Araxie Kilejian

Selective association of a fragment of the knob protein with spectrin, actin and the red cell membrane.

The knob protein of Plasmodium falciparum is essential for the formation of knob-like protrusions on the host erythrocyte membrane. A functional domain of the knob protein was identified. This peptide formed stable complexes with the two major red cell skeletal proteins, spectrin and actin. When introduced into resealed normal erythrocytes, the peptide associated selectively with the cytoplasmic surface of the membrane and formed knob-like electron dense deposits. Knobs are thought to play an important role in the immunopathology of P. falciparum infections. Our findings provide a first step towards understanding the molecular basis for selective membrane changes at knobs.

The primary structure of a Plasmodium falciparum polypeptide related to heat shock proteins.

A cDNA library constructed from ring-stage RNA isolated from Plasmodium falciparum FCR-3/Gambia was screened with immune human serum and two related positive clones were isolated. Nucleotide sequence analysis of these recombinant clones revealed an open translational reading frame for 681 amino acids with a calculated molecular weight of 74.3 kDa. The deduced amino acid sequence of the polypeptide shows extensive homology to several heat shock proteins (hsp) which have been described. Northern and Southern hybridization analysis indicates that P. falciparum has a second gene which shares common sequences with the hsp gene described in this study.

Structure of the knob protein (KP) gene of Plasmodium falciparum

We have determined the nucleotide sequence of the gene encoding the knob protein (KP) of Plasmodium falciparum (FCR-3/Gambia). The gene is interrupted by an intron which contains 34 imperfect tandemly repeated ATTTT sequences. The first exon encodes 33 amino acids with a hydrophobic core typical of signal peptides. The second exon has an open translational reading frame for 597 amino acids. The deduced protein sequence indicates that KP has multiple structural domains; unlike the N-terminal histidine-rich domain which we described previously, the C-terminal half is rich in lysine residues. Consistent with the apparent association of KP with the cytoplasmic surface of the host erythrocyte membrane, the protein is highly charged and hydrophilic.

The biosynthesis of the knob protein and a 65 000 dalton histidine-rich polypeptide of Plasmodium falciparum

Previous studies have shown the association of an 80 kDa polypeptide (KP) with the knobs which develop on the membranes of erythrocytes infected with Plasmodium falciparum. KP was also found to share antigenic determinants with the histidine-rich protein of Plasmodium lophurae. In this study, ring stages of knobby (K+) and knobless (K-) variants of P. falciparum were used in pulse-chase experiments to elucidate the temporal sequence of the biosynthesis of KP. Analysis of radiolabeled parasite-polypeptides on SDS-polyacrylamide gels indicated that pulse-labeled KP has the electrophoretic mobility of a 75 kDa polypeptide and is subsequently chased to an apparently 80-85 kDa form. In addition to KP, antibodies raised against HRP immunoprecipitated a 65 kDa histidine-rich polypeptide from K- as well as K+ parasites. Differential incorporation of selected amino acids into KP and the 65 kDa polypeptide revealed some distinct differences between these two polypeptides as well as from HRP.

Expression, partial purification and immunogenicity of fragments of the knob protein of Plasmodium falciparum

Three structural domains of the histidine-rich knob protein (KP) of Plasmodium falciparum were expressed in Escherichia coli. A single-step purification scheme was devised to obtain great enrichment of expressed polypeptides for use in subsequent experiments. Immune human sera from Africa, South-East Asia and South America were tested for reactivity with each of the expressed fragments. While the two fragments which represented the central and C-terminal regions of KP showed a strong reactivity with all the antisera which were tested, the N-terminal fragment which contains the repetitive histidine-rich sequences showed almost no reactivity.

Plasmodium falciparum: Comparison of the genomic organization of the knob protein gene in knobby and knobless variants

The genomic organization of the knob protein (KP) gene of knobby (K+) and knobless (K-) variants of the Thai isolate NT 108 and the Gambian isolate FCR-3 are compared. The restriction enzyme maps and the chromosomal location of the KP gene of K+ variants of both isolates are apparently identical. A comparison of the susceptibility of the KP gene to deletions and the extent of deletion of chromosomal DNA in K- variants of each geographical isolate suggests isolate-specificity of the stability of the deleted DNA sequences. With the exception of a mutant K- population of FCR-3, which could not be distinguished from K+ FCR-3, in all other K- variants of both geographical isolates the deletion of the KP gene was accompanied by the loss of several hundred base pairs of DNA from chromosome 2. The deletion resulted in the localization of the mutant KP gene in proximity to telomeric DNA sequences.

The biosynthesis of the histidine-rich protein of Plasmodium lophurae and the cloning of its gene in Escherichia coli

Uninucleate trophozoites and schizonts of Plasmodium lophurae were labeled metabolically with [3H]proline. Analysis of labeled parasites indicated that the histidine-rich protein (HRP) was the major polypeptide synthesized by both developmental stages; in trophozoites it represented a larger proportion of total labeled polypeptides. Polyadenylated RNA was prepared from trophozoites and translated in a rabbit reticulocyte lysate system in the presence of [3H]histidine. As compared to the approx. 50 kDa mature protein, in the cell-free system HRP was synthesized as an approx. 58 kDa precursor polypeptide. Size-selected, polyadenylated RNA was used to construct a complementary double-stranded DNA library in pBR322 and plasmids containing HRP sequences were identified by hybridization with a synthetic oligonucleotide probe.

Reactivity of a monoclonal antibody produced to the histidine-rich protein of Plasmodium lophurae with Plasmodium falciparum.

Monoclonal antibodies were produced against the histidine-rich protein of Plasmodium lophurae and tested for reactivity with Plasmodium falciparum antigens. One anti-histidine-rich protein monoclonal antibody showed immunological cross-reactivity with polypeptides of P. falciparum synthesized in vivo and in vitro.

The Knob Protein Gene of Plasmodium Falciparum

Membranes of erythrocytes infected with Plasmodium falciparum develop protrusions called knobs (Trager et al. 1966). These structures mediate the adhesion of parasitized cells to venous endothelium (Luse & Miller 1971) and are essential for the survival of parasites in natural infection; laboratory derived variants which lose their ability to produce knobs (K-) also lose their pathogenecity in susceptible hosts (Langreth & Peterson, 1985; Green et. al. 1985).