From the Department of Molecular Biology and
Biochemistry, University of California, Irvine, California 92697, the
§ Institute of Pathology, Case Western Reserve University,
Cleveland, Ohio 44106, the ¶ Diversa Corp., San Diego, California
92121, the
Naval Medical Research Center, Silver Springs,
Maryland 20910, and the ** Department of Microbiology and Immunology,
Albert Einstein College of Medicine, Bronx, New York 10461
Received for publication, April 16, 2001, and in revised form, May 11, 2001
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ABSTRACT |
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A novel Plasmodium falciparum gene,
MB2, was identified by screening a sporozoite cDNA
library with the serum of a human volunteer protected experimentally by
the bites of P. falciparum-infected and irradiated
mosquitoes. The single-exon, single-copy MB2 gene is
predicted to encode a protein with an Mr of
187,000. The MB2 protein has an amino-terminal basic
domain, a central acidic domain, and a carboxyl-terminal domain with
similarity to the GTP-binding domain of the prokaryotic translation
initiation factor 2. MB2 is expressed in sporozoites, the
liver, and blood-stage parasites and gametocytes. The MB2
protein is distributed as a ~120-kDa moiety on the surface of
sporozoites and is imported into the nucleus of blood-stage parasites
as a ~66-kDa species. Proteolytic processing is favored as the
mechanism regulating the distinct subcellular localization of the
MB2 protein. This differential localization provides
multiple opportunities to exploit the MB2 gene product as a
vaccine or therapeutic target.
Plasmodium falciparum is the most virulent etiological
agent of human malaria, responsible for over 90% of mortality due to the disease. Each year 300-500 million people are infected by malaria
parasites, and this results in 1.5-3 million deaths (1). Efforts to
eradicate malaria generally have failed, and currently the disease is
endemic in more than 90 countries throughout the tropics. Widespread
and increasing drug and insecticide resistance have exacerbated the
situation, undermining the effectiveness of existing malaria control
methods that depend on chemotherapy and vector control, respectively.
Novel means to fight the disease are needed urgently, and a vaccine is
predicted to have the greatest impact in addition to being the most
cost-effective control measure (2).
Experimental support for the development of a vaccine for human malaria
was provided first by the use of radiation-attenuated sporozoites as
immunogens (3). The success of this experimental vaccination provided
the impetus for the search for mechanisms of protective immune
responses and the target antigens involved. The circumsporozoite (CS)
protein was identified as the major surface antigen of
Plasmodium sporozoites (4-6). The CS protein has been a
leading vaccine candidate antigen because irradiated sporozoite-induced, protected human volunteers have high titers of
anti-CS1 antibodies (7), and
CS-specific monoclonal antibodies and cytotoxic T-lymphocytes could
adoptively transfer protection in a rodent malaria model system (8).
However, attempts to induce protection in humans using P. falciparum CS-based vaccines, despite recent improvement in their
immunogenicity, have repeatedly yielded only partial success
(9-13).
The inability to develop a vaccine based on the CS protein was
interpreted to indicate that additional antigens play a role in
irradiated sporozoite-mediated protection against infection (14). It
then becomes important to identify antigens that may act independently,
additively or synergistically with the CS protein in the development of
a multicomponent vaccine. Here we report the characterization of
MB2, a novel gene encoding a P. falciparum sporozoite surface antigen identified by screening a CS-depleted sporozoite expression cDNA library with serum from a human
volunteer protected by the bites of P. falciparum-infected
and irradiated mosquitoes. The MB2 gene is expressed in
sporozoites, the exoerythrocytic stages, the asexual blood stages, and
gametocytes, but the gene product is localized differentially in these
developmental stages. This differential localization provides multiple
opportunities to exploit the MB2 gene product as a vaccine
and drug target.
Library Construction--
A CS-depleted sporozoite
cDNA library was constructed from a P. falciparum
salivary gland sporozoite cDNA library (strain NF54; Ref. 15) using
a hydroxyapatite column-based subtractive hybridization technique (16).
Briefly, to prepare the target cDNA sense-strands, DNA from the
unsubtracted library was linearized with NotI and used as a
template to transcribe antisense cRNAs with T7 RNA polymerase
(Megascript, Ambion). Template DNA was removed by DNase treatment and
the antisense cRNA strands were used to generate cDNA sense strands
in a reaction using SuperScript (Life Technologies, Inc.) reverse
transcriptase. To prepare the driver cRNA, a CS clone, G89 (17), was
linearized with NotI. Digestion products were used to
generate antisense CS cRNA with T7 RNA polymerase (Megascript, Ambion).
The target cDNA sense strands were allowed to reassociate
with a 50-fold excess of the driver cRNA antisense strands. The reassociation mix was loaded onto a hydroxyapatite column and non-duplex, single-stranded target cDNA was separated from duplex cDNA/cRNA by elution with a high molarity phosphate buffer. Primers specific for the UniZap Library Screening--
Phage were plated and lifted onto
nitrocellulose membranes that were soaked in 10 mM
isopropylthio- DNA Sequencing of MB2--
The primary nucleotide sequences of
all clones were determined by the dideoxynucleotide chain termination
method (19) using a 33P nucleotide terminator kit (Amersham
Pharmacia Biotech). Specific oligonucleotide primers for sequencing
were made by Heligen Laboratories (Huntington Beach, CA). Contiguity of
clones was verified by gene amplification of genomic DNA using the
following primers: a, 5'-GGTGATGACATTGAAGATATGAATG-3'; b,
5'-CAATAGAATAGATATAATCACC; c, 5'-CTGGGTCATCATATGGAAAAGTG-3'; and d,
5'-CAATACACCCTGCAACCTTTCC-3'.
Southern and Northern Analyses--
P.
falciparum genomic DNA was isolated using a
phenol/chloroform-based procedure (18) from blood-stage parasites
(strain FCR3) cultured in vitro. The DNA was digested with
various restriction endonucleases, and Southern blots were prepared as
described (18). The probe for Southern blot analyses was prepared by
labeling the sporozoite cDNA clone, spz-MB2, with
radioactive [32P]ATP using the Megaprime DNA system
(Amersham Pharmacia Biotech). Total RNA was isolated from blood-stage
parasites of the same strain cultured in vitro using the
Trizol® reagent (Life Technologies, Inc.). 15-20 µg of
total RNA were electrophoresed and Northern blots were prepared as
described (18). Two 32P-labeled probes consisting of
nucleotides 1-580 and 2393-2836 of the coding sequence of
MB2 were used separately on filters to which RNA from
blood-stage parasites had been transferred.
Recombinant Protein Expression and Purification--
Segments of
the MB2 open reading frame (ORF) were expressed in bacteria as
GST-MB2-6xHis fusion proteins from the dual-affinity expression
vector, pAK1-6H (20). NcoI and SmaI
cloning sites were created for each insert by amplifying NF54 strain
genomic DNA. The primer pair, 5'-GATGCCATGGAATATAATAGAATATGCTCA-3' and 5'-GATCCCGGGTTTTTATTATTAGAAGAATCA-3', was used to amplify a sequence that encodes a peptide, designated MB2-B, that overlaps amino acids
(aa) 95-206. The primer pair, 5'-GATGCCATGGATTCTTCTAATAATAAAAAT-3' and
5'-ATGCATCCCCGGGTCATTTTTTATTTGAAGAATTCTC-3', was used to amplify a
sequence that encodes a second peptide, designated MB2-C, that overlaps
aa 200-316. The primer pair,
5'-GTATGCCATGGTCCACGAAAATAAAGAATATAATTCAAG-3' and
5'-GATCCCGGGTCATCGAGCGATTCATTTTGGTC-3', was used to amplify a sequence
encoding the peptide, MB2-FA, that overlaps aa 764-945. Finally, the
primer pair, 5'-GATGCCATGGATGG TAATAGAACAAATAATGAC-3' and
5'-GATCCCGGGTACGCTTCGATTATATCGTTTGGCTC-3', was used to amplify a
sequence that encodes the peptide, MB2-IF2, that overlaps aa 1337-1606. The amplification products were digested with
NcoI and SmaI and ligated into pAK1-6H. The
ligation mixture was used to transform Escherichia coli
DH10B, and transformants were selected. Bacterial cells were grown at
37 °C in SuperBroth (Life Technologies, Inc.) to an
A600 = 0.6 and induced in 1 mM final
concentration of isopropylthio- Rabbit Immunization--
400 µg of purified recombinant
protein were injected subcutaneously into a rabbit four times at 2-week
intervals. Ten days following the last injection, high titer sera were
obtained from the rabbit. The sera were depleted of anti-GST antibodies
by chromatography on GST-bound nickel columns.
Immunoelectron Microscopy--
P. falciparum
parasites and parasite-infected cells or tissues were fixed for 30 min
at 4 °C with 1% formaldehyde, 0.1% glutaraldehyde in a 0.1 M phosphate buffer, pH = 7.4. Fixed samples were
washed, dehydrated and embedded in LR White resin (Polysciences, Inc.). Thin sections (70-80 nm) were blocked in a phosphate buffer containing 5% w/v nonfat dry milk and 0.01% v/v Tween 20 (21). Grids were incubated at 4 °C overnight in solutions containing variable
concentrations of rabbit antiserum reactive to domain-specific
recombinant proteins diluted in the blocking buffer. Pre-immune sera
were used as negative controls. After washing, grids were incubated for
1 h in 15 nm gold-conjugated goat anti-rabbit IgG (Amersham
Pharmacia Biotech) diluted 1:40 in phosphate buffer containing 1%
bovine serum albumin and 0.01% Tween 20. Following the 1-h incubation,
grids were rinsed with phosphate buffer containing 1% bovine serum
albumin and 0.01% Tween 20 and fixed with glutaraldehyde to stabilize
the gold particles. Samples were stained with uranyl acetate and lead
citrate and examined by electron microscopy.
Immunoblot Analysis--
Protein extracts from parasites were
prepared by boiling them in sample buffer for 10 min (22). For the
sporozoite stage, parasites were isolated from dissected salivary
glands of infected mosquitoes. For the asexual blood stages, parasites
were obtained from a saponin lysis of infected red blood cells grown in
culture (23). Protein extracts were fractionated on 8%
SDS-polyacrylamide gel electrophoresis and transferred onto
nitrocellulose membranes. The membranes were incubated in rabbit
antiserum diluted 1:500 for 1 h. Horseradish peroxidase-conjugated
anti-rabbit IgG was used to detect positive signals using the ECL kit
(Amersham Pharmacia Biotech). Preimmune sera and lysates from
uninfected human red blood cells were used as negative controls.
Library Construction--
To evaluate the efficiency of CS
depletion in the subtracted cDNA library, 100 ng of the recovered
single-stranded cDNA were amplified with oligonucleotide primers
complementary to the cloning vector to generate a heterogeneous mixture
of fragments of 200-1000 bp in size. The products were amplified again
with gene-specific primers. Although two marker genes, TRAP
(24, 25) and P2 (26), were detected, no signal corresponding
to the CS gene was amplified, indicating that the subtraction technique
was highly efficient (data not shown). The amplification products were
subcloned into the UniZap vector and packaged in Library Screening, Southern and Northern Analyses of MB2--
A
screening of 1 × 104 primary phage with the human
volunteer serum led to the selection of 18 candidate phage clones.
These were rescreened, and 12 phage again were reactive for antibodies. Southern analyses showed that 1 of the 12 secondary clones hybridized specifically to P. falciparum genomic DNA and showed
patterns of hybridization consistent with a single-copy gene (Fig.
1A). This 496-bp sporozoite
cDNA clone was designated spz-MB2 and was selected for
further characterization. Northern analyses of RNA isolated from
blood-stage parasites cultured in vitro and hybridized with
probes derived from both the 5' and 3' ends of the complete ORF
(described below) produced a single positive signal at ~7.5 kilobases
(Fig. 1B).
Sequence Analysis of the MB2 cDNA and Gene--
A comparison
of the size of the spz-MB2 cDNA with the mRNA
detected in the Northern analyses indicates that it is not a
full-length cDNA. Furthermore, primary sequencing of
spz-MB2 showed that it lacked a translation termination
codon and represented an incomplete ORF. Sequence complementary to
MB2 was detected in an asexual blood-stage cDNA library
using specific gene amplification primers, and therefore the library
was screened with the spz-MB2 cDNA. Two overlapping
blood-stage cDNAs, c3-1-18 and c18-4-23,
were identified (Fig. 2A).
Nucleotide sequence analysis revealed that the reading frame of
spz-MB2 was contained entirely within a contig formed by
these two cDNAs. The c3-1-18 clone contained a putative translation initiation codon and a 435-bp 5' end untranslated region
(UTR). The 3' end termini of the c3-1-18 and
c18-4-23 cDNAs each have what appear to be
polyadenylation (poly(A)) sequences characteristic of the 3' end
termini of processed mRNAs. However, there were no translation
termination codons located to the 5' end of the poly (A) tracks in
either of the cDNAs, and the overlap of c3-1-18 with
c18-4-23 revealed that the 17 terminal A nucleotides in
c3-1-18 comprise an internal A-rich nucleotide stretch in
c18-4-23. Therefore, we concluded that the oligo(dT) primed
the mRNA for cDNA synthesis from within the coding region.
To obtain additional 3' end sequence of MB2, a
Sau3AI genomic library (strain ITO) was screened using as a
probe the 400 nucleotides at the 3' end of c18-4-23. A
genomic clone, g2-4-4#5, was identified having overlapping
and contiguous sequence with c18-4-23. The sequence of
g2-4-4#5 confirmed that the 17-A region at the 3' end of
c18-4-23 is an internal A-rich nucleotide track, supporting the conclusion that these A-rich internal nucleotide tracks were primed
by oligo(dT). To obtain additional 3' end cDNA sequence, the 600 nucleotides at the 3' end of g2-4-4#5 were used to screen the blood-stage cDNA library, resulting in the identification of
the cDNA clone, c3-4-29. Sequencing of
c3-4-29 revealed that it was contiguous with
c18-4-23. In addition, there are three stop codons at the
3' end of c-3-4-29, commencing at nucleotides 5266, 5272, and 5293, and there is a putative poly(A) region near the 3' end of the
last stop codon. The positions of the stop codons and the authenticity
of the poly(A) of the MB2 cDNA were supported by the
genomic clone, g6-2-2, identified by screening the genomic library with a probe derived from the 3' end of c3-4-29.
Similarly, the 5' end UTR and the start codon of MB2 also
were verified by the clone g2-6-8, isolated from the
genomic library using a probe derived from the 5' end of
c3-1-18. The overlapping primary sequences of the three
blood-stage cDNA clones and the contiguity of their reading frames
allowed us to assemble a complete ORF of MB2 that is 4830 nucleotides in length, of which 77% of the bases are A-T pairs (data
not shown). No nucleotide polymorphisms were observed among the
cDNA and genomic sequences, indicating that there is a single
allele of the MB2 gene encoded and expressed in the parasite strains used in our analyses.
Because the nucleotide sequences of the three genomic clones did not
overlap, we designed gene amplification primers a, b, c, and d (Fig.
2A) to assess the contiguity of the MB2 gene in the parasite genome. Amplification products produced by the primer pairs a+b and c+d with parasite genomic DNA as the template gave the
predicted product sizes, ~700 and ~1000 bp, respectively (data not
shown), indicating that MB2 is organized as a contiguous, single-exon gene in the parasite genome.
Sequence Analysis of the MB2 Putative Translation
Product--
MB2 encodes a putative translation product
that is 1610 aa in length with an approximate molecular mass of 187 kDa
(Fig. 2, B and C). The predicted protein is rich
in asparagine (15%) and lysine (13%) and is strongly basic with a
calculated net charge of +20 at pH 7 and a pI of 8.3. The primary amino
acid sequence can be separated into three distinct, linear domains, the
first of which is an amino-terminal basic domain of 490 residues (aa 1-490) with a calculated net charge of +30 and a pI of 9.4. We have
designated this the "B" domain. This domain contains a region of
six 9-aa imperfect repeats (aa 211-264) with the consensus sequence L,
N, S, K, K, N, D/N, N, T/S. The central acidic domain, designated
"A," encompasses 496 residues (aa 491-986) with a calculated net
charge of Ultrastructural Localization of MB2 Protein--
Immunoelectron
microscopy (IEM) was used to study the subcellular localization of the
MB2 antigen. All rabbit antisera prepared against
recombinant peptides derived from the B and A domains (Fig.
2A), and reacted with sectioned material containing
sporozoites, showed that MB2 protein was localized
predominantly to the surface (Fig. 3,
A-D). This was true of sporozoites in salivary glands (Fig.
3, A and B), as well as those that invaded
in vitro cells of the human liver cell line, HepG2-A16 (Fig.
3, C and D). No antibody reaction was detected
with sporozoites using the anti-G domain antibody (data not shown).
Preimmune control sera for all reagents were negative (data not
shown).
In contrast, the majority of the MB2 protein detected in
blood-stage parasites using both antisera against the B domain was localized in the nucleus, with some antibody reactivity detected in the
cytoplasm (Fig. 3, E and F) and data not shown).
Rabbit antisera against the A and G domains detected protein only in the cytoplasm of these parasites (Fig. 3G and data not
shown). Furthermore, the numbers of gold particles observed in sections of parasites exposed to antibodies against the A and G domains were low
when compared with the signal produced by the anti-B domain antisera,
indicating probably that the majority of MB2 protein present
at the blood stages does not contain the A and G domains.
MB2 protein was detected in the cytoplasm, nucleus, and
parasitophorous vacuole (PV) space of gametocyte-stage parasites using the anti-B domain antiserum (Fig. 3H). MB2
protein detected by the anti-A domain antiserum was localized only in
the PV space (Fig. 3I), indicating that the protein detected
in the nucleus and cytoplasm with the anti-B domain antiserum does not
contain the A domain. The anti-G domain antiserum produced a high
background signal, making it difficult to interpret any specific
localization pattern.
Finally, we attempted to use IEM to look at the localization of
MB2 protein in the exoerythrocytic stages of the parasite. A
section of the liver of an infected Aotus monkey was reacted with the anti-B domain antiserum. It is hard to locate the parasites in
these sections, but we were able to find some that revealed that the
protein is localized mostly in the cytoplasm with some in the PV space
(Fig. 3J). Sections reacted with the anti-A domain antiserum
had high backgrounds obscuring any evidence of a specific localization pattern.
Immunoblot Analyses--
A series of immunoblotting experiments
were performed with parasite protein extracts prepared from the
sporozoite and blood stages to determine the relative size of the
MB2 protein (Fig. 4). Both
anti-B and anti-A domain antisera detected a single polypeptide of
~120 kDa at the sporozoite stage (Fig. 4, A and
B). No immunoblot analyses were done on sporozoite
preparations with anti-G domain antiserum because of the negative
results obtained in the IEM analyses. Immunoblotting using anti-B
domain antibody detected a single polypeptide of ~66 kDa at the blood
stages (Fig. 4C). Furthermore, the 66-kDa polypeptide was
not detected with either the anti-A or anti-G domain antibodies (Fig.
4D and data not shown). These data are consistent with the
IEM study and indicate that the MB2 polypeptide located at
the surface of sporozoites consists of only the B and A domains, and
the polypeptide translocated into the nucleus of parasites at the blood
stages consists primarily of the B domain.
A major difficulty in immunoscreening of expression libraries to
identify P. falciparum sporozoite antigens is the abundant and immunodominant characteristics of the CS protein. The abundant expression of the CS gene results in its representation in high frequency in cDNA libraries (15), and its immunodominant repeat domain induces a high antibody titer in the host providing the screening antiserum (7). Thus, to identify novel, non-CS antigens, we
depleted the CS sequence from a sporozoite cDNA expression library
prior to screening. The effectiveness of this approach was demonstrated
by the isolation of MB2, a gene encoding a novel antigen
with stage-dependent localization.
The Southern analyses revealed that MB2 is present in the
parasite genome most likely as a single-copy gene. The nucleotide sequence data indicated that it consists of a single exon and is
represented most likely in our parasite samples by a single allele. The
Northern analyses with mRNA obtained from the blood-stage parasites
indicated that MB2 is expressed as a single large
transcript. We do not know if this size is common to the other
developmental stages of the parasite because of the difficulty in
obtaining sufficient mRNA for blotting experiments. However,
because the gene is single-copy and contains no intron, it is unlikely
that multiple transcripts are produced, resulting either from
expression of different genes or alternative splicing of a single gene.
Thus, it is likely that the single-transcript expression of
MB2 is common to other developmental stages.
The overall length of the reconstructed MB2 cDNA is 2.2 kilobases smaller than the RNA species detected in the Northern
analyses. This difference results most likely from large 5' end, and
perhaps 3' end, untranslated regions. Although we have neither a single genomic nor cDNA clone that spans the entire ORF of MB2,
based on the overlapping primary sequence of the cDNA clones, the
contiguity of their reading frames, and the gene amplification analyses
of the genomic clones, we are confident that we have the complete expressed sequence of the MB2 gene.
The complete ORF of MB2 predicts a full-length protein of
187 kDa. However, there are many predicted sites for post-translational modification by myristoylation, glycosylation and phosphorylation. Therefore, it is likely that the actual molecular weight of the primary
protein structure is increased by processing of individual amino acids.
The predicted MB2 protein is rich in asparagine (15%) and
lysine (13%), and therefore is strongly basic. Asparagine is the most
commonly used (~12%) amino acid in P. falciparum, followed by lysine and glutamic acid (~10%) (33, 34). Two other
sporozoite surface proteins, CS and the
sporozoite-threonine-and-asparagine-rich protein (STARP) (35), contain
29% and 25% asparagine, respectively. It has been speculated that
asparagine-rich motifs in the amino acid sequence might be targets of
opsonizing antibodies, promoting parasite phagocytosis by immune cells
(36-38). Whether the MB2 protein is a target of opsonizing
antibodies is not known, but it is recognized by the immune serum of a
human volunteer protected by the irradiated sporozoite vaccine.
Unlike a number of characterized Plasmodium genes that are
active only in certain stages, the MB2 gene is expressed in
many developmental stages of the parasite life cycle. However, the MB2 gene product has differential localization throughout
development. The stage-dependent differential localization
of the MB2 protein suggests strongly that it has a
multifunctional role during development of the parasites. It is
conceivable that it functions as a signal recognition molecule while it
is on the surface of the sporozoites. It then may transmit a signal to
the nucleus by migrating there during the blood stages. Once inside the
nucleus, it may function in the regulation of gene expression,
participating in the process of turning off genes that are not required
and activating genes that are required for blood stage infection.
Examples of genes that are known to be inactivated as the parasite
develops to the blood-stage are CS (39-40) and
TRAP (25), and genes that are activated are merozoite
surface protein genes, MSPs (41).
In gametocytes, the protein product is localized in the nucleus,
cytoplasm, and the PV space. This differential localization may
indicate that the MB2 gene product is in a transitional
phase from its functional role in the nucleus to the cell surface or it
may have a role in the development of the sexual stages of the
parasite. In the exoerythrocytic stage, the MB2 protein
detectable by anti-B domain antisera is localized mainly in the
cytoplasm, although some can be detected in the PV space. As with the
gametocytes, this expression may be a transitional phase in the
specific localization as the parasite develops in the liver. The
expression of MB2 in this stage is important potentially as
a vaccine target since the hepatocyte expresses major
histocompatibility complex molecules that can be recognized by T cells.
Research in the last 10 years has indicated that the infected
hepatocyte can be an important target for immune attack (11).
Although the MB2 protein is localized on the surface
membrane of sporozoites, the primary amino acid sequence contains no apparent transmembrane domain or glycosylphosphatidylinositol anchor
signal. However, the amino acid sequence does contain a polybasic motif
that was shown to function as plasma membrane localization signal as
well as CRS motif (31, 42). Many cytokines are retained on the membrane
surface of the producer cell in a process mediated by the CRS. Studies
have shown that, if the basic amino acids are deleted or mutated to
acidic or neutral amino acids, then the membrane localization of the
protein is affected (43, 44). Therefore, we predict that the polybasic
motif is the most likely domain used to localize the MB2
product to the membrane surface of sporozoites.
We provide evidence supporting the conclusion that protein processing
is the mechanism by which MB2 regulates its differential cellular
localization (Fig. 5). The MB2
protein is localized mostly to the surface membrane of sporozoites as a
~120-kDa species consisting of the B and A domains (Figs. 3
(A and B) and 5A). It is important to
note that the predicted molecular mass of the B and A domains combined
is ~116 kDa. This similarity in molecular mass of the actual protein
and the predicted domains is remarkably close and does not take into
consideration the effects of post-translational changes to specific
amino acids or the arbitrary boundaries assigned to the domains. As the
sporozoites invade hepatocytes represented by the human cultured liver
cells, the B and A domains of the MB2 protein are still
detectable on the membrane surface (Figs. 3 (C and
D) and 5B). We do not have immunoblot data for
this stage, but we infer from the IEM study that the size of the
protein is most likely ~120 kDa, similar to the size detected in free
sporozoites. As the parasite develops to the blood stages, the majority
of MB2 protein detected inside the parasite nucleus consists
only of the B domain and is represented in immunoblots by a ~66-kDa species (Figs. 3 (E and F) and 5C).
The predicted molecular mass, ~57 kDa, of the B domain selected by
our analysis of the amino acid primary structure, is consistent with
this smaller size polypeptide. As the parasite differentiates to
gametocytes, the MB2 protein is found in the PV space as
well as the nucleus and cytoplasm (Figs. 3 (H and
I) and 5D). Based on the different labeling
patterns seen with the anti-B and anti-A domain antisera, it is likely that the signal in the cytoplasm and nucleus originates from the ~66-kDa moiety. The protein in the PV space contains at least the B
and A domains, and may contain the G domain. However, as noted in the
results, the IEM study using the anti-G domain antibody is
inconclusive, and we do not have immunoblot data that would provide the
size of MB2 for the gametocyte stage.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage vector (Stratagene) were used to
amplify the subtracted cDNA, and the amplification products were
subcloned into the phage arms of the UniZap vector and packaged.
-D-galactoside and air-dried prior to use.
Membranes were incubated in the serum of human volunteer 52 at a 1:100 dilution, and
horseradish peroxidase-conjugated anti-human IgG+IgM were used to
detect positive antibody reactions by the ECL system (Amersham
Pharmacia Biotech). Positive phage were screened a second time to
isolate single phage clones. Additional cDNA and genomic clones
(strain ITO) were recovered using MB2-derived 32P-labeled probes and standard library-screening
techniques (18).
-D-galactoside for 3-4 h
to express recombinant proteins. Purification of recombinant proteins
was done using the ProBond resin (Invitrogen) modified by the inclusion
of imidazole at 85 mM final concentration in the washing
buffer. Eluted fractions were analyzed for the presence of recombinant
proteins by SDS-polyacrylamide gel electrophoresis and immunoblotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage, producing a
library of 1.45 × 106 primary phage.
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Fig. 1.
Southern and Northern analyses of the
MB2 gene and transcription product. A,
Southern blot of P. falciparum genomic DNA, strain FCR3,
digested with various restriction enzymes and hybridized with the
spz-MB2 cDNA clone. Lane 1,
EcoRI/HindIII; lane 2,
PstI/HindIII; lane 3,
PstI/EcoRI; lane 4,
PstI/EcoRV; lane 5,
PstI/NdeI. B, Northern blot of
P. falciparum blood-stage mRNA hybridized with a probe
derived from nucleotides 1-580 of the MB2 ORF. The
lane contained 20 µg of total RNA. The approximate
locations of molecular size markers in kilobases (kb) are
indicated to the right of each of the
panels.
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Fig. 2.
Structure of the MB2 gene
and expression products. A, schematic representation of
cDNA and genomic clones used to identify and assemble a cDNA
containing the complete ORF of the MB2 gene. The cDNAs,
spz-MB2, c3-1-18, c18-4-23, and
c3-4-29, are represented as horizontal
lines above a linear representation of the
full-length MB2 cDNA. The numbers
above each cDNA refer to the terminal nucleotide
positions in the completed cDNA. The As in
parentheses in the cDNA clones represent the internal
and terminal priming poly(A) sites of the oligo(dT) primers. The
full-length cDNA is represented as a horizontal
line numbered with the positions of the
translation initiation (ATG) and translation termination (TAA) codons,
and the beginning and end of the sequence. The 5' end untranslated
region (5'-UTR) and polyadenylation sequences (A)
also are indicated. The three horizontal lines at
the bottom denote the MB2 genomic clones,
g2-6-8, g2-4-4#5, and g6-2-2. The
locations of the terminal nucleotides with respect to the cDNA are
indicated above each line. Four horizontal
arrows (a-d) represent the orientation and
approximate location of gene amplification primers used to verify the
contiguity of the sequence in the parasite genome. B,
schematic representation of the MB2 protein sequence. The
three domains, basic (B), acidic (A), and
GTP-binding (G), are indicated as blocks with the
junctions of the domains numbered below. The
amino (H2N) and carboxyl (COOH) ends
are labeled. The four short horizontal
lines represent the approximate extents of the polypeptides,
MB2-B, MB2-C, MB2-FA, and MB2-IF2, used to generate antibodies.
C, primary amino acid sequence of the conceptual translation
of the MB2 gene. Amino acids in bold represent
the putative signal peptide; bold and boxed,
putative nuclear localization sequences; bold and
italicized, repeat regions with a single repeat unit
underlined; bold and underlined, cell-surface
retention sequence; italicized and boxed, motifs
conserved in the G domain.
6.2 and a pI of 6.1. The boundary between the B and A
domains was selected to maximize the basic and acidic properties of the
respective domains. The A domain contains two regions of imperfect
repeats of 5 amino acids. The first region (aa 493-542) contains 10 repeats with a consensus sequence of D, N, Q/P, N, Y. The second region
(aa 870-914) contains nine repeats with a consensus of I/M, N/D, V, Q,
D. No similarities to any sequences of known function deposited in the
data bases were detected for either the B or A domains. Finally, a
624-residue carboxyl-terminal domain (aa 987-1610) with sequence
similarity to the GTP-binding domain of the prokaryotic translation
initiation factor 2, IF2, as revealed by the BLAST search program (27)
has been designated "G." The boundary between the A and G domains
was selected based on the start of the regions of similarity of the
MB2 protein with known IF2 molecules. In contrast to its
overall hydrophilic nature, the MB2 polypeptide contains at
the amino terminus a strongly hydrophobic region (aa 1-25) mapped by a
Kyte-Doolittle hydrophobicity plot. The PSORT computer program (28)
predicted an uncleavable signal peptide in the hydrophobic
amino-terminal region of MB2. However, the SignalP program
(29) predicted that the signal peptide could be cleaved between a pair
of S-S residues at aa 27-28 (Fig. 2C). Currently, we have
no experimental data that support one alternative over the other. The
PSORT program also predicted a number of nuclear localization signals
(NLS), PKKK (aa 120-123), RRKK (aa 173-176), KKKKK (aa 652-656), and
a bipartite NLS, KKNKELPFNNKFKKIIK (aa 718-734), within the B and A
domains. Multiple putative sites for N-glycosylation,
N-myristoylation, and phosphorylation were detected by the
ScanProsite program (Ref. 30; data not shown). There is a polybasic
motif, KKKKKGKSRKK (aa 956-966), just before the start of the G
domain, that could function as a plasma membrane localization signal as
well as a cell-surface retention sequence (CRS) (31). This sequence
also could be a putative NLS, although PSORT failed to identify it as
such. The similarity of the G domain to the GTP-binding domains of the
prokaryotic IF2 proteins includes the conservation of sequence and
spacing of three motifs, GX4GK (aa 999-1005),
DX2K (aa 1046-1049), and NKXD (aa
1100-1104), common to this family of proteins (32). There is a small
variation in the third motif, TKXD, in MB2 as compared with
the consensus seen in other G proteins (Fig. 2C).
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Fig. 3.
Immunolocalization of the MB2
protein in different developmental stages of P. falciparum. A-D, sporozoite preparations. A
shows a cross-section of a sporozoite (S) in the mosquito
salivary gland (Sg) reacted with anti-B domain antiserum.
B is a cross-section of a free sporozoite reacted with
anti-A domain antiserum. C and D are
partial-oblique and cross sections (respectively) of sporozoites in
HepG2-A16 cells (He) reacted with anti-B and anti-A domain
antisera, respectively. PV is the parasitophorous vacuole
space. E-G, asexual stage parasite preparations.
E is a cross-section of a trophozoite in an erythrocyte
(E), showing localization principally to the parasite
nucleus (N) and some in the parasite cytoplasm
(Pc). F is a section of schizonts showing
MB2 localization to the nucleus and some cytoplasm. Hemazoin
(Hz) also is visible. Both E and F
were reacted with anti-B domain antiserum. G shows sections
of parasites at the merozoite (Mz) stage reacted with anti-A
domain antiserum, showing only cytoplasmic localization. H
and I, localization of MB2 in gametocytes (labeled
G) reacted with anti-B and anti-A domain antisera,
respectively. MB2 can be detected in the nucleus, cytoplasm, and the PV
space. Arrows indicate the location of gold particles.
J, localization of MB2 in the exoerythrocytic
(EE) stages of an Aotus monkey hepatocyte
(AH) reacted with anti-B domain antiserum. All
bars are 0.5 µm in length.
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Fig. 4.
Immunoblot analysis of protein extracts of
P. falciparum sporozoite and blood stages.
A and B, protein extracts prepared from
sporozoites recovered from salivary glands of infected mosquitoes.
C and D, proteins extracts prepared from asexual
blood-stage parasites. A and C were probed with
anti-B domain (MB2-B) antiserum; B and D were
probed with anti-A domain (MB2-FA) antiserum. The molecular size
markers (in kDa) are indicated to the left of each figure,
and arrows to the right mark the locations of the
MB2 polypeptides.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
Summary of expression and localization data
for the MB2 protein. Each panel
(A-D) lists the stage of the parasite (first
line) and the molecular size determination based on
immunoblotting (second line). The
third line indicates which domains were detected
in either the immunoblotting or immune electron microscopy experiments.
The immune electron micrographs are excerpted from Fig. 4. The
asterisks in B and D indicate that the
molecular size is not confirmed by immunoblotting analyses. The
question mark (?) in D
indicates that the presence of the G domain could not be unequivocally
confirmed. All abbreviations are as in Figs. 3 and 4.
We propose that the MB2 protein detected weakly in the cytoplasm of blood-stage parasites by the anti-A and anti-G domain antisera most likely represents the full-length, newly synthesized MB2 protein that has not been processed proteolytically into the ~66-kDa polypeptide. We propose further that the full-length MB2 protein is processed specifically at the sporozoite stage to the ~120-kDa polypeptide during synthesis and/or cellular trafficking. The ~120-kDa species contains the polybasic CRS-like motif, allowing it to be preferentially retained on the surface of the sporozoite. The secondary or higher-order structure of the ~120-kDa protein may conceal the NLS in the B and A domains. At the blood stage, the full-length MB2 protein is processed specifically into the ~66-kDa polypeptide as supported by the absence of the ~120-kDa species. The processing of the MB2 protein into the ~66-kDa polypeptide would remove the polybasic motif, thus removing the membrane targeting signal, and perhaps this processing exposes the nuclear localization signals allowing the ~66-kDa polypeptide to translocate to the nucleus. Finally, MB2 protein in the gametocyte stage may be processed into at least two forms, one of which consists of at least the B and A domains and is exported to the PV space. The other form, consisting most likely of only the B domain, is transported to the nucleus.
Another interesting feature of the MB2 protein is the G domain, which has significant sequence similarity to the prokaryotic IF2. Our data indicated that the G domain is not present in the MB2 protein detected on the sporozoite surface, nor is it present in the nucleus at the blood stages. It is conceivable that the cleavage of the MB2 protein requires energy, and this requirement is fulfilled by the G domain since it can bind to GTP. The cleavage process most likely includes removal of the G domain as evidenced by the inability to detect it with specific antiserum in most stages of the parasite. Alternatively, because MB2 can bind potentially to GTP, it is possible that there are conformational differences between the GTP-bound, GDP-bound, and unbound states that can regulate the distinct proteolytic processing of the MB2 protein.
In the last two decades, research efforts to develop recombinant
vaccines against malaria have yielded largely limited successes. Although we have made significant progress in understanding immune responses and identifying a number of antigens, we are still not certain as to which host immune mechanisms and target antigens are
relevant to protective immunity. In particular, the parasite antigenic
composition at the sporozoite stage is still largely unknown. Because
of the complexity of the Plasmodium species, we do not know
if we have reached the heart of its antigenic repertoire required for
identifying antigens that are relevant to protection or we are still
wandering at the periphery, being misled by a limited number of
identified antigens. However, vaccine development remains a viable
option because it is clear that sterile immunity can be experimentally
induced in humans (3). We believe that an optimal malaria vaccine would
need to target multiple stages of the parasite life cycle, including
the sexual stages developed inside the invertebrate host. As more novel
antigens are being characterized, it is our expectation that the
knowledge acquired from them can bridge the gap between recombinant and
sporozoite-attenuated vaccines.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Jacques Prudhomme and Irwin Sherman (University of California, Riverside, CA) for providing blood stage parasites, Michael Hollingdale (Leeds University, Leeds, United Kingdom) for the infected Aotus and HepG2 preparations, and John Sacci (Naval Medical Research Center, Silver Springs, MD) for sporozoites. The study protocol was approved by institutional review boards at Naval Medical Research Center and Walter Reed Army Institute of Research, 1989-1994 (Protocol HURRAO Log A-4839) in compliance with all federal regulations governing the protection of human subjects. The experiments reported herein were conducted according to the principles set forth in the "Guide for the Care and Use of Laboratory Animals."
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FOOTNOTES |
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* This work was supported by grants from the Burroughs-Wellcome Fund and the John D. and Catherine T. MacArthur Foundation (to A. A. J.) and by National Institutes of Health Grant AI35827 (to H. F.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF378132-AF378138.
To whom correspondence should be addressed: Dept. of
Molecular Biology and Biochemistry, 3205 Bio Sci II, University of
California, Irvine, CA 92697-3900. Tel.: 949-824-5930;
Fax: 949-824-2814; E-mail: aajames@uci.edu.
Published, JBC Papers in Press, May 22, 2001, DOI 10.1074/jbc.M103375200
2 W. O. Rogers, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CS, circumsporozoite; CRS, cell-surface retention signal; IEM, immunoelectron microscopy; GST, glutathione S-transferase; NLS, nuclear localization signal; ORF, open reading frame; PV, parasitophosporous vacuole; aa, amino acid(s); UTR, untranslated region; bp, base pair(s); MSP, merozoite surface protein; TRAP, thrombospondin related anonymous protein.
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