A quantitative assay for binding and inhibition of Plasmodium falciparum Erythrocyte Binding Antigen 175 reveals high affinity binding depends on both DBL domains

Abstract

Plasmodium falciparum Erythrocyte Binding Antigen 175 (PfEBA-175) engages Glycophorin A (GpA) on erythrocytes during malaria infection. The two Duffy binding like domains (F1 and F2) of PfEBA-175 that form region II (RII) are necessary for binding GpA, and are the target of neutralizing antibodies. Recombinant production of RII in Pichia pastoris and baculovirus has required mutations to prevent aberrant glycosylation or deglycosylation resulting in modifications to the protein surface that may affect antibody recognition and binding. In this study, we developed a recombinant system in Escherichia coli to obtain RII and F2 without mutations or glycosylation through oxidative refolding. The system produced refolded protein with high yields and purity, and without the need for mutations or deglycosylation. Biophysical characterization indicated both proteins are well behaved and correctly folded. We also demonstrate the recombinant proteins are functional, and develop a quantitative functional flow cytometry binding assay for erythrocyte binding ideally suited to measure inhibition by antibodies and inhibitors. This assay showed far greater binding of RII to erythrocytes over F2 and that binding of RII is inhibited by a neutralizing antibody and sialyllactose, while galactose had no effect on binding. These studies form the framework to measure inhibition by antibodies and small molecules that target PfEBA-175 in a rapid and quantitative manner using RII that is unmodified or mutated. This approach has significant advantages over current methods for examining receptor-ligand interactions and is applicable to other erythrocyte binding proteins used by the parasite.

Keywords: Adhesion; Duffy binding like domain; Erythrocyte invasion; Glycophorin A; Malaria; Oxidative refolding; PfEBA-175.

Figure 1. Purification of recombinant RII and F2 results in monodisperse samples

(A) Schematic showing the domains of PfEBA-175. F1 (green) and F2 (purple) are DBL domains that together form region II (RII). Signal sequence is in grey, C-terminal cysteine rich domain is in yellow, transmembrane domain is in dark blue and putative cytoplasmic domains are in light blue. (B) Size exclusion chromatography profile (left panel) and SDS-PAGE analysis (right panel) reveal single peaks and pure protein for (B) RII and (C) F2. Multi-angle static light scattering demonstrates (D) RII and (E) F2 are monodisperse and not crosslinked.

Figure 2. Small-angle x-ray scattering of RII and F2

The SAXS of (A) RII and (B) F2 indicate the recombinant proteins are correctly folded and match the theoretical scatter expected from crystal structures. The experimental (black) and theoretical SAXS profile (red) for the crystal structure of RII or F2 plotted as scattering intensity versus scattering momentum. The ab initio molecular envelopes (grey) are overlaid onto the crystal structure (F1 – green, F2 purple) in the insets. PDB entry 1ZRO was used to obtain structural models for both RII and F2.

Figure 3. A quantitative binding assay

(A) Erythrocytes were labeled with recombinant RII and stained with a FITC conjugated anti-6x-His antibody. Left panel: bright-field. Right panel: FITC channel. (B) Representative flow cytometry profile for erythrocytes. (C) Representative flow cytometry profile for erythrocytes labeled with recombinant RII. Gates used to quantify unlabeled and labeled populations are indicated at the top of the plot with percentages for each sample

Figure 4. Erythrocyte binding by RII measured by flow cytometry, plotting median fluorescence intensity (MFI) against enzyme treatment

Trypsin treatment of erythrocytes or addition of an anti-Glycophorin A antibody prevent binding of recombinant RII-His, while chymotrypsin treatment has no effect.

Figure 5. RII binds to erythrocytes with higher affinity than F2

Flow cytometry analysis for (A) RII-His binding in the presence of DMEM; (B) RII-His binding in the presence of DMEM supplemented with 100 mM NaCl; (C) F2-His binding in the presence of DMEM; and (D) F2-His binding in the presence of DMEM supplemented with 100 mM NaCl. (E) Quantification of binding by plotting median fluorescence intensity (MFI) against concentration of ligand in DMEM. (F) Quantification of binding by plotting median fluorescence intensity (MFI) against concentration of ligand in DMEM supplemented with 100 mM NaCl results in a linear range.

Figure 6. Inhibition of RII-His erythrocyte binding by α-2,3-sialyllactose and the inhibitory antibody R217

(A) Inhibition by α-2,3-sialyllactose results in a characteristic dose response curve with an IC50 of 13.9 mM. (B) Galactose is unable to inhibit binding. (C) Inhibition by R217 results in a characteristic dose response curve with an IC50 of 104 nM.