UAA is
used in the vast majority of the Dictyostelium genes. We
replaced the UAG stop codon of the CSP gene by a UAA codon (pEDII-CS
49), which resulted in enhanced CSP accumulation (data not shown). This
strain was used in further studies.
peptide. A hundredth equimolar amount of
(NANP)
peptide was sufficient to observe a significant
reduction in the binding of the monoclonal antibody to the 62-kDa CSP
(Fig. 2B, lane c), whereas complete inhibition
was obtained with a tenth equimolar amount (Fig. 2B,
lane b).
Integrity of the CSP Expressed in Dictyostelium
As
a first purification step, we took advantage of the presence of a
C-terminal hydrophobic segment
(32) , which should allow the
partitioning of the protein in Triton X-114. Indeed, most of the newly
synthesized CSP accumulated in the detergent phase (data not shown). To
monitor the subsequent purification steps, cells were pulse-labeled
with [S]methionine
(35) . Triton
X-114-soluble proteins were loaded on an anti-NANP affinity column.
After elution a major [
S]methionine-labeled
protein with an apparent molecular mass of 62 kDa was detected by
fluorography only in CSP-synthesizing cells (data not shown).
Secretion of CSP into the Medium
The presence of
the leader peptide, as well as of the C-terminal segment of CSP
resembling GPI anchor recognition sequences should have resulted in
cell surface expression of modified CSP. However, we only detected CSP
within the D. discoideum cells, and not at the cell surface or
secreted in the medium (data not shown). We treated pEDII-CS 49
extracts with GPI-PLD (Fig. 3A) to cleave the lipid
moiety of the GPI anchors. We used GPI-PLD instead of GPI-PLC, since
the GPI anchor of contact site A contains a ceramide in its lipid
moiety
(39) , which prevents its cleavage by most PLCs. Removal
of the lipidic portion of the anchor should have altered its
partitioning in Triton X-114. No change in partitioning could be
observed upon GPI-PLD treatment (Fig. 3A), indicating
that CS-49 is probably not modified by a GPI in D. discoideum.
Figure 3:
Intracellular and secreted forms of CSP.
A, cells producing CSP-49 (49) or CSP-150
(150), the protein encoded by pEDII-CS 150 (Fig. 1A),
were lysed in 1% Triton X-114 and subjected to the GPI-PLD present in
human serum (lanes +) or mock-incubated (lanes -) for 5 min at 30 °C in presence of protease
inhibitors. The samples were adjusted to 2% Triton X-114 and phase
separated. Proteins partitioning into the detergent (TX) or
aqueous (Aq) phases were separated by SDS-PAGE, blotted, and
reacted with the Sp3E9 antibody. B, cells producing CSP-49
(49) or CSP-150 (150) were centrifuged at 10,000
g and lysed, and then proteins were separated by
SDS-PAGE (about 2
10
cells/lane). An equivalent
amount of medium supernatant was precipitated by adding four volumes of
acetone. After centrifugation at 10,000
g, the
pelleted proteins were separated by SDS-PAGE electrophoresis (medium).
After transfer to nitrocellulose, multiple strips were cut out of each
lane and the CSP was revealed using the Sp3E9
antibody.
The non-processing of GPI anchors resulted in intracellular
retention in other
systems
(40, 41, 42, 43) . We thus
deleted the C-terminal 23 amino acids of CSP encompassing the putative
GPI anchoring sequence, leaving the last cysteine residue (residue 309)
followed by a serine to avoid possible misfolding. The modified coding
region was inserted in pVEII and the resulting plasmid (construct
pEDII-CS 150) introduced into D. discoideum cells. The CS-150
protein, on the contrary to CS-49, was detected solely in the aqueous
phase after Triton X-114 partitioning (Fig. 3A). GPI-PLD
treatment had no effect on the protein, as expected. Furthermore, the
CS-150 protein was secreted into the culture medium as seen by
immunoblotting after acetone precipitation (Fig. 3B). It
should be noted that we were unable to precipitate CS-150 from the
medium using trichloroacetic acid (10%), indicating the extreme acid
solubility of the CSP (data not shown).
Expression of CSP on the Dictyostelium Cell
Surface
For the development of a live vaccine, diagnostic test,
or hepatocyte binding studies, we wished to express CSP on the surface
of D. discoideum. We thus replaced the CSP C-terminal
hydrophobic segment by the last 49 amino acids of the contact site A
(CsA) polypeptide containing a GPI-anchoring domain
(31) . To
obtain inducible expression and to avoid possible toxicity problem, we
inserted the CSP/CsA fusion gene under the control of the ras promoter (25, 26). The construct was introduced into D.
discoideum cells (pERIV-CS) and CSP surface expression was
analyzed by immunofluorescence using anti-CSP antibodies
(Fig. 4). ras promoter expression is induced by addition
of external cAMP in only 20-40% of the cells, which are those
differentiating into prestalk cells
(24) . Consequently, we
observed CSP/CsA in a restricted number of cells (Fig. 4). The
staining was clearly restricted to the cell periphery (arrow)
and the punctuated appearance indicated the presence of patches of CSP
on the cell surface (arrowhead) as seen in the sporozoite of
P. falciparum.
Figure 4:
Cell surface expression CSP in
Dictyostelium. pERIV-CS cells were starved for 6 h and induced
for 1 h with 200 µM cAMP. The cells were reacted with
anti-(NANP) antibody, followed by fluorescein-conjugated
anti-mouse IgG at 4 °C to prevent phagocytosis and pinocytosis. A
restricted number of cells are stained, corresponding to the prestalk
cells. Prespore cells can be considered as negative control.
A, phase contrast. B, fluorescence. The bar represents 50 µm.
This result was confirmed by flow cytometry
using different antibodies. First, we used the mAb Sp3E9 directed
against the NANP epitope (Fig. 5A). Cells transformed
with the vector alone (P1) showed no fluorescence over background,
whereas pERIV-CS cells showed a second fluorescence peak corresponding
to about 40% of the cells (Fig. 5C). When cAMP addition
was omitted, the second peak mostly disappeared, indicating that the
expression of CSP was cAMP-inducible. In contrast, no signal could be
detected with a mouse antiserum directed against a peptide of the N
terminus of CSP corresponding to amino acids 22-125 (data not
shown). To exclude that the absence of recognition was due to a
proteolytic cleavage of the CSP N terminus, we performed immunoblots.
As seen in Fig. 6A, a 69-kDa CSP was detected in
pERIV-CS cells by human serum of infected patients (B10), and mouse
antisera raised against peptide 22-125 or peptide 289-390.
This result suggests that the CSP at the surface of D. discoideum cells adopts a configuration that influences its recognition by
specific antibodies.
Figure 5:
Surface fluorescence labeling. Labeled
cells were analyzed using a fluorescence activated cell sorter.
Fluorescence intensity in arbitrary units (x axis) is
represented against the number of cells (y axis). PanelA corresponds to pVEII-transfected cells (P1)
after cAMP induction, panelB to CSP-expressing cells
grown in absence of cAMP, and panelC to
CSP-expressing cells in presence of cAMP.
Figure 6:
Integrity and phospholipid modification of
CSP expressed at the surface of D. discoideum cells.
A, total cell proteins were analyzed as described in Fig. 2,
except that the human antiserum (B10), a mouse antiserum
against residues 22-125 of CSP, or a mouse antiserum against
residues 289-390 was used instead of anti-NANP antibody.
B, proteins extracted from about 2 10
cells were treated by GPI-PLD with 1 (lanes1)
or 5 units for 1 h (lanes5) and analyzed as
described in Fig. 3A. Aq, aqueous phase; Tx,
Triton X-114 detergent phase. 0, samples not treated by
GPI-PLD.
Finally we asked how the CSP might be anchored
in the membrane. The CSP/CsA protein has the amphiphilic character
expected for a GPI-anchored protein
(34, 40) since it
partitions in the Triton X-114 detergent phase
(Fig. 6B). To confirm the presence of a GPI anchor on
the CSP/CsA fusion protein, we lysed the cells by three cycles of
freezing and thawing in 0.008% Triton X-100 and treated the cell
lysates for 1 h with 1 or 5 units of with GPI-PLD. Removal of the
lipidic portion of the CSP/CsA with 5 units of GPI-PLD altered its
hydrophobic character and provoked its partitioning into the aqueous
phase (Fig. 6B, lane5). Incubation
with 1 unit had a reduced effect (Fig. 6B, lane1 and data not shown). These results show the presence of
GPI anchored CSP at the surface of Dictyostelium cells, thus
indicating that D. discoideum can produce, transport, and
process heterologous parasite proteins.
Immune Response to CSP Harboring Dictyostelium
Cells
To assess the ability of the GPI-modified CSP to induce an
immune response, BALB/c mice were immunized subcutanously or
intraperitonally with 2 10
whole cells mixed with
incomplete Freund's adjuvant. Ten days after a second injection,
the humoral immune response was analyzed by ELISA against synthetic
peptides from different regions of the CSP (). Antibodies
were detected against the immunodominant NANP repeat region, against
the C-terminal non-repetitive region (amino acids 289-390), but
not against the N-terminal 22-125 synthetic peptide. Antibody
titers did not vary as a result of the route of injection
(), and no specific antibodies were detected following
injection of live cells in absence of adjuvant (data not shown). Six of
these antisera raised against CSP produced in Dictyostelium were further assayed for recognition of P. falciparum air-dried, non-fixed sporozoites. All mice sera positive for
D. discoideum CSP were found to bind to the sporozoite as
scored by immunofluorescence using fluorescein isothiocyanate-linked
secondary anti-mouse IgG antibody. Furthermore, the limiting dilution
for air-dried sporozoites resembled that measured using synthetic
peptides in an ELISA () with a maximum of 1/5,000 for one
serum.
haplotype host. Additional experimental evidence is necessary to
define if the protein produced in D. discoideum shows the same
conformation as its native P. falciparum counterpart.
Table: ELISA and immunofluorescence assay (IFA)
reactivity of sera of pERIV-CS immunized mice with
(NANP), M1(289-390) peptides, and air-dried
fixed sporozoites
10
cells.
peptide, Dr. L. Kühn for synthesizing
oligonucleotides, M. Rousseaux and S. Pinaud for technical assistance,
S. Frütiger for amino acid sequencing, P. Zaech and C. Knabenhans
for FACS analysis, and P. Dubied and B. Allegrini for photographic
work.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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Molecular and Cellular Proteomics
Journal of Lipid Research
Biochemistry and Molecular Biology Education