Dataset Information

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.

SUBMITTER: Srivastava A

PROVIDER: S-EPMC2841952 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BACKGROUND: Infected humans make protective antibody responses to the PfEMP1 adhesion antigens exported by Plasmodium falciparum parasites to the erythrocyte membrane, but little is known about the kinetics of this antibody-receptor binding reaction or how the topology of PfEMP1 on the parasitized erythrocyte membrane influences antibody association with, and dissociation from, its antigenic target. METHODS: A Quartz Crystal Microbalance biosensor was used to measure the association and dissociation kinetics of VAR2CSA PfEMP1 binding to human monoclonal antibodies. Immuno-fluorescence microscopy was used to visualize antibody-mediated adhesion between the surfaces of live infected erythrocytes and atomic force microscopy was used to obtain higher resolution images of the membrane knobs on the infected erythrocyte to estimate knob surface areas and model VAR2CSA packing density on the knob. RESULTS: Kinetic analysis indicates that antibody dissociation from the VAR2CSA PfEMP1 antigen is extremely slow when there is a high avidity interaction. High avidity binding to PfEMP1 antigens on the surface of P. falciparum-infected erythrocytes in turn requires bivalent cross-linking of epitopes positioned within the distance that can be bridged by antibody. Calculations of the surface area of the knobs and the possible densities of PfEMP1 packing on the knobs indicate that high-avidity cross-linking antibody reactions are constrained by the architecture of the knobs and the large size of PfEMP1 molecules. CONCLUSIONS: High avidity is required to achieve the strongest binding to VAR2CSA PfEMP1, but the structures that display PfEMP1 also tend to inhibit cross-linking between PfEMP1 antigens, by holding many binding epitopes at distances beyond the 15-18 nm sweep radius of an antibody. The large size of PfEMP1 will also constrain intra-knob cross-linking interactions. This analysis indicates that effective vaccines targeting the parasite's vulnerable adhesion receptors should primarily induce strongly adhering, high avidity antibodies whose association rate constant is less important than their dissociation rate constant.

Project description:Plasmodium falciparum malaria remains one of the world's leading causes of human suffering and poverty. Each year, the disease takes 1-3 million lives, mainly in sub-Saharan Africa. The adhesion of infected erythrocytes (IEs) to vascular endothelium or placenta is the key event in the pathogenesis of severe P. falciparum infection. In pregnant women, the parasites express a single and unique member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family named VAR2CSA, which is associated with the ability of the IEs to adhere specifically to chondroitin sulphate A (CSA) in the placenta. Several Duffy-binding-like domains from VAR2CSA molecules have been shown in vitro to bind to CSA, but it has also been demonstrated that Duffy-binding-like domains from PfEMP1 proteins other than VAR2CSA can bind CSA. In addition, the specificity of the binding of VAR2CSA domains to glycosaminoglycans does not match that of VAR2CSA-expressing IEs. This has led to speculation that the domains of native VAR2CSA need to come together to form a specific binding site or that VAR2CSA might bind to CSA through a bridging molecule. Here, we describe the expression and purification of the complete extracellular region of VAR2CSA secreted at high yields from insect cells. Using surface plasmon resonance, we demonstrate that VAR2CSA alone binds with nanomolar affinity to human chondroitin sulphate proteoglycan and with significantly weaker affinity to other glycosaminoglycans, showing a specificity similar to that observed for IEs. Antibodies raised against full-length VAR2CSA completely inhibit recombinant VAR2CSA binding, as well as parasite binding to chondroitin sulphate proteoglycan. This is the first study to describe the successful production and functionality of a full-length PfEMP1. The specificity of the binding and anti-adhesion potency of induced IgG, together with high-yield production, encourages the use of full-length PfEMP1 in vaccine development strategies.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure