Late stage erythrocytic forms of Plasmodia synthesize glycoproteins that insert into the membranes of host red blood cells. A prominent such protein made by P. knowlesi is a Mr 74,000, pI 4.8 glycoprotein called GP74. This is exposed at the external surfaces of infected erythrocytes, reacts with antibodies from immune animals, shares antigenic determinants with P. falciparum proteins and, according to vaccination trials, can confer protection against otherwise lethal infections with P. knowlesi. This program aims to define mechanisms of biosynthesis, processing and membrane insertion of GP74; (b) find the extent of peptide and immunological homology between GP74, other P. knowlesi proteins and other parasite-synthesized host cell membrane proteins of Plasmodium, and (c) evaluate the functional and immunological roles of GP74 related proteins. We seek to answer the following specific questions: 1. Are there precursors for GP74 and how are they expressed? 2. How is GP74 inserted into membranes? 3. How is GP74 transferred to the host cell membrane? 4. What relationships exist between GP74, other P. knowlesi and P. falciparum proteins. Technical approaches include pulse-chase labeling of parasitzed erythrocytes with radioactive amino acid and sugar precursors, cell-free translation of plasmodial mRNA, without and with microsomal membrane acceptors (amino acid and saccharide labeling; kinetics of insertion, peptide exposure after insertion), surface-labeling of intact cells and inside-out vesicies (lactoperoxidase-catalyzed radioiodination; galactose oxidase/borotritride), protease treatment and protein/peptide analysis after metabolic of surface - labeling, cross-linking analyses of protein/protein interactions, metabolic labeling of membrane glycoproteins by isolated parasites, vs. cell-free translation and intact parasitized cells, surface-labeling of intraerythrocytic stages vs. intact infected erythrocytes, fractionation of infected cells after metabolic labeling to identify """"""""migration vesicies"""""""", analysis of labeled membrane proteins by immunoprecipitation (immune sera, monoclonal antibodies) followed by DS PAGE or IEF/DS PAGE, and peptide mapping.
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