The broad, long-term objectives of this project are to define the mechanisms involved in the interaction between hepatocytes and malaria sporozoites and to develop specific inhibitors that prevent the crucial hepatic stage of the disease.
The specific aims are to characterize the heparan sulfate proteoglycans (HSPG) from hepatocytes which bind to the malaria circumsporozoite protein (CS) by 1) defining the oligosaccharide which recognizes the ligand region II-plus of the CS protein, 2)characterizing athe core protein, 3) demonstrating at the electron microscopical level the morphology of the CS-binding HSPG, and 4) examining at the cellular level the consequences of the interaction between the HSPG and CS, in particular, whether there is CS interiorization and signal transduction. Heparan sulfate oligosaccharides with avidity to the CS region II-plus will be isolated, depolymerized an their structure characterized by HPLC. Definition of the core protein includes the determination of its Mr in SDS- PAGE, of its reactivity with various monoclonal antibodies to cell surface HSPGs, and cloning of the corresponding gene. We will study the turnover of the cell surface HSPGs of HepG2 cells in the absence of CS, and then determine the fate of CS and of the HSPG receptors following their interaction. For this purpose, the HSPGs will be radiolabeled with [35S]- sulfate, and the CS (or region II-plus peptides) radiolabeled with 125I]- iodine. Changes in the phosphorylation patterns of HepG2 cells upon CS, or region II-plus peptide binding will be examined by phosphoamino acid analysis, and by means of antibodies to phosphotyrosine. Small oligosaccharides (or mimics) which bind to region II-plus and inhibit attachment of sporozoites to hepatocytes may have medical applications in the prophylaxis of malaria infection, and in addition may be valuable tools to study the function of host proteins which display the region II-plus motif, such as thrombospondin, properdin, the terminal complement components and F-spondin, a molecule involved in the development of the nervous system.
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