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An erythrocyte cytoskeleton-binding motif in exported Plasmodium falciparum proteins.

ABSTRACT: Binding of exported malaria parasite proteins to the host cell membrane and cytoskeleton contributes to the morphological, functional, and antigenic changes seen in Plasmodium falciparum-infected erythrocytes. One such exported protein that targets the erythrocyte cytoskeleton is the mature parasite-infected erythrocyte surface antigen (MESA), which interacts with the N-terminal 30-kDa domain of protein 4.1R via a 19-residue sequence. We report here that the MESA erythrocyte cytoskeleton-binding (MEC) domain is present in at least 13 other P. falciparum proteins predicted to be exported to the host cell. An alignment of the putative cytoskeleton-binding sequences revealed a conserved aspartic acid at the C terminus that was omitted from the originally reported binding domain. Mutagenesis experiments demonstrated that this aspartic acid was required for the optimal binding of MESA to inside-out vesicles (IOVs) prepared from erythrocytes. Using pulldown assays, we characterized the binding of fragments encoding the MEC domains from PFE0040c/MESA and six other proteins (PF10_0378, PFA0675w, PFB0925w, PFD0095c, PFF1510w, and PFI1790w) to IOVs. All seven proteins bound to IOVs, with MESA showing the strongest affinity in saturation binding experiments. We further examined the interaction of the MEC domain proteins with components of the erythrocyte cytoskeleton and showed that MESA, PF10_0378, and PFA0675w coprecipitated full-length 4.1R from lysates prepared from IOVs. These data demonstrated that the MEC motif is present and functional in at least six other P. falciparum proteins that are exported to the host cell cytoplasm.

SUBMITTER: Kilili GK

PROVIDER: S-EPMC3209045 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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An erythrocyte cytoskeleton-binding motif in exported Plasmodium falciparum proteins.

Kilili Geoffrey K GK LaCount Douglas J DJ

Eukaryotic cell 20110909 11

Binding of exported malaria parasite proteins to the host cell membrane and cytoskeleton contributes to the morphological, functional, and antigenic changes seen in Plasmodium falciparum-infected erythrocytes. One such exported protein that targets the erythrocyte cytoskeleton is the mature parasite-infected erythrocyte surface antigen (MESA), which interacts with the N-terminal 30-kDa domain of protein 4.1R via a 19-residue sequence. We report here that the MESA erythrocyte cytoskeleton-binding ...[more]

PMID: 21908595

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Project description:Erythrocytes are reservoirs of important epoxide-containing lipid signaling molecules, including epoxyeicosatrienoic acids (EETs). EETs function as vasodilators and anti-inflammatory modulators in the bloodstream. Bioactive EETs are hydrolyzed to less active diols (dihydroxyeicosatrienoic acids) by epoxide hydrolases (EHs). The malaria parasite Plasmodium falciparum infects host red blood cells (RBCs) and exports hundreds of proteins into the RBC compartment. In this study, we show that two parasite epoxide hydrolases, P falciparum epoxide hydrolases 1 (PfEH1) and 2 (PfEH2), both with noncanonical serine nucleophiles, are exported to the periphery of infected RBCs. PfEH1 and PfEH2 were successfully expressed in Escherichia coli, and they hydrolyzed physiologically relevant erythrocyte EETs. Mutations in active site residues of PfEH1 ablated the ability of the enzyme to hydrolyze an epoxide substrate. Overexpression of PfEH1 or PfEH2 in parasite-infected RBCs resulted in a significant alteration in the epoxide fatty acids stored in RBC phospholipids. We hypothesize that the parasite disruption of epoxide-containing signaling lipids leads to perturbed vascular function, creating favorable conditions for binding and sequestration of infected RBCs to the microvascular endothelium. The malaria parasite exports hundreds of proteins into the erythrocyte compartment. However, for most of these proteins, their physiological function is unknown. In this study, we investigate two "hypothetical" proteins of the α/β-hydrolase fold family that share sequence similarity with epoxide hydrolases (EHs)-enzymes that destroy bioactive epoxides. Altering EH expression in parasite-infected erythrocytes resulted in a significant change in the epoxide fatty acids stored in the host cell. We propose that these EH enzymes may help the parasite to manipulate host blood vessel opening and inflame the vessel walls as they pass through the circulation system. Understanding how the malaria parasite interacts with its host RBCs will aid in our ability to combat this deadly disease.

Project description:Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity.

Dataset Information

An erythrocyte cytoskeleton-binding motif in exported Plasmodium falciparum proteins.

Publications

An erythrocyte cytoskeleton-binding motif in exported Plasmodium falciparum proteins.

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