Critical glycosylated residues in exon three of erythrocyte glycophorin A engage Plasmodium falciparum EBA-175 and define receptor specificity

Abstract

Erythrocyte invasion is an essential step in the pathogenesis of malaria. The erythrocyte binding-like (EBL) family of Plasmodium falciparum proteins recognizes glycophorins (Gp) on erythrocytes and plays a critical role in attachment during invasion. However, the molecular basis for specific receptor recognition by each parasite ligand has remained elusive, as is the case with the ligand/receptor pair P. falciparum EBA-175 (PfEBA-175)/GpA. This is due largely to difficulties in producing properly glycosylated and functional receptors. Here, we developed an expression system to produce recombinant glycosylated and functional GpA, as well as mutations and truncations. We identified the essential binding region and determinants for PfEBA-175 engagement, demonstrated that these determinants are required for the inhibition of parasite growth, and identified the glycans important in mediating the PfEBA-175-GpA interaction. The results suggest that PfEBA-175 engages multiple glycans of GpA encoded by exon 3 and that the presentation of glycans is likely required for high-avidity binding. The absence of exon 3 in GpB and GpE due to a splice site mutation confers specific recognition of GpA by PfEBA-175. We speculate that GpB and GpE may have arisen due to selective pressure to lose the PfEBA-175 binding site in GpA. The expression system described here has wider application for examining other EBL members important in parasite invasion, as well as additional pathogens that recognize glycophorins. The ability to define critical binding determinants in receptor-ligand interactions, as well as a system to genetically manipulate glycosylated receptors, opens new avenues for the design of interventions that disrupt parasite invasion.

Importance: Plasmodium falciparum uses distinct ligands that bind host cell receptors for invasion of red blood cells (RBCs) during malaria infection. A key entry pathway involves P. falciparum EBA-175 (PfEBA-175) recognizing glycophorin A (GpA) on RBCs. Despite knowledge of this protein-protein interaction, the complete mechanism for specific receptor engagement is not known. PfEBA-175 recognizes GpA but is unable to engage the related RBC receptor GpB or GpE. Understanding the necessary elements that enable PfEBA-175 to specifically recognize GpA is critical in developing specific and potent inhibitors of PfEBA-175 that disrupt host cell invasion and aid in malaria control. Here, we describe a novel system to produce and manipulate the host receptor GpA. Using this system, we probed the elements in GpA necessary for engagement and thus for host cell invasion. These studies have important implications for understanding how ligands and receptors interact and for the future development of malaria interventions.

FIG 1

Schematic of constructs for glycophorin A (GpA) expression. (A) Domain structure of the GpA constructs, including the signal sequence (GpA SS), the extracellular domain (GpA amino acids 23 to 84), and the Fc fusion protein with the PreScission protease (PP) and 6×His tag, (B) Amino acid sequence alignment of GpA constructs used and GpB for comparison. O-glycans are indicated by black circles, and the single N-glycan is indicated by a grey circle. Sequence similarity is indicated by grey shading.

FIG 2

Expression, purification, and analysis of recombinant GpA and variants expressed as fusions to an Fc domain and 6-His tag. (A) Reduced SDS-PAGE gel of recombinant GpA, recombinant GpA glycan mutants, and recombinant GpA exon 3Δ. Apparent molecular mass estimates by SDS-PAGE are consistent with heavily glycosylated samples. (B) SDS-PAGE gel of rGpA under oxidizing (−DTT) and reducing (+DTT) conditions reveal that rGpA is a disulfide linked dimer. (C) Size exclusion chromatography of recombinant GpA, RII-175 (73 kDa), and the Fc-His domain (54 kDa) alone show that rGpA elutes before RII-175 or Fc, consistent with a molecular mass of 110 kDa.

FIG 3

Recognition of untreated and enzyme-treated rGpA and rGpA exon 3Δ by antibodies and lectins. UT, untreated; NA, neuraminidase treated; PNGF, PNGase F treated; Fc, Fc domain alone. (A) Anti-GpA monoclonal antibody (Ab) binding to rGpA. The antibody binds to UT rGpA but not Fc. NA and PNGF treatment of rGpA did not affect binding to the antibody. (B) Anti-GpA monoclonal antibody binding to rGpA exon 3Δ. The antibody is unable to bind rGpA exon 3Δ, while it retains the ability to bind rGpA. A positive control for binding using an anti-His antibody binds to both rGpA and rGpA exon 3Δ, demonstrating that the inability of the anti-GpA antibody is not due to a lack of protein. (C) SBA from Glycine max binding to rGpA. Binding is observed upon NA treatment, since sialic acid removal exposes GalNAc residues predominantly found in O-glycans. (D) SBA from Glycine max binding to rGpA exon 3Δ. Again, binding is observed upon NA treatment. (E) Lectin PNA from Arachis hypogaea binding to rGpA. PNA binding is observed only on NA treatment, since removal of the sialic acid exposes the correct sugar residue for binding. PNA does not bind the Fc domain. (F) PNA from Arachis hypogaea binding to rGpA exon 3Δ. Again, binding is observed upon NA treatment. A.U. = absorbance units.

FIG 4

PfEBA-175 RII binding depends on glycans in exon 3. (A) Anti-PfEBA-175 RII Western blots after pulldown with rGpA wild type, mutant rGpAs, and rGpA exon 3Δ. The panels are three independent pulldowns, demonstrating experimental reproducibility. PfEBA-175 RII binds to rGpA (lane 1), and neuraminidase (NA) treatment of rGpA abolishes binding (lane 2). The single mutations S66A (lane 3), S69A (lane 4), and T72A (lane 5) have various effects on binding. The triple mutation S66A/S69A/T72A (lane 6) completely abolishes binding. rGpA exon 3Δ (lane 7) is unable to significantly bind PfEBA-175 RII, indicating that residues 23 to 49 of GpA are unable to support efficient binding. (B) Band intensities were quantified by densitometry and plotted as means ± SEM. Significance compared to result for GpA was determined by a one-way ANOVA. Asterisks denote P < 0.001 (***), P < 0.01 (**), and P < 0.05 (*).

FIG 5

Recombinant GpA is fully functional and can compete with endogenous GpA on erythrocytes to inhibit parasite invasion. Purified rGpA or Fc was serially diluted and tested for its ability to inhibit parasite growth. Representative inhibition curves are shown for the 3D7 (A), Dd2 (B), or FVO/FCR1 (C) strain. Growth inhibition by rGpA was determined by microscopy analysis of parasitemia, and the results are expressed as growth normalized to that of untreated control wells (0 µM).

FIG 6

rGpA triple mutant and rGpA exon 3Δ fail to inhibit parasite invasion of 3D7, Dd2, or FVO/FCR1. rGpA, rGpA triple mutant, or rGpA exon 3Δ was purified and used at 30 µM in a growth inhibition assay with the 3D7 (A), Dd2 (B), FVO/FCR1 (C), or HB3 (D) strain. Fc was included in the experiment as a control. HB3 showed no inhibition by rGpA, since this strain invades through sialic-acid-independent pathways that do not rely on PfEBA-175. Parasitemia at the end was analyzed by microscopy, and growth was normalized to that of untreated control wells (0 µM). Data shown are means ± SEM from three independent experiments, each done in triplicate. Asterisks denote P < 0.001 (***) and P < 0.01 (**), and “n.s.” denotes not significant.