Figure 1.
Structural analysis of the gene encoding Pf332.
A. Schematic structure of the Pf332 gene. The gene is composed of a 5′ exon I (PF11_0506) with a size of 1704 bp and a 3′ exon II (PF11_0507), separated by a short intron of 236 bp. The 3′-exon II is the gene fragment previously referred to as the gene coding for the antigen Pf332. The location of the primers is indicated as arrows. B. PCR amplification from cDNA and gDNA across the intron region of the Pf332 gene: Lane 1: PCR amplification with primers UP3 and UP4 from cDNA; lane 2: PCR amplification with the same primer pair from gDNA. C. Northernblot with probes located at 5′-end (UP1–UP2) and across the splicing site (UP3–UP4) of the Pf332 gene. Total RNA from 3D7AH1 iRBC was resolved in an agarose gel (lanes 1 and 3). Lane 2 shows the hybridisation with the first probe (UP1–UP2); lane 4 shows the hybridisation with the second probe (UP3–UP4) revealing a band of the same size as seen in the first hybridization.
Table 1.
Amino acid substitutions in the region encoded by exon I of Pf332
Figure 2.
Schematic illustration of the conserved structure of the erythrocyte-binding region of Pf332 and the EBL family.
The N-terminal region of the Pf332 encoded by exon I contains a Duffy-binding like domain homologous to the DBL-domain of the EBL-family. This domain of Pf332 is located closer to the N-terminal part of the molecule and lacks a signal leading sequence as compared to the other EBL family members. It aligns with the F2 region of EBA-175 of P. falciparum and the DBP of P. vivax. In addition, the carboxyl cysteine (c-cys)-rich domain present upstream of the transmembrane domain in EBA-175 and DBP is not found in Pf332.
Figure 3.
Transcription and expression of Pf332 in different P. falciparum clones/strains.
A: The level of Pf332 mRNA transcription, relative to the endogenous control seryl-tRNA synthetase, was determined by real-time quantitative PCR. Normalized Pf332 levels at different times p.i. are plotted for 3D7AH1 (•), FCR3S1.2 (○) and 7G8 (▾). Error bars represent the standard deviation of the quotient and * denotes significantly higher levels of transcription in FCR3S1.2 (p<0.001, One Way ANOVA with Tukey test). The amount of Pf332 mRNA transcripts in FCR3S1.2 is around two fold higher than in 3D7AH1 and 7G8 at the time of maximal transcription. B: Upper panel: Immunofluorescence staining with α-DBL/nDBL-domain antibodies of Pf332. α-DBL/nDBL-domain antibodies (green) visualize at early trophzoite stage (around 20 h p.i.) vesicles that carry Pf332 in the cytosol of the iRBC; when the iRBC reaches late trophozoite/schizont stage (40–47 h p.i.), Pf332 can be observed in association with the iRBC membrane. Lower panel: Additional staining with the α-Pf332 monoclonal antibody m33G2. Staining with previously raised sera against the Pf332 (m33G2, red) show that α-DBL/nDBL-domain antibodies (green) co-localize with sera raised against the polypeptide encoded by exon II of Pf332. Staining is shown for the parasite strain 7G8, DNA staining with Hoechst (blue); Scale bars = 1 µm. C: Immunoblot analysis confirmed that antibodies raised against the DBL/nDBL-domain of Pf332 reacted with the same high molecular weight polypeptide as the previously raised antibodies α-EB200 and m33G2 (compare Fig. S3). This verifies that the sequence of the Pf332-DBL/nDBL-domain is an additional exon of the same open reading frame as the previously described Pf332. The total amount of expressed Pf332 varies in between parasites, with FCR3S1.2 iRBC expressing the highest amount of Pf332. An α-glycophorin antibody was used compare the amount of loaded material in the different lanes. The bands corresponding to Pf332 are marked with an asterix.
Figure 4.
The Pf332 DBL-domain is iRBC surface associated.
Immunofluorescence with antibodies against the DBL-domain visualizes the surface association of the Pf332 DBL-domain as shown for HB3 iRBC (A+C) and FCR3S1.2 iRBC (B+D). A+B: Parasites are unstained; the RBC membrane is indicated with a white line/arrow, the intracellular parasite with a grey line/arrow. C+D: Parasites are stained orange with Ethidium-bromide. Scale bars = 1 µm. E: Western blot with the same α-Pf332 DBL-domain antibody as used for the immunofluorescence assays; the antibody recognizes only the molecule Pf332 and does not cross-react with any other proteins.
Figure 5.
The DBL domain of Pf332 binds to human RBC.
A: The protein Pf332 consists of a potentially extracellular region encoded by exon I and is composed of a DBL- and nDBL-domain followed by a predicted transmembrane domain. The region of the molecule originally described as Pf332 is encoded by a second exon and contains a Glutamic-acid rich part, which includes a Valin-rich area as well as the region EB200 build of numerous repeats of a XXXEEXXEEXX motif (X = hydrophobic aa). B: The Pf332-DBL- and Pf332nDBL-region were expressed as two individual GST-fusion proteins in E.coli. Binding assays, in which human RBC were incubated with the DBL-, nDBL- and an unrelated control protein were carried out. Binding of the proteins was visualized by subjecting the RBC to immunoblotting, where the bound protein was detected with an α-GST antibody. The DBL- but not the nDBL-domain or the unrelated control protein was able to bind to human RBC. C: Transient transfected CHO cells expressing either the DBL- or nDBL-domain on their surface were tested for their ability to bind to human RBC. CHO cells were stained red with PHK26 and incubated with RBC stained green with PKH67. CHO-cells expressing the DBL-domain avidly bound RBC, lower panel, while CHO cells expressing the nDBL-region or MOCK-transfected cells did not show any binding towards RBC (middle and upper panel).
Figure 6.
Antibodies directed against the DBL-domain of Pf332 inhibit parasite invasion.
RBC infected with the parasite clones/strains FCR3S1.2, 3D7AH1 and 7G8 were cultivated in the presence of α-Pf332-DBL/nDBL-antibodies for 24 h allowing reinvasion of the parasites into new RBC. The parasitemia was analysed by flow-cytometry and compared to a control cultivated in the presence of a serum raised against a non-related protein. Parasites displayed a decreased invasion rate in the presence of α-Pf332-DBL/nDBL-antibodies. The effect of α-Pf332-DBL/nDBL-antibodies (light bars) is slightly lower as compared to inhibition caused by antibodies against a hybrid protein of AMA1 and MSP1 (dark bars). Bars represent the mean of three experiments; error bars indicate the standard deviation.