Dataset Information

The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1.

ABSTRACT: Plasmodium falciparum extensively modifies the infected red blood cell (RBC), resulting in changes in deformability, shape and surface properties. These alterations suggest that the RBC cytoskeleton is a major target for modification during infection. However, the molecular mechanisms leading to these changes are largely unknown. To begin to address this question, we screened for exported P. falciparum proteins that bound to the erythrocyte cytoskeleton proteins ankyrin 1 (ANK1) and band 4.1 (4.1R), which form critical interactions with other cytoskeletal proteins that contribute to the deformability and stability of RBCs. Yeast two-hybrid screens with ANK1 and 4.1R identified eight interactions with P. falciparum exported proteins, including an interaction between 4.1R and PF3D7_0402000 (PFD0090c). This interaction was first identified in a large-scale screen (Vignali et al., Malaria J, 7:211, 2008), which also reported an interaction between PF3D7_0402000 and ANK1. We confirmed the interactions of PF3D7_0402000 with 4.1R and ANK1 in pair-wise yeast two-hybrid and co-precipitation assays. In both cases, an intact PHIST domain in PF3D7_0402000 was required for binding. Complex purification followed by mass spectrometry analysis provided additional support for the interaction of PF3D7_0402000 with ANK1 and 4.1R. RBC ghost cells loaded with maltose-binding protein (MBP)-PF3D7_0402000 passed through a metal microsphere column less efficiently than mock- or MBP-loaded controls, consistent with an effect of PF3D7_0402000 on RBC rigidity or membrane stability. This study confirmed the interaction of PF3D7_0402000 with 4.1R in multiple independent assays, provided the first evidence that PF3D7_0402000 also binds to ANK1, and suggested that PF3D7_0402000 affects deformability or membrane stability of uninfected RBC ghosts.

SUBMITTER: Shakya B

PROVIDER: S-EPMC5738903 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1.

Shakya Bikash B Penn Wesley D WD Nakayasu Ernesto S ES LaCount Douglas J DJ

Molecular and biochemical parasitology 20170613

Plasmodium falciparum extensively modifies the infected red blood cell (RBC), resulting in changes in deformability, shape and surface properties. These alterations suggest that the RBC cytoskeleton is a major target for modification during infection. However, the molecular mechanisms leading to these changes are largely unknown. To begin to address this question, we screened for exported P. falciparum proteins that bound to the erythrocyte cytoskeleton proteins ankyrin 1 (ANK1) and band 4.1 (4. ...[more]

PMID: 28627360

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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:BackgroundDuring erythrocytic schizogony, Plasmodium falciparum interacts with the human erythrocyte membrane when it enters into, grows within and escapes from the erythrocyte. An interaction between the P. falciparum M18 aspartyl aminopeptidase (PfM18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) PfM18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed.MethodsrPfM18AAP was produced as a hexahistidine-fusion protein in Escherichia coli and purified using magnetic bead technology. The pI of the enzyme was determined by two-dimensional gel electrophoresis and the number of subunits in the native enzyme was estimated from Ferguson plots. The enzymatic activity over a pH and temperature range was tested by a coupled enzyme assay. Blot overlays were performed to validate the spectrin-PfM18AAP interaction, as well as identify additional interactions between the enzyme and other erythrocyte membrane proteins. Sequence analysis identified conserved amino acids that are expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization.ResultsrPfM18AAP has a molecular weight of ~67 kDa and the enzyme separated as three entities with pI 6.6, 6.7 and 6.9. Non-denaturing gel electrophoresis indicated that rPfM18AAP aggregated into oligomers. An in vitro coupled enzyme assay showed that rPfM18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37 degrees C. The spectrin-binding region of PfM18AAP is not found in Homo sapiens, Saccharomyces cerevisiae and otherPlasmodium species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with rPfM18AAP against spectrin and erythrocyte membrane proteins indicated that rPfM18AAP binds to spectrin, as well as to protein 4.1, protein 4.2, actin and glyceraldehyde 3-phosphate dehydrogenase.ConclusionStudies characterizing rPfM18AAP showed that this enzyme interacts with erythrocyte spectrin and other membrane proteins. This suggests that, in addition to its proposed role in hemoglobin digestion, PfM18AAP performs other functions in the erythrocyte host and can utilize several substrates, which highlights the multifunctional role of malaria enzymes.

Dataset Information

The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1.

Publications

The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1.

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