Plasmodium falciparum-infected erythrocyte adhesion to the type 3 repeat domain of thrombospondin-1 is mediated by a modified band 3 protein

Abstract

Previously, the binding site for the Plasmodium falciparum-infected erythrocyte (PE) was determined to be the C-terminal 120 or 140 kDa region but not the N-terminal 25 kDa domain of thrombospondin (TSP). In this work, we have localized the TSP binding site for PE more precisely. PE adhered to glutathione-S-transferase-fusion proteins containing the type 3 repeat (T3) of TSP, but not to other functional domains of TSP (i.e. N-terminal domain, procollagen domain, type 1 and 2 repeat, and C-terminal domain). Soluble T3 inhibited PE binding to immobilized TSP. PE binding to immobilized T3 was inhibited by soluble TSP, a monoclonal antibody directed against the T3, glycine-arginine-glycine-aspartic acid-serine-proline (GRGDSP) peptide, and *cysteine-GRGDSP-cysteine*, where *cysteine and cysteine* form a disulfide linkage, suggesting involvement of an RGD-containing motif in the T3. In support of this, a fusion protein which excluded the RGD motif showed no PE binding activity. Earlier it was shown that the amino acid sequence of the band 3 protein, histidine-proline-leucine-glutamine-lysine-threonine-tyrosine (HPLQKTY), was exposed on PE and mediated PE binding to TSP. Monoclonal antibodies, which recognize HPLQKTY and inhibit PE binding to TSP, also inhibited PE binding to the T3. The involvement of the sequence was confirmed by the fact that an octamer of HPLQKTY-containing peptide bound to the T3 but not to the RGD motif-excluded fusion protein and the binding to T3 was inhibited by GRGDSP peptide. Thus, PE binding to the T3 domain of TSP is mediated by the peptidic sequence HPLQKTY of band 3 which is exposed on PE.

Introduction

Plasmodium falciparum infects between 300–500 million people, leading to more than 3 million deaths, affecting mostly African children under 5 years of age. Cerebral malaria (CM), the most common lethal complication in P. falciparum infections is marked by engorgement of cerebral capillaries and post-capillary venules with parasitized erythrocytes (PE) [1], [2]. On autopsy, the brain of CM victims is grayish in color, there are petechial hemorrhages, and although the endothelium is not damaged in any obvious way, immunohistochemical studies have shown that there is endothelial cell (EC) activation [3]. During the first 24 h of the P. falciparum asexual cycle, PE are found in the peripheral circulation, but during the second 24 h, the PE bearing the more mature, pigmented stages sequester, i.e. they adhere to the endothelium of the deep tissues including the brain, heart, lung, kidney and liver [4], [5]. The sequestered PE are protected from the filtering action of the spleen, and by being in a relatively hypoxic microenvironment parasite growth and reproduction are favored. Since PE sequestration may be a cause of CM, understanding the molecular basis of EC-PE adhesion could lead to more effective therapies.

Adherence to ECs occurs through a specific interaction between proteins on the PE surface and ligands on the endothelium. Most PE that bind to EC (or their in vitro cellular surrogates) have surface protuberances called knobs; the appearance of knobs is coincident with the time when PE sequester [6]. Below the elevated portion of the plasma membrane of the knob is an electron dense plaque consisting of histidine-rich protein [7], and the knob’s outer exposed surface contains a parasite-encoded protein, PfEMP1 [8], as well as modifications in the most abundant erythrocyte membrane protein, band 3, the anion transporter [9]. Several ligands (i.e. CD36 [10], intercellular cell adhesion molecule-1 (ICAM-1) [11], vascular cell adhesion molecule, E-selectin [12], platelet/endothelial cell adhesion molecule-1 [13], chondroitin sulfate A [14], [15] and TSP [16]) on the EC have been reported to bind to PE and some have claimed that PfEMP 1 alone is responsible for binding to all ligands [17], whereas others [18] have suggested that PfEMP1 mediates binding only to CD36 and ICAM-1 and the modified forms of band 3 bind to TSP. Previously we demonstrated that TSP is the ligand for the band 3-related adhesin [19]. To more precisely localize the TSP binding site for the band 3-related adhesin we have used glutathione-S-transferase (GST) fusion proteins, synthetic peptides, and monoclonal antibodies (MAb). In this paper, we report that the RGD sequence in the type 3 repeats (T3) of TSP is the PE recognition site.