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, 21 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 (²¹FLVNGRDVQNNIVDE³⁵), 3279 (²⁰¹FLVGCHPSDGKCNLY²¹⁵), 3287 (²⁴¹TASCGVWDEWSPCSV²⁵⁵), 3289 (²⁵¹SPCSVTCGKGTRSRK²⁶⁵), 3327 (⁴⁴¹ERKQSDPQSQDNNGNY⁴⁵⁵) and 3329 (⁴⁵¹DNNGNRHVPNSEDREY⁴⁶⁵). The HABPs show saturable binding and dissociation constants between 140 and 900 nM with 40 000-855 000 binding sites per cell. The 3279 (²⁰¹FLVGCHPSDGKCNLY²¹⁵), 3323 (⁴²¹NDKSDRYIPYSPLSP⁴³⁵) and 3331 (⁴⁶¹SEDRETRPHGRNNENY⁴⁷⁵) 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 (²⁴¹TASCGVWDEWSPCSV²⁵⁵) and 3289 (²⁵¹SPCSVTCGKGTRSRK²⁶⁵) 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.