Plasmodium falciparum:: binding studies of peptide derived from the sporozoite surface protein 2 to Hep G2 cells

Plasmodium falciparum sporozoite surface protein 2 (Pf SSP2), also called thrombospondin related anonymous protein (TRAP), is involved in the process of sporozoite invasion of hepatocytes. Pf SSP2/TRAP possesses two different adhesion domains sharing sequences and structural homology with von Willebrand factor A-domains and human repeat I thrombospondin (TSP). Pf SSP2/TRAP has also been implicated in sporozoite mobility and in mosquito salivary gland invasion processes. We tested 15-mer long synthetic peptides having five overlapping residues covering the complete protein Pf SSP2 sequence in binding assays to Hep G2 cells. In these 57 peptides, high-activity binding peptides (HABPs) were identified; five were in the adhesion domains already described and 16 were in two regions toward the protein's carboxy and middle terminal part. Six HABPs showed conserved amino acid sequences: 3243 ((21)FLVNGRDVQNNIVDE(35)), 3279 ((201)FLVGCHPSDGKCNLY(215)), 3287 ((241)TASCGVWDEWSPCSV(255)), 3289 ((251)SPCSVTCGKGTRSRK(265)), 3327 ((441)ERKQSDPQSQDNNGNY(455)) and 3329 ((451)DNNGNRHVPNSEDREY(465)). The HABPs show saturable binding 2 and dissociation constants between 140 and 900 nm with 40000-855 000 binding sites per cell. The 3279 ((201)FLVGCHPSDGKCNLY(215)), 3323 ((NDKSDRYIPYSPLSP435)-N-421) and 3331 ((461)SEDRETRPHGRNNENY(475)) HABPs have B epitopes in their sequences; these have previously been recognized by antibodies partially inhibiting hepatocyte invasion and development of the hepatic state. The 3287 ((241)TASCGVWDEWSPCSV(255)) and 3289 ((251)SPCSVTCGKGTRSRK(265)) HABPs share common sequences with the Pf SSP2/TRAP region II plus, which is present in a great number of adhesion proteins. Based on this information, six new peptides covering the high binding regions identified previously were synthesized and, using a competition assay, the amino acid involved in the binding were determined.