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Identification of a Plasmodium falciparum phospholipid transfer protein.

van Ooij C, Withers-Martinez C, Ringel A, Cockcroft S, Haldar K, Blackman MJ - J. Biol. Chem. (2013)

Bottom Line: Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes.In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles.Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.

ABSTRACT
Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte.

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PFA0210c and its orthologs possess phosphatidylcholine transfer activity.A, phosphatidylcholine transfer activity of PFA0210c and its orthologs, PKH_020910 and PCHAS_020730. Phospholipid transfer was measured by the transfer of radioactive phosphatidylcholine from donor vesicles to acceptor vesicles as described under “Experimental Procedures.” The phosphatidylinositol transfer protein PITP is included as a positive control. The MBP-LacZ fusion and the no protein samples serve as negative controls. B, PFA0210c can bind phospholipids directly. A transfer assay was set up as in A, except that no donor vesicles were added. At the concentration of protein used in the experiment in A, no accumulation of radioactivity in the supernatant is detected, but adding 10-fold more protein results in accumulation of radioactivity in the supernatant. Numbers below the axis indicate the concentration of MBP-PFA0210c-His6 in μg/ml. C, transfer activity of point mutants of PFA0210c. Phosphatidylcholine transfer of PFA0210c carrying a mutation at the indicated position was measured using the same assay as in panel A. Note that mutation of the highly conserved residue Arg-324 (see Fig. 1) does not fully inactivate the protein. D, time dependence of phosphatidylcholine transfer by PFA0210c. The concentration of PFA0210c used was 5 μg/ml. E, dose dependence of PFA0210c-dependent phosphatidylcholine transfer. Numbers below the axis indicate the concentration of MBP-PFA0210c-His6 in μg/ml.
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Figure 3: PFA0210c and its orthologs possess phosphatidylcholine transfer activity.A, phosphatidylcholine transfer activity of PFA0210c and its orthologs, PKH_020910 and PCHAS_020730. Phospholipid transfer was measured by the transfer of radioactive phosphatidylcholine from donor vesicles to acceptor vesicles as described under “Experimental Procedures.” The phosphatidylinositol transfer protein PITP is included as a positive control. The MBP-LacZ fusion and the no protein samples serve as negative controls. B, PFA0210c can bind phospholipids directly. A transfer assay was set up as in A, except that no donor vesicles were added. At the concentration of protein used in the experiment in A, no accumulation of radioactivity in the supernatant is detected, but adding 10-fold more protein results in accumulation of radioactivity in the supernatant. Numbers below the axis indicate the concentration of MBP-PFA0210c-His6 in μg/ml. C, transfer activity of point mutants of PFA0210c. Phosphatidylcholine transfer of PFA0210c carrying a mutation at the indicated position was measured using the same assay as in panel A. Note that mutation of the highly conserved residue Arg-324 (see Fig. 1) does not fully inactivate the protein. D, time dependence of phosphatidylcholine transfer by PFA0210c. The concentration of PFA0210c used was 5 μg/ml. E, dose dependence of PFA0210c-dependent phosphatidylcholine transfer. Numbers below the axis indicate the concentration of MBP-PFA0210c-His6 in μg/ml.

Mentions: To investigate the potential phospholipid transfer activity of PFA0210c, we expressed a portion of the protein, including the putative START domain (residues 144 to 418) as well as the corresponding region from the Plasmodium knowlesi (PKH_020910) and P. chabaudi (PCHAS_020730) orthologs. All three recombinant proteins were produced as fusions to MBP, and included a C-terminal His6 tag. The purified recombinant proteins were then tested for their capacity to transfer phosphatidylcholine using a previously described transfer assay in which donor vesicles consisting of phosphatidylcholine, phosphatidic acid, N-lactosylethanolamine, and a radiolabeled phospholipid are mixed with acceptor vesicles containing phosphatidylcholine and phosphatidic acid. After incubation with recombinant protein to allow transfer of phospholipids to occur, the donor and acceptor vesicle populations are separated by agglutination and centrifugation of the donor vesicles. The residual radioactivity in the supernatant, which contains just the acceptor vesicles and the recombinant protein, reflects the amount of phospholipid transferred. As a positive control for these assays we used the well described phospholipid transfer protein PITPα, which can transfer phosphatidylinositol and phosphatidylcholine (45). As shown in Fig. 3A, all three recombinant Plasmodium orthologs transferred PC at levels similar to that of the control protein PITP. No transfer activity was detected in the absence of acceptor vesicles, indicating that the observed activity reflects transfer to acceptor vesicles and is not the result of association of radiolabeled lipid with the input recombinant protein (Fig. 3B). However, when a 10-fold higher level of transfer protein was used in this assay, accumulation of radioactivity could be detected in the supernatant, at significantly higher levels than that accumulated when the same high concentration of MBP-LacZ was added, indicating that the protein can bind directly to radioactive lipids (Fig. 3B).


Identification of a Plasmodium falciparum phospholipid transfer protein.

van Ooij C, Withers-Martinez C, Ringel A, Cockcroft S, Haldar K, Blackman MJ - J. Biol. Chem. (2013)

PFA0210c and its orthologs possess phosphatidylcholine transfer activity.A, phosphatidylcholine transfer activity of PFA0210c and its orthologs, PKH_020910 and PCHAS_020730. Phospholipid transfer was measured by the transfer of radioactive phosphatidylcholine from donor vesicles to acceptor vesicles as described under “Experimental Procedures.” The phosphatidylinositol transfer protein PITP is included as a positive control. The MBP-LacZ fusion and the no protein samples serve as negative controls. B, PFA0210c can bind phospholipids directly. A transfer assay was set up as in A, except that no donor vesicles were added. At the concentration of protein used in the experiment in A, no accumulation of radioactivity in the supernatant is detected, but adding 10-fold more protein results in accumulation of radioactivity in the supernatant. Numbers below the axis indicate the concentration of MBP-PFA0210c-His6 in μg/ml. C, transfer activity of point mutants of PFA0210c. Phosphatidylcholine transfer of PFA0210c carrying a mutation at the indicated position was measured using the same assay as in panel A. Note that mutation of the highly conserved residue Arg-324 (see Fig. 1) does not fully inactivate the protein. D, time dependence of phosphatidylcholine transfer by PFA0210c. The concentration of PFA0210c used was 5 μg/ml. E, dose dependence of PFA0210c-dependent phosphatidylcholine transfer. Numbers below the axis indicate the concentration of MBP-PFA0210c-His6 in μg/ml.
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Figure 3: PFA0210c and its orthologs possess phosphatidylcholine transfer activity.A, phosphatidylcholine transfer activity of PFA0210c and its orthologs, PKH_020910 and PCHAS_020730. Phospholipid transfer was measured by the transfer of radioactive phosphatidylcholine from donor vesicles to acceptor vesicles as described under “Experimental Procedures.” The phosphatidylinositol transfer protein PITP is included as a positive control. The MBP-LacZ fusion and the no protein samples serve as negative controls. B, PFA0210c can bind phospholipids directly. A transfer assay was set up as in A, except that no donor vesicles were added. At the concentration of protein used in the experiment in A, no accumulation of radioactivity in the supernatant is detected, but adding 10-fold more protein results in accumulation of radioactivity in the supernatant. Numbers below the axis indicate the concentration of MBP-PFA0210c-His6 in μg/ml. C, transfer activity of point mutants of PFA0210c. Phosphatidylcholine transfer of PFA0210c carrying a mutation at the indicated position was measured using the same assay as in panel A. Note that mutation of the highly conserved residue Arg-324 (see Fig. 1) does not fully inactivate the protein. D, time dependence of phosphatidylcholine transfer by PFA0210c. The concentration of PFA0210c used was 5 μg/ml. E, dose dependence of PFA0210c-dependent phosphatidylcholine transfer. Numbers below the axis indicate the concentration of MBP-PFA0210c-His6 in μg/ml.
Mentions: To investigate the potential phospholipid transfer activity of PFA0210c, we expressed a portion of the protein, including the putative START domain (residues 144 to 418) as well as the corresponding region from the Plasmodium knowlesi (PKH_020910) and P. chabaudi (PCHAS_020730) orthologs. All three recombinant proteins were produced as fusions to MBP, and included a C-terminal His6 tag. The purified recombinant proteins were then tested for their capacity to transfer phosphatidylcholine using a previously described transfer assay in which donor vesicles consisting of phosphatidylcholine, phosphatidic acid, N-lactosylethanolamine, and a radiolabeled phospholipid are mixed with acceptor vesicles containing phosphatidylcholine and phosphatidic acid. After incubation with recombinant protein to allow transfer of phospholipids to occur, the donor and acceptor vesicle populations are separated by agglutination and centrifugation of the donor vesicles. The residual radioactivity in the supernatant, which contains just the acceptor vesicles and the recombinant protein, reflects the amount of phospholipid transferred. As a positive control for these assays we used the well described phospholipid transfer protein PITPα, which can transfer phosphatidylinositol and phosphatidylcholine (45). As shown in Fig. 3A, all three recombinant Plasmodium orthologs transferred PC at levels similar to that of the control protein PITP. No transfer activity was detected in the absence of acceptor vesicles, indicating that the observed activity reflects transfer to acceptor vesicles and is not the result of association of radiolabeled lipid with the input recombinant protein (Fig. 3B). However, when a 10-fold higher level of transfer protein was used in this assay, accumulation of radioactivity could be detected in the supernatant, at significantly higher levels than that accumulated when the same high concentration of MBP-LacZ was added, indicating that the protein can bind directly to radioactive lipids (Fig. 3B).

Bottom Line: Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes.In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles.Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.

ABSTRACT
Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte.

Show MeSH
Related in: MedlinePlus