Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes.
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.
Ocampo
Marisol
M
Fundación Instituto de Immunologia de Colombia and Universidad Nacional de Colombia, Avda. Calle 26 No. 50-00, Bogotá, Colombia. marisol_ocampo@fidic.org.co
Rodríguez
Luis E
LE
Curtidor
Hernando
H
Puentes
Alvaro
A
Vera
Ricardo
R
Valbuena
John J
JJ
López
Ramses
R
García
Javier E
JE
Ramírez
Luis E
LE
Torres
Elizabeth
E
Cortes
Jimena
J
Tovar
Diana
D
López
Yolanda
Y
Patarroyo
Manuel A
MA
Patarroyo
Manuel E
ME
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001483:Base Sequence; D015153:Blotting, Western; D018955:CD36 Antigens / Q000737:chemistry; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism* / Q000469:parasitology*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010963:Plasmodium falciparum / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D018955:CD36 Antigens; D015815:Cell Adhesion Molecules; D003432:Cross-Linking Reagents; D017931:DNA Primers; D018076:DNA, Complementary; D010455:Peptides; D011108:Polymers; D015800:Protozoan Proteins; C502072:cytoadherence-linked asexual protein 3, Plasmodium falciparum