Plasmodium falciparum-CD36 Structure-Function Relationships Defined by Ortholog Scanning Mutagenesis

Abstract

Background: The interaction of Plasmodium falciparum-infected erythrocytes (IEs) with the host receptor CD36 is among the most studied host-parasite interfaces. CD36 is a scavenger receptor that binds numerous ligands including the cysteine-rich interdomain region (CIDR)α domains of the erythrocyte membrane protein 1 family (PfEMP1) expressed on the surface of IEs. CD36 is conserved across species, but orthologs display differential binding of IEs.

Methods: In this study, we exploited these differences, combined with the recent crystal structure and 3-dimensional modeling of CD36, to investigate malaria-CD36 structure-function relationships and further define IE-CD36 binding interactions.

Results: We show that a charged surface in the membrane-distal region of CD36 is necessary for IE binding. Moreover, IE interaction with this binding surface is influenced by additional CD36 domains, both proximal to and at a distance from this site.

Conclusions: Our data indicate that subtle sequence and spatial differences in these domains modify receptor conformation and regulate the ability of CD36 to selectively interact with its diverse ligands.

Keywords: host-parasite interaction; infected erythrocytes; ortholog swap mutagenesis; scavenger receptor.

Figure 1.

Human CD36 model. (A) Three-dimensional model based on the crystal structure of lysosomal integral membrane protein (LIMP)-2 [16]. Structure depicted in gray and domains of interest color coded. Domain I in red: SHIQQVM; domain II in green: YADVSDGNR; and other amino acids of interest in blue: T, LLKK. Model generated using PyMol. Helical bundle and apex region marked. (B) The cationic patch on the surface of CD36 viewed from the top. Cationic residues in the area depicted in magenta, whereas flanking domain I (red) and domain II (green) are highlighted. The rest of the structure is shown in gray. (C) Domain I and II alignment between human and bovine CD36 sequences.

Figure 2.

Infected erythrocyte (IE) binding by human CD36 mutants. (A) Quantification of IE binding assays on mutant CD36-green fluorescent protein (GFP). Infected erythrocyte binding of a selected experiment in triplicate is shown (mean + standard deviation), and statistical significance compared with hCD36 wild type (**, P < .01) is marked. Binding experiments were fixed and mounted for analysis using Zeiss Apotome. Tiles (10 × 10) were recorded using ×25 objective. (B) Confocal images (×63) of representative IE binding experiments of GFP fusion expressing cell lines (green). For fluorescent visualization, IEs were stained with phycoerythrin (PE)-conjugated antiglycophorin A as surface marker (red) and 4’,6-diamidino-2-phenylindole (DAPI) to stain the nucleus (blue). A 10-μm size bar is shown. Surface expression was normalized to BD555455 monoclonal antibody binding.

Figure 3.

Infected erythrocyte binding by human and bovine CD36. (A) Quantification of IE binding of a representative experiment in triplicate (mean + standard deviation). Statistical significance compared with hCD36 (blue) and bCD36 (purple) wild type are shown (*, P < .05; ***, P < .0001). (B) Confocal images (×63) of representative parasite binding experiments of green fluorescent protein (GFP) fusion expressing cell lines (blue, 4’,6-diamidino-2-phenylindole [DAPI]; green, GFP fusion construct; red, antiglycophorin A-phycoerythrin [PE]). A 10-μm size bar is shown. hCD36 surface expression was normalized to FA6-152 monoclonal antibody binding, and bCD36 surface expression was normalized to CIDRα6_D3_IT4var12 binding (Supplementary Figure 2).

Figure 4.

Infected erythrocyte (IE) binding to human- and bovine-based CD36 mutants. (A) Human-based constructs (blue). Quantification of IE binding (mean + standard deviation [SD]) of experiments done in triplicate with statistical significance based on comparison to hCD36 wild type (*, P < .05). (B) Bovine-based constructs (purple). Quantification of IE binding (mean + SD) of experiments done in triplicate, and statistical significance compared with bCD36 wild type (*, P < .05 and ***, P < .0001). hCD36 mutants surface expression were normalized to FA6-152 monoclonal antibody binding, and bCD36 mutants surface expression were normalized to CIDRα6_D3_IT4var12 binding (Supplementary Figure 2). GFP, green fluorescent protein.

Figure 5.

CIDR peptides binding to CD36 mutants. (A) Quantification of peptide binding to hCD36 and point mutants by flow cytometry performed in duplicate (mean + standard deviation). Statistical significance compared with hCD36 wild type (*, P < .05). (B) Flow cytometry charts for selected constructs. (C) Quantification of peptide binding to hCD36-based constructs (blue). Statistical significance compared with hCD36 wild type (*, P < .05). (D) Quantification of peptide binding to bCD36-based constructs (purple). Statistical significance compared with bCD36 wild type (*, P < .05). Surface expression of A was normalized to BD555455 monoclonal antibody binding, hCD36 mutants in C were normalized to FA6-152 monoclonal antibody binding, and bCD36 mutants in D were normalized to CIDRα6_D3_IT4var12 binding (Supplementary Figure 2). GFP, green fluorescent protein.

Figure 6.

DiI_OxLDL binding. (A) Quantification of oxidized low-density lipoprotein (oxLDL) binding to human-based (blue) and bovine-based (purple) constructs by flow cytometry performed in duplicate (mean + standard deviation) (*, P < .05). (B) Flow cytometry charts for selected constructs. hCD36 mutant results were surface normalized to FA6-152 monoclonal antibody binding, and bCD36 mutants were normalized to CIDRα6_D3_IT4var12 binding (Supplementary Figure 2). GFP, green fluorescent protein.