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.

Genetic conflicts with Plasmodium parasites and functional constraints shape the evolution of erythrocyte cytoskeletal proteins

“…Ankirin is also targeted by KAHRP 6 and other P . falciparum proteins 47 , 48 . Indeed, a previous study that analyzed the evolution of genes that interact with Apicomplexa parasites indicated that SPTB and ANK1 display features suggestive of adaptive evolution in mammals 49 .…”

The Plasmodium falciparum MESA erythrocyte cytoskeleton-binding (MEC) motif binds to erythrocyte ankyrin

“…Resolved protein bands were visualized after staining with Coomassie Brilliant Blue (CBB). Bands were excised and prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described in [35] and Shakya et al (submitted). Erythrocyte cytoskeleton proteins that bound to purified MBP-PF3D7_0402100 were identified as described in Shakya et al (submitted).…”

“…Split-luciferase assays were performed as previously described ( [35,39] and Shakya et al submitted). Kinetic split-luciferase assays were performed by adding N-FLuc-ankyrin to samples containing C-FLuc-MESA MEC construct and luciferase substrate in binding buffer.…”

“…An N-terminal fragment of 4.1R interacted with PF3D7_0402100, but not the MESA MEC, suggesting that the binding site for PF3D7_0402100 may reside in a different domain. Indeed, the PHIST domain has been reported to bind to 4.1 R (Shakya et al, submitted, and [35,43]).…”

Investigation of NOT1 proteins in regulating gene expression in plasmodium falciparum

“…NOT1.2 is predominantly detected in the cytoplasm at schizont stage as shown in facilitates the interaction between spectrin and actin. PHISTa also interacts with erythrocyte ankyrin ANK1, which links spectrin to the erythrocyte membrane and reduces the deformability of iRBCs [262,263]. The function of the third exported protein, PF3D7_0831400 is unknown.…”