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J. Biol. Chem., Vol. 277, Issue 9, 7092-7098, March 1, 2002
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,
From the Growth and Development Section, Laboratory
of Malaria and Vector Research, NIAID, National Institutes of Health,
Bethesda, Maryland 20892-0425, the § Malaria Program, Naval
Medical Research Center, Silver Spring, Maryland 20910, and the
¶ Department of Microbiology and Immunology, School of Medicine,
University of Maryland, Baltimore, Maryland 21201
Received for publication, July 20, 2001, and in revised form, December 17, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Plasmodium sporozoites display
circumsporozoite (CS) protein on their surface, which is involved in
the attachment of sporozoites to liver cells. CS protein is a member of
the thrombospondin type I repeat (TSR) domain family and possess a
single copy of TSR domain toward its carboxyl terminus. We show by a
direct measurement the correlation between the binding activity of
various segments of the CS protein and their ability to inhibit the
invasion of liver cells by the sporozoites. We made eight truncated
versions of Plasmodium falciparum CS protein to elucidate
the role of various regions in the binding and invasion process.
Deletion of the TSR domain actually enhanced binding activity by
2-3-fold without the loss of receptor specificity, indicating that TSR
may not be the only domain in defining the specificity of binding.
These same deletions blocked invasion of live sporozoites more
efficiently than proteins that include the TSR domain. Deletion of as
little as six amino acids from amino terminus of the protein, however, renders it incapable of binding to liver cells and as an inhibitor of
sporozoite invasion. Hence, the binding of CS protein to liver cells
and its ability to inhibit the invasion process are affected in a
parallel manner, both positively and negatively, by sequence changes in
the encoded CS gene. This indicates that both assays are
measuring interrelated phenomenon and points to the essential involvement for the amino-terminal portion of the CS protein in these processes.
The circumsporozoite
(CS)1 protein is the
predominant surface antigen of Plasmodium sporozoites and
performs numerous functions for the parasite. It is vital for the
development of sporozoites (1), helps parasite to invade salivary
glands in mosquitoes (2), and has also been implicated in the binding
of sporozoites to liver cells (3, 4). Once inside, CS has been shown to inactivate the protein synthesis machinery of the host cell (5). CS
protein is one of the key targets recognized by the host immune system
and has emerged as one of the prime candidates for an anti-malarial vaccine (6-8).
CS protein can be roughly segmented into three parts, namely (i) the
amino terminus region-containing region I, (ii) a central repeat
region, and (iii) a carboxyl terminus segment containing TSR domain
(Fig. 1A). Region I has been implicated in the invasion of
salivary glands in mosquitoes and in the inhibition of protein synthesis in liver cells (2, 5). The central repeat segment is a low
complexity region containing multiple copies of species-specific repeat
peptides (NANP in case of P. falciparum) with no obvious sequence homology to any known protein. The carboxyl terminus of CS
demonstrates sequence conservation across the species (9) and its
region II-plus has been implicated in liver cell binding and subsequent
inhibition of protein synthesis (3-5). Region II plus is an 18-amino
acid region and is a part of a larger TSR domain, which was initially
discovered in thrombospondin (10).
TSR is an ancient eukaryotic domain (11) now known to be present in
more than 300 different proteins (12), including surface antigens of
pathogenic microorganisms (13) as well as metalloproteases of mammalian
origin (14). Although the role of TSR domain in these diverse proteins
has largely not been defined, in a few cases it has been shown to
interact with cellular and extracellular matrix (15, 16), specifically
heparan sulfate, a ubiquitous cell surface polysaccharide, CD36 and
various other GAGs (17).
Sporozoites, after being injected into the circulation, traverse
through the cytosol of several cells before finally invading a
hepatocyte (18). Given that the TSR domain receptors are ubiquitous on
host cells, it is difficult to envision that the TSR domain alone is
responsible for specific binding and invasion of hepatocytes. To
address this issue, we have constructed variants of the CS protein
lacking either the TSR domain or residues from amino terminus or both.
Characterization of these mutants uncovers the crucial role of amino
terminus of CS protein in these processes.
Materials
Escherichia coli strain BL21( Construction of CS Deletion Mutants
Carboxyl Terminus Deletions--
Plasmid pCS1 was used as
template for the construction of these deletion mutants. Plasmid pCS1
contains DNA sequence encoding for a mature CS protein in pET11a, a T7
promoter-based E. coli expression vector (19). Two CS DNA
sequences lacking 90 and 109 amino acids from the carboxyl terminus
were amplified by PCR. The oligonucleotides used for amplification were
designed such that an NdeI and a BamHI
restriction site was introduced at 5' and 3' ends, respectively. The
insert was digested and cloned in pET11a, giving rise to plasmids pCS20
and pCS21 lacking 109 and 90 amino acids from the carboxyl terminus,
respectively. Plasmid pCS27 only encodes for the first 97 amino acids
from the amino terminus and has been described elsewhere (20).
Amino Terminus Deletions--
DNA encoding truncated versions of
CS, lacking 6, 14, 23, 28, and 90 amino acids from the amino terminus,
were amplified by PCR from plasmid pCS1 and cloned in vector pET11a
giving rise to plasmids pCS19, pCS16, pCS17, pCS6, and pCS7, respectively.
Carboxyl and Amino Terminus Deletion--
Plasmid pCS20 was used
as a template to amplify a fragment of CS lacking 28 amino acids from
the amino terminus and 109 amino acids from the carboxyl terminus. The
truncated fragment was cloned in pET11a, giving rise to plasmid pCS25.
Expression, Localization, and Purification of Recombinant
Proteins
All the constructs were expressed in bacterial strain BL21,
containing T7 RNA polymerase gene under the control of lac
promoter, as described (19). Briefly, cells were transformed with the desired plasmid and the culture was expanded in super broth (pH 7.2),
containing 100 µg/ml ampicillin. At A600 of
1.0, cells were induced for 4 h with 2 mM
isopropyl-1-thio- Cell Binding Assay
Hepatoma cell line HepG2 was used to evaluate the binding
activity, and the assay was performed as described (3). Briefly, cells
were fixed with 4% paraformaldehyde followed by blocking with
Tris-buffered saline containing 1% bovine serum albumin. Proteins were
incubated with cells for 1 h, followed by anti-CS monoclonal
antibody for 30 min and alkaline phosphatase coupled anti-mouse IgG for
30 min. 1 mM 4-methylumbelliferyl phosphate was used as
substrate, and fluorescence was measured in a fluorometer with
excitation at 350 nm and emission at 460 nm. The sensitivity of the
fluorometer was adjusted such that the fluorescence activity in a given
assay was within the maximum detection limit of the instrument.
Binding Activity of CS Mutants in the Presence of GAGs
For evaluating the effect of various GAGs on the binding
activity of the carboxyl terminus deletions, 50 nM
recombinant proteins were co-incubated with different concentrations of
heparin, fucoidan, chondroitin sulfate A, chondroitin sulfate B, or
dextran sulfate for 15 min at 37 °C before the addition of this mix
onto the cells. For amino-terminal deletions, 50 nM protein
was co-incubated with 5 nM heparin. The assay was continued
as described above.
Effect of Heparinase I and Chondroitinase ABC on the Binding
Activity of CS-Heparin Complex
HepG2 cells were treated with log dilutions of heparinase I or
chondroitinase ABC or buffer for 2 h at 37 °C. The cells were washed three times with Tris-buffered saline, followed by addition of
50 nM recombinant protein and incubation at 37 °C for
1 h. Fluorescence was measured as described above.
Competition Binding Analysis
For competition analysis, HepG2 cells after blocking were
incubated with different concentrations of CS27 or peptide
(MRGSSSNTRVLNELNYDNAG) for 60 min before adding the protein. The
experiment was continued as described above.
Sporozoite Invasion Assay
The assay was performed as described by Shi et al.
(21) and is a modification of the method first described by Hollingdale et al. (22). HepG2 cells at a density of 5 × 104/well were seeded in eight-well chamber slides 3 days
before the experiment. P. falciparum (NF54) sporozoites were
isolated from mosquito salivary glands using a discontinuous gradient
as described (23). Different concentrations of CS proteins were added
to triplicate wells immediately followed by the addition of 25,000 sporozoites. Sporozoites were incubated with cells for 3 h
following which unattached sporozoites were removed by washing. The
cells were then fixed with ice-cold methanol and incubated overnight at
4 °C. The slides were blocked with 3% bovine serum albumin and
incubated with 10 µg/ml NFS1 antibody (anti-NANP repeat monoclonal) for 30 min at 37 °C. Unbound antibodies were removed by washing with
phosphate-buffered saline, followed by the addition of anti-mouse IgG-horseradish peroxidase conjugate. Bound enzyme was revealed by
using diaminobenzidine as substrate. Slides were mounted with Permount
and read using a 20× objective lens. The percentage of inhibition was
determined by the following formula: [(control Cloning, Expression, Localization, and Purification of Deletion
Mutants of CS--
Eight different deletion constructs of CS lacking
residues from amino or carboxyl terminus or both were created (Fig.
1A). Plasmids pCS19, pCS16,
pCS17, pCS6, and pCS7 lacked the first 6, 14, 23, 28, and 90 amino
acids from the amino terminus, respectively. Plasmids pCS21 and pCS20
encoded truncated versions of CS where 90 and 109 amino acids from the
carboxyl terminus were deleted. Plasmid pCS25 had deletions on both
ends and lacked 28 and 109 amino acids from amino and carboxyl termini,
respectively. Authenticity of all the clones was verified by sequencing
(data not shown). All the constructs were expressed in BL21( The Effect of Deletions on the Binding Activity of CS
Protein--
Binding activity of deletion mutants was evaluated on
HepG2 cells. Carboxyl terminus mutants CS20 and CS21, though without the TSR domain, demonstrated an 2-3-fold enhancement in the binding activity in comparison to the native protein CS1 (Fig.
2A). The binding activity was
dose-dependent and specific, as it showed saturation at
higher concentrations. Although deletion of the TSR domain led to an
increase in activity, truncations from the amino terminus rendered CS
inactive. Loss of the first six amino acids (CS19) led to a 75%
decrease in the binding activity of the protein (Fig. 2B).
Deletion of more than six amino acids (proteins CS16, CS17, CS6, and
CS7) resulted in a complete loss of activity (Fig. 2B),
although these proteins could still bind to the heparin column (data
not shown). Protein CS25, where the first 28 amino acids of the active
protein CS20 were removed, also became inactive (Fig. 2B).
These mutants remained inactive even when tested at higher
concentration (data not shown). The difference in binding activity of
various constructs was not because of a difference in recognition of
the bound protein by the antibody, as monoclonal antibody used in all
these experiments recognize the central repeat region of the protein,
which was not mutated, and on a Western blot recognized all the mutant
proteins with equal efficiency (data not shown).
Binding Specificity of TSR Domain Deletion Mutants--
CS protein
binds to a GAG moiety of liver cell surface proteoglycan (4, 27). The
first GAG binding site to be discovered in CS protein was the TSR
domain in the carboxyl-terminal region. To determine whether TSR domain
deletion mutants (CS20 and CS21) were also specifically binding to a
GAG-based cell surface receptor, their binding activity was evaluated
in the presence of five different GAGs (heparin, fucoidan, dextran
sulfate, chondroitin sulfate A, and chondroitin sulfate B). For both
the TSR deletion mutants (CS20, CS21) and the native protein, heparin
was the most potent inhibitor of binding with an IC50 of
2-3 µM (Fig.
3A, Table
I). The involvement of nonspecific and
low-affinity receptors in binding was ruled out because complete
inhibition of binding of both the truncated and full-length protein was
obtained at 50 µM heparin (Fig. 3A). Dextran
sulfate had a similar inhibitory effect on the truncated proteins
(IC50 2.2-3.8 µM), but a 10-fold excess of
dextran sulfate was needed to inhibit the activity of the native protein (IC50 = 32 µM), indicating that
dextran sulfate could be binding to both the amino terminus and the
carboxyl terminus of CS1(Fig. 3B, Table I).
Fucoidan had an IC50 of 250 µM for all the
three proteins (Fig. 3C, Table I). In contrast, chondroitin sulfate A and chondroitin sulfate B failed to inhibit the binding of
all the three proteins even at the highest concentration tested (Table
I). We have recently shown that chondroitin sulfate A, B, and E are
ineffective in inhibiting the binding of full-length CS protein to
HepG2 cells (28). The inhibition profile of various GAGs on the binding
of TSR deletion mutants was similar to the full-length protein,
suggesting that all the three proteins are binding to the identical
receptor.
Previously we have shown that, at substoichiometric concentrations,
heparin was able to substantially enhance binding of CS protein to
HepG2 cells (29, 30). A similar profile was seen with the mutants,
where submolar concentrations of heparin improved the binding activity
by 3-fold (Fig. 3A). Dextran sulfate also showed a similar
profile for enhancement, whereas fucoidan promoted the binding even
when present at molar excess (Fig. 3, B and
C).
Binding Activity of TSR Deletion Mutant on Heparinase I- or
Chondroitinase ABC-treated Cells--
As TSR domain deletions were
binding through a GAG-based receptor, the GAG moiety of the receptor
was characterized. Binding activity of TSR deletion mutants and the
full-length protein was evaluated on HepG2 cells that had been treated
with either heparinase I or chondroitinase ABC. Heparinase I primarily
cleaves heparin-like regions of heparin-like GAGs (31, 32), whereas
chondroitinase has been widely used in depolymerization of GAGs and
proteoglycan chondroitin sulfates (33). Treatment of cells with
heparinase I resulted in 50-80% decrease in the binding activity of
both TSR deletion mutants (CS20, CS21) and the full-length protein (CS1). This indicates that both the TSR deletions and the full-length protein are specifically interacting with heparin-based GAGs. The
treatment of HepG2 cells with chondroitinase ABC did not affect the
binding of either of the three proteins (Fig.
4).
Activation of Amino Terminus CS Deletion Mutants by
Heparin--
Recently we have shown that the binding activity to HepG2
cells in otherwise inactive CS proteins can be restored in presence of
substoichiometric concentrations of heparin (30). To evaluate the
capacity of heparin to restore the binding on HepG2 cells, 50 nM inactive amino terminus deletions (CS6, CS7, and CS19)
were pre-incubated with 5 nM heparin before being added to
the cells. Although heparin did show partial activation of CS19, which
lacks only six amino acids, it was unable to activate the other
inactive amino terminus deletions (Fig.
5). This indicates that the residues involved in the activation process are localized in the amino terminus
of the protein.
Competition of CS Protein Binding by Peptide--
Deletion of the
first six amino acids resulted in a loss of binding activity; thus, a
peptide representing the first 20 amino acids of CS1 was synthesized
and used as a competitor to inhibit the binding activity of CS1
protein. The peptide failed to inhibit the binding activity (data not
shown). On the other hand, CS27, representing the amino terminus of CS
protein, inhibited the binding of CS1 and CS20 by 65 and 35%,
respectively (Fig. 6), suggesting that
the residues involved in binding are distributed across the amino
terminus region of the protein and might be present close together in
the tertiary structure of the protein.
Inhibition of Sporozoite Invasion--
In a double-blinded
experiment, full-length (CS1), and truncated proteins (CS21, CS19, and
CS25) were used to compete with P. falciparum sporozoites
during liver cell invasion. Only intracellular sporozoites that
successfully invaded the liver cells in the presence of various
concentrations of the full-length or the deletion mutants were counted.
The results of this assay corroborated the findings of the binding
studies, as at comparable concentrations, CS21, protein lacking the TSR
domain, effectively stopped P. falciparum sporozoites from
invading HepG2 cells and showed very significant inhibition (89%) at
the lowest concentration (1 µg/ml) tested. Full-length protein CS1
showed only 36% inhibition at the same concentration (Table
II). At the same time, CS19, lacking the first six amino acids, showed minimal inhibition (11%), whereas CS25
was unable to inhibit invasion at all the concentrations tested. CS19
was able to inhibit invasion by 88% at the highest concentration
tested (20 µg/ml), whereas similar levels of inhibition were obtained
by CS1 at 5 µg/ml concentration, suggesting that CS19 is 4-fold less
efficient in inhibiting invasion with respect to the full-length
protein.
The CS protein has long been of interest because of its putative
role in parasite targeting and invasion of the liver. We constructed
eight different deletion mutants of mature CS protein to study what
role its various regions might play in the binding and invasion of
liver cells by the parasite.
Once the proteins were purified, the contrast between CS deletions and
the full-length protein in cell binding was evaluated, and the results
were subsequently compared with their potential to inhibit invasion of
liver cells by live sporozoites. Surprisingly, in the absence of TSR
domain, the truncated protein not only bound to HepG2 cells, it
actually showed a 2-3-fold enhancement over the full-length protein
(Fig. 2A). In contrast, deletion of the first six residues
from amino terminus alone (CS19) resulted in a major loss of binding
activity and deletion of more than six residues totally inactivated the
protein (Fig. 2B). Binding activity of active TSR deletion
mutant (CS20) was also lost when the first 28 amino acids were deleted
(CS25), clearly demonstrating that a major binding motif of CS is
located in the amino terminus region of the protein. Curiously,
although deletion of the first few amino acids (CS19, CS16, CS17, CS6)
resulted in a loss of binding to HSPG on HepG2 cells, these proteins
had bound to the heparin column. Heparan sulfate, although structurally
related to heparin, has low levels of sulfation and contains a
glucuronic acid residue in place of the flexible iduronic acid residue
found in heparin. Both heparin and heparan sulfate, because of their
negative charge, interact with positively charged amino acids in a
given protein. In protein CS6 (lacks 28 amino terminus residues), 22%
of the remaining amino terminus residues (15 of 67) are positively
charged. Binding of CS6, CS16, CS17, CS19, and CS25 to heparin column
can be attributed to the flexibility and high sulfate content of
heparin, which would have allowed it to interact with positively
charged residues of CS protein that otherwise may not be a part of the binding motif involved in interaction with HSPG-based receptor on liver cells.
Could the binding of TSR domain deletions be the result of nonspecific
interaction of these truncated proteins with liver cells? To
investigate this proposition, we competed the binding of TSR domain
deletions (CS20 and CS21) with five different GAGs. These GAGs have
been regularly used as competitors by several investigators to evaluate
the specificity of CS protein binding to liver cells (4, 27, 28).
Reduction in binding activity of these TSR domain deletions in the
presence of heparin was comparable with the inhibition profile of the
full-length protein CS1 (Fig. 3A). This indicates that, like
the full-length protein, the TSR deletion mutants bind through a
GAG-based receptor on hepatocytes. Additionally, both the deletion
mutant and full-length CS protein lost their binding activity when
HepG2 cells were pre-treated with heparinase I (Fig. 4). These
experiments indicated that, like the full-length protein, the TSR
domain truncated proteins were binding through a heparin-based receptor
on hepatocytes.
We have recently shown that heparin, at substoichiometric
concentrations, enhances the binding activity of CS protein presumably by cross-linking a large number of CS molecules (29, 30). An
enhancement in the binding of TSR domain deletion mutants in the
presence of submolar concentrations of heparin indicated that residues
involved in enhancement did not relate to the TSR domain. In contrast,
proteins truncated at proximal end of the amino terminus, as CS19,
lacking the first six amino acids, showed only partial activation.
Proteins with deletions extending beyond those six amino acids could
not be activated (Fig. 5), indicating that the residues involved are
located toward the amino terminus of the protein and this phenomenon is
not because of nonspecific aggregation of the proteins by GAGs.
An enhancement of CS binding at substoichiometric concentrations of
GAGs is not an isolated biological event. Previously, heparin has been
shown to mediate adhesion of Leishmania amistigotes to
cellular receptors (34), and, recently, McCormick et al. (35) have also demonstrated that heparin can enhance the binding of
P. falciparum infected erythrocytes to human microvascular endothelial cells. Similarly, T. gondii tachyzoites show an
increased infectivity in the presence of low concentration of sulfated
glycoconjugates (36). Hence, a carefully modulated response to varying
concentrations of GAGs could be a fine-tuned mechanism that these
pathogens have adapted for their propagation and survival.
A peptide including the entire amino terminus (CS27) served to compete
with the entire protein for binding to cells. To further elucidate the
contribution of this amino-terminal region in binding, we tried to
inhibit binding of the CS protein with a peptide containing only the
first 20 amino acids of mature P. falciparum CS protein (Fig. 6). The 20-mer peptide did not inhibit binding, which may indicate that the residues involved in interaction are not contiguous in the primary structure of the amino terminus, although in a tertiary
conformation they could be present close to one another and an intact
amino terminus might be essential for providing the correct structural conformation.
A similar situation exists in fibroblast growth factor, another
heparin-binding protein, where eight amino acids involved in heparin
binding are widely distributed (Asn-27, Arg-120, Thr-121, Lys-125,
Lys-129, Gln-134, Lys-135, and Ala-136) but are present close to one
another in the tertiary structure of the protein (37, 38). A multiple
sequence alignment of the amino terminus of CS protein from various
species does suggest the presence of at least four conserved positively
charged residues (Arg-9, Lys-34, Lys-43, Arg-46), which could be
responsible for the binding activity of CS protein.
An intact amino terminus is required for the binding of CS protein to
HepG2 cells. Is an intact amino terminus a prerequisite for invasion?
To investigate this proposition, full-length and truncated proteins
were evaluated for their capacity to inhibit the invasion of HepG2
cells by P. falciparum sporozoites. The results, where
sporozoites that successfully invaded the hepatocytes were counted,
were consistent with the binding profiles. The TSR domain deletion
(CS21) completely inhibited invasion of sporozoites at the lowest
concentration. The truncated protein, missing only six amino acids at
the amino terminus, was required in a 20-fold excess to achieve the
same level of inhibition. More extensive deletions of the amino
terminus were totally ineffective as competitors, clearly showing that
an intact amino terminus is not only required for binding, it is also a
prerequisite for the invasion process. Recently it has been shown that,
prior to invasion, attachment of sporozoites to liver cells requires
binding of CS protein to cell surface HSPG (39). The inhibition of
invasion by TSR deletion mutants could have occurred by effectively
competing out sporozoites from attaching on to the liver cells,
although blocking subsequent steps that result in invasion could not be
ruled out.
We have recently shown that the TSR domain could be effectively
neutralized, by converting a conserved cysteine of TSR domain at
position 395 to alanine, without losing the binding activity of the
protein (30). A similar mechanism has been reported in thrombospondin-related anonymous protein (TRAP), another sporozoite protein, where this domain could be eliminated without affecting the
ability of sporozoites to infect mice (40). Our results necessarily
alter our vision of sporozoite entry into the liver cells. We propose
that, in CS, involvement of TSR domain could be a part of a multistage
process of interaction where TSR domain could be acting as a low
affinity molecular anchor for the parasite, whereas amino terminus
mediates a high affinity and specific cell surface binding. A similar
mechanism exists in protease nexin 1, where retention of the protein
complex and cell surface binding is mediated by two different regions
of the protein (41). Presence of both the motifs in the protein could
reflect an optimal equilibrium between the two activities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DE3) and vector
pET11a were obtained from Novagen (Madison, WI). Heparin, fucoidan,
chondroitin sulfate A, chondroitin sulfate B, dextran sulfate,
chondroitinase ABC, and heparinase I of Flavobacterium
heparinum was obtained from Sigma. Restriction enzymes and
cell culture supplies were purchased from Invitrogen.
Heparin-Sepharose column was obtained from Amersham Biosciences,
Inc. The hepatoma cell line (HepG2) was purchased from ATCC
(Manassas, VA). Anti-mouse-alkaline phosphatase conjugate was from
Pierce. Monoclonal antibody 2A10 directed against the
(NANP)n repeat domain was a kind gift from Dr.
Robert Wirtz (Centers for Disease Control and Prevention, Atlanta, GA).
-D-galactopyranoside. Cells were
harvested, and the periplasm was separated by a gentle osmotic shock
without disrupting the cell membrane. Except for pCS7, CS proteins from
all the constructs were purified on a heparin-Sepharose affinity column
as described (19). Periplasmic extract containing CS7 was purified on a
Q-Sepharose (anion exchange) column. Subsequently, all the proteins
were subjected to gel filtration chromatography.
test)/control] × 100.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DE3) as
described (19). On localization, all the proteins were found to be
secreted into the periplasm (data not shown). Numerous recombinant
proteins, when secreted into the periplasm, have been shown to fold
correctly by the periplasmic protein folding machinery of the E. coli (24, 25). Periplasm was used as source to purify the
proteins. CS protein possess heparin binding properties, which have
been attributed to region I and the TSR domain of the protein (26). As
all the mutants had at least one of the two domains intact,
heparin-Sepharose column was used for purification. All the deletion
mutants except CS7 bound onto the heparin-Sepharose column. Protein
CS7, although containing both Region I and TSR domain, did not bind to
the heparin-Sepharose column and had to be purified onto an anion
exchange column. This suggested that the CS-heparin interaction is not
solely dependent on region I and TSR domain of the protein. Repeats are
not responsible for these interactions as CS27, a repeat-less protein,
coding only for the first 97 amino acids from amino terminus also bound to the heparin column with equal efficiency. Protein containing fractions were pooled and purified to apparent homogeneity by size
exclusion chromatography (Fig. 1B). The proteins eluted from the column within the range of their stipulated sizes, indicating that
they were maintaining a monomeric configuration. Authenticity of the
proteins was verified by amino terminus sequencing and mass
spectrometry (data not shown).
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Fig. 1.
A, schematic representation of different
constructs of CS. CS1 encodes the full-length protein. CS20, CS21, and
CS27 are carboxyl terminus deletions of CS. CS6, CS7, CS16, CS17, and
CS19 represent amino terminus deletions of CS. CS25 is a double
deletion where 28 and 109 amino acids from amino and carboxyl terminus,
respectively, have been deleted. B and C,
analysis of purified CS mutants. Purified proteins were run on a
4-20% SDS-polyacrylamide gel under reducing conditions and stained
with Coomassie. Lane 1 in each panel represents
unmutated protein CS1. Panel B, amino terminus deletions.
Lanes 2-6 represent CS6, CS7, CS16, CS17, and CS19,
respectively. Panel C, carboxyl terminus and double
deletions. Lanes 2, 3, and 4 represent
protein CS21, CS20, and CS25, respectively. Molecular size markers are
shown in kDa.
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Fig. 2.
Binding activity of CS mutants. HepG2
cells were incubated with different concentrations of CS mutants for
1 h, followed by addition of anti-CS antibody and alkaline
phosphatase-conjugated IgG. Bound enzyme was revealed by a fluorescent
substrate. The x axis represents the recombinant protein
concentrations, whereas the y axis shows the binding
activity of recombinant proteins in arbitrary fluorescence units.
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Fig. 3.
Binding activity of carboxyl terminus
deletions in the presence of GAGs. 50 nM of CS1
(circle), CS20 (triangle), and CS21
(square) was incubated with different concentrations of
heparin (A), dextran sulfate (B), and fucoidan
(C) before being added onto HepG2 cells. Fluorescence was
measured as described.
IC50 (µM) values of various GAGs on CS mutants
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Fig. 4.
Binding activity of CS mutant in presence of
heparinase I and chondroitinase ABC. HepG2 cells were treated with
different concentration of heparinase I (open
symbols) or chondroitinase ABC (filled
symbols) for 2 h followed by addition of CS1
(circle), CS20 (triangle), and CS21
(square) protein. Fluorescence was measured as described.
Inhibition, % Control represents the decrease in
binding activity of CS proteins on enzyme-treated cells with respect to
nontreated cells.
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Fig. 5.
Binding activity of amino terminus deletions
in the presence of heparin. 50 nM each of the CS
protein mutants was co-incubated with 5 nM heparin
(shaded bars) for 15 min followed by its addition
on HepG2 cells. Binding activity was compared with the protein
incubated with buffer (black bars).
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Fig. 6.
Binding activity of CS mutant in presence of
CS27. HepG2 cells were pre-incubated with different concentrations
of polypeptide CS27 for 1 h. This was followed by the addition of
CS1 ( 
) or CS20 (
) and incubation for 1 h. The binding
assay was continued as described.
Inhibition of sporozoite invasion by recombinant CS proteins
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENTS |
|---|
We thank Carl Hammer for mass spectrometry analysis and Mark Garfield for amino terminus sequencing of the recombinant proteins.
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FOOTNOTES |
|---|
* 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.
To whom correspondence should be addressed: Rm. 126, Bldg. 4, Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, 4 Center Dr. MSC 0425, Bethesda, MD 20892-0425. Tel.: 301-496-6149; Fax: 301-402-0079; E-mail:
tmccutchan@niaid.nih.gov.
Published, JBC Papers in Press, December 20, 2001, DOI 10.1074/jbc.M106862200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: CS, circumsporozoite; TSR, thrombospondin type I repeat; GAG, glycosaminoglycan; HSPG, heparan sulfate proteoglycan.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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