The Epitope of Monoclonal Antibodies Blocking Erythrocyte Invasion by Plasmodium falciparum Map to The Dimerization and Receptor Glycan Binding Sites of EBA-175

Xavier Ambroggio,Joan Aebig,Lubin Jiang,Harold Obiakor,Jan Lukszo,David L Narum,Photini Sinnis

doi:10.1371/journal.pone.0056326

Abstract

The malaria parasite, Plasmodium falciparum, and related parasites use a variety of proteins with Duffy-Binding Like (DBL) domains to bind glycoproteins on the surface of host cells. Among these proteins, the 175 kDa erythrocyte binding antigen, EBA-175, specifically binds to glycophorin A on the surface of human erythrocytes during the process of merozoite invasion. The domain responsible for glycophorin A binding was identified as region II (RII) which contains two DBL domains, F1 and F2. The crystal structure of this region revealed a dimer that is presumed to represent the glycophorin A binding conformation as sialic acid binding sites and large cavities are observed at the dimer interface. The dimer interface is largely composed of two loops from within each monomer, identified as the F1 and F2 β-fingers that contact depressions in the opposing monomers in a similar manner. Previous studies have identified a panel of five monoclonal antibodies (mAbs) termed R215 to R218 and R256 that bind to RII and inhibit invasion of erythrocytes to varying extents. In this study, we predict the F2 β-finger region as the conformational epitope for mAbs, R215, R217, and R256, and confirm binding for the most effective blocking mAb R217 and R215 to a synthetic peptide mimic of the F2 β-finger. Localization of the epitope to the dimerization and glycan binding sites of EBA-175 RII and site-directed mutagenesis within the predicted epitope are consistent with R215 and R217 blocking erythrocyte invasion by Plasmodium falciparum by preventing formation of the EBA-175– glycophorin A complex.

Highlights

Malaria as a clinical disease is due to the complex cyclical process of merozoite invasion of red blood cells (RBCs)
Binding of EBA-175 region II (RII) mutants to human erythrocytesa athe rosette number was an average counted from two biological repeats in each experiment. berythrocyte-binding assay was performed with normal erythrocytes in two independent experiments. cerythrocyte-binding assay was performed with neuraminidase-treated erythrocytes Nm, neuraminidase
P479, Y559, and W560 (P335, Y415, and W416 of rEBA-175 RII), were predicted by PepSurf and/or Mapitope to belong to the epitopes of monoclonal antibodies (mAbs) R215, R217, and R256 (Figure 1, Text S1, and Figure S1)

Summary

Introduction

Malaria as a clinical disease is due to the complex cyclical process of merozoite invasion of red blood cells (RBCs). Merozoite proteins function during invasion in a coordinated manner while subject to an active immune response and phenotypic variation of human erythrocytes. The 175 kDa erythrocyte binding antigen (EBA-175) was one of the earliest micronemal proteins identified that bound human erythrocytes and whose function could be blocked by antibodies [1]. Within EBA-175, a region identified as RII contains two Duffy Binding Like (DBL) domains called F1 and F2 that are responsible for binding glycophorin A in a sialic acid dependent manner [2,3,4]. A recombinant EBA-175 RII protein [5] was crystallized in the form of a dimer [6], indicating that EBA-175 dimerization may be biologically important for receptor binding and RBC invasion

Methods

Results

Conclusion