Subdomain 3 of Plasmodium falciparum VAR2CSA DBL3x Is Identified as a Minimal Chondroitin Sulfate A-binding Region

Abstract

Molecular interactions between the VAR2CSA protein, expressed on the surface of Plasmodium falciparum-infected erythrocytes, and placental chondroitin sulfate A (CSA) are primarily responsible for pregnancy-associated malaria (PAM). Interrupting these interactions may prevent or ameliorate the severity of PAM. Several of the Duffy binding-like (DBL) domains of VAR2CSA, including the DBL3x domain, have been shown to bind CSA in vitro, but a more detailed understanding of how DBL domains bind CSA is needed. In this study, we demonstrate that subdomain 3 (S3), one of the three subdomains of VAR2CSA DBL3x by itself, is the major contributor toward CSA binding. NMR spectroscopy and flow cytometry analyses show that S3 and the intact DBL3x domain bind CSA similarly. Mutations within the S3 portion of DBL3x markedly affect CSA binding. Both recombinant molecules, S3 and DBL3x, are recognized by antibodies in the plasma of previously pregnant women living in malaria-endemic regions of Mali, but much less so by plasma from men of the same regions. As the S3 sequence is highly conserved in all known VAR2CSA proteins expressed by different parasite isolates obtained from various malaria endemic areas of the world, the identification of S3 as an independent CSA-binding region provides a compelling molecular basis for designing interventions against PAM.Each year, pregnancy-associated malaria (PAM) 3 poses a risk to millions of women worldwide. Although women living in malaria-endemic areas eventually develop immunity to malaria after repeated exposure to Plasmodium falciparum, they become susceptible to severe disease during pregnancy. PAM is caused by the massive sequestration of P. falciparum-infected erythrocytes (IEs) that selectively bind to CSA in the placenta, leading to severe inflammation and life-threatening outcomes for both mother and child (1).VAR2CSA, a member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, is primarily responsible for the binding of IEs to CSA (2). The var2csa gene is found in nearly all P. falciparum isolates (3) and is unusually conserved compared with other members of the var gene family that encode PfEMP1 molecules (4). VAR2CSA is specifically up-regulated in infected erythrocytes that are either isolated from the placenta (5) or selected in vitro for binding to CSA (2). On disruption of the var2csa gene, the ability of the infected erythrocytes to adhere to CSA is lost (6) or greatly reduced (7).The risk of PAM is highest in the first pregnancy due to lack of VAR2CSA-specific antibodies, but with increasing parity, women acquire such antibodies that block the binding of IEs to CSA in the placenta. The naturally acquired antibodies from multiparous women collected from different malaria-endemic regions of the world recognize CSA-binding IEs in the placenta (8 -12). The ability of such antibodies to block adhesion of IEs to CSA by recognizing conserved epitopes among different parasite clones, suggests that VAR2CSA-based vaccination against PAM is possible.Expressing...

Identification of VAR2CSA Domain-Specific Inhibitory Antibodies of the Plasmodium falciparum Erythrocyte Membrane Protein 1 Using a Novel Flow Cytometry Assay

“…In this study, we investigated whether DBL2X or DBL3X single-domain or subdomain 3 constructs, is the minimal CSA binding region (33), could elicit inhibitory antibodies. Previous work has suggested that the DBL2X and DBL3X domains contain CSA-binding activity, and binding activity has been mapped to the DBL3 subdomain 3 (12,24,40). However, it has been questioned whether single domains accurately recapitulate the CSA binding site in native VAR2CSA (41), because single domains have much lower binding activity and more promiscuous binding specificity than fulllength recombinant proteins (19,20).…”

“…5). Previous nuclear magnetic resonance (NMR) studies have shown that the DBL3X-S3 domain can bind CSA (40), suggesting that on its own this region may have very weak binding affinity for CSA. These weakly interacting DBL3X-S3 antibodies may be sufficiently specific to effectively block parasite-IE binding to CSA.…”

Functional Comparison of Plasmodium falciparum Transmission-Blocking Vaccine Candidates by the Standard Membrane-Feeding Assay

“…Plasma samples from Malian adults (n ϭ 45) were collected from a study approved by the Ethics Committee of the Faculty of Medicine, Pharmacy and Odontostomatology, University of Bamako, and the Institutional Review Board (IRB) of the National Institute of Allergy and Infectious Diseases (NIAID) (13). Individual written informed consent was obtained from all participants.…”

Discovery of GAMA, a Plasmodium falciparum Merozoite Micronemal Protein, as a Novel Blood-Stage Vaccine Candidate Antigen

“…Human plasma samples were collected from adults living in three areas of malaria endemicity in Mali (29). The study was approved by the ethical review committees of the Faculty of Medicine, Pharmacy, and Dentistry at the University of Bamako (Mali) and the NIAID, National Institutes of Health (Bethesda, MD).…”