Genetic Evidence That Dimerization of Glycophorin a Is Critical for Red Cell Invasion By Plasmodium Falciparum but Not for the Binding of EBA-175

Introduction: Genome-wide association studies showed that resistance to malaria may be linked to the GYPA/B/E locus (Leffler et al, Science, 2017) and it is assumed that GYPA is the receptor for P. falciparum EBA-175 ligand (Sim et al, Science, 1994). The crystal structure of the erythrocyte binding domain of EBA-175 (RII) suggests that its binding to GYPA induces dimerization of RII by assembling GYPA extracellular domain (Tolia et al, Cell, 2005), a model consistent with studies pointing out the importance of the dimer status of GYPA in this interaction (Durainsingh et al, PNAS, 2003). Thanks to the description of a new GYPA variant, we investigated the role of GYPA dimerization in RBC invasion by P. falciparum.

Results: Whole exome sequencing in patient with an alloantibody to a high-prevalence antigen of unknown specificity led to the finding of eight candidate variants including a homozygous 284T>C GYPA mutation resulting in ILE95THR substitution and previously reported only at heterozygous state (frequency 0.0038 % in the Genome Aggregation Database). Others RBCs with rare blood type including Dantu and En(a-) were as reactive as the standard panel cells and the antibody was claimed to be of unknown specificity. Regarding the importance of Ile95 for GYPA dimerization (Trenker et al, JACS, 2015), we analyzed GYPA organization in RBC membranes from the proband and his homozygous brother. Indeed, immunoblotting with a specific anti-GYPA antibody showed the absence of the dimeric form of GYPA in the Ile95Thr and Dantu RBCs. Flow cytometry showed a slight decrease of GYPA but normal expression of Band3 and Wr^b antigen in both variants. However, a second anti-GYPA antibody recognizing another epitope of this protein showed a strong decrease of GYPA in these variants. These results indicate a structural alteration of the GYPA in both Ile95Thr and Dantu RBCs. Interestingly, flow cytometry revealed that recombinant PfEBA-175 is able to bind the monomeric form of GYPA in Ile95Thr but not in Dantu RBCs. Finally, ex vivo invasion assays showed a drastic decreased invasion for all Ile95Thr, Dantu, En(a-) and M^k/M^k RBCs, suggesting a crucial role of GYPA dimerization for RBC invasion by P.falciparum.

Conclusion: We reported a new Ile95Thrh variant of GYPA organized in a monomeric form at the RBC membrane. Although EBA-175 binding is normal, this variant seems to be resistant to P. falciparum invasion, as it is the case for Dantu and En(a-) variants. Our findings provide genetic evidence that dimerization of GYPA is critical for RBC invasion by Plasmodium falciparum but not for the binding of EBA-175. This outcome could explain the poorly elucidated mechanism of Dantu RBC resistance to P. falciparum invasion.