The circumsporozoite protein (CSP) is the major surface protein of Plasmodium sporozoites, the infective stage of malaria. Although CSP has been extensively studied as a malaria vaccine candidate, little is known about its structure. Here, we show that CSP is proteolytically cleaved by a papain family cysteine protease of parasite origin. Our data suggest that the highly conserved region I, found just before the repeat region, contains the cleavage site. Cleavage occurs on the sporozoite surface when parasites contact target cells. Inhibitors of CSP processing inhibit cell invasion in vitro, and treatment of mice with E-64, a highly specific cysteine protease inhibitor, completely inhibits sporozoite infectivity in vivo.Malaria infection is initiated when an infected Anopheline mosquito injects sporozoites during a blood meal. After injection, sporozoites enter the bloodstream and go to the liver, where they invade hepatocytes and develop into exoerythrocytic forms. The circumsporozoite protein (CSP) is the major surface protein of the sporozoite and forms a dense coat on the parasite's surface. Studies have shown that CSP mediates sporozoite adhesion to target cells (for review see reference 1) and that it is required for sporozoite development in the mosquito (2). In addition, CSP has been extensively studied as a vaccine candidate and, thus far, is the only Plasmodium protein shown to confer protection to immunized individuals (for review see reference 1).Comparison of the deduced amino acid sequences of CS proteins from all species of Plasmodium shows that they have a similar overall structure (see Fig. 1 A and reference 1). They all contain a central repeat region whose amino acid sequence is species specific and two conserved regions: a five amino acid sequence called region I, immediately before the repeats, and a known cell-adhesive sequence with similarity to the type I thrombospondin repeat (TSR; reference 3). CSP has a canonical glycosylphosphatidyl inositol (GPI) anchor addition sequence in its COOH terminus; however, the presence of a GPI anchor has not been demonstrated.It was noted 20 yr ago that CSP immunoprecipitated from sporozoite lysates consists of one to two high MW bands (that differ by ‫ف‬ 1 kD) and a low MW band that is 8-10 kD smaller (4, 5). Biosynthetic studies showed that the initial label is incorporated into the top bands and the lower MW band appears later as a processed product (4, 5). The precise nature of this processing, as well as its functional significance, have remained unknown. In this report, we have determined the structural basis for this conserved feature of CSPs and have explored its role during sporozoite invasion of hepatocytes. RESULTS AND DISCUSSIONThe NH 2 -terminal portion of CSP is proteolytically cleaved by a cysteine protease To study the structure of the high and low MW CSP forms, we made polyclonal antisera to peptides representing the entire NH 2 -terminal and COOH-terminal thirds of CSP from Plasmodium berghei , a rodent malaria parasite. These antisera ...

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...