Critical glycosylated residues in exon three of erythrocyte Glycophorin A engage Plasmodium falciparum EBA-175 and define receptor specificity

Erythrocyte invasion is an essential step in the pathogenesis of malaria. The erythrocyte binding-like (EBL) family of Plasmodium falciparum proteins recognizes glycophorins (Gp) on erythrocytes and plays a critical role in attachment during invasion. However, the molecular basis for specific receptor recognition by each parasite ligand has remained elusive, as is the case with the ligand/receptor pair P. falciparum EBA-175 (PfEBA-175)/GpA. This is due largely to difficulties in producing properly glycosylated and functional receptors. Here, we developed an expression system to produce recombinant glycosylated and functional GpA, as well as mutations and truncations. We identified the essential binding region and determinants for PfEBA-175 engagement, demonstrated that these determinants are required for the inhibition of parasite growth, and identified the glycans important in mediating the PfEBA-175–GpA interaction. The results suggest that PfEBA-175 engages multiple glycans of GpA encoded by exon 3 and that the presentation of glycans is likely required for high-avidity binding. The absence of exon 3 in GpB and GpE due to a splice site mutation confers specific recognition of GpA by PfEBA-175. We speculate that GpB and GpE may have arisen due to selective pressure to lose the PfEBA-175 binding site in GpA. The expression system described here has wider application for examining other EBL members important in parasite invasion, as well as additional pathogens that recognize glycophorins. The ability to define critical binding determinants in receptor-ligand interactions, as well as a system to genetically manipulate glycosylated receptors, opens new avenues for the design of interventions that disrupt parasite invasion

Structural and functional basis for inhibition of erythrocyte invasion by antibodies that target Plasmodium falciparum EBA-175

Disrupting erythrocyte invasion by Plasmodium falciparum is an attractive approach to combat malaria. P. falciparum EBA-175 (PfEBA-175) engages the host receptor Glycophorin A (GpA) during invasion and is a leading vaccine candidate. Antibodies that recognize PfEBA-175 can prevent parasite growth, although not all antibodies are inhibitory. Here, using x-ray crystallography, small-angle x-ray scattering and functional studies, we report the structural basis and mechanism for inhibition by two PfEBA-175 antibodies. Structures of each antibody in complex with the PfEBA-175 receptor binding domain reveal that the most potent inhibitory antibody, R217, engages critical GpA binding residues and the proposed dimer interface of PfEBA-175. A second weakly inhibitory antibody, R218, binds to an asparagine-rich surface loop. We show that the epitopes identified by structural studies are critical for antibody binding. Together, the structural and mapping studies reveal distinct mechanisms of action, with R217 directly preventing receptor binding while R218 allows for receptor binding. Using a direct receptor binding assay we show R217 directly blocks GpA engagement while R218 does not. Our studies elaborate on the complex interaction between PfEBA-175 and GpA and highlight new approaches to targeting the molecular mechanism of P. falciparum invasion of erythrocytes. The results suggest studies aiming to improve the efficacy of blood-stage vaccines, either by selecting single or combining multiple parasite antigens, should assess the antibody response to defined inhibitory epitopes as well as the response to the whole protein antigen. Finally, this work demonstrates the importance of identifying inhibitory-epitopes and avoiding decoy-epitopes in antibody-based therapies, vaccines and diagnostics

Regulation of Marginal Zone B-Cell Differentiation by MicroRNA-146a.

B-cell development in the bone marrow is followed by specification into functional subsets in the spleen, including marginal zone (MZ) B-cells. MZ B-cells are classically characterized by T-independent antigenic responses and require the elaboration of distinct gene expression programs for development. Given their role in gene regulation, it is not surprising that microRNAs are important factors in B-cell development. Recent work demonstrated that deficiency of the NFκB feedback regulator, miR-146a, led to a range of hematopoietic phenotypes, but B-cell phenotypes have not been extensively characterized. Here, we found that miR-146a-deficient mice demonstrate a reduction in MZ B-cells, likely from a developmental block. Utilizing high-throughput sequencing and comparative analysis of developmental stage-specific transcriptomes, we determined that MZ cell differentiation was impaired due to decreases in Notch2 signaling. Our studies reveal miR-146a-dependent B-cell phenotypes and highlight the complex role of miR-146a in the hematopoietic system

Multimeric assembly, clustered interactions, and molecular complexes between parasite ligands and host-cell receptors for invasion.

<p>(<b>A</b>) PfTRAP engagement with heparan sulphate proteoglycans (HSPGs) on the hepatocyte surface; (<b>B</b>) proteolytic processing and shedding of PfMSP1 exposes the 19 kDa fragment (MSP1₁₉) that forms an invasion complex with MSP9 and the band 3 homodimer; (<b>C</b>) assembly of two PfEBA-175 monomers around dimeric glycophorin A of erythrocytes; (<b>D</b>) stepwise multimeric assembly of two PvDBP with two Duffy antigen/receptor for chemokines on reticulocyte surface; (<b>E</b>) monomeric interaction between PfEBA-140 and glycophorin C on erythrocytes; (<b>F</b>) proposed complexes of TgMIC2 and TgM2AP and of TgMIC1, TgMIC4, and TgMIC6 on the parasite surface; (<b>G</b>) variations in oligomeric states of GPI-anchored surface antigens (SAGs) create distinct interaction sites.</p

Multimeric assembly, clustered interactions, and molecular complexes between parasite ligands and host-cell receptors for invasion.

<p>(<b>A</b>) PfTRAP engagement with heparan sulphate proteoglycans (HSPGs) on the hepatocyte surface; (<b>B</b>) proteolytic processing and shedding of PfMSP1 exposes the 19 kDa fragment (MSP1₁₉) that forms an invasion complex with MSP9 and the band 3 homodimer; (<b>C</b>) assembly of two PfEBA-175 monomers around dimeric glycophorin A of erythrocytes; (<b>D</b>) stepwise multimeric assembly of two PvDBP with two Duffy antigen/receptor for chemokines on reticulocyte surface; (<b>E</b>) monomeric interaction between PfEBA-140 and glycophorin C on erythrocytes; (<b>F</b>) proposed complexes of TgMIC2 and TgM2AP and of TgMIC1, TgMIC4, and TgMIC6 on the parasite surface; (<b>G</b>) variations in oligomeric states of GPI-anchored surface antigens (SAGs) create distinct interaction sites.</p

Antibody epitope identification by immunofluorescence assay.

<p>DBL domain proteins were surface expressed on HEK293 cells and probed with R217 or R218 as primary and Alexafluor-546 labeled anti-IgG₁ as secondary. Green channel shows GFP tagged expressed protein. Red channel shows Alexafluor-546 labeled proteins on HEK293 cell surface. Merged channel shows overlap between green and red channels.</p

Direct antibody-inhibition of GpA binding by RII.

<p>(A) RII binds to GpA (lane 1), but not to neuraminidase treated GpA (GpA-NA – lane 2). Addition of R217 IgG or Fab prevents RII from binding GpA (lane 3 and 4). Neither R218 IgG (lane 5), R218 Fab (lane 6), control IgG (lane 7) nor control Fab (lane 8), block RII/GpA receptor binding. Binding is specific as neuraminidase treatment prevents binding in all cases (lanes 2 and 9–14). Note that an increased signal is observed with R218 IgG over R218 Fab (compare lanes 5 and 6) suggesting bivalent binding. This assay was performed with RII at 3 µM and antibody or Fab fragments at 6 µM. (B) Titration of R217 demonstrates the specificity of interaction as all concentrations around and above the available RII concentration (3 µM) prevents binding (lanes 6–10). At concentrations below the available RII binding occurs as not all the RII is bound by R217 (lanes 3–5). (C) Titration of R218 demonstrates that R218 is unable to directly prevent GpA binding at any concentration.</p

Crystal structure of F1/R218 Fab complex.

<p>(A) Overall structure of the F1/R218 Fab complex shown in ribbon representation. The F2 domain of RII is colored in green. The Fab heavy chain (VH) is in blue and the light chain (VL) in pink. (B) Ribbon representation of F1 mapping the R218 epitope. Residues contacted by the Fab are show in stick. Residues contacted by the heavy chain are colored blue, residues contacted by the Fab light chain are colored orange, and residues contacted by both chains are in beige. Residues not contacted by the antibody are in green. (C) Surface representation of F2 mapping the R217 epitope. Color scheme as in B. (D) Surface representation of the R218 Fab, mapping heavy chain residues (blue) that contact F2 (green). The light chain is shown in white. (E) Surface representation of the R218 Fab, mapping light chain residues (orange) that contact F2 (green). The heavy chain is shown in white.</p