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 DBL6ε) 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 ¼ 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. malaria | pregnancy | plasmodium | chondroitin | structure

Structural and Functional Insight into How the Plasmodium falciparum VAR2CSA Protein Mediates Binding to Chondroitin Sulfate A in Placental Malaria

“…Srivastava et al (20) showed the same binding characteristics for DBL1X-DBL3X. This, and small-angle x-ray scattering (SAXS) data proposing that VAR2CSA exhibits an overall compact structure (14), suggested a CSA-binding site dependent on multiple domain and interdomain regions.…”

“…Such studies revealed that the full-length VAR2CSA ectodomain binds specifically to CSA with an affinity in the nanomolar range (13,14,19). In addition, we have recently shown that the core CSAbinding site lies within the DBL2X-CIDR PAM region.…”

“…VAR2CSA interacts with an unusually low-sulfated form of chondroitin sulfate A (CSA), attached to proteoglycans in the intervillous spaces of the placenta (11)(12)(13)(14). Immunity to placental malaria is acquired over multiple pregnancies (15).…”

“…The expression of the full-length VAR2CSA ectodomain (FV2) from FCR3 (13,19) and 3D7 (14,20) type parasites has enabled us to study both the binding properties and the structural characteristics of the VAR2CSA protein. Such studies revealed that the full-length VAR2CSA ectodomain binds specifically to CSA with an affinity in the nanomolar range (13,14,19).…”

Towards an anti-disease malaria vaccine

“…(A) The architecture of PfEMP1 ectodomains displayed on the surface of erythrocytes infected with P. falciparum. Classical PfEMP1s are rigid and elongated in structure [51], whereas VAR2CSA is the only PfEMP1 known to have an overall globular structure [52]. The two domain types in PfEMP1, CIDR domains (yellow) and DBL domains (green), are indicated.…”