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

Fundación Instituto de Inmunologia de Colombia and Universidad Nacional de Colombia, Bogotá, Columbia

(RECEIVED May 25, 2004; FINAL REVISION October 12, 2004; ACCEPTED October 18, 2004)

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.

Keywords: Plasmodium falciparum; cytoadherence; C32 cells; peptides

Abbreviations: CLAG, cytoadherence-linked asexual protein • HABPs, high activity binding peptides • PRBCs, parasitized red blood cells • HBS, HEPES buffered saline

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04883905.

Reprint requests to: Marisol Ocampo, Avda. Calle 26 No. 50-00, Bogotá, Colombia; e-mail: marisol_ocampo{at}fidic.org.co; fax: +57-1-324-4672.

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