Biochemical, Biophysical, and Functional Characterization of Bacterially Expressed and Refolded Receptor Binding Domain ofPlasmodium vivax Duffy-binding Protein

Abstract

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes. The P. vivax Duffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includes Plasmodium falciparum sialic acid binding protein and Plasmodium knowlesi Duffy binding protein. The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli. Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate for P. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.The invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions between host receptors and parasite ligands (1). Plasmodium vivax and the related simian malaria parasite Plasmodium knowlesi require interaction with the Duffy blood group antigen to invade human erythrocytes (2, 3). P. knowlesi can also invade rhesus erythrocytes using alternative Duffy-independent receptors (4). P. falciparum commonly uses sialic acid residues of glycophorin A as invasion receptors (5-9). Like P. knowlesi, P. falciparum also invades erythrocytes by multiple pathways and is not completely dependent on sialic acid residues of glycophorin A (8,10,12,13).Parasite ligands that bind host receptors to mediate erythrocyte invasion include P. vivax and P. knowlesi Duffy-binding proteins, P. knowlesi ␤ and ␥ proteins, which bind Duffy-independent receptors on rhesus erythrocytes, and P. falciparum sialic acid-binding protein (also known as EBA-175), which binds sialic acid residues on glycophorin A (4, 14 -18). These parasite ligands share similar features and belong to a family of erythrocyte-binding proteins (19). The extracellular domain of each erythrocyte-binding protein contains two conserved cysteine-rich regions, regions II and VI, at the amino and carboxyl ends, respectively. P. falciparum EBA-175 contains a tandem duplication (F1 and F2) of the N-terminal, conserved, cysteine-rich region. The functional receptor binding domain of each erythrocyte-binding protein lies in region II (20, 21). In the case of EBA-175, region F2 was found to have receptor b...

Functional and Immunological Characterization of a Duffy Binding-Like Alpha Domain from Plasmodium falciparum Erythrocyte Membrane Protein 1 That Mediates Rosetting

“…This is in contrast to a previous study (25) showing that bacterially produced DBL␣ domains were unable to disrupt rosettes. This discrepancy suggests that recombinant proteins produced through denaturation of inclusion bodies may not reflect the correct conformation and that methods to refold the recombinant protein are needed to obtain functional DBL domains (3,33). Recombinant refolded DBL␣ was recognized by plasmas from individuals residing in regions of India and Mozambique with high rates of malaria transmission, indicating that DBL␣ contains B-cell epitopes that are naturally exposed to the host immune system.…”

“…Bound protein was eluted from the erythrocytes with 300 mM NaCl, separated by SDS-PAGE, and detected by Western blotting using a mouse monoclonal antibody against pentahistidine (Qiagen). Plasmodium vivax region II (PvRII) of the Duffy binding protein, which was previously shown to bind Duffy antigen receptor for chemokines on erythrocytes by this method (33), was used as a positive control. The intensities of the bands were quantified by densitometry scanning.…”

“…Antibody responses were expressed in arbitrary units (AU) to account for day-to-day variation and were calculated as follows: (OD sample / OD positive control ) ϫ 100. The cutoff for positivity was defined as the AU value 3 standard deviations above the arithmetic mean for the negative control plasmas (33…”

Biochemical and Immunological Characterization of Bacterially Expressed and RefoldedPlasmodium falciparum42-Kilodalton C-Terminal Merozoite Surface Protein 1

“…These differences may be due to the type of glycosylation and the extent of glycosylation on molecules produced in the two different expression systems (30). E. coli is thus well suited for the production of nonglycosylated malarial parasite antigens as vaccine candidates (23,28), and in this report we provide the first description of methods developed for refolding and purification of functional E. coli MSP1 42 produced in a bacterial expression system.…”

Antibody Responses and Avidity of Naturally Acquired Anti-Plasmodium vivax Duffy Binding Protein (PvDBP) Antibodies in Individuals from an Area with Unstable Malaria Transmission

“…29 In addition, different studies suggested that stronger humoral and cellular immune responses to PvDBP-II develop progressively with increasing age, 22,24,29,30 showing a boosting effect that was likely because of repeated exposures to the infection. 28 Also, the anti-PvDBP-II antibodies in populations living in areas endemic for P. vivax could block PvDBP-II ligand DARC-positive erythrocytes 21,31 and inhibit erythrocyte invasion in vitro . 28,32 As shown by several studies, selection of an antigen for vaccination requires a detailed understanding of natural immune responses elicited by the protein from different malariaendemic regions with various epidemiology, and the result of similar studies from one malaria-endemic region cannot be extrapolated to other areas of malaria-endemic regions in the world.…”