Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes

Abstract

Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to the vascular endothelium is a critical event in the pathology of Plasmodium falciparum malaria. It has been suggested that the clag gene family is essential in cytoadherence to endothelial receptors. Primers used in PCR and RT-PCR assays allowed us to determine that the gene encoding CLAG 3 (GenBank accession no. NP_473155) is transcribed in the Plasmodium falciparum FCB2 strain. Western blot showed that antisera produced against polymerized synthetic peptides from this protein recognized a 142-kDa band in P. falciparum schizont lysate. Seventy-one 20-amino-acid-long nonoverlapping peptides, spanning the CLAG 3 (cytoadherence-linked asexual protein on chromosome 3) sequence were tested in C32 cell and erythrocyte binding assays. Twelve CLAG peptides specifically bound to C32 cells (which mainly express CD36) with high affinity, hereafter referred to as high-affinity binding peptides (HABPs). Five of them also bound to erythrocytes. HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. Affinity constants were between 100 nM and 800 nM. Cross-linking and SDS-PAGE analysis allowed two erythrocyte binding proteins of around 26 kDa and 59 kDa to be identified, while proteins of around 53 kDa were identified as possible receptor sites for C-32 cells. The HABPs' role in Plasmodium falciparum invasion inhibition was determined. Such an approach analyzing various CLAG 3 regions may elucidate their functions and may help in the search for new antigens important for developing antimalarial vaccines.

Figure 1.

Plasmodium falciparum gDNA cytoadherence-linked asexual protein (CLAG) amplification products using A5/A7 or B1/B3 primer sets (lanes 1 and 4, respectively). Plasmodium falciparum cDNA amplification products using A5/A7 or B1/B3 primer sets (lanes 2 and 5, respectively). Negative controls (lanes 3,6). M, 100-bp molecular weight markers.

Figure 2.

Nucleotide sequence alignment between the 474-bp amplified product (Clone 7A) and the reported CLAG nucleotide sequence (NP_473155). Sequences where primers anneal are highlighted in gray. Dots and dashes represent identities and gaps, respectively.

Figure 3.

CLAG recognition by polyclonal antibodies. Western blotting was done with preimmune and final blood sera of goat against a P. falciparum lysate (lanes 1–4,7). (Lanes 1,2) Preimmune goat sera, (lanes 3,4) final blood sera. MW markers are shown on the right-hand side.

Figure 4.

C-32 cell- and erythrocyte-binding assays using PfCLAG 3.2 peptides. (A) Each of the black bars represents the slope of the specific binding graph, which is named specific binding activity. Peptides with 2% were considered as having high specific erythrocyte binding (HABPs). (B) Specific binding activity for HABP peptide analogs; jumbled peptide sequences are shown to the right, and the bars on the left represent specific binding. The numbers in the first column represent FIDIC’s internal code number for those peptides synthesized.

Figure 4.

C-32 cell- and erythrocyte-binding assays using PfCLAG 3.2 peptides. (A) Each of the black bars represents the slope of the specific binding graph, which is named specific binding activity. Peptides with 2% were considered as having high specific erythrocyte binding (HABPs). (B) Specific binding activity for HABP peptide analogs; jumbled peptide sequences are shown to the right, and the bars on the left represent specific binding. The numbers in the first column represent FIDIC’s internal code number for those peptides synthesized.

Figure 5.

PfCLAG 3.2 HABP circular dichroism analysis. (A) Helical structural elements present in most HABPs. (B) Two of the HABPs presented random coil structural elements. (C) Peptide 30373 (presenting high binding to two cell types) clearly showed a helical conformation.

Figure 6.

Autoradiograph from HABPs cross-linking assays. C-32 cell (A) and erythrocyte membrane (B) proteins were cross-linked with radiolabeled PfCLAG 3.2 peptides. (A) (Lane 1) molecular weight marker, (lane 2) SDS-PAGE for C-32 cells, (lanes 3,5) total binding, (lanes 4,6) binding inhibited in the presence of unlabeled peptide. (B) Odd-numbered lanes show the total binding (i.e., cross-linking in the absence of unlabeled peptide), and even-numbered lanes show inhibited binding (i.e., cross-linking in the presence of unlabeled peptide). Due to space constraints, only some cross-linking with C-32 cells is shown.