title: 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.
authors.affiliation: Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, Saint Louis, MO, USA.
authors.forename: Nichole D
authors.initials: ND
authors.lastname: Salinas
authors.affiliation: Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, Saint Louis, MO, USA. Electronic address: tolia@wustl.edu.
authors.forename: Niraj H
authors.initials: NH
authors.lastname: Tolia
mesh_terms: D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000235:genetics*; D011485:Protein Binding; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C065668:erythrocyte-binding antigen 175, Plasmodium
keywords: Adhesion; Duffy binding like domain; Erythrocyte invasion; Glycophorin A; Malaria; Oxidative refolding; PfEBA-175