Infected erythrocytes expressing DC13 PfEMP1 differ from recombinant proteins in EPCR-binding function

Abstract

Recent advances have identified a new paradigm for cerebral malaria pathogenesis in which endothelial protein C receptor (EPCR) is a major host receptor for sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the brain and other vital organs. The parasite adhesins that bind EPCR are members of the IE variant surface antigen family Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) containing specific adhesion domains called domain cassette (DC) 8 and DC13. The binding interaction site between PfEMP1 and EPCR has been mapped by biophysical and crystallography studies using recombinant proteins. However, studies examining the interaction of native PfEMP1 on the IE surface with EPCR are few. We aimed to study binding to EPCR by IEs expressing DC8 and DC13 PfEMP1 variants whose recombinant proteins have been used in key prior functional and structural studies. IE binding to EPCR immobilized on plastic and on human brain endothelial cells was examined in static and flow adhesion assays. Unexpectedly, we found that IEs expressing the DC13 PfEMP1 variant HB3var03 or IT4var07 did not bind to EPCR on plastic and the binding of these variants to brain endothelial cells was not dependent on EPCR. IEs expressing the DC8 variant IT4var19 did bind to EPCR, but this interaction was inhibited if normal human serum or plasma was present, raising the possibility that IE-EPCR interaction may be prevented by plasma components under physiological conditions. These data highlight a discrepancy in EPCR-binding activity between PfEMP1 recombinant proteins and IEs, and indicate the critical need for further research to understand the pathophysiological significance of the PfEMP1-EPCR interaction.

Keywords: EPCR; PfEMP1; cell adhesion; endothelium; malaria.

Fig. 1.

DC8 IT4var19, but not DC13 HB3var03 and IT4var07, IEs bind to rEPCR. IT4var19 IE (A), HB3var03 IE (B), and IT4var07 IE (C) binding to 50 μg/mL receptors absorbed onto plastic dishes tested under static conditions is shown. The filled circles are IEs that stained positive with DC8 or DC13 PfEMP1 homologous antibodies, and the empty circles did not stain with the DC8 or DC13 PfEMP1 antibodies. Half-filled circles are IEs that were not successfully stained with antibodies due to loss of bound IEs during staining. The difference in mean binding values compared with the PBS control from n ≥ 3 independent experiments was analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) Binding of IT4var19, HB3var03, and IT4var07 IEs to 50 μg/mL rEPCR coated onto a microslide under flow conditions at a shear stress of 1 dyne/cm². The dotted line shows the background levels of binding seen with an uncoated microslide. Each data point is from an independent experiment, and the mean and SEM are shown.

Fig. 2.

DC8 IT4var19, but not DC13 HB3var03 and IT4var07, IE binding to HBECs is reduced by EPCR antibodies and soluble recombinant protein (rEPCR). IT4var19 IE (A), HB3var03 IE (B), and IT4var07 IE (C) binding to the immortalized HBEC-5i line under static conditions with 20 μg/mL antibody or soluble recombinant protein is shown. (D) Binding of the three parasite lines to primary HBMECs under static conditions with 20 μg/mL soluble rEPCR. Each data point is from an independent experiment, and the mean and SEM are shown. The difference in mean values compared with control (with no added antibody or protein) was analyzed by one-way ANOVA with Tukey’s multiple comparisons test (A–C) and paired t test (D). **P < 0.01. The vertical dotted line indicates experiments performed separately. mAb, monoclonal antibody; pAb, polyclonal antibody.

Fig. 3.

DC8 IT4var19, but not DC13 HB3var03 and IT4var07, IE binding to the HBEC line HBEC-5i is reduced by EPCR siRNA knockdown. (A) Expression of EPCR on HBEC-5i by immunostaining and flow cytometry. HBEC-5i was transfected with EPCR siRNA (orange) or a negative control siRNA (blue). Forty-eight hours after transfection, HBEC-5i was stained with 1 μg/mL goat polyclonal antibody to EPCR (orange/blue) or goat IgG-negative control (red), followed by Alexa Fluor 488 donkey anti-goat IgG at a 1:1,500 dilution. Data shown are representative of four similar experiments. (B–D) Binding of IEs to HBEC-5i transfected with control siRNA or EPCR siRNA under static conditions. The data shown are the mean and SEM from four independent experiments. The difference in mean values compared with the siRNA control was analyzed by a two-tailed paired t test. *P < 0.05. NS, not significant.

Fig. 4.

DC8 IT4var19, but not DC13 HB3var03 and IT4var07, IE binding to the HBEC cell line HBEC-5i is reduced by recombinant PfEMP1 CIDR proteins. IT4var19 IE (A), HB3var03 IE (B), and IT4var07 IE (C) binding to HBEC-5i under static conditions with 50 μg/mL soluble recombinant PfEMP1 protein is shown. The data shown are the mean and SEM from three independent experiments. The difference in mean binding values compared with control (with no added protein) was analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. *P < 0.05. NS, not significant.

Fig. 5.

DC8 IT4var19, but not DC13 HB3var03 and IT4var07, IE binding to the HBEC line HBEC-5i is abolished by human serum. IT4var19 IE (A), HB3var03 IE (B), and IT4var07 IE (C) binding to HBEC-5i under static conditions with pooled human or FBS is shown. (D) Binding of IT4var19 IEs to HBEC-5i with sEPCR-depleted pooled human serum. Each data point is from an independent experiment, and the mean and SEM are shown. The difference in mean values compared with the “No Serum” control (A–C) or “0.1% BSA” control (D) was analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 6.

Human serum inhibits adhesion of DC8 IT4var19-expressing IEs to rEPCR. IT4var19 IE binding to 50 μg/mL receptors absorbed onto plastic dishes tested under static conditions in binding medium with 10% pooled human serum or 0.1% BSA (No serum) is shown. The mean and SEM from three independent experiments are shown. The differences in mean values compared with the No serum controls were analyzed by a two-tailed paired t test for each receptor. *P < 0.05; **P < 0.01. NS, not significant.