A leading cause of human cerebral malaria which claims millions of lives each year is thought to be the attachment of Plasmodium falciparum- infected erythrocytes to brain microvascular endothelial cells. In view of developing a novel therapeutic strategy for blocking and/or reversing the sequestration of parasitized erythrocytes, the objective of this project is to identify erythrocyte membrane protein interactions which may have deleterious consequences in malaria parasite sequestration. Cytoadherence of P. falciparum-infected erythrocytes is attributed to parasite-derived var gene products (VARs), a family of antigenically variant receptors localized to knob structures on the surface of parasitized erythrocytes. Despite their high antigenic diversity, VARs have a remarkably conserved cytoplasmic domain. Our preliminary findings that the cytoplasmic domain of a var gene product (termed VARCD) binds to knob-associated histidine-rich protein (KAHRP), a major protein involved in the assembly of knob structures, to actin filaments, and to spectrin dimers are consistent with our hypothesis that VARs are anchored to surface knobs by interacting with knob-associated proteins and host cytoskeleton.
Our specific aims are as follows: (a) As an extension of our preliminary studies, we will focus on elucidating the binding interactions of VARCD with KAHRP and erythrocyte cytoskeletal proteins. Studies will include the quantitation of binding interactions using a surface plasmon resonance detection technology, the characterization of specific binding segments by employing in vitro mutagenesis and synthetic peptides, and the x-ray crystallographic determination of binding interfaces. (b) We will develop peptide inhibitors that will disrupt the binding between VARCD and KAHRP by tightly associating to VARCD. Studies will involve the use of a conformationally-constrained peptide display library, a BIAcore biosensor instrument, and x-ray crystallography. Disrupting a key intracellular binding interaction such as VARCD approximately KAHRP may have a profound prohibitory effect on sequestration in vivo. (c) Extracellular regions of band 3 protein in parasitized erythrocytes have been correlated with cytoadherence and rosetting as putative adhesins. We will examine whether adhesion properties of parasitized erythrocytes are modulated using erythroid band 3-null mice which we have recently developed. Studies will include pathological examinations of infected band 3-null mice and quantitative measurements of the effect of erythroid band 3 deficiency on parasitized erythrocyte sequestration by employing in vivo and ex vivo methods.
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