The Binding of the Circumsporozoite Protein to Cell Surface Heparan Sulfate Proteoglycans Is Required for PlasmodiumSporozoite Attachment to Target Cells

Abstract

The major surface protein of malaria sporozoites, the circumsporozoite protein, binds to heparan sulfate proteoglycans on the surface of hepatocytes. It has been proposed that this binding event is responsible for the rapid and specific localization of sporozoites to the liver after their injection into the skin by an infected anopheline mosquito. Previous in vitro studies performed under static conditions have failed to demonstrate a significant role for heparan sulfate proteoglycans during sporozoite invasion of cells. We performed sporozoite attachment and invasion assays under more dynamic conditions and found a dramatic decrease in sporozoite attachment to cells in the presence of heparin. In contrast to its effect on attachment, heparin does not appear to have an effect on sporozoite invasion of cells. When substituted heparins were used as competitive inhibitors of sporozoite attachment, we found that sulfation of the glycosaminoglycan chains at both the Nand O-positions was important for sporozoite adhesion to cells. We conclude that the binding of the circumsporozoite protein to hepatic heparan sulfate proteoglycans is likely to function during sporozoite attachment in the liver and that this adhesion event depends on the sulfated glycosaminoglycan chains of the proteoglycans.Protozoans of the genus Plasmodium are the causative agents of malaria. Malaria infection is initiated when an infected anopheline mosquito injects sporozoites during a blood meal. The injected sporozoites travel to the liver and invade hepatocytes where they develop into exoerythrocytic forms. The speed and specificity of sporozoite localization to hepatocytes suggest a receptor-mediated event. Previous studies have shown that the major sporozoite surface protein, the circumsporozoite protein (CS), 1 binds to heparan sulfate proteogly- (3, 4). However, the inhibitory effect on sporozoite infectivity, while demonstrating that the CS-HSPG interaction is important, does not indicate if the glycan is required for sporozoite attachment, invasion, or subsequent development in hepatocytes.In vitro assays (5, 6) have been used to determine whether the CS-HSPG interaction is critical for cell invasion. Frevert et al. (6) found that removal of the majority of cell surface HSPGs had a minimal inhibitory effect on sporozoite invasion of cells. One interpretation of these data is that the binding of CS to HSPGs does not function during sporozoite invasion. Another possibility, however, is that CS binding to HSPGs functions in the more dynamic conditions found in the blood circulation and leads to arrest of sporozoites in the liver sinusoids. In this paper we modify the standard sporozoite invasion assay and provide evidence that the interaction between CS and cell surface HSPGs functions during the initial attachment of sporozoites to cells under conditions that mimic flow. In addition, we show that the sulfate moieties of the HSPG glycosaminoglycan chains (GAGs) are important for attachment of sporozoites to cells. MATERIALS AND METHODSSpo...

A Binding Site for Highly Sulfated Heparan Sulfate Is Identified in the N Terminus of the Circumsporozoite Protein

“…This is supported by an earlier study in which soluble heparin required both these side groups for efficient inhibition of recombinant CSP binding to HepG2 cells (29). Furthermore, the spacing of charged groups on HS was important for binding to region I-plus because CS and DS were poor competitors even though they both have higher sulfate contents than HS.…”

“…It is also clear that at least one of the cell surface ligands for CSP is HS (6,29,30), and there have been a number of studies suggesting that region II-plus (region II plus eight downstream residues) serves as the actual HS binding site (21,22,31). However, in these studies, the HS binding activity of region II-plus was determined indirectly in cell culture after HS-lyase digestion by showing diminished binding in Chinese hamster ovary cells deficient in proteoglycan synthesis and by heparin and HS prevention of proteolytic digestion of CSP.…”

A long and winding road: The Plasmodium sporozoite's journey in the mammalian host

“…The addition of sulfate moieties to HSPG GAGs is one of these modifications and as stated above, liver HSPGs are more highly sulfated compared to HSPGs from other organs [43], suggesting that the degree of GAG chain sulfation could account for the selective targeting of sporozoites to the liver. Indeed it has been found that CSP binds preferentially to cells expressing HSPGs with highly sulfated GAGs [38,39] and that sporozoite attachment to cells decreases as the degree of GAG chain sulfation decreases [38]. One confounding issue is that these highly sulfated HSPGs are found behind the endothelial cell barrier.…”

“…Several lines of evidence suggest that this arrest is mediated by the interaction between the major surface protein of sporozoites, the circumsporozoite protein (CSP) and the highly sulfated HSPGs of hepatocytes [35][36][37][38][39]. Recombinant CSP binds specifically to hepatic HSPGs and in vivo, the physiologic ligands for hepatic HSPGs, namely lactoferrin and lipoprotein remnants, delay CSP clearance from the circulation and inhibit sporozoite infection of hepatocytes [40].…”

A new release on life: emerging concepts in proteolysis and parasite invasion

“…1B) whereas capping is observed for apical invasion proteins. Shaving occurs continuously as the parasite penetrates and is (Dubremetz et al ., 1985;Grimwood and Smith, 1995) Yes (Mineo and Kasper, 1994;He et al ., 2002) Unknown TgMIC1/MIC4/MIC6 (Meissner et al ., 2002b) Yes (Fourmaux et al ., 1996;Brecht et al ., 2001) MPP1 TgMIC2/M2AP Yes (Carruthers et al ., 1999;Harper et al ., 2004) MPP1 Zhou et al ., 2004) TgMIC3/MIC8 (Meissner et al ., 2002b) Yes (Garcia-Reguet et al ., 2000;Meissner et al ., 2002b) MPP1 TgAMA1 (Donahue et al ., 2000;Hehl et al ., 2000) Unknown MPP1 (S. A. Howell, M. J. Blackman and V. B. Carruthers, unpublished) Plasmodium PfTRAP and PbTRAP (Bhanot et al, 2003;Silvie et al ., 2004) Yes (McCormick et al ., 1999;Akhouri et al ., 2004) Serine protease (Silvie et al .. 2004) PfCSP (Stewart and Vanderberg, 1988; Yes (Pinzon-Ortiz et al ., 2001) Unknown PfMSP1/MSP6/MSP7 (Stafford et al ., 1994) Yes (Goel et al ., 2003;Li et al ., 2004) MESH (Howell et al ., 2003) PkAMA1 and PfAMA1 (Deans et al ., 1984;Howell et al ., 2001) Yes (Fraser et al ., 2001) MESH (Howell et al ., 2003) EBL-DBP (Camus and Hadley, 1985;Haynes et al ., 1988) Yes (Camus and Hadley, 1985;Haynes et al ., 1988) Unknown Py235 (Ogun and Holder, 1996) Yes (Ogun and Holde...…”