Scientists suggest blocking PfRh5 interaction with erythrocyte protein BSG may represent a new therapeutic target.

Researchers have identified a malaria parasite ligand that appears to be vital for all strains of the Plasmodium falciparum to infect red blood cells. The protein PfRh5 interacts with the Ok blood group antigen basigin (BSG, also known as CD147, EMMPRIN, and M6), says the Wellcome Trust Sanger Institute investigators who led the research.

Their subsequent studies showed that it was possible to block erythrocyte invasion by all P. falciparum strains tested, using relatively low concentrations of anti-BSG antibodies. Gavin J. Wright, Ph.D., and colleagues, report their findings in Nature in a paper titled “Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.”

Most research focused on identifying P. falciparum merozoite proteins that are essential to erythrocyte invasion has focused on two major parasite protein families, the EBAs and RhS, the researchers explain. However, while erythrocyte receptors have been found for a number of the parasite ligands, none of the receptor-ligand pairs identified to date are essential for erythrocyte invasion of all parasite strains tested.

PfRh5, on the other hand, appears unique amongst the EBAs and Rhs because it can’t be deleted in any P. falciparum strain and is thus apparently essential for parasite blood growth in blood cells. Prior research has indicated that both native and recombinant PfRh5 bind to an unknown glycosylated receptor.  

In order to identify an erythrocyte receptor for PfRh5, the Sanger team compiled a library of 40 human erythrocyte proteins for which the whole ectodomain was expected to be expressed as a soluble recombinant protein by mammalialn cells and screened these systematically for interactions with a recombinant PfRh5 protein, also produced by mammalian cells. To do this they used a screening technology developed at the Sanger, known as Avexis (Avidity-based Extracellular Interaction Screen), which is designed to detect direct low-affinity protein interactions between ectodomain fragments expressed as either biotin-tagged baits or highly avid pentameric β-lactamase-tagged preys.

The results identified just one erythrocyte receptor bait, BSG, which interacted with the PfRh5 prey. BSG is a member of the immunoglobulin superfamily (IgSF) and exists in both long (three IgSF domains, BSG-L) and short (two IgSF domains, BSG-S) splice isoforms. Although the screening work was carried out using BSG-L, the researchers subsequently confirmed that PfRh5 could also bind with BSG-S, which is believed to be the major isoform expressed on erythrocytes, as long as both the BSG-S IgSF domains were intact.

Confirmation that PfRh5 interacts directly with BSG-S and BSG-L was carried out using purified proteins and surface plasmon resonance (SPR). Interestingly, changing or removing the glycan residues on BSG had no effect on PfRh5 binding, indicating that the binding site is located in the BSG protein core.   

The team then used two approaches to determine whether PfRh5-BSG interaction is an absolute requirement for erythrocyte invasion by the parasite. In the first they added soluble BSG-S to invasion assays to specifically compete with the blood cell-bound receptor. In the second they knocked down BSG protein expression in erythrocytes.

Addition of the soluble BSG-S protein into an invasion assay strongly inhibited the ability of P. falciparum to gain entry to red blood cells across all strains of parasite evaluated, in a dose-dependent manner, relative to controls, which included each of the two, non-binding BSG-SIgSF domains added individually. The researchers confirmed that the inhibitory effects of the soluble BSG-S protein weren’t indirect by instead adding to the invasion assay two independent purified anti-BSG mAbs, which also resulted in a potent invasion-blocking effect even at low antibody concentrations.

P. falciparum isolates vary widely in terms of their ability to invade erythrocytes treated with different receptor-modifying enzymes such as trypsin, chymotrypsin, and neuraminidase, which demonstrates differential dependencies on erythrocyte receptors for invasion, the authors continue. To see if BSG represented a critical invasion receptor across P. falciparum lines known to use different invasion pathways, one of the anti-BSG antibodies, MEM-M6/6, was tested on nine culture-adapted strains representing seven different PfRh5 sequence variants along with six freshly isolated P. falciparum strains.  As hoped, MEM-M6/6 potently inhibited the invasion of all the strains tested.

The team then used the knockdown approach to independently verify that the PfRh5-BSG interaction is critical to erythrocyte invasion. Differentiating erythrocytes were isolated from hematopoietic stem cells transduced with lentiviruses containing either a short hairpin RNA targeting BSG or a scrambled shRNA control (pLKO).

The knockdown led to a roughly 50% to 60% reduction in cell surface BSG levels in the resulting mature erythrocytes, relative to the pLKO control cells. When the knockouts were tested in invasion assays, the erythrocyte BSG deficiency significantly inhibited invasion of different P. falciparum strains.

“The interaction between BSG and PfRh5 is essential for parasite entry and may perform a fundamentally different function to the other EBA and Rh proteins, which are involved in redundant, partially overlapping invasion pathways,” the authors conclude. “The dependence on a single receptor-ligand pair across many P. falciparum strains may provide new possibilities for therapeutic intervention.”

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