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PubMed1980

Labelling of membrane glycoproteins of cultivated Plasmodium falciparum.

Membrane glycoprotein synthesis by Plasmodium falciparum was determined by metabolic labelling in the presence of 74 kBq/ml (2.5 muCi/ml) glucosamine-(3)H. Five major glycoprotein bands and four minor ones were demonstrated. A control experiment using normal, outdated, human erythrocytes indicates that there was no incorporation of the labelled glucosamine into the erythrocyte membrane. It was also demonstrated that the rate of membrane glycoprotein synthesis by mature parasites of the trophozoite and schizont stages was twice that of the ring-stage parasites. Cytochemical surface-labelling experiments had led to the conclusion that the membrane of malaria parasites contains little or no glycoprotein. Our studies indicate, however, that there is significant synthesis of membrane glycoprotein by the parasite and that this can be metabolically labelled and measured by using radioactive glucosamine as precursor of the glycoprotein.

Carbon Radioisotopes↗

PubMed1994

Characterization of membrane proteins exported from Plasmodium falciparum into the host erythrocyte.

Plasmodium falciparum is an intracellular parasite of the red blood cell. During development it exports proteins which are transported to specific locations within the host erythrocyte. We have begun to identify and characterize exported membrane proteins of P. falciparum in order to obtain specific marker molecules for the study of the mechanisms involved in the distribution of parasite-derived proteins within the host cell. In this report we describe the characterization of a 35 kDa protein which is recognized by a monoclonal antibody. The protein is tightly associated with membranes isolated from infected erythrocytes; it is resistant to extraction with alkali and soluble after treatment with detergents. It is located at the membrane of the parasitophorous vacuole and in membrane-bound compartments which appear in the cytoplasm of the infected erythrocyte. The protein co-localizes with the previously described exported protein-1 (exp-1). Considering its localization and physical similarities to exp-1, we name the 35 kDa protein the exported protein-2 (exp-2).

Animals↗

PubMed1990

Genetic diversity of Plasmodium falciparum shows geographical variation.

Sixty Plasmodium falciparum isolates, 20 each from Thailand, Zimbabwe, and Brazil, were characterized for 20 variant genetic markers, including the enzymes glucose phosphate isomerase, adenosine deaminase and peptidase, 11 other proteins detected by 2-dimensional electrophoresis (2D-PAGE), 2 merozoite surface antigens (MSA-1 and MSA-2), one exported antigen (Exp-1), and sensitivity to the drugs chloroquine, pyrimethamine, and mefloquine. The study examines the extent of diversity between individual isolates and the differences in the frequency of certain variants of the markers between the 3 countries. The principal conclusions to be drawn from the study are that there is extensive polymorphism in many of the genetically determined characters of this parasite, multiple infections with greater than 1 genetically distinct parasite are common, and there are geographical variations in the frequencies with which variant forms of certain markers occur.

Alleles↗

PubMed1999

A shotgun optical map of the entire Plasmodium falciparum genome.

The unicellular parasite Plasmodium falciparum is the cause of human malaria, resulting in 1.7-2.5 million deaths each year. To develop new means to treat or prevent malaria, the Malaria Genome Consortium was formed to sequence and annotate the entire 24.6-Mb genome. The plan, already underway, is to sequence libraries created from chromosomal DNA separated by pulsed-field gel electrophoresis (PFGE). The AT-rich genome of P. falciparum presents problems in terms of reliable library construction and the relative paucity of dense physical markers or extensive genetic resources. To deal with these problems, we reasoned that a high-resolution, ordered restriction map covering the entire genome could serve as a scaffold for the alignment and verification of sequence contigs developed by members of the consortium. Thus optical mapping was advanced to use simply extracted, unfractionated genomic DNA as its principal substrate. Ordered restriction maps (BamHI and NheI) derived from single molecules were assembled into 14 deep contigs corresponding to the molecular karyotype determined by PFGE (ref. 3).

Animals↗

PubMed1984

Purification and characterization of an aminopeptidase from Plasmodium falciparum.

A soluble aminopeptidase from Plasmodium falciparum was purified by high performance liquid chromatography. The enzyme has a molecular weight of 100 000 and pI 6.8. Activity can be monitored conveniently with L-alanine-p-nitroanilide or L-leucine-p-nitroanilide at 405 nm or with L-leucine-7-amido-4-methylcoumarin in a fluorescence assay. The enzyme is inhibited by bestatin and phosphoramidone but not by leupeptin, chymostatin, antipain or pepstatin. pH-rate studies indicated the presence of a group on the free enzyme, pKa = 6.6, which must be in the conjugate base form for activity. The aminopeptidase has an essential sulfhydryl group at the active site which is rapidly modified by Hg2+ or Zn2+, is slowly modified by p-hydroxymercuribenzoate, but is not accessible to iodoacetamide or N-ethylmaleimide. The aminopeptidase is inhibited noncompetitively by chloroquine, mefloquine and quinacrine (Ki = 410, 280 and 20 microM, respectively) but is not inhibited by quinine or primaquine. Hemin does not inhibit. Complexation of hemin with quinacrine prevents inhibition by quinacrine.

Aminopeptidases↗

PubMed2003

Identification of nuclear proteins that interact differentially with Plasmodium falciparum var gene promoters.

The Plasmodium falciparum virulence factor PfEMP1 is responsible for both antigenic variation and cytoadherence of infected erythrocytes in malaria. Approximately 50 var genes per parasite genome code for this highly polymorphic surface protein. We showed recently that chromosome-central and subtelomeric var genes are controlled by different promoters. Here, we report that transcriptional repression of var genes located in different chromosomal regions occurs by different mechanisms. Subtelomeric var gene transcription is repressed 4-8 h before that of chromosome-central var genes. Both repression events coincide with the shifted expression of two distinct nuclear proteins binding specifically to conserved sequence motifs, SPE1 and CPE, present in the respective promoter. Furthermore, a reiterated and highly conserved subtelomeric var promoter element (SPE2) interacts with a nuclear factor exclusively expressed during S-phase. Promoter analysis by transient transfection suggested direct involvement of these interactions in var gene repression and silencing, and identified regions implicated in transcriptional activation of var genes.

Animals↗

PubMed1987

Optimal concentration of p-aminobenzoic acid and folic acid in the in vitro assay of antifolates against Plasmodium falciparum.

In vitro tests for Plasmodium falciparum sensitivity to pyrimethamine, sulfadoxine, and both drugs in combination were performed in four kinds of culture medium, each differing in p-amino benzoic acid (PABA) and folic acid concentrations. Results of the tests using pyrimethamine-sensitive and pyrimethamine-resistant isolates indicated that drug activity was reduced proportionally to the concentrations of these two growth factors in the medium. The optimal concentrations of PABA and folic acid for parasite growth and drug susceptibility, as evaluated by microscopic examination and by the extent of incorporation of radioactive 14C-pyrimethamine and 14C-sulfadoxine, were 10 ng/ml and 2 ng/ml, respectively. Depletion of PABA and folic acid from the medium had no effect on drug-resistant parasites but multiplication of drug-sensitive isolates was markedly reduced. Medium containing 0.5 ng/ml PABA and 10 ng/ml folic acid was the best for parasite growth regardless of the degree of drug sensitivity. Results obtained by using this medium agreed most closely with results from in vivo observations.

4-Aminobenzoic Acid↗

PubMed1988

Variability in drug susceptibility amongst clones and isolates of Plasmodium falciparum.

Heterogeneity within isolates of Plasmodium falciparum in regard to drug susceptibility is described from studies with three Thai isolates and some clones derived from them. One isolate (T9), which before cloning was resistant in vitro to chloroquine and pyrimethamine, contained a diverse assortment of clones, some of which were sensitive to one or other, or both, of these drugs. Another isolate (CH150) was initially sensitive to mefloquine in vitro, but, on recrudescence of infection in the patient, developed a number of clones all of which had diminished susceptibility to mefloquine. Drug pressure in a laboratory culture of CH150 resulted in a similar change in susceptibility. Hence resistant clones are thought to have been present as a minority component of the original isolate CH150. On testing uncloned isolates at different times after growth in culture, drug susceptibility showed considerable variability, but clones remained stable. Reaction in vitro of these isolates to some other drugs (amodiaquine, Fansidar, quinine) is also described, and the results are discussed in relation to changes in drug resistance of malaria parasites which may occur in laboratory cultures and under field conditions.

Animals↗

PubMed2005

A potential novel mechanism for the insertion of a membrane protein revealed by a biochemical analysis of the Plasmodium falciparum cytoadherence molecule PfEMP-1.

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is exposed on the surface of infected erythrocytes where it both acts as an important pathogenicity factor in malaria and undergoes antigenic variation as a means of immune evasion. Because the mammalian erythrocyte lacks a protein secretory machinery there has been much interest in elucidating the mechanism whereby this protein is transferred from its site of synthesis within the parasite to its final destination. Current opinion favours a mechanism whereby PfEMP-1 becomes cotranslationally inserted into the endoplasmic reticulum of the parasite and is subsequently transported as an integral part of an erythrocyte cytoplasmic membrane system derived from the parasite. Here we show that the solubility characteristics of this protein during several stages of its transport pathway are inconsistent with this view. Instead we propose that the protein is synthesized as a peripheral membrane protein which only when it arrives at its final destination assumes a transmembrane topology. Even in this state, the extractability of the protein with urea suggest that it is anchored in the membrane by protein-protein rather than by protein-lipid interaction.

Amino Acid Sequence↗

PubMed2004

Identification of a mitochondrial superoxide dismutase with an unusual targeting sequence in Plasmodium falciparum.

The intraerythrocytic stages of Plasmodium falciparum are exposed to oxidative stress and require functional anti-oxidant systems to survive. In addition to the parasite's known iron-dependent superoxide dismutase PfSOD1, a second SOD gene (PfSOD2) interrupted by 8 introns was identified on chromosome 6. Molecular modelling shows that the structure of PfSOD2 is similar to other iron-dependent SODs and phylogenetic analysis suggests PfSOD1 and PfSOD2 are the result of an ancestral gene duplication. The deduced amino acid sequence of PfSOD2 is similar to PfSOD1 but has a long N-terminal extension. Immunofluorescence studies show that PfSOD1 is cytosolic, whereas the N-terminal extension of PfSOD2 targets a green fluorescent protein fusion into the parasite's mitochondrion. Both SOD genes are transcribed during the erythrocytic cycle with PfSOD1 mRNA levels up to 35-fold higher than those of PfSOD2. Northern blots demonstrated that the mRNA levels of both SOD genes are up-regulated upon exposure to oxidative stress.

Adaptation, Psychological↗

PubMedDate not supplied

The alanine-rich heptad repeats are intact in the processed form of Plasmodium falciparum MSP3.

The potential of Plasmodium falciparum merozoite surface protein 3 as a component of an asexual-stage malaria vaccine is currently being assessed. The precursor form of MSP3 undergoes cleavage during schizogony to generate a mature processed form. It is unknown if this cleavage event is necessary for MSP3 function, but it may be an important consideration for assessing and developing MSP3 as an asexual-stage vaccine candidate. We have therefore determined the cleavage site in MSP3 by sequencing the N-terminus of the processed form of MSP3, which was isolated from parasite material. The position of the cleavage site indicates that the processed form of MSP3 retains the three blocks of alanine-rich heptad repeats, which are predicted to provide the structural framework for an intramolecular coiled-coil. The cleavage-site motif has many features in common with the published cleavage sites of MSP1(30), MSP6(36), and MSP7(22), which are all located on the merozoite surface and are implicated in the erythrocyte invasion process. The common cellular location and similar cleavage-site motifs suggest that these merozoite proteins may be cleaved by the same or related proteases.

Alanine↗

PubMed2004

Overproduction, purification, and characterization of the Plasmodium falciparum heat shock protein 70.

Plasmodium falciparum heat shock protein (PfHsp70) has been proposed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. However, the biochemical and chaperone properties of PfHsp70 have not been elucidated. The heterologous overproduction of P. falciparum proteins in Escherichia coli is problematic because of its AT-rich genome and the usage of codons that are rarely used in E. coli. In this paper, we describe the successful overproduction of (His)(6)-PfHsp70 in E. coli using the pQE30 expression vector system. Initial experiments with E. coli [pQE30/PfHsp70] resulted in the overproduction of the full-length protein and truncated derivatives. The RIG plasmid, which encodes tRNAs for rare codons, was engineered into the E. coli [pQE30/PfHsp70] strain, resulting in significant reduction of the truncated (His)(6)-PfHsp70 derivatives and improved yields of the full-length protein. (His)(6)-PfHsp70 was successfully purified using nickel-chelating Sepharose affinity chromatography and its biochemical properties were determined. The V(max), K(m), and k(cat) for the basal ATPase activity of (His)(6)-PfHsp70 were found to be 14.6 nmol/min/mg, 616.5 microM, and 1.03 min(-1), respectively. Gel filtration studies indicated that (His)(6)-PfHsp70 existed largely as a monomer in solution. This is the first study to biochemically describe PfHsp70 and establishes a foundation for future studies on its chaperone properties.

Adenosine Triphosphatases↗

PubMed1990

Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum demonstrates variability in its dependence upon erythrocyte sialic acid residues for invasion. Some lines of P. falciparum invade neuraminidase-treated or glycophorin-deficient red blood cells poorly, or not at all, while other lines invade such cells at substantial rates. To explore the molecular basis of non-sialic acid dependent invasion, we selected parasite lines from a clone (Dd2) that initially exhibited low invasion of neuraminidase-treated erythrocytes. After maintaining Dd2 for several cycles in neuraminidase-treated erythrocytes, parasite lines were recovered that invaded both untreated and neuraminidase-treated erythrocytes at equivalently high rates (Dd2/NM). The change in phenotype was maintained after removal of selection pressure. Four subclones of Dd2 were isolated and each readily converted from sialic acid dependence to non-sialic acid dependence during continuous propagation in neuraminidase-treated erythrocytes. The neuraminidase-selected lines and the Dd2 clone demonstrated identical restriction fragment length polymorphism markers indicating that the Dd2 clone was not contaminated during the selection process. Parasite proteins that bound to neuraminidase-treated and untreated erythrocytes were indistinguishable among the parent Dd2 clone and the neuraminidase-selected lines. The ability of the Dd2 parasite to change its invasion requirements for erythrocyte sialic acid suggests a switch mechanism permitting invasion by alternative pathways.

Animals↗

PubMed1994

Brefeldin A inhibits transport of the glycophorin-binding protein from Plasmodium falciparum into the host erythrocyte.

Plasmodium falciparum, a protozoan parasite of the human erythrocyte, causes the most severe form of malaria. During its intraerythrocytic development, the parasite synthesizes proteins which are exported into the host cell. The compartments involved in the secretory pathway of P. falciparum are still poorly characterized. A Golgi apparatus has not been identified, owing to the lack of specific protein markers and Golgi-specific post-translational modifications in the parasite. The fungal metabolite brefeldin A (BFA) is known to inhibit protein secretion in higher eukaryotes by disrupting the integrity of the Golgi apparatus. We have used the parasite-encoded glycophorin-binding protein (GBP), a soluble protein found in the host cell cytoplasm, as a marker to investigate the effects of BFA on protein secretion in the intracellular parasite. In the presence of BFA, GBP was not transported into the erythrocyte, but remained inside the parasite cell. The effect caused by BFA was reversible, and the protein could be chased into the host cell cytoplasm within 30 min. Transport of GBP from the BFA-sensitive site into the host cell did not require protein synthesis. Similar observations were made when infected erythrocytes were incubated at 15 degrees C. Incubation at 20 degrees C resulted in a reduction rather than a complete block of protein export. The relevance of our findings to the identification of compartments involved in protein secretion from the parasite cell is discussed.

Animals↗

PubMed2005

Structural metal dependency of the arginase from the human malaria parasite Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum possesses a single gene with high similarity to the metalloproteins arginase and agmatinase. The recombinant protein reveals strict specificity for arginine, and it has been proposed that its function in ornithine production is as a precursor for polyamine biosynthesis. The specific activity of the plasmodial arginase was found to be 31 micromol min(-1) mg(-1) protein and the k(cat) was calculated as 96 (s-1) . The Km value for arginine and Ki value for ornithine were determined as 13 mM and 19 mM, respectively. The active arginase is a homotrimer of ca. 160 kDa. Dialysis of the arginase against EDTA results in monomers of approximately 48 kDa; however, the quaternary structure can be restored by addition of Mn 2+ . Mutagenic analyses of all the amino acid residues proposed to be involved in metal binding led to complex dissociation, except for the His-193-Ala mutant, which was also inactive but retained the trimeric structure. Substitution of His-233, which has been suggested to be in charge of proton shuttling within the active site, disrupted the trimeric structure and thereby the activity of the Pf arginase. Northern blot analysis identified a stage-specific expression pattern of the plasmodial arginase in the ring/young trophozoite stage, which guarantees the provision of ornithine for essential polyamine biosynthesis.

Amino Acid Sequence↗

PubMed2006

Hypervariability within the Rifin, Stevor and Pfmc-2TM superfamilies in Plasmodium falciparum.

The human malaria parasite, Plasmodium falciparum, possesses a broad repertoire of proteins that are proposed to be trafficked to the erythrocyte cytoplasm or surface, based upon the presence within these proteins of a Pexel/VTS erythrocyte-trafficking motif. This catalog includes large families of predicted 2 transmembrane (2TM) proteins, including the Rifin, Stevor and Pfmc-2TM superfamilies, of which each possesses a region of extensive sequence diversity across paralogs and between isolates that is confined to a proposed surface-exposed loop on the infected erythrocyte. Here we express epitope-tagged versions of the 2TM proteins in transgenic NF54 parasites and present evidence that the Stevor and Pfmc-2TM families are exported to the erythrocyte membrane, thus supporting the hypothesis that host immune pressure drives antigenic diversity within the loop. An examination of multiple P.falciparum isolates demonstrates that the hypervariable loop within Stevor and Pfmc-2TM proteins possesses sequence diversity across isolate boundaries. The Pfmc-2TM genes are encoded within large amplified loci that share profound nucleotide identity, which in turn highlight the divergences observed within the hypervariable loop. The majority of Pexel/VTS proteins are organized together within sub-telomeric genome neighborhoods, and a mechanism must therefore exist to differentially generate sequence diversity within select genes, as well as within highly defined regions within these genes.

Amino Acid Sequence↗

PubMed1981

Partial purification and characterization of lactate dehydrogenase from Plasmodium falciparum.

Lactate dehydrogenase (LDH) from Plasmodium falciparum was partially purified by two different procedures. In the first procedure, parasitized erythrocytes (80% parasitemia) were lysed, and the soluble fraction was purified on DEAE-Sephadex to separate the parasite LDH(LDH-P) from the LDH isoenzymes present in the human erythrocytes. LDH-P was then purified by high-performance liquid chromatography (HPLC) on a TSK-G-3000 SW protein column. This two-step procedure gave LDH-P with specific activity 85 micromol/min/mg protein; this represented a 700-fold increase in specific activity relative to the starting lysate. Alternatively, parasites of P. falciparum were isolated by mechanical rupture of infected erythrocytes followed by differential centrifugation. The 100,000 X g supernatant obtained after lysis of these parasites showed LDH-P specific activity 3.6 micromol/min/mg protein. This activity was free of contaminating erythrocyte LDH as determined by electrophoresis and specific staining for LDH. Further purification of LDH-P by HPLC, as before, gave material with specific activity 98 micromol/min/mg protein. Recoveries of activity on HPLC were more than 90%, demonstrating the usefulness of this procedure for the partial purification of small quantities of parasite protein. The kinetic properties of LDH-P were compared with those of two of the human isozymes, LDH-H4 and LDH-M4 . LDH-P resembles LDH-H4 in its kinetic properties: KM (NADH) is 7, 8.3 and 1.3 microM for LDH-P, LDH-H4 and LDH-M4, respectively; KM (pyruvate) is 30, 60 and 180 microM for LDH-P, LDH-H4 and LDH-M4. LDH-P differs significantly from LDH-H4 and LDH-M4 in that LDH-P is not sensitive to inhibition by high pyruvate nor sensitive to inhibition by the complex between NAD+ and pyruvate. LDH-P is inactivated within seconds by sodium deoxycholate at concentrations that do not affect LDH-H4 and slowly inactivate LDH-M4.

Animals↗

PubMed2001

Proteolytic processing and primary structure of Plasmodium falciparum apical membrane antigen-1.

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a malaria merozoite integral membrane protein that plays an essential but poorly understood role in invasion of host erythrocytes. The PfAMA-1 ectodomain comprises three disulfide-constrained domains, the first of which (domain I) is preceded by an N-terminal prosequence. PfAMA-1 is initially routed to secretory organelles at the apical end of the merozoite, where the 83-kDa precursor (PfAMA-1(83)) is converted to a 66-kDa form (PfAMA-1(66)). At about the time of erythrocyte invasion, PfAMA-1(66) selectively translocates onto the merozoite surface. Here we use direct microsequencing and mass spectrometric peptide mass fingerprinting to characterize in detail the primary structure and proteolytic processing of PfAMA-1. We have determined the site at which processing takes place to convert PfAMA-1(83) to PfAMA-1(66) and have shown that both species possess a completely intact and unmodified transmembrane and cytoplasmic domain. Following relocation to the merozoite surface, PfAMA-1(66) is further proteolytically cleaved at one of two alternative sites, either between domains II and III, or at a membrane-proximal site following domain III. As a result, the bulk of the ectodomain is shed from the parasite surface in the form of two soluble fragments of 44 and 48 kDa. PfAMA-1 is not detectably modified by the addition of N-linked oligosaccharides.

Amino Acid Sequence↗