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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 Plasmodium orthologs can efficiently transfer several different phospholipids.A, phospholipid transfer from radiolabeled HL60 cells to acceptor vesicles by PKH_020910 and PCHAS_020730. Transfer of phospholipids from the cells to the acceptor vesicles was set up as described under “Experimental Procedures” and the lipids transferred were identified by analytical thin layer chromatography, followed by exposure of the TLC plate to a phosphorimager screen. B, quantitation of the phospholipid transfer detected in panel A. Background values of the No protein sample were used as baseline and were subtracted from the values of the transfer by the Plasmodium proteins and the control protein. Quantitation was performed as described under “Experimental Procedures.” C, transfer of phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin by PFA0210c was measured using the same transfer assay as in Fig. 3A, except that radioactive phosphatidylethanolamine, phosphatidylinositol, or sphingomyelin was incorporated into the donor vesicles. The higher background levels of transfer seen in the negative controls in the case of phosphatidylethanolamine and phosphatidylinositol likely reflect the presence of impurities within the preparations of the radioactive lipids (see “Experimental Procedures”).
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Figure 5: PFA0210c and its Plasmodium orthologs can efficiently transfer several different phospholipids.A, phospholipid transfer from radiolabeled HL60 cells to acceptor vesicles by PKH_020910 and PCHAS_020730. Transfer of phospholipids from the cells to the acceptor vesicles was set up as described under “Experimental Procedures” and the lipids transferred were identified by analytical thin layer chromatography, followed by exposure of the TLC plate to a phosphorimager screen. B, quantitation of the phospholipid transfer detected in panel A. Background values of the No protein sample were used as baseline and were subtracted from the values of the transfer by the Plasmodium proteins and the control protein. Quantitation was performed as described under “Experimental Procedures.” C, transfer of phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin by PFA0210c was measured using the same transfer assay as in Fig. 3A, except that radioactive phosphatidylethanolamine, phosphatidylinositol, or sphingomyelin was incorporated into the donor vesicles. The higher background levels of transfer seen in the negative controls in the case of phosphatidylethanolamine and phosphatidylinositol likely reflect the presence of impurities within the preparations of the radioactive lipids (see “Experimental Procedures”).

Mentions: Most phospholipid transfer proteins display a profound preference for specific phospholipids, although some can transfer several different phospholipids (49). To test the ability of PFA0210c and the P. knowlesi and P. chabaudi orthologs to transfer phospholipids other than PC, we initially tested which phospholipids could be transferred by PKH_020910 and PCHAS_020730 in a permeabilized cell assay. In this assay, the test protein is incubated with permeabilized cells that have been labeled with [14C]acetate, and therefore contain a wide variety of radiolabeled phospholipids, in the presence of acceptor vesicles. After separation of the cells and the acceptor vesicles, the identity of the phospholipids transferred to the vesicles can be determined by analytical TLC. This revealed that PKH_020910 and PCHAS_020730 can transfer phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine in addition to phosphatidylcholine (Fig. 5, A and B). Minimal transfer activity was detected in the presence of MBP-LacZ or in the absence of protein. To verify that PFA0210c can also transfer phosphatidylethanolamine and phosphatidylinositol, we produced donor vesicles containing purified 14C-labeled phosphatidylethanolamine or phosphatidylinositol. PFA0210c was able to transfer both of these phospholipids (Fig. 5C), whereas the control protein PITP was able to transfer only PI, as expected (45). In addition, PFA0210c was also able to transfer sphingomyelin, similar to PITP, which has previously been shown to transfer sphingomyelin (50). This result strongly suggests that PFA0210c and its Plasmodium orthologs have the capacity to transfer multiple phospholipids.


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 Plasmodium orthologs can efficiently transfer several different phospholipids.A, phospholipid transfer from radiolabeled HL60 cells to acceptor vesicles by PKH_020910 and PCHAS_020730. Transfer of phospholipids from the cells to the acceptor vesicles was set up as described under “Experimental Procedures” and the lipids transferred were identified by analytical thin layer chromatography, followed by exposure of the TLC plate to a phosphorimager screen. B, quantitation of the phospholipid transfer detected in panel A. Background values of the No protein sample were used as baseline and were subtracted from the values of the transfer by the Plasmodium proteins and the control protein. Quantitation was performed as described under “Experimental Procedures.” C, transfer of phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin by PFA0210c was measured using the same transfer assay as in Fig. 3A, except that radioactive phosphatidylethanolamine, phosphatidylinositol, or sphingomyelin was incorporated into the donor vesicles. The higher background levels of transfer seen in the negative controls in the case of phosphatidylethanolamine and phosphatidylinositol likely reflect the presence of impurities within the preparations of the radioactive lipids (see “Experimental Procedures”).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814793&req=5

Figure 5: PFA0210c and its Plasmodium orthologs can efficiently transfer several different phospholipids.A, phospholipid transfer from radiolabeled HL60 cells to acceptor vesicles by PKH_020910 and PCHAS_020730. Transfer of phospholipids from the cells to the acceptor vesicles was set up as described under “Experimental Procedures” and the lipids transferred were identified by analytical thin layer chromatography, followed by exposure of the TLC plate to a phosphorimager screen. B, quantitation of the phospholipid transfer detected in panel A. Background values of the No protein sample were used as baseline and were subtracted from the values of the transfer by the Plasmodium proteins and the control protein. Quantitation was performed as described under “Experimental Procedures.” C, transfer of phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin by PFA0210c was measured using the same transfer assay as in Fig. 3A, except that radioactive phosphatidylethanolamine, phosphatidylinositol, or sphingomyelin was incorporated into the donor vesicles. The higher background levels of transfer seen in the negative controls in the case of phosphatidylethanolamine and phosphatidylinositol likely reflect the presence of impurities within the preparations of the radioactive lipids (see “Experimental Procedures”).
Mentions: Most phospholipid transfer proteins display a profound preference for specific phospholipids, although some can transfer several different phospholipids (49). To test the ability of PFA0210c and the P. knowlesi and P. chabaudi orthologs to transfer phospholipids other than PC, we initially tested which phospholipids could be transferred by PKH_020910 and PCHAS_020730 in a permeabilized cell assay. In this assay, the test protein is incubated with permeabilized cells that have been labeled with [14C]acetate, and therefore contain a wide variety of radiolabeled phospholipids, in the presence of acceptor vesicles. After separation of the cells and the acceptor vesicles, the identity of the phospholipids transferred to the vesicles can be determined by analytical TLC. This revealed that PKH_020910 and PCHAS_020730 can transfer phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine in addition to phosphatidylcholine (Fig. 5, A and B). Minimal transfer activity was detected in the presence of MBP-LacZ or in the absence of protein. To verify that PFA0210c can also transfer phosphatidylethanolamine and phosphatidylinositol, we produced donor vesicles containing purified 14C-labeled phosphatidylethanolamine or phosphatidylinositol. PFA0210c was able to transfer both of these phospholipids (Fig. 5C), whereas the control protein PITP was able to transfer only PI, as expected (45). In addition, PFA0210c was also able to transfer sphingomyelin, similar to PITP, which has previously been shown to transfer sphingomyelin (50). This result strongly suggests that PFA0210c and its Plasmodium orthologs have the capacity to transfer multiple phospholipids.

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