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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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The phosphatidylcholine transfer activity of PFA0210c resides within the START domain.A, phosphatidylcholine transfer activity of N-terminal truncations. The transfer activity of versions of MBP-PFA0210c-His6 containing a portion of PFA0210c beginning at the indicated N-terminal residue were tested using the same transfer assay as described in the legend to Fig. 3A. PFA0210cN99, PFA0210cN123, and PFA0210cN144 contain the entire START domain; PFA0210cN168 through PFA0210cN290 lack increasingly large portions of the START domain. B, phosphatidylcholine transfer activity of C-terminal truncations. The transfer activity of the MBP-PFA0210c-His6 containing the indicated region of PFA0210c was tested. The versions (or constructs) containing residues 144–384 and 144–418 contain the entire START domain.
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Figure 4: The phosphatidylcholine transfer activity of PFA0210c resides within the START domain.A, phosphatidylcholine transfer activity of N-terminal truncations. The transfer activity of versions of MBP-PFA0210c-His6 containing a portion of PFA0210c beginning at the indicated N-terminal residue were tested using the same transfer assay as described in the legend to Fig. 3A. PFA0210cN99, PFA0210cN123, and PFA0210cN144 contain the entire START domain; PFA0210cN168 through PFA0210cN290 lack increasingly large portions of the START domain. B, phosphatidylcholine transfer activity of C-terminal truncations. The transfer activity of the MBP-PFA0210c-His6 containing the indicated region of PFA0210c was tested. The versions (or constructs) containing residues 144–384 and 144–418 contain the entire START domain.

Mentions: All orthologs of PFA0210c contain a polypeptide sequence in addition to the annotated START domain, although as observed above this sequence is not highly conserved between the orthologs (supplemental Fig. S1). To explore whether the additional sequence plays a part in phospholipid transfer activity and to determine the minimal domain required for phospholipid transfer, we produced C-terminal and N-terminal truncations of PFA0210c and assessed them using the radioactive phospholipid transfer assay. The N terminus of the protein contains the signal sequence (amino acids 1–24) and the HT motif (amino acids 60–65), which are likely removed during the course of export (46). Similarly, as the region immediately following the HT has been shown to be important for export (14, 15, 47), it is unlikely to have a role in the function of the exported protein. Furthermore, the segment between amino acid residues 65 and 99 is not conserved between PFA0210c and its Plasmodium orthologues, making it unlikely that it has a conserved function. For our analysis of the functional domain of PFA0210c, we therefore used recombinant constructs starting at amino acid residue 99. Our assays showed that recombinant proteins containing amino acid residues 99, 123, or 144 (PFA0210cN99, PFA0210cN123, and PFA0210cN144, respectively) as the N-terminal residue can transfer phospholipids, although the transfer activity of PFA0210cN144 was slightly lower than that of PFA0210cN99 and PFA0210N123. When PFA0210c was truncated further, as in the cases of PFA0210cN168 through PFA0210ccN290, no transfer activity was detected. PFA0210cN99, PFA0210cN123, and PFA0210cN144 contain the entire annotated START domain, whereas PFA0210cN168 through PFA0201cN290 lack increasingly large portions of the START domain (Fig. 4A). The slightly decreased activity of PFA0210cN144 could be the result of the close proximity of the MBP portion of the fusion protein.


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)

The phosphatidylcholine transfer activity of PFA0210c resides within the START domain.A, phosphatidylcholine transfer activity of N-terminal truncations. The transfer activity of versions of MBP-PFA0210c-His6 containing a portion of PFA0210c beginning at the indicated N-terminal residue were tested using the same transfer assay as described in the legend to Fig. 3A. PFA0210cN99, PFA0210cN123, and PFA0210cN144 contain the entire START domain; PFA0210cN168 through PFA0210cN290 lack increasingly large portions of the START domain. B, phosphatidylcholine transfer activity of C-terminal truncations. The transfer activity of the MBP-PFA0210c-His6 containing the indicated region of PFA0210c was tested. The versions (or constructs) containing residues 144–384 and 144–418 contain the entire START domain.
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Related In: Results  -  Collection

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Figure 4: The phosphatidylcholine transfer activity of PFA0210c resides within the START domain.A, phosphatidylcholine transfer activity of N-terminal truncations. The transfer activity of versions of MBP-PFA0210c-His6 containing a portion of PFA0210c beginning at the indicated N-terminal residue were tested using the same transfer assay as described in the legend to Fig. 3A. PFA0210cN99, PFA0210cN123, and PFA0210cN144 contain the entire START domain; PFA0210cN168 through PFA0210cN290 lack increasingly large portions of the START domain. B, phosphatidylcholine transfer activity of C-terminal truncations. The transfer activity of the MBP-PFA0210c-His6 containing the indicated region of PFA0210c was tested. The versions (or constructs) containing residues 144–384 and 144–418 contain the entire START domain.
Mentions: All orthologs of PFA0210c contain a polypeptide sequence in addition to the annotated START domain, although as observed above this sequence is not highly conserved between the orthologs (supplemental Fig. S1). To explore whether the additional sequence plays a part in phospholipid transfer activity and to determine the minimal domain required for phospholipid transfer, we produced C-terminal and N-terminal truncations of PFA0210c and assessed them using the radioactive phospholipid transfer assay. The N terminus of the protein contains the signal sequence (amino acids 1–24) and the HT motif (amino acids 60–65), which are likely removed during the course of export (46). Similarly, as the region immediately following the HT has been shown to be important for export (14, 15, 47), it is unlikely to have a role in the function of the exported protein. Furthermore, the segment between amino acid residues 65 and 99 is not conserved between PFA0210c and its Plasmodium orthologues, making it unlikely that it has a conserved function. For our analysis of the functional domain of PFA0210c, we therefore used recombinant constructs starting at amino acid residue 99. Our assays showed that recombinant proteins containing amino acid residues 99, 123, or 144 (PFA0210cN99, PFA0210cN123, and PFA0210cN144, respectively) as the N-terminal residue can transfer phospholipids, although the transfer activity of PFA0210cN144 was slightly lower than that of PFA0210cN99 and PFA0210N123. When PFA0210c was truncated further, as in the cases of PFA0210cN168 through PFA0210ccN290, no transfer activity was detected. PFA0210cN99, PFA0210cN123, and PFA0210cN144 contain the entire annotated START domain, whereas PFA0210cN168 through PFA0201cN290 lack increasingly large portions of the START domain (Fig. 4A). The slightly decreased activity of PFA0210cN144 could be the result of the close proximity of the MBP portion of the fusion protein.

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