Role of Plasmodium falciparum thrombospondin-related anonymous protein in host-cell interactions
© Akhouri et al. 2008
Received: 05 December 2007
Accepted: 22 April 2008
Published: 22 April 2008
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© Akhouri et al. 2008
Received: 05 December 2007
Accepted: 22 April 2008
Published: 22 April 2008
Thrombospondin-related anonymous protein (TRAP) is essential for sporozoite motility and for liver cell invasion. TRAP is a type 1 membrane protein that possesses multiple adhesive domains in its extracellular region.
Plasmodium falciparum TRAP (PfTRAP) and its subdomains were expressed in a mammalian expression system, and eleven different mutants generated to study interaction of PfTRAP with liver cells. Binding studies between HepG2 cell extracts and PfTRAP were performed using co-immunoprecipitation protocols.
Five different amino acid residues of PfTRAP that are involved in liver cell binding have been identified. These PfTRAP mutants bound to heparin like the wild type PfTRAP thereby suggesting a non-heparin mediated binding of PfTRAP to liver cells. Three Src family proteins -Lyn, Lck and CrkL which interact with PfTRAP are also identified. Liver cell extracts and immunoprecipitated Src family kinases phosphorylated PfTRAP at multiple sites. An analysis of multiple TRAP sequences revealed Src homology 3 domain (SH3) binding motifs.
Binding of PfTRAP to SH3-domain containing proteins like Src-family kinases and their ability to phosphorylate PfTRAP suggests a novel role for PfTRAP in cell signaling during sporozoite invasion and homing inside the liver cells. These data shed new light on TRAP-liver cell interactions.
Malaria is a major parasitic disease that claims more than two million lives and causes more than 500 million clinical cases every year . Despite continuous efforts to control malaria, it remains a major health problem in the tropical world, mainly due to Plasmodium falciparum. Since the search for a malaria vaccine remains elusive, there is a constant and pressing need to identify new drug and vaccine targets. Malaria infection in humans starts when infected female Anopheles mosquitoes take their blood meal and inject sporozoites into the host (human) skin. Although, it remains unclear how sporozoites reach hepatocytes, experimental evidence suggests role of various molecules for successful infection that includes components both from the host and the parasite. Apicomplexans lack a classical locomotory organelles like flagellum or cilium but show an actin based locomotion called gliding motility [2, 3]. Secretory organelles, including rhoptries and dense granules, are present in apicomplexans which secrete a number of proteins required for infectivity, motility and invasion . Two important surface molecules that play key roles in liver cell infection are the circumsporozoite protein (CS) and the thrombospondin-related anonymous protein (TRAP). CS is a multifunctional protein that is involved in sporozoite development in mosquitoes, invasion into mosquito salivary gland and into human liver cells [5–11]. TRAP is stored in the micronemes  and becomes surface exposed at the sporozoite anterior tip when parasite comes in contact with host cells . TRAP also plays an important role in liver cell invasion of sporozoites by helping sporozoites in gliding motility and in recognition of host receptors on the mosquito salivary gland and hepatocytes [14–18].
The P. falciparum TRAP (PfTRAP) extracellular domain (ECD) consists of three domains/motifs that include the A-domain (similar to the A-or I-domain which is found in integrins), the TSP (thrombospondin repeat motif, a heparin-binding module, also called the RII region) and a proline-rich segment at the C-terminus. Sequence analysis of the proline-rich segment revealed the presence of SH3-domain binding PxxP motifs in Plasmodium TRAPs. To understand the role(s) for multiple adhesive domains of PfTRAP, its extracellular domain (ECD) and subdomains were expressed using a mammalian expression system. Five crucial amino acid residues -C55, D162, C205, R307 (of RGD) and S416, that are likely to be involved in TRAP-liver cell interactions, were mapped. Surprisingly, most of these mutants did not show reduction in heparin-binding abilities, suggesting that the significant reduction in TRAP mutants binding to HepG2 cells was not due to loss of heparin binding ability. Interaction of PfTRAP with host SH3-domain containing tyrosine kinases -CrkL, Lck and Lyn that belong to the c-Src kinase family was also studied. Immunoprecipitation experiments indicated that each of these three tyrosine kinases and total HepG2 cell extract were able to phosphorylate PfTRAP at multiple sites. Together, these data indicates non-heparin based interaction of PfTRAP to liver cells and also suggests a role for PfTRAP in signaling during sporozoite invasion of hepatocytes.
PfTRAP A-domain, A+RII, A+RII+RGD and ECD were PCR amplified using forward primer 5'GCACATACCGGTAGAGATGTGCAAAACAATATAGTG 3' and reverse primers 5'GCACCCTCGAGACTATCCACTACCACTACCACTACCTTTGCTGTCCATGCAGAATCAGCATAC 3' (for A domain), 5'GCACCCTCGAGACTATCC ACTACCACTACCACTACCTTAACATCTAATGGTTCCCATTTTGG3' (forA+RII), 5'GCACCCTCGAGACTATCCACTACCACTACCACTACCTTTGGTTTTTGGACAGAAGAATTATC3' (forA+RII+RGD) and 5'GCACCCTCGAGACTATCCACTACCACTACCACTACCTTTTTATATTTATTATCTGATTCTCCTTTTTT 3' (for ECD). The amplified PCR fragments were digested with Age I and Xho I and fragments were cloned in pHL vector . Recombinant clones were identified by restriction digestion of plasmid DNA obtained from colonies after transformation of the ligation mix. The clones were further confirmed by automated DNA sequencing.
Three clones each of PfTRAP and its fragments were selected. DNA (2 μg) of each of these were added to 100 μl of incomplete DMEM. PEI (6 μg) was then added to each mix and incubated at room temperature for 15 min. In parallel, HEK 293 cells at 60–70 percent confluency (in 35-mm tissue culture plates) were washed with PBS to remove serum. Cells in the incomplete DMEM were incubated with transfection mix for 3–4 h at 37°C in a humidized CO2 incubator, and 2 ml of expression media (iDMEM pH7.2+2%FBS+1× non-essential amino acid+ 1× antibiotic and antimycotic+2 mM L-glutamine) were added to each well. The media that contained secreted protein were harvested four days post-transfection and cleared by centrifugation at 5,000 rpm. Expression of recombinant proteins was detected using western blotting with anti-penta-his and anti-PfTRAP antibodies.
Large-scale transfections (~4 litres) were carried out for pHL-PfTRAP constructs. The media were harvested from each plate and centrifuged at 5,000 rpm for 20 min at 4°C. Supernatants were filtered through 0.2 μ-filter membrane and concentrated in a diafiltration unit (Millipore) containing filtration cartridge of 10 kDa cut off. The media were dialyzed against PBS supplemented with 500 mM NaCl (pH 8.0) to remove chelators from the media. The harvested media containing PfTRAP proteins were passed through chelating column (Amersham Biosciences) that had been charged with Ni2+ and pre-equilibrated with the dialysis buffer (PBS supplemented with 500 mM NaCl pH 8.0). Columns were washed with 10 column volumes of dialysis buffer, and then with 30 and 50 mM imidazole buffer. The bound proteins were eluted in 200 mM imidazole elution buffer. Eluted protein fractions were further concentrated in a 10 kDa centriprep (vivaspin) and subjected to gel exclusion chromatography (GPC). Purity of the proteins was assessed on 12% SDS-PAGE. Similar protocols were used for PfTRAP subdomains and mutants.
1 × 105HepG2 cells were seeded in each well in a 96 well cell culture plate and were allowed to grow for 24 h. Media were discarded and cells were fixed with 4% (w/v) paraformaldehyde in PBS for 10 min. Fixed cells were washed and blocked with 2% BSA for two hours. Cells were incubated with equimolar (250 nmoles) amounts of PfTRAP ECD and its subdomains for one hour. Cells were washed and incubated with PfTRAP ECD antibody raised in rabbit followed by incubation with HRP conjugated anti-rabbit antibody. Binding was detected at 490 nm using OPD as substrate.
Hepatoma cells (HepG2 cells, Huh-7 cells) and HEK 293 cells (kidney cell line) at a confluency of 60–70% were dislodged using an enzyme free dissociation buffer (Gibco Life Sciences). Cells were washed in PBS and viability assay was performed to check for the percentage cell viability. FACS analysis was performed with hepatocytes and kidney cells HEK 293 cells. From 90–95% viable cell population, 1 × 106 cells were added to each microfuge tubes and washed thrice in FACS buffer and suspended in 50 μl of FACS buffer (PBS, pH 7.4). Cells were then incubated in FACS buffer supplemented with 1% BSA and incubated on ice for one hour. Biotinylated PfTRAP ECD protein (prepared using Pierce reagent Sulfo-NHS LC-LC Biotin) was added in equimolar amounts to each tube and allowed to bind with cells for one hour on ice. The cells were washed three times with FACS buffer, and streptavidin-PE (Calbiochem) was added to each tube at a dilution of 1: 2000. It was allowed to incubate for one hour on ice in dark. The unbound dye was washed with FACS buffer and stained cells were fixed in PBS containing 0.1% paraformaldehyde. The number of stained cells were counted using FACS and the mean value of stained cells was obtained using FACS analysis software. Stained cells alone and biotinylated-BSA bound to cells were used as controls in these experiments.
Sequence of oligos used for site directed mutagenesis of PfTRAP
Oligos used in the sets of forward and reverse primers to generate mutations in the PfTRAP polypeptide.
HepG2 cells were cultured in complete DMEM at 37°C in a humidified CO2 incubator. 1 × 105 cells were seeded in a 96-well cell-culture plate and were allowed to grow for 24 h. HepG2 cells were fixed with 4% (w/v) paraformaldehyde for 10 min at room temperature and blocked with 2% BSA for two hours at room temperature. Equal amounts of PfTRAP ECD and its mutants were incubated with HepG2 cells for one hour and bound proteins were quantified using anti-PfTRAP antibodies. Wild type PfTRAP ECD was used as positive and quantitative control for the binding of mutant proteins to HepG2 cells.
HepG2 cells and HEK 293 cells were scraped from the monolayer culture flasks using enzyme free cell dissociation buffer (Gibco). Cells were washed with PBS to get rid of any other contaminant elements. Washed cells were lysed using Down's homogenizer and the cell extracts were solubilized in 250 mM (final concentration) n-octyl-beta-D-glucopyranoside buffer. The extracts were further ultracentrifuged at 100,000 g using 60Ti (Beckmann) rotor to remove insoluble matter.
PfTRAP ECD, the A-domain and the A+RII domains (2–5 μg) were incubated with HepG2 or HEK 293 whole cell extracts in the presence of ATP at 37°C for 30 min. These PfTRAP fragments were immunoprecipitated using polyclonal anti-PfTRAP antibodies that had been pre-adsorbed on protein A Sepharose beads. Beads were washed in PBS supplemented with 50 mM n-octyl-β-D-glucopyranoside and 500 mM NaCl. The sample was again washed in PBS thrice, boiled and resolved by SDS-PAGE. PfTRAP protein bands were cut and sent for LC-MS/MS analysis. Sample containing [γ-32P] ATP in gels were dried and phosphorylation was detected by autoradiography. HepG2 cells extract alone was taken as a negative control and PfTRAP alone with ATP was taken as a nonspecific and autophosphorylation control.
PfTRAP ECD protein was incubated with whole cell extract of HepG2 and HEK 293 cells and in presence of cold ATP at 37°C for 30 min. The reaction mixture was immunoprecipitated using anti-PfTRAP antibody pre-adsorbed on protein A-sepharose beads. Samples were boiled and resolved on SDS-PAGE. Phosphorylation of PfTRAP was detected using phosphospecific antibodies against serine, threonine and tyrosine residues. Negative controls of HepG2 cells extract (with ATP without PfTRAP) and PfTRAP (with ATP without HepG2 cell extract) were used in these experiments.
HepG2 cell membrane extracts were incubated with 5 μg of purified PfTRAP ECD for 30 min at 4°C in the presence of protease inhibitor cocktail and ATP. To determine the molecule(s) interacting with PfTRAP ECD, the mixture was immunoprecipitated using anti-PfTRAP antibody pre-adsorbed on beads. The beads were then washed in PBS supplemented with 50 mM n-octyl-β-D-glucopyranoside and 500 mM NaCl, and samples were boiled, resolved on 12% SDS-PAGE. Presence or absence of tyrosine kinases was detected using specific antibodies against c-Src, Grb2, Lyn and Lck and CrkL. Similar experiments with cytoplasmic and membrane extracts were performed to detect any serine and threonine kinase interactors.
Antibodies coupled to protein A beads were used to pull down CrkL, Lck, and Lyn from HepG2 membrane extracts. The PfTRAP ECD protein (20 μg) was incubated with above beads in the presence of [γ-32P] ATP at 37°C for 1 hr. PfTRAP was eluted from the mixture using 1M NaCl, immunoprecipitated using anti-PfTRAP antibodies, resolved on SDS-PAGE and detected by autoradiography after two days of exposure.
The modeling suggested that T131 may be in an exposed loop, while D162 may be part of MIDAS motif. Mutants of T131 and D162 have been previously shown to affect sporozoite infectivity to salivary gland and liver cells . A mutant R307 was also generated as it is part of RGD motif that has been shown to be involved in integrin interactions [21, 22]. Purified mutants and wild type ECD were incubated with hepatocytes and their binding ability was assessed by ELISA assays described previously . As shown in Figure 3c, two mutants C205-A and D162-A showed ~71 and ~70% reduced binding to HepG2 cells in comparison to wild type PfTRAP ECD. Mutants C43 and C55 (in A-domain) showed reduced binding by up to ~32% and 54% respectively. Intriguingly, arginine 307, which is part of conserved RGD motif and for which no specific biological significance in PfTRAP has so far been documented, appeared important for TRAP binding to HepG2 cells. Mutation of R307, which remains conserved even in field isolates of P. falciparum TRAP, showed ~47% reduction in binding to HepG2 cells (Figure 3c). Surprisingly, most of the TRAP mutants except C55 bound to heparin in a similar fashion as the wild type PfTRAP (Figure 3b). TRAP mutant C55 showed about 50% reduction in heparin binding. These results suggest a potential role for these five residues in non-heparin mediated binding to HepG2 cells.
PfTRAP has been previously suggested to bind to HepG2 cells by interacting with sulfated proteoglycans. These studies were carried out with multimeric or soluble aggregates of recombinant PfTRAP [15, 16, 23, 24]. In the present study, recombinant PfTRAP and its subdomains were produced in monomeric, properly folded forms using a mammalian expression system. The expressed molecules were secreted out of the mammalian cells due to the presence of signal sequence at the N-terminus of the expressed proteins. Eleven different mutant TRAP proteins were generated to map the amino acid residues involved in PfTRAP binding to HepG2 cells. Results of HepG2 cell binding assays with mammalian expressed PfTRAP and its subdomains showed differential binding abilities. PfTRAP ECD bound to HepG2 cells more strongly than its other two fragments (PfTRAP A+RII and PfTRAP A). The HepG2 cell binding of TRAP was specific as neither PfTRAP ECD nor its two recombinant subdomains were able to bind HEK 293 cells (kidney cells). These results were in agreement with a previous study where a baculo-expressed PfTRAP A domain was expressed in monomeric forms .
Amino acids involved in binding of PfTRAP proteins to HepG2 cells were evaluated. Five mutants (D162, C205, C55, R307 and S416) exhibited 40–70% reduced binding to HepG2 cells. Specifically, the reduced binding of R307 (a part of the RGD triplet) indicates that this conserved sequence plays an important role in binding of PfTRAP to HepG2 cells via yet unidentified receptor(s) on liver cells. Surprisingly, the-T131A mutant that has been earlier shown to be important for hepatocyte invasion and salivary gland infection in mosquitoes [14, 25] showed only ~17% reduction in HepG2 cell binding in this study in comparison to wild type. The results of site-directed mutagenesis experiments in this paper thus suggested that amino acid residues other than those present in RII region play an important role in TRAP-HepG2 cell interaction. These mutants were also analysed for heparin binding, where no significant reduction in binding was observed. Together, these results suggest that PfTRAP binds to HepG2 cells using multiple modes; both heparin and non-heparin mediated interactions play roles in mediating TRAP interaction to HepG2 cells. Similar multiple interactions have been reported for a number of proteins involved in cell-cell attachment . For example, extracellular matrix protein fibronectin simultaneously binds to heparin sulfate proteoglycans of syndecans and one or more integrins to induce cell spreading and focal adhesion formation . Also a recent study showed that the extracellular domains of proteoglycans could bind to the protein ligands independent of heparin sulfate chains . In the present study, an arginine residue (R281) in the RxRKR motif was also mutated, but the mutant protein failed to get secreted in the supernatant raising question about the misfolding of TRAP mutant molecule as suggested by Tossavainen and coworkers .
PxxP sequence motifs in Plasmodium TRAP C-terminal regions
RDC PPKP; PVIP IK; PDVP VK; PILP IK; PEIP SK
PSNP NK; RDC PQIP; PVIP NK; RRN PNKP; PIIP QK; KDE PEIP
Results presented in this study show that PfTRAP is a multifaceted molecule that plays important roles in sporozoite binding to HepG2 cells and possibly in signal transduction. At present, it is difficult to say whether the modification of PfTRAP occurs while it is still on the sporozoite surface (thereby supporting the sporozoite activation model of transmigration) [30–32] or once it has been shed along with heparan sulfate and localized into the cytoplasm of the host cell. However, these observations open new avenues for investigating the role of PfTRAP during the transmigration of sporozoite through the host cells or during the homing of sporozoites inside the HepG2 cells.
We wish to thank all past and present members of ICGEB for their assistance in this work. We thank Dr. Ghata Gupta for assistance in making some of the clones. P.M. laboratory is supported by a grant from DBT, India. R.R.A. and Ashwani Sharma are supported by CSIR and UGC Research Fellowships. A.S. was supported by a Wellcome Trust International Senior Research Fellowship. This work is supported by an Indo-Finland grant from the Department of Biotechnology, India.
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