Identification of glycosaminoglycan binding regions in the Plasmodium falciparum encoded placental sequestration ligand, VAR2CSA

Abstract

Background: Pregnancy malaria is caused by Plasmodium falciparum-infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.

Methods: To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.

Results: The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.

Conclusion: Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.

Figure 3

A DBL2 peptide binds to CSA and not to CSC. (A) Peptide binding assay to CSA: P2b (red) binds to CSA (■) in a peptide concentration-specific manner and not to the plate (▲). The control peptide (blue) does not bind to neither the plate (×) nor CSA (◆). (B) Peptide binding assay to CSC: P2b (red) does not bind to either CSC (■) or to the plate (▲). The control peptide (blue) does not bind to neither the plate (×) nor CSC (◆).

Figure 1

Frequency of identifed phages sorted according to the identity of the VAR2CSA region and the method of biopanning. The var2csa phage display library was biopanned four rounds on each of the following: biotinylated CSA (A, red); Chondroitin sulfate proteoglycans of human placenta (CSPG) (A, yellow); bovine CSA (A, green); CHO cells expressing CSA on the surface (B, pink) and human placental choriocarcinoma cell line BeWo (B, blue). Control biopannings were done on ELISA plates coated with BSA (A, blue) and the original library was sequenced prior to selection to confirm the presence of sequences belonging to all domains (A, black). For each assay 15 CSA-binding clones were sequenced. Each coloured bar indicates the VAR2CSA sequence expressed by the selected phage on the x-axes and the frequency by which the sequence was detected by sequencing on the y-axes. The length of the coloured bar indicates the length of the VAR2CSA sequence in the selected phage. The VAR2CSA regions most often expressed by the CSA-binding phages are shaded in grey (P1-P5).

Figure 2

Structural models of VAR2CSA DBL domains showing surface-exposed regions and GAG binding regions. The surface-exposed epitopes previously determined by depleting female IgG plasma on parasites expressing VAR2CSA are shown in blue. The GAG binding regions identified by phage display assays are shown in yellow. The overlap of surface-exposed regions and GAG binding regions is shown in green.

Figure 4

Anti-P2 antibodies react with native VAR2CSA expressed on the surface of infected erythrocytes. The histogram (A) shows staining of red blood cells infected with late stages of NF54^var2csa. The IE reacted with rabbit affinity purified antibodies against P2c peptide (red) and DBL5 (blue). The rabbit prebleed is shown in solid grey. The picture (B) shows an IFA image of IE double stained with anti-P2c antibodies (green) and DNA (DAPI) staining (blue).