Plasmodium falciparum domain mediating adhesion to chondroitin sulfate A: a receptor for human placental infection

Abstract

Malaria during the first pregnancy causes a high rate of fetal and neonatal death. The decreasing susceptibility during subsequent pregnancies correlates with acquisition of antibodies that block binding of infected red cells to chondroitin sulfate A (CSA), a receptor for parasites in the placenta. Here we identify a domain within a particular Plasmodium falciparum erythrocyte membrane protein 1 that binds CSA. We cloned a var gene expressed in CSA-binding parasitized red blood cells (PRBCs). The gene had eight receptor-like domains, each of which was expressed on the surface of Chinese hamster ovary cells and was tested for CSA binding. CSA linked to biotin used as a probe demonstrated that two Duffy-binding-like (DBL) domains (DBL3 and DBL7) bound CSA. DBL7, but not DBL3, also bound chondroitin sulfate C (CSC) linked to biotin, a negatively charged sugar that does not support PRBC adhesion. Furthermore, CSA, but not CSC, blocked the interaction with DBL3; both CSA and CSC blocked binding to DBL7. Thus, only the DBL3 domain displays the same binding specificity as PRBCs. Because protective antibodies present after pregnancy block binding to CSA of parasites from different parts of the world, DBL-3, although variant, may induce cross-reactive immunity that will protect pregnant women and their fetuses.

Figure 1

Cloning and expression of the FCR3.varCSA gene. (A) Overlapping clones of the varCSA gene were isolated from genomic FCR3-CSA parasites and were sequenced. Regions amplified by reverse transcription–PCR from FCR3-CSA trophozoites mRNA confirm that the genomic varCSA gene sequence is contiguous with the exception of the intron region. (B) Schematic domain organization of the FCR3.varCSA gene. An unusually small intron of 230 bp separates exon I and exon II of the varCSA gene. The amino acid boundaries of the different DBL and CIDR-1 domains are indicated. (C) Domain regions that have been expressed on the surface of CHO-745 cells with their amino acid boundaries.

Figure 2

(A) A large var transcript is detected in total RNA of FCR3-CSA trophozoites. Lanes: 1, FCR3.varCSA DBL-1 sequence probe; 2, Hsp70-specific probe hybridizing to the P. falciparum heat shock gene transcript of ≈3 kilobases. (B) Identification of a high molecular weight ¹²⁵I-labeled and trypsin-sensitive erythrocyte surface molecule. Shown is the separation of SDS extracts of surface iodinated FCR3-CSA (first lane) and FCR3-CD36 (third lane) trophozoites. The labeled high molecular mass proteins of ≈400 and 250 kDa are sensitive to trypsinization (second lane and fourth lane). (C) CSA carrying human thrombomodulin (hTM)-coated Dynabeads affinity-purifies a high molecular weight molecule in SDS extracts from FCR3-CSA but not from FCR3-CD36 trophozoites. The purified molecule is sensitive to trypsin treatment. ¹²⁵I-PfEMP-1 molecules are indicated by arrows.

Figure 3

Binding of Biot-CSA to transfected CHO-745 cells. (A) Binding of anti-biotin-coated Dynabeads to CHO-745 cells expressing domains of FCR3varCSA incubated with CSA-biotin. (B) Percentage of transfected cells that bound four or more beads. (C) Inhibition of binding of CSA and CSC to DBL-3 and DBL-7 transfectant. Transfected cells were incubated with Biot-CSA or Biot-CSC without (control) or after preincubation with 200 μg/ml CSA (+CSA) or CSC (+CSC). Binding is given as number of positive cells (B) or number of beads (C) per 100 cells. Error bars represent the standard deviation from three different experiments.