A cDNA clone expressing a rhoptry protein of Plasmodium falciparum

Rl, Coppel; Bianco, Angelica; Culvenor, JG; Pe, Crewther; Gv, Brown; Rf, Anders; Dj, Kemp

doi:10.1016/0166-6851(87)90020-x

Cited by 43 publications

(20 citation statements)

References 26 publications

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“…gDNA extracted from trophozoites (24-36 hours) was analyzed by Southern blot hybridization using standard procedures and the KAHRP gene as probe. 25 The trypsin-cleavage assay to determine surface PfEMP1 was performed as described using synchronized trophozoites (20-28 hours after invasion) harvested by magnetic cell sorting (CS columns; Mitenyi Biotec, Auburn, CA). 26 The SDS-soluble fractions of the preparations were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed with polyclonal rabbit anti-ATS antiserum (1:1000) for PfEMP1.…”

Section: Methodsmentioning

confidence: 99%

The role of KAHRP domains in knob formation and cytoadherence of P falciparum-infected human erythrocytes

Rug

Prescott

Fernandez

et al. 2006

Blood

143

153

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Surface protrusions of Plasmodium falciparum-infected erythrocytes, called knobs, display focal aggregates of P falciparum erythrocyte membrane protein 1 (PfEMP1), the adhesion ligand binding endothelial-cell receptors. The resulting sequestration of infected erythrocytes in tissues represents an important factor in the course of fatalities in patients with malaria. The main component of knobs is the knob-associated histidine-rich protein (KAHRP), and it contributes to altered mechanical properties of parasiteinfected erythrocytes. The role of KAHRP domains in these processes is still elusive. We generated stable transgenic P falciparum-infected erythrocytes expressing mutant versions of KAHRP. Using atomic force and electron microscopy we show that the C-terminal repeat region is critical for the formation of functional knobs. Elasticity of the membrane differs dramatically between cells with different KAHRP mutations. We propose that the 5 repeat region of KAHRP is important in cross-linking to the host-cell cytoskeleton and this is required for knob protrusion and efficient adhesion under physi- IntroductionPlasmodium falciparum is the most lethal malaria parasite of humans. 1 An important aspect in virulence of P falciparum is the ability of infected erythrocytes to sequester in and obstruct the microvasculature of different organs. 2 These abnormal circulatory properties of erythrocytes involve parasite-induced alterations in their biomechanical and adhesive properties and are important for survival and pathogenicity of P falciparum. 3 Cytoadhesion is mediated by the antigenically variant P falciparum erythrocyte membrane protein-1 (PfEMP1), which can bind to host receptors including CD36 and chondroitin sulfate A (CSA). 4 PfEMP1 is concentrated on electron-dense elevations of the membrane referred to as knobs, 5-7 providing a platform for adherence under physiologic flow conditions. 8 Increased erythrocyte rigidity and adhesiveness result in dramatically augmented hemodynamic resistance observed in microvasculature perfused with P falciparum-infected erythrocytes. 9 Knobs consist predominantly of the knob-associated histidine-rich protein (KAHRP), 10,11 assembling on the cytoplasmic face of the membrane. KAHRP is required for knob formation. 8 KAHRP has a signal sequence with a recessed hydrophobic core that directs the protein into the parasitophorous vacuole via the endomembrane system. 12 A second signal, we termed the "Plasmodium export element" (PEXEL), 13 is required for transfer across the parasitophorous vacuole membrane to the erythrocyte cytosol. 13,14 The PEXEL is followed by a histidine-rich region containing sequences responsible for interaction with Maurer clefts, 15 structures assembled in the P falciparum-infected erythrocyte cytosol important in protein sorting and trafficking. 12 There are also 2 blocks of highly charged repeats, designated the 5Ј and 3Ј repeats according to their location. 10,11 Current evidence suggests KAHRP interacts with various cytoskeletal components of the erythr...

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Section: Methodsmentioning

confidence: 99%

The role of KAHRP domains in knob formation and cytoadherence of P falciparum-infected human erythrocytes

Rug

Prescott

Fernandez

et al. 2006

Blood

143

153

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“…Protein concentration was determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. The C-terminal fragment of RhopH3 protein, Ag44 (19), expressed as a GST fusion, was kindly provided by F. Motorina.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Membrane-associated Rhoptry Protein of Plasmodium falciparum

Topolska

Lidgett

Truman

et al. 2004

Journal of Biological Chemistry

117

136

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Invasive forms of apicomplexan parasites contain secretory organelles called rhoptries that are essential for entry into host cells. We present a detailed characterization of an unusual rhoptry protein of the human malaria parasite Plasmodium falciparum, the rhoptryassociated membrane antigen (RAMA) that appears to have roles in both rhoptry biogenesis and host cell invasion. RAMA is synthesized as a 170-kDa protein in early trophozoites, several hours before rhoptry formation and is transiently localized within the endoplasmic reticulum and Golgi within lipid-rich microdomains. Regions of the Golgi membrane containing RAMA bud to form vesicles that later mature into rhoptries in a process that is inhibitable by brefeldin A. Other rhoptry proteins such as RhopH3 and RAP1 are found in close apposition with RAMA suggesting direct protein-protein interactions. We suggest that RAMA is involved in trafficking of these proteins into rhoptries. In rhoptries, RAMA is proteolytically processed to give a 60-kDa form that is anchored in the inner face of the rhoptry membrane by means of the glycosylphosphatidylinositol anchor. The p60 RAMA form is discharged from the rhoptries of free merozoites and binds to the red blood cell membrane by its most C-terminal region. In early ring stages RAMA is found in association with the parasitophorous vacuole.Plasmodium falciparum malaria is one of the most important infectious diseases of humans, accounting for ϳ2 million deaths each year. The stages of the parasite that grow and multiply in red blood cells (RBCs) 1 cause all the pathological effects associated with the disease, and accordingly invasion of red blood cells is one of the most important steps in the parasite life cycle. Three sets of secretory organelles, the rhoptries, micronemes, and dense granules are involved in and are essential for the invasion process. The understanding of the role of these organelles provides important knowledge about the basic biology of malaria and potential therapeutical targets.Rhoptries of Plasmodium parasites are paired club-shaped organelles located at the apical end of merozoites, the form of the parasite that invades RBCs. Following the attachment of merozoites to the RBC surface, rhoptries discharge their contents onto the RBC membrane (1). Rhoptry organelles disappear after internalization of merozoites and thus are formed de novo with each erythrocytic cycle. Rhoptry formation occurs late in the erythrocytic stages of the parasite, and elucidation of rhoptry biogenesis of malaria parasites has been hindered by the lack of early organelle markers. Most of our knowledge is based on microscopic examinations, which suggest that rhoptry biogenesis follows the secretory pathway route, with rhoptry organelles being formed by sequential fusion of post-Golgi vesicles (2, 3), although why particular vesicles are selected is unclear.Rhoptry contents include both protein and lipid components, which assemble to form membrane-like structures. Protein constituents of the rhoptry contents are stil...

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“…Nucleic Acid Analysis-Genomic DNA was isolated from mixed trophozoite/schizont stage parasites as described (34). Manipulation of recombinant DNA and analysis of nucleic acids by Southern blot hybridization were carried out using standard procedures (35).…”

Section: Methodsmentioning

confidence: 99%

A Subset of Plasmodium falciparum SERA Genes Are Expressed and Appear to Play an Important Role in the Erythrocytic Cycle

Miller¹,

Good

Drew³

et al. 2002

Journal of Biological Chemistry

160

173

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The Plasmodium falciparum serine repeat antigen (SERA) has shown considerable promise as a blood stage vaccine for the control of malaria. A related protein, SERPH, has also been described in P. falciparum. Whereas their biological role remains unknown, both proteins possess papain-like protease domains that may provide attractive targets for therapeutic intervention. Genomic sequencing has recently shown that SERA and SERPH are the fifth and sixth genes, respectively, in a cluster of eight SERA homologues present on chromosome 2. In this paper, the expression and functional relevance of these eight genes and of a ninth SERA homologue found on chromosome 9 were examined in blood stage parasites. Using reverse transcriptase-PCR and microarray approaches, we demonstrate that whereas mRNA to all nine SERA genes is synthesized late in the erythrocytic cycle, it is those genes in the central region of the chromosome 2 cluster that are substantially up-regulated at this time. Using antibodies specific to each SERA, it was apparent that SERA4 to -6, and possibly also SERA9, are synthesized in blood stage parasites. The reactivity of antibodies from malaria-immune individuals with the SERA recombinant proteins suggested that SERA2 and SERA3 are also expressed at least in some parasite populations. To examine whether SERA genes are essential to blood stage growth, each of the eight chromosome 2 SERA genes was targeted for disruption. Whereas genes at the periphery of the cluster were mostly dispensable (SERA2 and -3 and SERA7 and -8), those in the central region (SERA4 to -6) could not be disrupted. The inability to disrupt SERA4, -5, and -6 is consistent with their apparent dominant expression and implies an important role for these genes in maintenance of the erythrocytic cycle.

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A cDNA clone expressing a rhoptry protein of Plasmodium falciparum

Cited by 43 publications

References 26 publications

The role of KAHRP domains in knob formation and cytoadherence of P falciparum-infected human erythrocytes

The role of KAHRP domains in knob formation and cytoadherence of P falciparum-infected human erythrocytes

Characterization of a Membrane-associated Rhoptry Protein of Plasmodium falciparum

A Subset of Plasmodium falciparum SERA Genes Are Expressed and Appear to Play an Important Role in the Erythrocytic Cycle

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