Receptor-Based Identification of Novel Peptide Ligands as Inhibitors of Blood Stage Malaria.

Abstract

Malaria parasite Plasmodium falciparum uses multiple receptors on the surface of human red blood cells to attach and invade host cells during blood stage infection. Glycophorins, including glycophorin A, B, and C have been implicated as host receptors and play an important role during Plasmodium falciparum invasion in human erythrocytes, particularly in the sialic acid-dependent parasite strains. To identify the parasite proteins that could bind to human glycophorins, we screened a phage display cDNA library of P. falciparum (FCR3 strain, a sialic acid-dependent strain) using human glycophorins and native intact human erythrocytes as bait. After four rounds of library screening and panning, 35 phage clones were identified that bound to purified glycophorins immobilized on the plastic surface. DNA sequencing of 12 phage clones revealed that they encode the same 7-amino acid sequence, ETTLKSF. Using immobilized intact human erythrocytes, a similar screening strategy led to the isolation of additional 20 phage clones, and the DNA sequencing of 6 such clones again revealed the same 7-amino acid sequence, ETTLKSF. In vitro binding of synthetic ETTLKSF peptide to purified glycophorins and intact erythrocytes was confirmed by ELISA and indirect immunofluorescence assays. Pull-down experiments demonstrated that the ETTLKSF peptide specifically interacts with glycophorin C, but not with glycophorin A and B, on human erythrocytes. The synthetic ETTLKSF peptide blocked merozoite invasion of P. falciparum in human erythrocytes in a dose-dependent manner, whereas the control peptides were without any effect. We have named the ETTLKSF peptide as GBL-1, the Glycophorin Binding Ligand-1. We propose that further characterization of GBL-1, the first peptide ligand that specifically binds to human glycophorin C, could lead to the development of novel anti-malarial agents that prevent P. falciparum invasion in human erythrocytes via the glycophorin C receptor.