Monoclonal antibody epitope mapping of Plasmodium falciparum rhoptry proteins.
Plasmodium falciparum rhoptry proteins of the 140/130/110-kDa high molecular weight complex (HMWC) are secreted into the erythrocyte membrane during merozoite invasion. Epitopes of membrane-associated HMWC proteins can be detected using rhoptry-specific antibodies by immunofluorescence assays. Phospholipase treatment of ring-infected intact human erythrocytes, membrane ghosts, and inside-out vesicles results in the release of the HMWC as demonstrated by immunoblotting. We characterized the membrane-associating properties of the 110-kDa protein in more detail. PLA2 from three different sources; bee venom, Naja naja venom, and porcine pancreas, were examined and all were equally effective in releasing the 110-kDa protein. Furthermore, PLA2 activity was inhibited by o-phenanthroline, quinacrine, maleic anhydride, and partially by p-bromophenacyl bromide, indicating that the activity of PLA2 is specific. Using sequential protease and phospholipase digestion experiments to map the immunoreactive and functional epitopes of the 110-kDa protein, a 35-kDa protease-resistant protein associated with mouse and human erythrocyte membranes was identified. Limited proteolysis of the 110-kDa protein and analysis by immunoblotting demonstrated several immunoreactive cleavage products, including a highly protease-resistant peptide fragment of approximately 35-kDa which corresponds to the membrane-associated protein. Epitope mapping of the 130-kDa rhoptry protein resulted in a different pattern of cleavage products. Stage-specific metabolic labeling of P. falciparum with [3H] palmitate and [3H] myristate was performed to determine the lipophilic properties of the HMWC. Results showed the incorporation of label into proteins of approximate molecular weight 200 and 45-kDa, predominantly in the late schizont stage. Interestingly, proteins of 140 and 110/100-kDa, corresponding to [35S] methionine-labeled proteins were labeled with [3H]palmitate in ring-infected erythrocyte membranes. However, these proteins were not immunoprecipitated by a rhoptry protein-specific monoclonal antibody, 1B9. Similar label incorporation was not obtained with [3H]myristate. In Triton X-114 solubility studies, the HMWC proteins partitioned into the aqueous phase, suggesting that they are not integral membrane proteins. In addition, the proteins were extracted by 100 mM Na2CO3, pH 11.5, and immunoprecipitated by rhoptry-specific antibody. These results suggest that the HMWC proteins may exist in a soluble and membrane bound form. The latter may participate in membrane expansion and the formation of the parasitophorous vacuole during merozoite invasion.
Sam-Yellowe
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Department of Biology, Cleveland State University, Ohio 44115-2403.
Ndengele
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D000818:Animals; D000911:Antibodies, Monoclonal; D002254:Carbonates / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology; D000939:Epitopes / Q000276:immunology*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes; D006801:Humans; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009024:Morphogenesis; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D017830:Octoxynol; D015388:Organelles / Q000187:drug effects / Q000276:immunology*; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D054467:Phospholipases A2; D010963:Plasmodium falciparum / Q000276:immunology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D015800:Protozoan Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002254:Carbonates; D000939:Epitopes; D009227:Myristic Acids; D010169:Palmitic Acids; D015800:Protozoan Proteins; D019814:Myristic Acid; D019308:Palmitic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D008715:Methionine; D010741:Phospholipases A; D054467:Phospholipases A2; D010450:Endopeptidases