Wenlu Li

The accumulation and metabolism of a fluorescent ceramide derivative inPlasmodium falciparum-infected erythrocytes

We have examined the accumulation and metabolism of N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]aminocaproyl sphingosine (C6-NBD-cer) in Plasmodium falciparum FCR-3/A2-infected erythrocytes. C6-NBD-cer transferred to live infected erythrocytes at 2 degrees C to label the infected red cell surface and intracellular parasite membranes. Subsequent incubation for 30 min at 2 degrees C, resulted in a depletion of the ceramide label from the red cell membrane and an accumulation of fluorescence in parasite membranes, by an energy independent process. When the cells were subsequently warmed to 37 degrees C for 30 min, virtually all of the ceramide was converted to N-[7-(4-nitrobenzo-2-oxa-1,3- diazole)]aminocaproyl sphingosine-1-phosphocholine (C6-NBD-Sm). Uninfected erythrocytes were incapble of sphingomyelin synthesis. By fluorescence microscopy, sphingomyelin synthesis in infected erythrocytes occurred in compartments morphologically similar to those accumulating ceramide. To examine the intracellular sites of ceramide accumulation glutaraldehyde fixed cells were labeled with C6-NBD-ceramide and subsequently back extracted to remove excess probe. This resulted in a depletion of label at the red cell membrane but prominent fluorescence remained associated with the parasite. Photobleaching in the presence of diaminobenzidine resulted in precipitates in intraerythrocytic cisternae and the vacuolar membrane surrounding the parasite, rather than a perinuclear Golgi apparatus within the organism. The results support a novel organisation of plasmodial membranes regulating the accumulation and metabolism of C6-NBD-cer in infected erythrocytes.

Stage-specific expression of plasmodial proteins containing an antigenic marker of the intraerythrocytic cisternae.

A monoclonal antibody, LWLI, recognized 3 proteins of 45, 50 and 102 kDa in Plasmodium falciparum-infected erythrocytes. The 45- and 50-kDa proteins were parasite-encoded and displayed markedly different peptide maps, indicating that they were distinct plasmodial polypeptides with a common antigenic epitope rather than differentially processed forms of a primary translational product. The 45-kDa protein was present throughout intraerythrocytic growth, while the 50-kDa molecule was not detected earlier than 11 h in the life cycle. The 102-kDa protein was only expressed in trophozoite- and schizont-infected red cells: its structural relationship to the 45- and 50-kDa proteins, if any, remains undefined. By indirect immunofluorescence and immunoelectron microscopy, LWLI bound to flattened intraerythrocytic cisternae exported into the erythrocyte cytoplasm. The results support the theory that proteins recognized by the antibody were concentrated in these compartments and their common antigenic epitope may serve as a marker for the cisternae. Stage-specific expression of LWLI reactive proteins implicates developmental regulation of cisternal functions during asexual parasite development.

RECOGNITION OF A 170 KD PROTEIN IN MAMMALIAN GOLGI COMPLEXES BY AN ANTIBODY AGAINST MALARIAL INTRAERYTHROCYTIC LAMELLAE

Abstract Human erythrocytes infected with the malarial parasite Plasmodium falciparum contain flattened membrane lamellae. It has been suggested that the lamellae may be involved in the sorting of malarial proteins to the cytoplasm and the cell membrane of the host erythrocyte. We have previously shown that the lamellae accumulate sphingolipids by virtue of their lipid composition in a manner similar to the trans-Golgi and the trans-Golgi network in mammalian cells. In this paper, we show by immunofluorescence microscopy that a monoclonal antibody to the lamellae labelled a perinuclear organelle that colocalized with WGA and the mannose-6-phosphate receptor in cultured mammalian cells. Immunoelectron microscopy experiments revealed that LWLI labels cisternae of the trans-face and the trans-Golgi network. Western blot analysis of subcellular fractions using LWLI detected a 170 kD protein which is associated with the luminal side of Golgi membranes of rat liver and is conserved in all cell lines studied. Our results indicate that (i) the 170 kD protein is a novel marker of the mammalian trans-Golgi and the trans-Golgi network and (ii) in addition to similarities in their morphological and lipid characteristics, the lamellae induced by P. falciparum in erythrocytes share proteinaceous determinants with the Golgi apparatus of mammalian cells.