Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA

Abstract

Pregnancy-associated malaria (PAM) is a serious consequence of sequestration of Plasmodium falciparum-parasitized erythrocytes (PE) in the placenta through adhesion to chondroitin sulfate A (CSA) present on placental proteoglycans. Recent work implicates var2CSA, a member of the PfEMP1 family, as the mediator of placental sequestration and as a key target for PAM vaccine development. Var2CSA is a 350 kDa transmembrane protein, whose extracellular region includes six Duffy-binding-like (DBL) domains. Due to its size and high cysteine content, the full-length var2CSA extracellular region has not hitherto been expressed in heterologous systems, thus limiting investigations to individual recombinant domains. Here we report for the first time the expression of the full-length var2CSA extracellular region (domains DBL1X to DBL6epsilon) from the 3D7 parasite strain using the human embryonic kidney 293 cell line. We show that the recombinant extracellular var2CSA region is correctly folded and that, unlike the individual DBL domains, it binds with high affinity and specificity to CSA (K(D) = 61 nM) and efficiently inhibits PE from binding to CSA. Structural characterization by analytical ultracentrifugation and small-angle x-ray scattering reveals a compact organization of the full-length protein, most likely governed by specific interdomain interactions, rather than an extended structure. Collectively, these data suggest that a high-affinity, CSA-specific binding site is formed by the higher-order structure of the var2CSA extracellular region. These results have important consequences for the development of an effective vaccine and therapeutic inhibitors.

Fig. 1.

Recombinant 3D7-DBL1X-6ε is expressed in HEK293 cells. (A). Schematic view of the var2CSA domain organization and sequence limits of the recombinant domains studied (FCR3-DBL3X, FCR3-DBL6ε, 3D7-DBL4ε-6ε, and 3D7-DBL1X-6ε). It comprises six Duffy-binding-like domains (DBL1X to DBL6ε) and four interdomain regions (INT1-4) in the extracellular region, a transmembrane segment and acidic C terminus sequence (ATS). DBL1X, DBL2X, and DBL3X are green; DBL4ε, DBL5ε and DBL6ε are orange; N-terminal sequence (NTS) and interdomain regions (INT) are gray; the transmembrane and ATS regions are blue. The length of each bar corresponds to the domain size. (B). Purification of var2CSA derived proteins expressed in HEK293 and E. coli. SDS-PAGE Criterion XT Precast 4–12% Bis-Tris gel under reducing and nonreducing conditions was loaded with purified FCR3-DBL3X, FCR3-DBL6ε, 3D7-DBL4ε-6ε and 3D7-DBL1X-6ε. Protein was visualized with Coomassie blue (left) or transferred to a nitrocellulose membrane to detect recombinant protein using antiHis tag antibodies (right). Lanes 1-4: nonreduced FCR3-DBL3X, FCR3-DBL6ε, 3D7-DBL4ε-6ε, 3D7-DBL1X-6ε; lanes 5–8: reduced FCR3-DBL3X, FCR3-DBL6ε, 3D7-DBL4ε-6ε, 3D7-DBL1X-6ε.

Fig. 2.

Global shape of 3D7-DBL1X-6ε obtained from SAXS. (A). Plot of x-ray intensity log(I) against the scattering angle (s = 4π sin Θ/λ). The data points are black, calculated 3D7-DBL1X-6ε ab initio model curve is green and that corresponding to the theoretical extended model scattering curve is a red dashed line. (B). Orthogonal views of the 3D7-DBL1X-6ε ab initio model. (C). The theoretical extended model of the extracellular region of var2CSA, constructed as described in SI Text, shown to the same scale as the ab initio model in (B). DBL domains and interdomain regions are colored as in Fig. 1A.

Fig. 3.

3D7-DBL1X-6ε binds specifically to CSA. (A). ELISA-based direct binding assay of the 3D7-DBL1X-6ε to different sulfated glycosaminoglycans. Increasing concentrations of recombinant 3D7-DBL1X-6ε at serial dilutions of 0.31–20 μg/mL were added to wells previously coated with different glycosaminoglycans: decorin, CSA, CSC, heparan sulfate (HS). (B). Competitive inhibition of binding of recombinant DBL domains to decorin using various glycosaminoglycans. Recombinant proteins at 1 μg/mL were premixed with increasing amounts of GAG, 0.156–100 μg/mL (CSA, CSC, or HS) and incubated in plates previously coated with decorin.

Fig. 4.

3D7-DBL1X-6ε binds with high affinity to human placental CSPG. From surface plasmon resonance binding experiments using soluble 3D7-DBL1X-6ε and immobilized placental CSPG, the variation in the steady-state SPR signal as a function of protein concentration was used to calculate the dissociation constant K_D. The kinetic association curves used to derive K_D are shown in the insert. Protein concentrations ranged from 20 nM to 1,280 nM. The K_D constants of the other proteins were determined in the same way (Table 3).

Fig. 5.

3D7-DBL1X-6ε inhibits PE adhesion. Recombinant proteins DBL3X, DBL6ε, DBL4ε-6ε and DBL1X-6ε (0.2 mg/mL in PBS) were incubated on decorin-coated spots prior to addition of FCR3-CSA PE. Bound parasites were fixed with 2% glutaraldehyde and were counted to measure percentage inhibition. Percentage inhibition is indicated for each individual protein in the bottom right corner.