Interactions between Plasmodium falciparum skeleton-binding protein 1 and the membrane skeleton of malaria-infected red blood cells

Abstract

During development inside red blood cells (RBCs), Plasmodium falciparum malaria parasites export proteins that associate with the RBC membrane skeleton. These interactions cause profound changes to the biophysical properties of RBCs that underpin the often severe and fatal clinical manifestations of falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is one such exported parasite protein that plays a major role in malaria pathogenesis since its exposure on the parasitised RBC surface mediates their adhesion to vascular endothelium and placental syncytioblasts. En route to the RBC membrane skeleton, PfEMP1 transiently associates with Maurer's clefts (MCs), parasite-derived membranous structures in the RBC cytoplasm. We have previously shown that a resident MC protein, skeleton-binding protein 1 (SBP1), is essential for the placement of PfEMP1 onto the RBC surface and hypothesised that the function of SBP1 may be to target MCs to the RBC membrane. Since this would require additional protein interactions, we set out to identify binding partners for SBP1. Using a combination of approaches, we have defined the region of SBP1 that binds specifically to defined sub-domains of two major components of the RBC membrane skeleton, protein 4.1R and spectrin. We show that these interactions serve as one mechanism to anchor MCs to the RBC membrane skeleton, however, while they appear to be necessary, they are not sufficient for the translocation of PfEMP1 onto the RBC surface. The N-terminal domain of SBP1 that resides within the lumen of MCs clearly plays an essential, but presently unknown role in this process.

Keywords: Cell adhesion and mechanics; Malaria; Plasmodium falciparum; Protein 4.1R; Red blood cell; Spectrin.

Fig. 1

Association of the C-terminal domain of SBP1 with the RBC membrane skeleton. A. Schematic representation of SBP1. Repeat regions (light grey) and the trans-membrane domain (TM) are indicated. B. Binding of the SBP1 C-terminal domain (SBP1-C) to the RBC skeleton. RBCs resealed with recombinant SBP1-C appended to YFP (SBP1-C–YFP; lane 1), RBCs resealed with YFP alone (YFP; lane 2), intact RBCs incubated with SBP1-C–YFP then washed (lane 3) or untreated RBCs (lane 4) were solubilised in TX-100. The TX-100 soluble and insoluble fractions were resolved by SDS-PAGE and immuno-blotted using an anti-YFP antibody. C. IOV binding assay with EMP3F1a as a positive control and EMP3F5 and MBP as negative controls.

Fig. 2

Binding of SBP1 C-terminal domain to protein 4.1R. A. GST-tagged or N- or C-terminal SBP1 was used to pull down 4.1R purified from RBCs. Binding was detected by immunoblotting using specific anti-4.1R antibodies. This result is a representative example of 3 independent binding experiments all of which showed identical results. B. Schematic representation of protein 4.1R, showing the defined structural regions that were expressed as recombinant fragments as previously described [25]. The Coomassie-stained gel shows the purified recombinant proteins. C. Binding of the SBP1 C-terminal domain to protein 4.1R domains. GST-tagged 4.1R domains (expressed in pGEX-4T-2) were used to pull down His-tagged SBP1 C-term (expressed in pET24a). SBP1 binding was detected by immuno-blotting using an anti-hexahistidine antibody. D. Inhibition of binding of SBP1 C-terminal domain to protein 4.1R by the 16 kDa domain of protein 4.1. His-tagged SBP1-C was pre-incubated with increasing concentrations of either GST-tagged 16 kDa protein 4.1 domain or GST-tagged 22/24 kDa domain at room temperature for 30 min. Mixtures were then added to a 96-well plate that had been coated with 4.1R. The binding of His-tagged SBP1 C-term was detected using an anti-His antibody. Note the progressive decrease of SBP1-C binding to protein 4.1R with the increasing concentrations of 16 kDa but not with 22/24 kDa domains of protein 4.1. Data are the mean ± S.E.M. from three separate experiments.

Fig. 3

Binding of SBP1 C-terminal domain to spectrin. A. GST-tagged or N- or C-terminal SBP1 was used to pull down full-length spectrin purified from RBCs. Binding was detected by immunoblotting using specific anti-spectrin antibodies. A representative example of 3 independent binding experiments is shown but all 3 experiments showed essentially identical results. B. Schematic representation of the spectrin α- and β-chains; regions expressed as recombinant fragments are indicated [24]. Western blot showing the binding of SBP1-C to spectrin fragments, specifically the α-N5 repeats. C. α-N5 repeats were divided into the individual repeats expressed as GST fusion proteins and used to pull down the hexahistidine-tagged SBP1 C. Binding was detected by western blotting using anti-hexahistidine antibody. GST was used as negative control in all experiments. SBP1 binding was detected by immuno-blotting with an anti-hexahistidine antibody. D. Inhibition of binding of SBP1-C to the α4 repeat region of α-spectrin. Competitive inhibition was measured by an enzyme-linked immunosorbent assay (ELISA). SBP1-C was coated on 96-well plates then pre-incubated with various concentrations of the α4 repeat region of α-spectrin prior to the addition of purified spectrin dimer. The binding of spectrin dimer to SBP1-C decreased with progressively increasing concentrations of the α4 repeat region of α-spectrin. Data are the mean ± S.E.M. from three separate experiments.

Fig. 4

Localisation of SBP-1 in normal and protein 4.1R-deficient red blood cells. Representative confocal immunofluorescence images of normal (A) and 4.1R-deficient (C) RBCs immuno-labelled for Maurer's clefts (SBP1; green) and the RBC membrane skeleton (Glycophorin A, GpA; red). Co-localisation of SBP1 and GpA was analysed using ImageJ and the fraction of MCs (SBP1) co-localised with the RBC membrane skeleton in 73 normal (B) and 102 protein 4.1R-deficient (D) RBCs plotted. A significant shift in the mean co-localisation of SBP1 with GpA is observed between normal and 4.1R-deficient RBCs.

Fig. 5

Localisation of SBP1 C-terminal mutants. A. Southern blot analysis of the SBP1–AMA1-C parasite clones. The left panel is a schematic representation of the expected integration event. The restriction enzymes and the size of the expected bands are indicated. The right panel is the southern blot probed with an SBP1 gene-specific probe. The corresponding bands from the wild type (3D7), transfected plasmid, and integration events in the SBP1–AMA1-C clones (1C9, 1D7 and 1G3) are shown. B. Immunofluorescence analysis of RBCs infected with either normal (3D7) or transgenic parasites expressing a chimeric SBP1 protein in which the C-terminal domain of SBP1 was replaced with the C-terminal domain of P. falciparum AMA1 (SBP1–AMA1-C).

Fig. 6

Functional analysis of the SBP1 C-terminal domain. A. Representative images from immunofluorescence analysis RBCs infected with wild type 3D7 parasites or the three clonal parasite lines for the SBP1–AMA1-C chimeras (1C9, 1D7 and 1G3). Green represents SBP1 and red is spectrin. B. Scatter plot of the co-localisation analysis. Each data point represents the Manders’ coefficient calculated from multiple individual images. The horizontal line represents the mean and the error bars are the SEM. C. Representative scanning electron micrographs of RBCs infected with wild type 3D7 (top panel) or SBP1–AMA1-C (bottom panel) parasites showing the similarity in knob number and morphology between the parasite lines. D. The level of adhesion of PRBCs to platelet-expressed CD36 under flow conditions (0.1 Pa). Adherent PRBCs represent the number of 3D7- or SBP1–AMA1-C- (1C9, 1D7 and 1G3) PRBCs that adhered per 10⁷ PRBCs perfused through platelet-coated flow chambers. Data represent the mean + SEM for 3 independent experiments. E. Trypsin cleavage assay to determine the surface exposure of PfEMP1 in RBCs infected with SBP1–AMA1-C clonal parasite lines. The cleavage product at 75 kDa detected by anti-VARC (PfEMP1) antibody in wild type 3D7 and all SBP1–AMA1-C clones indicate that trafficking and surface exposure of PfEMP1 is similar for all parasite lines. Trypsin-mediated cleavage of glycophorin A (GpA) was used as a positive control for trypsin cleavage activity.